The Mulberry Genome (Compendium of Plant Genomes) 3031284771, 9783031284779

Table of contents :
Foreword
Preface to the Series
Preface
Contents
About the Editors
1 Botanical Features and Economic Significance of Mulberry
1.1 Introduction
1.2 Origin, Distribution, and Domestication
1.3 Taxonomy of Morus
1.4 Cytology
1.5 Biodiversity
1.5.1 Morphological Variations in Mulberry
1.6 Conservation of Biodiversity
1.6.1 Conservation Practices
1.7 Conclusion
References
2 Cultivation, Utilization, and Economic Benefits of Mulberry
2.1 Introduction
2.2 Indian Sericulture Scenario
2.3 Mulberry Cultivation
2.3.1 Agro-Climatic Regions and Sericultural Zones of India
2.3.2 Mulberry Species and Varieties Under Cultivation—An Overview
2.3.3 Mulberry Cultivation Practices
2.3.3.1 Climate and Soil Type
2.3.3.2 Planting Material, Propagation, and Planting Schedule
2.3.3.3 Planting Distance
2.3.3.4 Training and Pruning System
2.3.3.5 Irrigation
2.3.3.6 Nutrients and Fertilizers
2.3.3.7 Management of Disease and Pest
2.3.3.8 Leaf Harvest
2.4 Factors Affecting Mulberry Leaf Productivity
2.5 Utilization of Leaf
2.5.1 Utilization in Sericulture
2.5.2 Mulberry Leaf for Livestock
2.5.3 Mulberry in Food and Tea Industry
2.5.4 Mulberry Leaf for Medical Use
2.5.5 For Environmental Remediation
2.6 Economics of Mulberry Leaf Production
2.7 Conclusion and Future Prospective
References
3 Mulberry Breeding for Higher Leaf Productivity
3.1 Introduction
3.2 Breeding Objectives for Evolving Superior Mulberry Varieties
3.3 Prerequisites for Mulberry Improvement Breeding Programs
3.3.1 Germplasm and Genetic Diversity
3.3.2 Pre-breeding Study
3.3.3 Floral Biology and Anthesis
3.3.4 Crossability Study
3.3.5 Study of Genetics of Mulberry for Designing a Breeding Scheme
3.4 Conventional Breeding Approaches
3.4.1 Importation or Introduction of Mulberry Varieties
3.4.2 Screening and Selection from Natural Open-Pollinated Populations or Varieties
3.4.3 Cross-Breeding/Controlled Pollination by Hybridization
3.4.3.1 Method of Pollination
3.4.3.2 Types of Hybridization/Controlled Pollination
3.4.3.3 Synchronization of Flowering Time and Pollen Collection
3.4.3.4 Hybridization Through Cuttings
3.4.3.5 Modification of Flowering
3.4.4 Polyploidy Breeding
3.4.4.1 Artificial Induction of Polyploidy
Colchicine Treatment for Seeds
Colchicine Treatment for Bud
Induction of Tetraploidy by Radiation
Development of Tetraploids Through Hybridization
3.4.4.2 Cutting Back Method
3.4.4.3 Induction of Triploids Through Hybridization
3.4.4.4 Salient Features of Polyploids
3.4.4.5 Identification of Mulberry Polyploids
3.4.5 Mutation Breeding in Mulberry
3.4.5.1 Advantages of Mutation Breeding:
3.4.5.2 Common Mutagens Used in Mulberry
3.4.5.3 Handling of Mutated Plant Materials
3.4.5.4 Mutation in Cell and Tissue Culture
3.4.5.5 Mutation as a Tool in Cell Fusion
3.4.5.6 Application of Mutation Breeding
3.4.6 Utilization of Heterosis through F1 Hybrid Seed Complex
3.4.6.1 Advantages of Hybrid Seed Complex Varieties
3.4.6.2 Disadvantages
3.4.6.3 The Important Parameters for Parental Selection
3.4.6.4 Breeding Program
3.4.6.5 Production of Hybrid Seeds in the Garden
3.4.6.6 Cultivation of Hybrid Seed Variety for Leaf Production and Silkworm Rearing
3.4.7 Breeding for Stress-Tolerant Mulberry Varieties
3.4.7.1 Breeding for Drought/Soil Moisture Stress Tolerance
3.4.7.2 Breeding for Cold Tolerance
3.4.7.3 Breeding for Salt Tolerance
3.4.7.4 Breeding for Alkalinity Tolerance
3.4.7.5 Development of Varieties Suitable for Sub-optimal Irrigation and Inputs
3.4.7.6 In Vitro Screening for Abiotic Stress Tolerance
3.4.8 Breeding for Disease and Pest Resistance
3.4.8.1 Mulberry Dwarf Disease
3.4.8.2 Bacterial Blight
3.4.8.3 Bacterial Wilt
3.4.8.4 Powdery Mildew
3.4.8.5 Dieback Disease
3.4.8.6 Root rot
3.4.8.7 Root-Knot Nematodes (RKN)
3.5 Mulberry Variety Development: Screening, Selection and Evaluation
3.5.1 Transplantation of Seedlings and the First Selection
3.5.2 The First Selection/First Stage Observations
3.5.3 First Selection/Second Stage Observations/Primary Yield Evaluation
3.5.4 Second Selection or Final Yield Evaluation
3.5.5 Experimental Design for Mulberry Breeding Experiments
3.5.6 Multi Locational Trial and Variety Authorization
3.6 Mulberry varieties developed through Conventional Breeding Approaches
3.6.1 Major Mulberry Varieties Cultivated in India
3.6.2 Major Mulberry Varieties Cultivated in China
3.6.3 Major Mulberry Varieties Cultivated in Japan
3.6.4 Major Mulberry Varieties Cultivated in Thailand
3.6.5 Major Mulberry Varieties Cultivated in Brazil
3.7 Conclusion and Future Prospects
References
4 Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective
4.1 Introduction
4.2 Current Status and Challenges
4.3 Comparative Study Between M. Notabilis, M. Alba, and M. Indica Genome
4.3.1 Morus Notabilis
4.3.2 Morus Alba
4.3.3 Morus Indica
4.4 Chloroplast Genome
4.4.1 Importance of Study of Chloroplast Genome in Plant Biotechnology
4.4.2 Studies on the Chloroplast Genome of Morus Species
4.5 Population Genomics and Diversity Analysis
4.6 Application of Genomics Resources for Mulberry Breeding
4.7 Conclusion and Future Perspectives
References
5 Relationship Between Genome Size and Ploidy Level in Mulberry
5.1 Introduction
5.1.1 Estimation of Nuclear DNA Content
5.1.2 Cytogenetics Toward Estimation of Genome Size and Ploidy Level
5.1.3 Flow Cytometry (FCM) Toward Estimation of Genome Size and Ploidy Level
5.1.4 Intra- and Inter-specific Nuclear DNA Content Variation of Morus spp.
5.1.5 Relation Between Phenotypic Trait Plasticity and Genome Size (GS)
5.1.6 Molecular Analysis/Genetic Variation and Population Structure
5.2 Conclusion
Acknowledgements
References
6 Transcriptomics: Current Status and Future Prospects for Identifying Trait-Specific Genes in Mulberry
6.1 Introduction
6.2 Transcriptomics in Mulberry
6.2.1 Growth-Linked Traits in mulberry—An Overview
6.2.1.1 RNA-Seq to Understand Growth-Linked Traits
6.2.1.2 Prospecting Growth-Linked Genes—Leads from Other Systems
6.2.2 Stress-Adaptive Traits—Prospecting Genes and Key Pathways
6.2.2.1 RNA-Seq to Understand Abiotic Stress Tolerance
Drought
Salinity
Cold
Heavy Metals
6.2.2.2 RNA-Seq to Understand Biotic Stress Tolerance
6.2.3 Other Important RNA-Seq Studies
6.2.4 Targeted Studies in Prospecting Trait-Linked Genes
6.2.5 RNA-Seq Studies Indicated Many Proteins of Unknown Functions (PUFs) in Mulberry
6.3 Conclusion and Perspectives
Acknowledgements
References
7 Proteomics in Mulberry
7.1 Introduction
7.2 Proteomics of Different Mulberry Tissues
7.2.1 Leaf
7.2.2 Branch
7.2.3 Root
7.2.4 Flower and Fruit
7.2.5 Other Tissues
7.3 Mulberry Proteomics Under Stress Condition
7.3.1 Biotic Stress
7.3.1.1 Mulberry Yellow Type Dwarf Disease (MD)
7.3.1.2 Mulberry Fruit Sclerotiniosis
7.3.2 Abiotic Stress
7.3.2.1 Salt
7.3.2.2 Alkaline Stress
7.3.2.3 Low Temperature Stress
7.3.2.4 Drought
7.3.2.5 Other Stresses
7.4 Interaction Between Mulberry and Insect
7.5 Future and Prospects
References
8 Importance and Current Status of DUS Testing in Mulberry
8.1 Introduction
8.2 Genesis of Plant Variety Protection System
8.3 Protection of Plant Varieties and Farmers Right Act 2001
8.4 Novelty, Distinctness, Uniformity, and Stability
8.5 DUS Guidelines in Mulberry (Morus Spp.)
8.6 Characteristics Used in DUS Testing
8.7 States of Expression of the Characteristics
8.8 Example Varieties for States of Expression of the Characteristics
8.9 Reference varieties
8.10 Candidate Varieties
8.11 Plant Material Required
8.12 Conduct of Tests
8.13 Reasons for Two Location
8.14 Methods and Observation
8.15 Recommendation and Grant
8.16 Explanation on the Table of Characteristics
8.17 DUS Test Plot
8.18 Application for Registration of Mulberry Varieties Under PPVFRA 2001
8.19 Use of Molecular Markers in DUS Testing
8.20 Conclusion
References
9 Molecular Diagnostics of Soil-Borne and Foliar Diseases of Mulberry: Present Trends and Future Perspective
9.1 Introduction
9.2 Soil-Borne Diseases
9.2.1 Root Rot Disease
9.2.1.1 Black Root Rot (BRR)
9.2.1.2 Dry Root Rot
9.2.1.3 Rhizopus Rot:
9.2.1.4 Charcoal Root Rot
9.2.2 Root-Knot Nematodes (RKN)
9.3 Foliar Diseases
9.3.1 Leaf Spot
9.3.1.1 Cercospora moricola
9.3.1.2 Setosphaeria rostrata
9.3.1.3 Nigrospora sphaerica
9.3.1.4 Curvularia lunata
9.3.1.5 Pseudocercospora mori
9.3.1.6 Paramyrothecium roridum
9.3.1.7 Cladosporium pseudocladosporioides
9.3.1.8 Xanthomonas campestris Pv. Mori
9.3.2 Powdery Mildew
9.3.3 Leaf Rust
9.3.3.1 Black Leaf Rust
9.3.3.2 Brown Leaf Rust
9.3.3.3 Red Rust
9.3.3.4 Yellow Rust
9.3.4 Anthracnose Disease
9.3.5 Blight
9.3.5.1 Twig Blight
9.3.5.2 Bacterial Blight
9.3.6 Sooty Mold
9.3.7 Canker and Twig Dieback
9.4 Molecular Diagnostic Methods for the Detection of Plant Pathogens
9.4.1 Progress in the Application of Diagnostics Tools in Mulberry
9.4.1.1 Polymerase Chain Reaction (PCR)
9.4.1.2 End-Point PCR
9.4.1.3 ITS-Conventional PCR
9.4.1.4 Non-Internal Transcribed Spacers Nuclear Genes: PCR-Based Traditional Ways
9.4.2 Future Molecular Diagnostic Methods for Detection of Mulberry Pathogens
9.4.2.1 DNA or RNA Probe-Based Assays
9.4.2.2 In Situ Hybridization
9.4.2.3 FISH (Fluorescent in Situ Hybridization)
9.4.2.4 High Throughput Sequencing:
9.4.2.5 Isothermal Amplification-Based Methods
RCA or Rolling Circle Amplification
Loop-Mediated Amplification (LAMP)
9.5 Conclusions and Future Prospects
Acknowledgements
References
10 Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges
10.1 Introduction
10.2 Rationale
10.3 Regeneration
10.4 Genetic Transformation in Mulberry
10.5 Development of Stable Transgenic Mulberry Plants
10.6 Characterization of the Transgenic Mulberry Plants
10.7 Abiotic Stress Tolerance in Transgenic Mulberry
10.8 Biotic Stress Tolerance in Transgenic Mulberry
10.9 Transient Expression System in Mulberry
10.10 Nutritional Attributes of Transgenic Mulberry
10.11 Bioassay of Silkworm and Mulberry Pests
10.12 Conclusions
References
11 Application of Mulberry and Mulberry Silkworm By-Products for Medical Uses
11.1 Introduction
11.1.1 Anti-Diabetic Properties of Mulberry Leaf
11.1.2 Antioxidative Properties of Mulberry Leaf
11.1.3 Role of Mulberry Leaf in Prevention of Cardiovascular Diseases
11.1.4 Anticancer Effects of Mulberry Leaf
11.1.5 Anti-Inflammatory Effects of Mulberry Leaf
11.1.6 Neurological Disorders, Skin Diseases, Gastrointestinal Disorders
11.1.7 Antimicrobial Effects of Mulberry Leaf
11.2 Sericin Properties and Biomedical Applications
11.2.1 Immunological Response of Sericin
11.2.2 Antioxidant Role of Sericin
11.2.3 Sericin in Cosmetology
11.2.4 Supplement of Sericin in Culture Media and Cryopreservation
11.2.5 Sericin in Wound Healing
11.2.6 Antitumour Effect of Sericin
11.3 Wound Healing Applications of Silk Fibroin Protein
References
12 Application of Green Synthesized Nanoparticles in Sustainable Mulberry Production: Current Trends and Opportunities
12.1 Introduction
12.2 Green Synthesis of Nanoparticles and Their Use in Mulberry Production
12.3 Green Synthesized Nanofertilizers
12.3.1 Macronutrients
12.3.2 Micronutrients
12.4 Green Synthesized Nanopesticides
12.5 Nanofungicides and Bactericides
12.6 Nanoherbicides
12.7 Role of Green Synthesized Nanoparticles in Abiotic Stress Tolerance
12.8 Impact Assessment on Silkworm
12.9 Future Perspectives and Conclusion
References
13 Future Perspectives of Mulberry Genomic Research
13.1 Introduction
13.2 Improvement of Mulberry Through Genomic Research
13.2.1 Next-Generation Sequencing and Genotyping
13.2.2 Linkage Mapping and Genome-Wide Association Studies (GWAS)
13.2.3 Genomic Research for Understanding Complex Traits in Mulberry
13.2.4 Pangenome
13.3 Functional Genomics
13.4 Gene-Editing Technologies (CRISPR/Cas9)
References

Citation preview

Compendium of Plant Genomes

Belaghihalli N. Gnanesh Kunjupillai Vijayan   Editors

The Mulberry Genome

Compendium of Plant Genomes Series Editor Chittaranjan Kole, President, International Climate Resilient Crop Genomics Consortium (ICRCGC), President, International Phytomedomics & Nutriomics Consortium (IPNC) and President, Genome India International (GII), Kolkata, India

Whole-genome sequencing is at the cutting edge of life sciences in the new millennium. Since the first genome sequencing of the model plant Arabidopsis thaliana in 2000, whole genomes of about 100 plant species have been sequenced and genome sequences of several other plants are in the pipeline. Research publications on these genome initiatives are scattered on dedicated web sites and in journals with all too brief descriptions. The individual volumes elucidate the background history of the national and international genome initiatives; public and private partners involved; strategies and genomic resources and tools utilized; enumeration on the sequences and their assembly; repetitive sequences; gene annotation and genome duplication. In addition, synteny with other sequences, comparison of gene families and most importantly potential of the genome sequence information for gene pool characterization and genetic improvement of crop plants are described.

Belaghihalli N. Gnanesh Kunjupillai Vijayan

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Editors

The Mulberry Genome

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Editors Belaghihalli N. Gnanesh Central Sericultural Research and Training Institute Mysuru, Karnataka, India Sampoorna International Institute of Agri. Science and Horticultural Technology Maddur, Karnataka, India

Kunjupillai Vijayan International Sericultural Commission Central Silk Board Complex, BTM Layout Madiwala, Bangalore, Karnataka, India

ISSN 2199-4781 ISSN 2199-479X (electronic) Compendium of Plant Genomes ISBN 978-3-031-28477-9 ISBN 978-3-031-28478-6 (eBook) https://doi.org/10.1007/978-3-031-28478-6 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

This book series is dedicated to my wife Phullara and our children Sourav and Devleena Chittaranjan Kole

Foreword

Mulberry (Morus spp.) is an economically important crop being cultivated mainly to feed the silkworm Bombyx mori L. and has several other uses as well. However, compared to many other crops, the genetics of mulberry still remains obscure due to lack of adequate genomic resources. Nevertheless, with the help of the rapid advancements in the Next-Generation Sequencing, the first draft genome sequence of Morus notabilis could be done in the year 2013 and subsequent efforts led to sequencing of the whole genome of two other species (M. indica and M. alba). This has significantly increased the scope of understanding the genome of mulberry and to provide excellent opportunities to expedite genomics-assisted mulberry breeding. The present book entitled The Mulberry Genome presents a comprehensive picture covering domestication, conservation, genetic resources and diversity, traditional breeding, and utilization and economic benefits of mulberry. Genome analysis, current status and challenges, relationship between genome size and ploidy level, and prospecting trait-specific genes using transcriptomic and proteomics approaches in mulberry have been well described. Importance of distinctiveness, uniformity, and stability (DUS) analysis in mulberry for the genetic resource protection, molecular diagnostics of soil borne and foliar diseases, transgenic mulberry for stress tolerance, and significance of mulberry and mulberry silkworm by-products in medical use and application of green synthesized nanoparticles in sustainable mulberry production have also been narrated well. Thus, this book is expected to give quite useful information to students, teachers, and researchers in the academia as well as industries related to mulberry, especially to the sericulture. I congratulate and appreciate the efforts made by the editors, Drs. B. N. Gnanesh and K. Vijayan, in bringing out this book collating the all available information on mulberry to facilitate

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Foreword

better understanding the genetics of this important to improve its genetic potentiality for higher yield and better adaptation, in the light of the fast changing socio-climatic conditions.

October 2022

Prof. S. Ayyappan Chancellor Central Agricultural University Imphal, Manipur, India

Preface to the Series

Genome sequencing has emerged as the leading discipline in the plant sciences coinciding with the start of the new century. For much of the twentieth century, plant geneticists were only successful in delineating putative chromosomal location, function, and changes in genes indirectly through the use of a number of “markers” physically linked to them. These included visible or morphological, cytological, protein, and molecular or DNA markers. Among them, the first DNA marker, the RFLPs, introduced a revolutionary change in plant genetics and breeding in the mid-1980s, mainly because of their infinite number and thus potential to cover maximum chromosomal regions, phenotypic neutrality, absence of epistasis, and codominant nature. An array of other hybridization-based markers, PCR-based markers, and markers based on both facilitated construction of genetic linkage maps, mapping of genes controlling simply inherited traits, and even gene clusters (QTLs) controlling polygenic traits in a large number of model and crop plants. During this period, a number of new mapping populations beyond F2 were utilized and a number of computer programs were developed for map construction, mapping of genes, and for mapping of polygenic clusters or QTLs. Molecular markers were also used in the studies of evolution and phylogenetic relationship, genetic diversity, DNA fingerprinting, and map-based cloning. Markers tightly linked to the genes were used in crop improvement employing the so-called marker-assisted selection. These strategies of molecular genetic mapping and molecular breeding made a spectacular impact during the last one and a half decades of the twentieth century. But still they remained “indirect” approaches for elucidation and utilization of plant genomes since much of the chromosomes remained unknown and the complete chemical depiction of them was yet to be unraveled. Physical mapping of genomes was the obvious consequence that facilitated the development of the “genomic resources” including BAC and YAC libraries to develop physical maps in some plant genomes. Subsequently, integrated genetic–physical maps were also developed in many plants. This led to the concept of structural genomics. Later on, emphasis was laid on EST and transcriptome analysis to decipher the function of the active gene sequences leading to another concept defined as functional genomics. The advent of techniques of bacteriophage gene and DNA sequencing in the 1970s was extended to facilitate sequencing of these genomic resources in the last decade of the twentieth century. ix

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As expected, sequencing of chromosomal regions would have led to too much data to store, characterize, and utilize with the-then available computer software could handle. But the development of information technology made the life of biologists easier by leading to a swift and sweet marriage of biology and informatics, and a new subject was born—bioinformatics. Thus, the evolution of the concepts, strategies, and tools of sequencing and bioinformatics reinforced the subject of genomics—structural and functional. Today, genome sequencing has traveled much beyond biology and involves biophysics, biochemistry, and bioinformatics! Thanks to the efforts of both public and private agencies, genome sequencing strategies are evolving very fast, leading to cheaper, quicker, and automated techniques right from clone-by-clone and whole-genome shotgun approaches to a succession of second-generation sequencing methods. The development of software of different generations facilitated this genome sequencing. At the same time, newer concepts and strategies were emerging to handle sequencing of the complex genomes, particularly the polyploids. It became a reality to chemically—and so directly—define plant genomes, popularly called whole-genome sequencing or simply genome sequencing. The history of plant genome sequencing will always cite the sequencing of the genome of the model plant Arabidopsis thaliana in 2000 that was followed by sequencing the genome of the crop and model plant rice in 2002. Since then, the number of sequenced genomes of higher plants has been increasing exponentially, mainly due to the development of cheaper and quicker genomic techniques and, most importantly, the development of collaborative platforms such as national and international consortia involving partners from public and/or private agencies. As I write this preface for the first volume of the new series “Compendium of Plant Genomes,” a net search tells me that complete or nearly complete whole-genome sequencing of 45 crop plants, eight crop and model plants, eight model plants, 15 crop progenitors and relatives, and three basal plants is accomplished, the majority of which are in the public domain. This means that we nowadays know many of our model and crop plants chemically, i.e., directly, and we may depict them and utilize them precisely better than ever. Genome sequencing has covered all groups of crop plants. Hence, information on the precise depiction of plant genomes and the scope of their utilization are growing rapidly every day. However, the information is scattered in research articles and review papers in journals and dedicated Web pages of the consortia and databases. There is no compilation of plant genomes and the opportunity of using the information in sequence-assisted breeding or further genomic studies. This is the underlying rationale for starting this book series, with each volume dedicated to a particular plant. Plant genome science has emerged as an important subject in academia, and the present compendium of plant genomes will be highly useful to both students and teaching faculties. Most importantly, research scientists involved in genomics research will have access to systematic deliberations on the plant genomes of their interest. Elucidation of plant genomes is of interest not only for the geneticists and breeders, but also for practitioners of an array of plant science disciplines, such as taxonomy, evolution, cytology,

Preface to the Series

Preface to the Series

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physiology, pathology, entomology, nematology, crop production, biochemistry, and obviously bioinformatics. It must be mentioned that information regarding each plant genome is ever-growing. The contents of the volumes of this compendium are, therefore, focusing on the basic aspects of the genomes and their utility. They include information on the academic and/or economic importance of the plants, description of their genomes from a molecular genetic and cytogenetic point of view, and the genomic resources developed. Detailed deliberations focus on the background history of the national and international genome initiatives, public and private partners involved, strategies and genomic resources and tools utilized, enumeration on the sequences and their assembly, repetitive sequences, gene annotation, and genome duplication. In addition, synteny with other sequences, comparison of gene families, and, most importantly, the potential of the genome sequence information for gene pool characterization through genotyping by sequencing (GBS) and genetic improvement of crop plants have been described. As expected, there is a lot of variation of these topics in the volumes based on the information available on the crop, model, or reference plants. I must confess that as the series editor, it has been a daunting task for me to work on such a huge and broad knowledge base that spans so many diverse plant species. However, pioneering scientists with lifetime experience and expertise on the particular crops did excellent jobs editing the respective volumes. I myself have been a small science worker on plant genomes since the mid-1980s and that provided me the opportunity to personally know several stalwarts of plant genomics from all over the globe. Most, if not all, of the volume editors are my longtime friends and colleagues. It has been highly comfortable and enriching for me to work with them on this book series. To be honest, while working on this series I have been and will remain a student first, a science worker second, and a series editor last. And, I must express my gratitude to the volume editors and the chapter authors for providing me the opportunity to work with them on this compendium. I also wish to mention here my thanks and gratitude to Springer staff, particularly Dr. Christina Eckey and Dr. Jutta Lindenborn, for the earlier set of volumes and presently Ing. Zuzana Bernhart for all their timely help and support. I always had to set aside additional hours to edit books beside my professional and personal commitments—hours I could and should have given to my wife, Phullara, and our kids, Sourav and Devleena. I must mention that they not only allowed me the freedom to take away those hours from them but also offered their support in the editing job itself. I am really not sure whether my dedication of this compendium to them will suffice to do justice to their sacrifices for the interest of science and the science community. New Delhi, India

Chittaranjan Kole

Preface

Mulberry is a fast-growing deciduous, woody perennial plant that has been believed to have originated in sub-Himalayan tracts and spread into Africa, Asia, South America, Europe, and North and South America. It is being cultivated widely across Asian countries for its leaves to feed the silk producing insect Bombyx mori. Besides leaf, other parts of mulberry such as fruits, matured stem, and roots are also used for several economical purposes, which include human consumption and medical use. Taxonomically, Morus L. belonged to the family Moraceae under the order Urticales; molecular phylogenic studies placed Moraceae under the order Rosales. The species delimitations of the genus Morus are yet to be solved as the morphological traits vary considerably under different growing conditions and stages of development. Due to out crossing and natural hybridization among the species, most of the accessions being maintained by different institutes are of natural hybrids. The greater success rate with cross-pollination of species indicates that these species have comparable genetic relationships, and therefore, “species” status needs to be studied additionally. Based on the molecular characterization and phylogeny studies, the Morus genus has been classified into eight species, including M. alba, M. celtidifolia, M. insignis, M. mesozygia, M. nigra, M. notabilis, M. rubra, and M. serrata. Mulberry grows luxuriously in a flat land with fertile, well-drained, deep, and clayey to loamy, porous soil with good moisture holding capacity and a pH ranging from 6.5 to 6.8. Mulberry is reported to have a moderate level of tolerance to salinity and drought (abiotic stresses). Mulberry is propagated mainly through vegetative means, and plants in different ploidy such as haploid M. notabilis, with a chromosome number 14, diploid (M. indica and M. alba: 2n = 28), triploid (M. bombysis: 2n = 42), tetraploid (M. boninensis, M. cathayana and M. laevigata: 2n = 56), hexaploid (M. serrata and M. tilaefolia: 2n = 84), octoploid (M. cathayana: 2n = 112), and decasoploid (M. nigra: 2n = 308) are present in nature. However, diploids and triploids are preferred for cultivation, especially for sericulture purposes. Although mulberry is moderately tolerant to major abiotic stresses such as drought and salinity, the leaf yield and quality are affected by different types of stresses leading to the expression of damages at different levels. Different climatic condition insists on developing climate-resilient mulberry, which is a prerequisite for the future sustainability of the sericulture industry. Since mulberry is a highly heterozygous, cross-pollinated tree species, development of climate-smart varieties through conventional breeding is considered xiii

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to be difficult. Besides that, a high level of environmental adaptability and first-growing nature offer a suitable platform for an advanced breeding program. Since modern biotechnological tools have the potential to circumvent many of the constraints of traditional breeding, attempts have recently been made to employ biotechnological tools for mulberry crop improvement. The whole-genome sequencing data of mulberry has been completed in three species, and also transgenic mulberry with enhanced tolerance to drought and cold has been developed. Molecular markers have been used to assess the genetic diversity and mapping of genes. The Mulberry Genome book provides a detailed account of the efforts and achievements made through both traditional plant breeding and modern biotechnological techniques for the improvement of the mulberry to provide both leaf and fruits for the welfare of humanity. The book comprises a total of 13 chapters, begins with the Background of Mulberry and ends with forthcoming opportunities in Genome Research. Chapter 1 tells all about the evidence of the origin and domestication of mulberry along with its current taxonomic classifications and different conservation strategies being adopted to conserve the genetic pool for different purposes around the world. Chapters 2 and 3 deal with the utilization and economic benefits of mulberry along with the history, development, and achievements of classical breeding for higher leaf productivity. Chapter 4 focuses on current status, challenges, and genome analysis (nuclear and chloroplast) information available in public domain (M. notabilis, M. alba and M. indica) and also emphasized the application of integrated omics approaches through utilizing available genomics resources for population genomic analysis. Understanding the genome size, ploidy level, and its functional relationship with plant performance is one of the most essential parts of plant biology. To capitalize on the benefit of ploidy-associated traits through ploidy breeding, estimation of genome size, inter-specific ploidy level variation, and ploidy-associated traits have been discussed in Chap. 5. A comprehensive database of genomic, transcriptomic, metabolomic, and proteomic is available, which would serve as valuable resources to understand the biological processes in mulberry. Chapters 6 and 7 provide valuable information about transcriptomics and proteomics intending to prospect genes linked with growth and stress tolerance traits aiming to provide insights for future molecular biology research and enumerate the prospects for diversified breeding of mulberry trees. Chapter 8 describes the current status and importance of DUS testing for accurate documentation of true genetic variation to safeguard the mulberry variety by registering under Protection of Plant Varieties and Farmers’ Right’s Act (PPV& FR Act, 2001). Early monitoring of plant health and detecting the pathogen are important to decrease the disease intensity and spread and eco-friendly management practices, and Chap. 9 focuses on the use of inexpensive and easy-to-use molecular diagnostics tools to detect soil borne and foliar diseases in mulberry. Chapter 10 summarizes the status of in vitro regeneration, genetic transformation protocols and characterization of transgenic mulberry lines, future research directions, and their utility under field conditions. Mulberry is also a valuable medicinal plant that provides a wide range of

Preface

Preface

xv

bioactive compounds, including antioxidants like flavonoids and phenolics, as well as dietary fibre. Chapter 11 specifies the application of mulberry and mulberry silkworm by-products for medical use, and Chap. 12 addresses the green synthesized nanoparticles that have gained greater importance in the scientific community towards the development of novel nano-materials and application of nanopesticides, nanofertilizers, nanoherbicides, and other nanoscale materials for the sustainable sericulture industry. Lastly, Chap. 13 provides information on the future perspectives of mulberry genome research through the implementation of integrated omics study, advanced breeding program, genetic engineering, biotechnological techniques, and bioinformatics for mulberry genetic improvement towards stress tolerance. These chapters have been authored by eminent scientists from different countries, and we express our thanks to them for their contributions and cooperation from inception until the completion of this book project. As the book contains all information from genetic resources to the gene and genome sequences, we feel this The Mulberry Genome book will serve as valuable resource material and will be very much useful to researchers, breeders, students, and mulberry tree growers who are working on mulberry. We thank Prof. Chittaranjan Kole, Series Editor of the Compendium of Plant Genomes, for giving his constant support and encouragement during the editing of this book on The Mulberry Genome. The editors also acknowledge the help from all the staff of Springer Nature at all stages. Mysuru, Karnataka, India Thiruvananthapuram, Kerala, India

Dr. Belaghihalli N. Gnanesh Dr. Kunjupillai Vijayan

Contents

1

2

Botanical Features and Economic Significance of Mulberry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kunjupillai Vijayan, Belaghihalli N. Gnanesh, and Amalendu Tikader Cultivation, Utilization, and Economic Benefits of Mulberry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pawan Saini, Gulab Khan Rohela, Jalaja S. Kumar, Aftab A. Shabnam, and Amit Kumar

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13

57

3

Mulberry Breeding for Higher Leaf Productivity . . . . . . . . . . Thallapally Mogili, Tanmoy Sarkar, and Belaghihalli N. Gnanesh

4

Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Raju Mondal, Gulab Khan Rohela, Prosanta Saha, Prashanth A. Sangannavar, and Belaghihalli N. Gnanesh

5

Relationship Between Genome Size and Ploidy Level in Mulberry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Belaghihalli N. Gnanesh, Raju Mondal, H. B. Manojkumar, M. R. Bhavya, Pradeep Singh, G. S. Arunakumar, and Thallapally Mogili

6

Transcriptomics: Current Status and Future Prospects for Identifying Trait-Specific Genes in Mulberry . . . . . . . . . . 149 K. H. Dhanyalakshmi, Shivasharanappa S. Patil, Tinu Thomas, H. V. Chaitra, Hari Singh Meena, M. Savitha, and Karaba N. Nataraja

7

Proteomics in Mulberry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 Liu Yan, Lin Tianbao, Zhang Cankui, and Lv Zhiqiang

8

Importance and Current Status of DUS Testing in Mulberry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 M. R. Bhavya, P. Sowbhagya, Belaghihalli N. Gnanesh, G. S. Arunakumar, and H. B. Manojkumar

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9

Contents

Molecular Diagnostics of Soil-Borne and Foliar Diseases of Mulberry: Present Trends and Future Perspective . . . . . . . 215 Belaghihalli N. Gnanesh, G. S. Arunakumar, A. Tejaswi, M. Supriya, Anil Pappachan, and M. M. Harshitha

10 Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . 243 Tanmoy Sarkar, M. K. Raghunath, Vankadara Sivaprasad, and Babulal 11 Application of Mulberry and Mulberry Silkworm By-Products for Medical Uses . . . . . . . . . . . . . . . . . . . . . . . . . . 261 Ravindra M. Aurade, Y. Thirupathaiah, V. Sobhana, Dhaneshwar Padhan, B. Kishore Kumar, and Babulal 12 Application of Green Synthesized Nanoparticles in Sustainable Mulberry Production: Current Trends and Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 G. S. Arunakumar, Akhil Suresh, P. M. N. R. Nisarga, M. R. Bhavya, P. Sowbhagya, and Belaghihalli N. Gnanesh 13 Future Perspectives of Mulberry Genomic Research . . . . . . . 293 Belaghihalli N. Gnanesh, Raju Mondal, and Kunjupillai Vijayan

About the Editors

Dr. Belaghihalli N. Gnanesh is working as Professor and Dean at Sampoorna International Institute of Agri. Science and Horticultural Technology, recognized by the University of Mysore, India. He has more than 16 years of research experience in the field of germplasm resources, genomics, and disease-resistant breeding. He has worked with Agriculture Agri-Food Canada (AAFC), Winnipeg, as Natural Sciences and Engineering Research Council of Canada (NSERC) Visiting Fellow on marker development and molecular breeding of oats and also worked at the University of Manitoba, Canada, on canola blackleg disease resistance. He has been awarded the prestigious Science and Engineering Research Board, Ramanujan fellowship, instituted by the Government of India. His major focus at Central Sericultural Research Training Institute, Mysore, was on mulberry transcriptomes, genome sequencing, SNP discovery, linkage mapping, and molecular identification of Fusarium species complex associated with root rot of mulberry. He has published more than 40 quality research articles and several chapters. Dr. Kunjupillai Vijayan is Renowned Research Scientist with research achievements in plant breeding, genetics, cytogenetics, genomics, plant tissue culture, and germplasm development. He has more than 35 years of research experience in national and international research institutes. His research findings were published in more than 130 journals, proceedings, and book publications. He worked in Central Silk Board as Senior Scientist and at Academia Sinica, Taipei, Taiwan, as Postdoctoral Research Associate. He is Excellent Expert on genomic relationships of the genus Morus. He collaborated with many mulberry researchers in the world. He has been involved in the development of several mulberry varieties suitable for different agro-climatic conditions. He has also worked out, for the first time, a very comprehensive phylogeny of the genus Camellia.

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1

Botanical Features and Economic Significance of Mulberry Kunjupillai Vijayan, Belaghihalli N. Gnanesh , and Amalendu Tikader

1.1

Introduction

Mulberry (Morus; Moraceae) is a highly heterozygous plant widely seen in Asian countries (Fig. 1.1). Its leaves are mainly used for feeding the silkworm, Bombyx mori L., to produce the lustrous silk fiber. Mulberry fruit, a small berry, has a high nutrient value and is thus used for human consumption. The timber of mulberry is used for making furniture and other household items. Further, the bark of root and stem contains several phytochemicals useful for medical industries (Ghosh et al. 2017). Being a perennial tree, mulberry is also used for soil and water conservation as well as for landscaping.

1.2

Origin, Distribution, and Domestication

Mulberry is believed to have originated in the foothills of the Himalayan region, and later it extended to the tropics of Southern Hemisphere

K. Vijayan (&)
A. Tikader Central Silk Board, BTM Layout, Madiwala, Bangalore, Karnataka 560068, India e-mail: [email protected] B. N. Gnanesh Central Sericultural Research and Training Institute, Manandavadi Road, Srirampura, Mysuru, Karnataka 570008, India

(Benavides et al. 1994). Vavilov (1951). This theory of South Asian origin of mulberry has been well supported by early Tertiary Moraceae fossils excavated recently (Collinson 1989) and molecular phylogenetic studies (Zerega et al. 2005). Today, mulberry exists in Asia, Europe, North and South America, and Africa continents at an altitude ranging from sea level to as high as 4000 m (Tutin 1996; Machii et al. 1999; Le Houerou 1980). The vernacular names that are attached to some of the species also indicated their origin or morphological distinctiveness. For example, Morus alba is called ‘white mulberry’ because of the color of the fruit. According to Sharma et al. (2000), white mulberry is native to China but has spread to several other countries through human intervention. In India, mainly four species of mulberry M. alba, Morus indica, Morus serrata, and Morus laevigata made a very prominent presence (Vijayan et al. 2022a; Vijayan and Gnanesh 2022). Similarly, Morus nigra, the ‘black mulberry’ has black color fruit and is seen in Southern Europe, Southwest Asia, and the Mediterranean countries (Tutin 1996; Yaltirik 1982). The Morus rubra is called ‘red mulberry’ and is native to North America, and it has been cultivated in America since colonial times for fruits to prepare wine. Likewise, based on the place of origin Morus tartarica is termed as ‘Russian mulberry’, Morus serrata—the ‘Himalayan mulberry’, Morus mesozygia—‘African mulberry’, Morus celtidifolia—‘Mexican mulberry’, Morus microphylla—‘Texas mulberry’,

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_1

1

2

K. Vijayan et al.

Fig. 1.1 Panel of Diverse Mulberry Germplasm (PDMG) maintained at CSRTI, Mysuru

and Morus australis designated as the ‘Chinese mulberry’.

1.3

Taxonomy of Morus

The Moraceae family comprised 37 genera consisting of approximately 1100 species, which include known plants like mulberry, breadfruit (Artocarpus altilis), fig (Ficus carica), banyan (Ficus benghalensis), and upas (Clement and Weiblen 2009; He et al. 2013). The classification of the genus Morus started as early as 1753 by Linnaeus (1753) who recognized seven species such as M. alba L., M. rubra L., M. nigra L., M. indica L., M. tartarica L., M. papyrifera L., and M. tinctoria L. The taxonomy of the genus later underwent several revisions depending on the evolution of systematic classification from the sexual systems to the modern molecular phylogenetic system. For instance, Hooker (1885) under the natural system of classification placed the genus, Morus L., in the tribe Moreae of the family Moraceae under the order Urticales. Similarly, Takhatajan (1980) also placed Morus

under the family Moraceae of the order Urticales, as it was considered an advanced order among the woody flowering plants. However, the molecular phylogenies (Angiosperm Phylogeny Group 2003) placed the family Moraceae in the order Rosales. Similarly, the species within the genus Morus have also undergone several classifications and revisions (Koidzumi 1917; Engler and Prantl 1924; Katsumata 1972). Koidzumi (1917) divided the genus Morus into two sections, the Dolichostylae (long style) and the Macromorus (short style) and under each section two groups, viz. Papillosae and Pubescentae, embrace a total of 24 species and one subspecies (Table 1.1). Later, Morus was separated into two sections by Hotta (1954), viz. the Dolychocystolithiae and the Brachycystolithiae based on the shape and position of cystolith cells in the leaf and they recognized 35 species. More than 150 species appeared in the Index Kewensis, though majorities of them are considered either synonyms or varieties. It was also found that some of the earlier recognized species were even transferred to another genus as in the case of M. tinctoria and M. zanthoxylon which were

1

Botanical Features and Economic Significance of Mulberry

Table 1.1 Mulberry species recognized by Koidzumi (1917)

Sl. No.

Species

Sl. No.

Species

1

M. bombycis Koidz

13

M. latifolia Poir

2

M. alba L.

14

M. acidosa Griff

3

M. indica L.

15

M. rotunbiloba Koidz

4

M. kagayamae Koidz

16

M. notabilis C. K. Schn

5

M. boninensis Koidz

17

M. nigriformis Koidz

6

M. atropurpurea Roxb

18

M. serrata Roxb

7

M. laevigata Wall

19

M. nigra L.

8

M. formosensis Hotta

20

M. rubra L.

9

M. messozygia Stapf

21

M. celtidifolia Kunth

10

M. cathayana Hemsl

22

M. tiliaefolia Makino

11

M. microphylla Bickl

23

M. macroura Miq

12

M. rabica Koidz

24

M. multicaulis Perr

transferred to the genus Maclura Nuttal. Thus, proper revision of Morus is yet to be done to remove the controversies wrapped around it (Berg 2001). One of the major reasons for the difficulties of the delimitation of species in Morus is the natural hybrids developed through natural cross-hybridization (Das and Krishnaswami 1965; Dwivedi et al. 1989; Tikader and Dandin 2007). Thus, using modern techniques such as molecular data along with classical taxonomic tools, the ‘species’ delimitation of this genus needs to be revised (Wang and Tanksley 1989). Nepal and Purintun (2021) surveyed appropriate taxonomic work and studied herbarium samples for morphology and examined native species of Morus in North America. They summarized that there are thirteen spp. belonging to genus Morus including eight in Asia, one in Africa, and four in the New World.

1.4

Cytology

3

vegetatively. Even a natural haploid species, Morus notabilis, was reported from China (Maode et al. 1996). Mulberry with higher level of ploidy, like Morus tiliaefolia, Morus cathyana, M. nigra, M. serrata, and M. laevigata, is also present in nature (Vijayan et al. 2022b). In addition to them, a large number of artificial tetraploids and triploids have also been developed from better mother parents.

1.5

Biodiversity

1.5.1 Morphological Variations in Mulberry Mulberry holds huge morphological variations in the natural populations due to the presence of large number of natural hybrids. This variability among the species as well as within the species can be described with a few commonly available species such as M. alba, M. rubra, M. nigra, M. serrata, and M. laevigata as examples (Table 1.2).

Mulberry has different ploidy levels such as diploids (2x, 2n = 28), triploids (3x, 3n = 42), tetraploids (4x, 4n = 56), hexaploids (6x, (a) Leaf: Leaves of the white mulberry (M. 6n = 84), and docasaploid (22x, 22n = 308) alba) are simple, alternate, stipulate, petio(Basavaiah and Rajah 1989). This presence of late, entire, or lobed (Fig. 1.2). The number different ploidy in the natural population of of lobes varies from one to five. Leaves of mulberry is due to its ability to propagate the red mulberry (M. rubra) are larger,

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K. Vijayan et al.

Table 1.2 Morphological characters of domesticated (M. alba) and wild (M. laevigata, M. serrata, M. nigra and M. rubra) species of mulberry Morphological characters

M. alba

M. laevigata

M. serrata

M. nigra

M. rubra

Bud color

Brown

Brown

Dark brown

Black

Black

Bud size (mm )

16.50–39.90

24.10–79.55

19.40–79.20

18.00–75.10

15.50–38.00

Branch color

Gray or grayish yellow

Gray or grayish yellow

Gray colored

Dark colored

Dark colored

Branching

Erect

Semi-erect

Semi-erect

Erect

Erect

Leaf lobation

Lobed to unlobed

Lobed to unlobed

Lobed to unlobed

Lobed to unlobed

Lobed to unlobed

Leaf color

Pale green

Dark green

Dark green

Dark green

Dark green

Leaf surface

Smooth

Rough

Rough

Smooth

Rough

Leaf margin

Larger round serration

Shallow lobed

Large round serration

Large round serration

Smaller more pointed serration

Leaf length (cm)

10–15

28–32

20–25

10–15

7–10

Inflorescence length (cm)

3–4

5–12

2–5

3–5

2–4

Fruit color

White–red

White–red

Red

Dark black

Red

2

Petiole groove

Present

Present

Absent

Present

Present

Lenticel size (mm2)

0.41–2.00

0.70–2.25

1.44–8.00

1.40–7.50

0.38–1.95

Adapted from Vijayan et al. (2011)

thicker, blunt toothed, and often lobed. They are rough on their upper surfaces and pubescent underneath. The smaller black mulberry leaves are similar to those of the red mulberry morphologically but with sturdier twigs and fatter buds. The shape of the leaf may vary according to the age of the plant, growth, positions in the branches, period of growth, etc. Leaves of wild mulberry species such as M. laevigata, M. serrata, and M. tiliaefolia are big, and the lobation varies according to the age of the plant as well as the geography of the area.

(b) Flower: The inflorescence of mulberry, a catkin with pendent or drooping peduncle bearing unisexual flowers, shows considerable variations among species and genotypes (Fig. 1.3a, b). Male catkins are usually longer than female catkins and are loosely arranged, and after shedding the pollen, the

inflorescence dries and falls off. There are four persistent perianth lobes and four stamens with incurved filaments in the bud. The female inflorescence is usually short except for that of M. laevigata where it is longer than any other species, and the florets are arranged compactly. There are four persistent perianth lobes. The ovary is single celled, and the stigma is bifid. The ovules are pendulous. Although mulberry is predominantly dioecious, monoecious plants are also available in plenty as the sex of mulberry changes depending on environmental and physical conditions (Das and Mukherjee 1986; Tikader et al. 1995). Applications of chemicals also change the sex as it is considered that both male- and female-determining genes are present in mulberry, but their expression is determined by external stimuli such as climate or internal stimuli such as physiochemical factors (Minamizawa 1963; Tiku et al. 1988). Mukherjee (1965) observed that

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Botanical Features and Economic Significance of Mulberry

5

Fig. 1.2 Variability in the leaf of mulberry (Morus spp.)

rubra these substances concentrate in all the dioecism evolved from monoecism in mulcells of drupelets. The white mulberry fruits berry and sex reversal using hormones, are usually very sweet, but red mulberry chemicals, and growth regulators were also remains savor with higher contents of riboobserved (Jaiswal and Kumar 1980, 1981a, flavins and is usually deep red or almost b; Ogure et al. 1980; Kumar et al. 1985; Das black. Similarly, the fruits of black mulberry and Mukherjee 1986; Sikdar et al. 1988). are eye-catching, big, and juicy with a balTikader et al. (1995) observed sexual reverance of sweetness and tartness, which craft sal due to physical injury like pruning. them as best-flavored fruits in mulberry. The (c) Fruit: The fruit of mulberry is a sorosis mulberry fruits of all the species which are containing individual fruits, developed from ripened are delicate and should be very florets, called Achens. Once the female careful in harvesting and processing. flowers are pollinated, the white stigma turns into brownish color and finally dries (d) Seed: The seed of mulberry is small, light yellow to brown, oval-shaped with a nearly off. Subsequently, the fleshy bases of the flat surface at the micropylar region (Fig. 3 perianth begin to swell and become comd). The seed coat contains two layers: the pletely altered in texture and color. The outer hard and brittle one is called the testa ripened fruit is succulent, fatty, and full of and the inner thin papery and slightly juice. The color of the ripened fruit varies brownish one is called the tegmen. Inside the greatly from white to black with different seed coat lies the kernel containing outer color shades (Fig. 3c). The color of mulendosperm and inner embryo. The size and berry fruits is determined by coloring comweight of the seed vary from species to pounds accumulated in the ripened perianth species and varieties to varieties. Generally, as Ercisli and Orhan (2007) observed that the seeds retain viability only for few weeks the coloring compounds tend to concentrate at room temperature, but if stored under in the outer drupelet cells in M. alba, controlled temperature and humidity, the whereas in the fruits of M. nigra and M.

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K. Vijayan et al.

Fig. 1.3 Mulberry inflorescence (a), pollen grains observed under the compound microscope (40) (b), fruits (c), and seeds (d)

viability may be extended up to 3–6 months. The optimum temperature for germination is 28–30 °C.

1.6

Conservation of Biodiversity

Conservation of biodiversity is the measures being taken to protect and manage natural variability of species. Biodiversity of a species is the reflection of the total genetic variations present among the individuals in the species as a whole,

and it includes the total genetic assets of traditional varieties, landraces, elite lines, and special varieties developed by breeders and other researchers and their wild relatives (Tikader and Dandin 2007). Habitat degradation due to increased land conversion into human habitats, mining, deforestation, climatic changes, global warming, etc., has put extra pressure on the survival of species. Thus, concerted efforts have to be made to conserve the variability among the individuals and species for their better survival and also to make available valuable genetic resources of the crop species for the breeder to

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Botanical Features and Economic Significance of Mulberry

Table 1.3 Species-wise distribution of mulberry germplasm accessions available in Japan, China, India, and Korea

7

Species

Japan

China

India

Korea

M. bombycis Koidz

583

22

15

97

M. latifolia Poir

349

750

19

128

M. alba L.

259

762

93

105

M. acidosa Griff

44

–

–

1

M. wittorium Hand-Mazz

–

8

–

–

350

M. indica L.

30

–

M. mizuho Hotta

–

17

M. rotundiloba Koidz

24

4

2

–

M. kagayamae Koidz

23

–

–

1

M. australis Poir

–

37

2

–

M. notabilis C.K. Schn

14

–

–

–

55

–

–

11

–

–

–

M. mongolica Schneider M. boninensis Koidz

5 –

M. nigriformis Koidz

3

–

–

–

M. atropurpurea Roxb

3

120

–

–

M. serrata Roxb

3

–

18

–

M. laevigata wall

3

19

32

1

M. nigra L.

2

1

2

3

M. formosensis Hotta

2

–

–

–

M. rubra L.

1

–

1

–

M. mesozygia Stapf

1

–

–

–

M. celtifolia Kunth

1

–

–

–

M. cathayana Hemsl

1

65

1

–

M. tiliaefolia Makino

1

–

1

14

M. microphylla Bickl

1

–

–

–

M. macroura Miq

1

–

–

–

M. multicaulis Perr

–

–

15

–

Morus spp. (Unknown)

15

–

106

259

develop new varieties with higher potential for yield to cater to the need of the future. Mulberry is a highly important crop for the development of rural areas of Asian countries, and several measures were taken to conserve the genetic resources of mulberry by adopting different techniques as described hereunder.

1.6.1 Conservation Practices The conservation techniques adopted for a particular group of plant are dependent to a large

extent on the nature of the plant as well as its propagation abilities. Mulberry being a tree crop mostly propagated through vegetative means, and the conservation techniques being adopted for mulberry gene pools vary from natural habitat protection to ex situ conservation to cryopreservation of seeds, pollen, stem segments, and even DNA. The initiation of beginning conservation of biodiversity is by collecting as much information on the extent and availability of variability through field surveys, information available in the literature, and personal contacts. Once sufficient information is collected, based on

8

it, appropriate conservation strategies such as (i) in situ, (ii) ex situ, (iii) field gene bank, (iv) on-farm participatory, and (v) cryopreservation are to be formulated and implemented. (a) In Situ Conservation

K. Vijayan et al.

crop species, clonally propagated crops like mulberry with long juvenile periods and high genetic heterozygosity are conserved mainly through ex situ gene banks. Ex situ gene banks are advanced through stem cuttings or by grafting buds on suitable rootstocks. Ex situ gene banks have several components like the active collection for evaluation of accessions for economic traits and distribution of genetic resources for breeders and other research groups and the base collection for long-term preservation. Since the germplasm banks are being maintained in the field as live plants and are always under the mercy of environmental and other external perils, it is always advisable to maintain duplicate base collections in geographically different locations, though it has huge economic implications. Sericulturally important countries like China, India and Japan have been maintaining large number of germplasm accessions from different species in their germplasm banks (Table 1.3).

On site conservation is called in situ conservation. The area of natural population having higher genetic variability is well marked as biosphere reserves and protected from all destructive and disturbing activities. The major advantage of in situ conservation is that it permits evolutionary forces to act continuously to make the plant adapt well to changing climatic conditions. In situ conservation of mulberry genetic resources in India is done in biosphere reserves, national parks and botanical gardens, wildlife sanctuaries, etc. (Rao 2002). Also, in Canada efforts are being made to conserve red mulberry which has been declared an endangered tree species by the Committee on Status of Endangered Wildlife in Canada (COSEWIC). In the USA, red mulberry (c) On-farm Participatory Conservation is also listed as ‘threatened’ or ‘rare’ in the three northern states, and efforts are made to conserve this species with strong support from land man- On-farm conservation is the continuous cultivaagers and naturalists. A recovery plan has been tion and management of a diverse set of popuchalked out to conserve M. rubra in Hamilton’s lation with the help of progressive farmers Royal Botanical Gardens, Ball’s Falls Conser- (Eyzaguirre and Iwanaga 1996). Since plenty of vation Area, Niagara Glen, Rondeau Provincial mulberry genetic materials are available in the Park, Point Pelee National Park, Fish Point backyards, kitchen gardens, farmhouses, hortiProvincial Nature Reserve, Pelee Island, Middle cultural gardens, agricultural lands, and roadside Island, and East Sister Island. plantations, it is easy to maintain them through the participation of farmers and rural folks, who (b) Ex Situ Conservation use them for various purposes. Wild species like M. laevigata, M. serrata, M. tartarica, M. cathOff-site conservation is called ex situ conser- ayana, and many others usually get less attention vation wherein the conservation efforts are done from researchers and farmers engaged in serioutside the natural habitat by bringing the plant culture activities as the leaves of these species are materials to botanical gardens, experimental not useful for the rearing of silkworms. Neverstations, research institutes, etc. Historically, theless, the wild species have been regularly used botanical gardens and arboreta have played in horticulture and agro-forestry. Since these significant roles in the conservation of wild wild species carry many valuable genes and species of crop plants. Although, seed conser- traits, the genetic resources need to be conserved vation at low temperatures is the ideal and well and it is being done through the on-farm chosen method of conserving germplasm of participation of aboriginals and farmers.

1

Botanical Features and Economic Significance of Mulberry

(d) Cryopreservation Cryopreservation is the process of preserving cells or tissues in frozen conditions under very low temperatures using deep freezers or liquid nitrogen. In general, as stated elsewhere, the conservation of genetic resources in the field has the risk of losing either the whole materials through destruction by natural calamities, pests, and diseases or the genetic variability through genetic drift. (Withers and Engelmann 1997). Thus, it is necessary to adopt a method that could conserve a large number of plant species for a long period without losing variability and viability. Cryopreservation is one such technique where large quantities of genetic materials can be conserved for a long period without subjecting to many genetic changes as the freezing temperature inactivates the metabolic activities of the cell, Further, it requires less space, labor, and is also cost effective. Cryopreservation has four distinct stages such as freezing, storing, thawing, and reculturing. Freezing can be done in two ways: the classical one and the vitrification (Engelmann 2000). Under the classical cryopreservation method, the material is cooled slowly at a controlled rate (usually 0.1–4 °C/min) to about − 40 °C and subsequently immerses rapidly in the liquid nitrogen. This method is operationally complex and also requires sophisticated and expensive programmable freezers. However, in the vitrification method, a cell dehydration process is done before freezing the material through the application of chemicals like glycerol, ethylene, dimethylsulfoxide, propylene, etc. Then the material is subjected to ultra-rapid freezing, to make vitrification of intracellular solutes, i.e., formation of an amorphous glassy structure without the occurrence of ice crystals, which are detrimental to cellular structural integrity. This method is less complex and requires no programmable freezer. Therefore, it is suitable even for laboratories with only basic tissue culture facilities. Experiments showed that the ideal material for cryopreservation of mulberry is the winter buds, embryonic axes, pollen, and synthetic seeds (Niino and Sakai 1992; Niino

9

et al. 1993; Niino 1995). A general procedure for shoot tips cryopreservation before transferring into liquid nitrogen is first shoot segments were pre-frozen at − 3 °C for 10 days, − 5 °C (3 days) − 10 °C (1 day), and − 20 °C for one day. Buds were cultured on Murashige and Skoog’s (MS) medium after thawing in the air at 0 to 20 °C. The survival rate was 55 to 90%. Before pre-freezing at − 20 °C, partial dehydration of the bud up to 38.5% has been found to improve the recovery rates. The survivability of winter buds kept in liquid nitrogen for up to 3– 5 years is significantly not changed. Synthetic seeds developed from winter-hardened shoot tips were also cryopreserved successfully. Yakua and Oka (1988) demonstrated the potential of cryopreservation of intact vegetative buds of mulberry (M. bombycis) by pre-freezing and storing them in liquid nitrogen. The buds thawed later and cultured on MS media added with 1 mg I−1 BA regenerated successfully. It was observed that either pre-freezing at − 10 or − 20 °C together with rapid thawing at 37 °C or prefreezing at − 20 or − 30 °C along with gentle thawing at 0 °C is a proper condition for a high % of survivability and shoot regeneration.

1.7

Conclusion

Since mulberry plays a significant role in the economy of sericulturally important countries, efforts have been made consistently to understand the botanical features, origin, the process of domestication and conservation, and utilization of mulberry. Although quite a lot of information has been obtained recently, still it needs to be acquired especially the species delimitations, genomic, and genetic control of traits. These missing links have to be found to develop proper strategies for the effective and efficient conservation of this economically very valuable crop. International cooperation in this regard is urgently required; otherwise, many precious genes may get lost due to the increasing human interventions and the consequent climatic changes.

10

References Angiosperm Phylogeny Group (2003) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APGII. Bot J Linnean Soc 141:399–436 Basavaiah DSB, Rajan MV (1989) Microsporogenesis in Hexaploid Morusserrata Roxb. Cytologia 54:747–751 Benavides JE, Lachaux M, Fuentes M (1994) Efecto de la aplicación de estiércol de cabra en el suelo sobre la calidad y producción de biomasa de Morera (Morus sp.). In: Benavides JE (ed) Arboles y arbustosforrajeros en América Central. Volume II. CATIE, Turrialba, Costa Rica, pp 495–514 Berg CC (2001) Moreae, Artocarpeae, and Dorstenia (Moraceae). Flora Neotropica. Monogr 83, New York Botanical Garden, Bronx, New York, USA, pp 24–32 Clement WL, Weiblen GD (2009) Morphological evolution in the mulberry family (Moraceae). Syst Bot 34 (3):530–552 Collinson ME (1989) The fossil history of the Moraceae, Urticaceae (including Cecropiaceae), and Cannabaceae. In: Crane PR, Blackmore S (eds) Evolution, systematics, and fossil history of the Hamamelidae. Vol 2: ‘Higher’ Hamamelidae. Clarendon Press, Oxford, UK, pp 319–339 Das BC, Krishnaswami S (1965) Some observations on interspecific hybridization in mulberry. Indian J Seric 4:1–8 Das BK, Mukherjee SK (1986) Promotion of femaleness in Morus alba (L.) by multiple application of plant growth regulators. Geobios 13:272–273 Dwivedi NK, Suryanarayana N, Susheelamma BN, Sikdar AK, Jolly MS (1989) Interspecific hybridisation studies in mulberry. Sericologia 29:147–149 Engelmann F (2000) Importance of cryopreservation for the conservation of plant genetic resources. In Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research progress and application. IPGRI, Rome, Italy, pp 8–20. Engler A, Prantl K (1924) Dinaturlichen Pflanzen familien, 2nd edn (Leipzig) Ercisli S, Orhan E (2007) Chemical composition of White (Morus alba), Red (Morus rubra) and Black (Morus nigra) mulberry fruits. Food Chem 103(4):1380–1384 Eyzaguirre P, Iwanaga M (1996) Participatory plant breeding. In: Proceedings of workshop on participatory plant breeding, 26–29 July, Wageningen, Netherlands, IPGRI, Rome, Italy, p 164 Ghosh A, Gangopadhyay D, Chowdhury T (2017) Economical and environmental importance of mulberry: a review. Int J Plant Environ 3(02):51–58 He N, Zhang C, Qi X, Zhao S, Tao Y, Yang G, Lee TH, Wang X, Cai Q, Li D, Lu M (2013) Draft genome sequence of the mulberry tree Morus notabilis. Nat Commun 4(1):1–9 Hooker JD (1885) Flora of British India. L. Reeve and Co., Ltd., The East Book House, Ashford, Kent, UK, pp 491–493

K. Vijayan et al. Hotta T (1954) Taxonomical study on the cultivated mulberry in Japan. Faculty of Textile Fibres, Kyoto University, Kyoto, Japan, p 94 Jaiswal VS, Kumar A (1980) Induction of male inflorescence on the female plants of M. nigra L. by GA3. Indian J Exp Biol 18:911–913 Jaiswal VS, Kumar A (1981a) Activity and isozymes peroxidases inMorus nigra L. during sex differentiation. Z Pflanzenphysiol 102:299–302 Jaiswal VS, Kumar A (1981b) Modification of sex expression and fruit formation on male plants of Morus nigra L. by chloroflurenol. Proc Indian Acad Sci (Plant Sci) 90(5):395–400 Katsumata F (1972) Mulberry species in West Jawa and their peculiarities. J Seric Sci 41(3):213–222 Koidzumi G (1917) Taxonomy and phytogeography of the genus Morus. Bull Imperial Seric Station 11:1–50 Kumar R, Dandin SB, Rabindran S (1985) Modification of sex expression in mulberry (Morus alba L.) by Silver Nitrate. Indian J Exp Biol 23:288–289 Linnaeus C (1753) Morus. Species plantarum. Stockholm Impensis Laurentii Salvii, pp 968 Le Houerou HN (1980) The role of browse in management of natural grazing lands. In: Le Houerou HN (ed) Browse in Africa. The current state of knowledge. International Livestock Centre for Africa (ILCA), Addis Ababa, Ethiopia, 355 p Machii H, Koyama A, Yamanauchi H (1999) A list of genetic mulberry resources maintained at National Institute of Sericultural and Entomological Science. Misc Publ Natl Inst Seric Entomol Sci 26:1–77 Maode Y, Zhonghuai X, Lichun F, Yifu K, Xiaoyong Z, Chengjun J (1996) The discovery and study on a natural haploid Morusnotabilis Schneid. Acta Sericol Sin 22:67–71 Minamizawa K (1963) Experimental studies on the sex differentiation in mulberry. Bull Facul Agri 7:4–47 Mukherjee SK (1965) On some morphological evidence in floral structure towards the development of unisexuality in mulberry. Indian J Seric 4:1–7 Nepal MP, Purintun JM (2021) Systematics of the genus Morus L. (Moraceae). Mulberry: Genetic improvement in context of climate change, vol 25. CRC Press, p1 Niino T (1995) Cryopreservation of germplasm of mulberry (Morus sps.). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 32. SpringerVerlag, Berlin, pp 102–106 Niino T, Sakai A (1992) Cryopreservation of alginatecoated in-vitro grown shoot tips of apple, pear and mulberry. Plant Sci 87:199–206 Niino T, Koyaman A, Shirata K, Ohuchi S, Suguli M, Sakai A (1993) Long-term storage of mulberry winter buds by cryopreservation. J Seric Sci 62:431–434 Ogure M, Harashima N, Naganuma K, Matsushima M (1980) Effect of etheral and gibberellin on sex expression in mulberry Morus spp. J Seric Sci Japan 49:335–341 Rao AA (2002) Conservation status of mulberry genetic resources in India. Paper contributed to expert

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consultation on promotion of global exchange of sericulture germplasm satellite session of 19th International Sericulture Congress, 21–25 Sept Bangkok, Thailand. http://www.fao.org/DOCREP/005/AD107E/ ad107e0m.htm Sharma AC, Sharma R, Machii H (2000) Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers. Theor Appl Genet 101:1049–1055 Sikdar AK, Jolly MS, Dwivedi NK (1988) Polyploidization and modification of sex in mulberry by colchicine. Curr Sci 57(13):736–737 Takhatajan AL (1980) Outline of the classification of flowering plants (Magnoliophyta). Bot Rev 46 (3):225–359 Tikader A, Dandin SB (2007) Pre-breeding efforts to utilize two wild Morus species. Curr Sci 92:1072–1076 Tikader A, Vijayan K, Raghunath MK, Chakroborti SP, Roy BN, Pavankumar T (1995) Studies on sexual variation in mulberry (Morusspp.). Euphytica 84:115– 120 Tiku AK, Bindroo BB, Pandit RK (1988) Flowering process and anthesis of mulberry under temperate climatic conditions (Kashmir valley). Sericologia 28 (1):49–56 Tutin GT (1996) Morus L. In: Tutin GT, Burges NA, Chater AO et al (eds) Flora Europa, psilotaceae to platanaceae, 2nd ed, vol 1. Cambridge University Press, Australia Vavilov NI (1951) The origin, variation, immunity and breeding of cultivated plants. Soil Sci 72:482 Vijayan K, Gnanesh BN (2022) Genomic research in mulberry for higher silk productivity. In: Seritech, The new concepts in sericulture, The 26th International

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Sericultural Commission Congress, 7–11th September 2022, Cluj-Napoca, Romania, pp 49–74 Vijayan K, Gnanesh BN, Shabnam AA, Sangannavar PA, Sarkar T, Zhao W (2022a) Genomic designing for abiotic stress resistance in mulberry (Morus spp.). In: Genomic designing for abiotic stress resistant technical crops. Springer Nature. https://doi.org/10.1007/ 978-3-031-05706-9_7 Vijayan K, Arunakumar GS, Gnanesh BN, Sangannavar PA, Ramesha A, Zhao W (2022b) Genomic designing for biotic stress resistance in mulberry (Morus spp.). In: Genomic designing for biotic stress resistant technical crops. Springer Nature. https://doi. org/10.1007/978-3-031-09293-0_8 Vijayan K, Saratchandra B, Jaime A. Teixeira da Silva (2011) Germplasm conservation in mulberry. Scientia Horticulturae 128: 371–379 Wang ZY, Tanksley SD (1989) Restriction fragment length polymorphism in Oryza sativa L. Genome 32:1113–2111 Withers LA, Engelmann F (1997) In vitro conservation of plant genetic resources. In: Altman A (ed) Biotechnology in agriculture. Marcel Dekker, New York, USA, pp 57–88 Yakua H, Oka S (1988) Plant regeneration through meristem cultures from vegetative buds of mulberry (Morus bombycis Koidz.) stored in liquid nitrogen. Ann Bot 62:79–82 Yaltirik F (1982) Morus. In: Davis PH (ed) Flora of Turkey. Edinburgh University Press, Edinburgh, p 641 Zerega NJC, Clement WL, Datwyler SL, Weiblen GD (2005) Biogeography and divergence times in the mulberry family (Moraceae). MolPhylogenetEvol 37:402–416

2

Cultivation, Utilization, and Economic Benefits of Mulberry Pawan Saini, Gulab Khan Rohela, Jalaja S. Kumar, Aftab A. Shabnam, and Amit Kumar

Abbreviations

MSL MT ha °C ET RH OPH VAM cm N P

Mean sea level Metric tons Hectare (ha) Degree centigrade Evapotranspiration Relative Humidity Open pollinated hybrids Vesicular Arbuscular Mycorrhiza Centimeter Nitrogen Phosphorous

P. Saini Mulberry Breeding and Genetics Section, Host Plant Division, Central Sericultural Research and Training Institute, Central Silk Board, Ministry of Textiles, Government of India, Pampore, Jammu and Kashmir 192121, India e-mail: [email protected] G. K. Rohela Biotechnology Section, Host Plant Division, Central Sericultural Research and Training Institute, Central Silk Board, Ministry of Textiles, Government of India, Pampore, Jammu and Kashmir 192121, India J. S. Kumar (&) Central Muga Eri Research and Training Institute, Lahdoigarh, Jorhat, Assam 785700, India e-mail: [email protected] A. A. Shabnam
A. Kumar Host Plant Division, Central Muga Eri Research and Training Institute, Lahdoigarh, Jorhat, Assam 785700, India

K DAP MOP UCP AMPK PGC AMP ATP ILG LDL HDL DNJ GABA

2.1

Potassium Diammonium phosphate Murate of potash Uncoupling protein AMP-activated protein kinase Peroxisome proliferator-activated receptor gamma coactivator Adenosine monophosphate Adenosine tri-phosphate Insulin-like growth factors Low Density Cholesterol High Density Cholesterol Deoxynojirimycin c-Amino butyric acid

Introduction

India is an agricultural country where seventy percent of the population is involved in farming activities and living in rural and semi-urban areas. During the last two decades, so much emphasis is given to the promotion of agriculture and allied activities through various schemes and mission projects to double the income of farmers. Besides agriculture, there are other agro-based enterprises such as apiculture, mushroom cultivation, poultry farming, floriculture, and sericulture, which act as a secondary source of

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_2

13

14

additional income in a short period of time. Among the various agro-based enterprises, sericulture is an economically viable and easily adoptable yet labor-intensive activity that is perfectly suited to countries like India where a majority of the farmers are small and marginal. It provides round the year employment to the farmers. Sericulture, the art and science of producing silk, involves four important activities, i.e., host plant cultivation, silkworm rearing, production of raw silk (reeling), and weaving/ printing/dyeing of fabrics. India is the only country in the world where all four types of commercial silks, viz. mulberry, eri, muga, and tasar (tropical and oak), silk are produced. Among these four silks, mulberry silk is the most important and largely produced. Mulberry sericulture is a land-based activity and an admixture of agriculture, forestry, and livestock industry for employment generation for poor and weaker sections of society (Chauhan et al. 2018). Mulberry (Morus spp.) is a hardy, fast growing deciduous perennial plant that belongs to the family Moraceae (Pan and Lou 2008; Yang et al. 2010a, b). It grows well under varied edaphoclimatic conditions ranging from temperate to tropical conditions between 50° N latitude and 10° S latitude from 4000 m above mean sea level (MSL) height (Vijayan et al. 2018; Sarkar et al. 2018). It is mainly grown in Asian countries, i.e., India, China, Japan, Korea, Bangladesh, Uzbekistan, Nepal, and Afghanistan. In sericulture, mulberry foliage is primarily utilized in silkworm rearing (Vijayan et al. 2009; Vijayan 2009). From an economical point of view, it is an economically important multipurpose tree plant with multiple utilization such as feed, fodder, timber, bioremediation, pharmacy, jam, jelly, squash, tea, and dye (Rohela et al. 2020b). Mulberry leaves can be fed to cattle, goats, sheep, rabbits, fish, and even poultry. The fruits can be eaten fresh, preserved, vinified, or dried for winter use in semi-arid conditions. It provides timber also which can be used for making sports goods and furniture. Its fuel value is much superior to most agricultural residues. Mulberry grows faster than other woody plants, and its branches can be used as paper manufacturing raw material. Mulberry

P. Saini et al.

stem and stem poser are found to be good source of media for mushroom production. It can be an integral part of social forestry, different agroforestry systems like agri-silviculture, agri-silvihorticulture, silvopasture, energy plantation, boundary plantation, alley cropping, and perennial cropping, intercropping and can be grown as a dwarf tree in unutilized wastelands (Jhansilakshmi et al. 2009). Among the above economic uses, sericulture is the most important one as the silkworm (Bombyx mori L.) feeds on mulberry leaves and converts the leaf protein into silk protein (sericin and fibroin) after feeding on mulberry leaves (Ghosh et al. 2017). Mulberry leaf yield and quality are the two important aspects that play a major role in the sustainability of the sericulture industry in the region. The suitability of a mulberry cultivar for commercialization depends upon two aspects. Firstly, it should be agronomically superior with desired characteristics such as high rooting ability, fast growth, high yielding, resistance to biotic stresses (diseases and pests), and adaptability to a wide spectrum of edapho-climatic conditions. Secondly, it should be superior with respect to leaf quality and its suitability for both young and late age silkworm rearing for different rearing seasons and methods. Mulberry leaves are suitable as food for young age silkworms and must have favorable physical features such as suitable tenderness, thickness, and tightness to be eaten by silkworms. The leaves should be soft and rich in water content, protein, carbohydrate, and minerals. The quality and quantity of mulberry leaf fed during the silkworm rearing decide the success of the cocoon crop. According to Miyashita (1986), quality mulberry leaf contributes 38.2% toward successful bivoltine silkworm cocoon production. Mulberry leaf production has a direct relationship with successful silkworm rearing and provides about 60% of cocoon production cost (Yokoyama 1962; Das and Krishnaswami 1965). Despite mulberry cultivation across varied environmental conditions, the nutritional content, leaf palatability, and digestibility vary with the leaf age, type of cultivation, pruning, fertilizer and irrigation schedules, harvesting methods, storage duration, and

2

Cultivation, Utilization, and Economic Benefits of Mulberry

season (Chakravarty et al. 2018). The quality of the leaves has a pragmatic impact on the quality of cocoons, which has a direct impact on the quality of the silk yarn (Machii et al. 2000).

2.2

Indian Sericulture Scenario

In the global silk statistics list, India ranks the second largest silk producer after China with 38,530 metric tons (MT) of raw silk production during 2019–2020 (Anonymous 2020). Of the total raw silk production, mulberry silk contributes more than 70%. In the total raw silk production of India, mulberry silk share is 25,239 MT and vanya silk is 10,124 MT. The raw vanya silk includes 3136 MT of tasar silk, 7204 MT of eri, and 241 MT of muga silk during 2019–2020 (Anonymous 2020). The total area under mulberry cultivation in India is 239967 ha. In India, mulberry silk is mainly produced in Karnataka, Andhra Pradesh, Telangana, Tamil Nadu, West Bengal, and Jammu-Kashmir regions which accounts for approximately 95% of total raw mulberry silk production. Karnataka has a 106,384-hectare area under mulberry cultivation followed by Andhra Pradesh (44,607 ha), Tamil Nadu (23,268 ha), West Bengal (15,734 ha), and Jammu-Kashmir (8183 ha). Presently, mulberry sericulture is practiced in non-traditional states like Madhya Pradesh (2018 ha), Maharashtra (7154 ha), Uttarakhand (3478 ha), Uttar Pradesh (3711 ha), Manipur (3291 ha), Tripura (2064 ha), etc. In the eastern and north-eastern parts of India, the sericulture farmers have scattered and small land holdings, and total raw silk production has declined from 16.44 (1951–52) to 13.89% (2016–17). During the year 2019–20, the eastern and north-eastern Indian regions produced about 11% of total mulberry raw silk. In the north and north-west Indian states due to scattered mulberry plantations and more revenue from horticultural crops, there is a reduction in silk production and about 1.77% of total mulberry raw silk was accounted for. Despite the decrease in mulberry plantations, southern regions still produced more than 80% of total mulberry raw silk. The reason behind the reduction in mulberry

15

acreage may be attributed to faster urbanization, depleting groundwater resources, shortage of irrigation water, climate change, labor intensiveness, and cost, less revenue generation compared to horticultural crops (apple, walnut, almond, mango, papaya, etc.), new disease, and pest emergence. During 2011–2020, there has been a steady expansion of mulberry acreage in India (Table 2.1), and to improve production, productivity, and silk quality, it is indispensable to expand targeted mulberry acreage as per the set target of Central Silk Board 2030 vision documents (Table 2.2).

2.3

Mulberry Cultivation

2.3.1 Agro-Climatic Regions and Sericultural Zones of India In terms of major climate and growing season, an agro-climatic zone is a land unit that is climatically suitable for a specific range of crops/cultivars (Chauhan et al. 2018). India was divided into 11 agro-climatic zones based on parameters of homogeneity in agro-characteristics such as water surplus and water deficit, which were further categorized into 15 agro-climatic zones with the addition of one more criterion, the cropping method. A distinct ecological response to macro-climates articulated by soils, plants, fauna, and aquatic systems characterizes an ecological region on the earth's surface. Agroecological is the land unit cut out of the agroclimatic region when superimposed on landform and the kind of soils and soil conditions that act as modifiers to climate and length of the growing period. At present, India has been divided into 21 agro-ecological zones which are further grouped into 5 physiographic regions and 8 ecological subdivisions based upon the physiographic, ecological, and phytobiographical diversity. Further, under the national agriculture project, as per the agro-climatic regional approach and degree of commonality with basic features of soil, topography, altitude, climate and water resources, rainfall, and existing cropping patterns, these 15

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P. Saini et al.

Table 2.1 Area under mulberry during last 10 years in India Year

Mulberry plantation (ha)

Percent increase/decrease in area

2011–2012

181,089

–

2012–2013

186,015

2.6

2013–2014

203,023

9.1

2014–2015

219,819

8.3

2015–2016

208,947

− 4.9

2016–2017

216,810

3.8

2017–2018

223,926

3.3

2018–2019

235,001

4.9

2019–2020

239,967

2.1

2020–2021 (upto Dec. 2020)

247,000

2.84

Source Annual Reports, Central Silk Board

Table 2.2 Target fixed as per CSB vision document 2030

Production parameters

2017

2023

2030

Expansion of mulberry area (lakh ha)

2.30

3.00

3.86

Mulberry leaf productivity (mt/ha/yr)

45–50

48–52

55–60

Seed production (lakhs)

2210

–

5804

Cocoon yield improvement (kg/100dfls)

55–60

58–62

65–70

Renditta

7.32

7.00

6.5

Raw silk productivity (kg/ha/yr)

98.10

102.00

110.00

Employment (lakh persons)

85.10

100.00

150.00

Reduction in silk import (mt/yr)

2022

1050

0.00

Export earnings per year (Cr)

2093

2250

4000

agro-climatic zones were divided into 120 subzones. Being the host plant of the silkworm, it is cultivated under different spacing, pruning systems, agronomical practices in the bush, dwarf, and tree modes of plantation in various agroclimatic conditions and altitudes ranging from the temperate, sub-tropical, and tropical regions. Generally, 95% of mulberry is grown in rainfed areas worldwide while in India about 700 mm of rainfall is received in silk tracks of the country and about 800 mm in near hill areas. Accordingly, the mulberry leaf yield and quality levels, and harvesting pattern of mulberry leaf vary. In the temperate region, it is generally cultivated as monocrop and however, in the traditional sericulture belt of India, it is cultivated in multiple

cropping systems. Based on the mulberry cultivation in varied agro-climatic conditions, three major Sericultural zones have been established in India, i.e., temperate, sub-tropical, and tropical zones. An atmospheric temperature, ranging from 24 to 28 °C, is conducive to the good growth of mulberry. Growth and sprouting of buds cannot be obtained at a temperature below 13 °C and above 38 °C. In the temperate climate, mulberry does not sprout during the winter season, while in the tropics the growth is continuous. In temperate regions, mulberry sprouts in the spring season (March to April), when the temperature reaches 13 °C and continues to grow up to autumn (October). The plants remain dormant due to low atmospheric temperature from

2

Cultivation, Utilization, and Economic Benefits of Mulberry

November to March. Thus, in temperate regions, mulberry leaves are available for silkworm rearing from May to October. The climatic conditions throughout the year in India are ideal for mulberry and silkworm production. The temperature in Karnataka, India’s main silk-producing state, varies from 21.1 to 30.0 °C, while West Bengal experiences extremes in temperature, with lows of 15.5 °C in winter and highs of 36.6 °C in summer. Climatic conditions in Kashmir are favorable to rear silkworms from May to October. In most tropical countries, favorable climate exists throughout the year for the growth of mulberry. It is shown that the annual production of mulberry could be almost double that of temperate countries. Limitations of irrigation water in tropical countries appear to cause the low returns of mulberry leaves. Whereas mulberry is grown in dry farming conditions, the yield of the leaf is poor and the production of a quality leaf is also limited to the rainfall received. The atmospheric humidity is also low, which indirectly affects the succulence and growth of mulberry leaf (Rangaswami et al. 1995). The states of Jammu-Kashmir, Punjab, Himachal Pradesh, Haryana, Rajasthan, Uttar Pradesh, and Uttarakhand represent approximately India's north and north-western region. These states have their old history of mulberry cultivation along with acts as major hotspots for conserving region-specific mulberry biodiversity and their evaluation for better utilization in mulberry breeding. The north-western regions represent a diverse agro-climatic region ranging from cold arid zone to sub-tropical. The varied agro-climatic regimes, with temperatures ranging from as low as − 20 °C in the Ladakh area to as high as 46 °C in the plains of Punjab and Jammu, offer a wide scope of development of cold and thermo-tolerant mulberry genotypes. From the topographical point of view, the north-western Indian states have a high altitude of 1000–5200 ft above the mean sea level with varied soil types, i.e., sandy, silt loam, clay loam, alluvial, and black cotton soils. The whole of the North-west region and the states are broadly identified as temperate and

17

sub-tropical sericulture zones which are having salubrious climate sand that are ideally suited for bivoltine sericulture as well as for mulberry cultivation (Chauhan et al. 2018) (Table 2.3). The eastern and north-eastern Indian states constitute a composite climatic belt having subtropical with temperate climatic conditions in hilly regions. The eastern and north-eastern Sericultural regions of India include Odisha, Bihar, Madhya Pradesh, Sikkim, West Bengal, and seven sister states of the north-east (Table 2.4). These regions exhibited a wide range of variations with respect to soil, temperature, rainfall, and topography. Based on these variations, these regions are classified into four distinct Sericultural zones, viz. tropical humid, tropical dry, temperate dry, and temperate wet, each of which has a great influence on mulberry. The soil texture varied from alluvial to laterite to clay loamy to sandy loam. The pH of the soil is slightly alkaline to acidic. Saline soil with varying EC of 26.9 to 43.0 mmhos/cm could also be observed. Annual rainfall, evapotranspiration (ET), relative humidity (RH), wind velocity, temperature, and sunshine hours also vary widely from place to place as well as the season (Chakraborti et al. 2003). The southern Indian states are classified into five areas based upon the cultivation of mulberry in varied soil conditions which include high altitude hilly regions, plain-irrigated, semiarid rainfed, regions with saline and alkaline soils (Table 2.5).

2.3.2 Mulberry Species and Varieties Under Cultivation—An Overview Globally, mulberry is cultivated in more than 20 countries, viz. Japan, China, the Republic of Korea, the U.S.S.R., Sri Lanka, Hungary, Syria, India, Greece, Brazil, France, Italy, Spain, Yugoslavia, Poland, Turkey, Iran, Bulgaria, Lebanon, Burma, Thailand, Afghanistan, Romania, Cyprus, Cambodia, and the Republic of South Vietnam, across the world. In India, mulberry is cultivated throughout the year due to diversified and favorable climatic conditions

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Table 2.3 Agro-climatic zones of North-Western region of India Sl. No.

State

Area under mulberry plantation (ha)

No. of agro-climatic zones

Mean annual rainfall (mm)

Altitude (m)

Soil type

1

Jammu and Kashmir

8183

04 (Outer plains, Outer hills, Middle mountain and Inner mountain)

600–2000

360– 5200

Slitty loam to Clay loam and brown pH 6.5–7.5

2

Punjab

1164

05 (Hot humid, Rainfed, Subhumid, Semi-arid, Shivalik hills)

300–1400

180–600

Alluvium of Indus system pH 6.0–8.5

3

Himachal Pradesh

3183

06 (Sub-tropical, Sub-humid, High hills, Cold and dry, Alpine high land and fridged arid zone)

800–2000

365– 4500

Clay loam pH 5.5–5.7

4

Haryana

214

04 (Shivalik hills, Tertiary rocks, Indo-Gangetic alluvial plains, Aravalli Delhi wedge— Arid, Semi-arid and humid)

300–1500

300–800

Clay loam Reddish and brown pH 7.4–7.9

5

Uttar Pradesh

3711

03 (North hill, South hill/Plateau, Gangetic plains

750–1200

70–300

Alluvial sandy and clay loam pH

6

Uttarakhand

3478

05 (North-west hill area, mid height, high height, Bugyal area and snow clat)

1100–2000

600– 4500

Sandy loam and forest brown pH 6.0–6.5

7

Rajasthan

–

09 (Hyper arid, arid, semi-arid, humid, sub-humid, plain, Aravalli landscape, eastern upland, and Luni river basin)

180–1500

300– 1000

Black cotton sandy pH 6.5–9.5

Chauhan et al. (2018)

making sericulture a part time to full time occupation in non-traditional to traditional states of the country. There are more than 68 Morus species that have been reported in the literature and of which few species, viz. Morus alba, Morus indica, Morus multicaulis, Morus nigra, Morus latifolia, and Morus bombycis, are in cultivation (Datta 2000). Japan, China, and India are the major Sericultural countries, and a large number of mulberry varieties have been developed through several breeding methods (Tables 2.6, 2.7 and 2.8). About 24 and 19 Morus species are present in China and Japan, respectively. Genus Morus has a huge diversity in Continental America. Very

little diversity of Morus exists in Africa, Europe, and the Near East and is almost absent in the Australian continent. In China, more than 1000 cultivated varieties belong to four Morus species, i.e., M. multicaulis Perr. (Lu mulberry), M. alba L. (white mulberry), M. bombycis Koidz (mountain mulberry), and M. atropurpurea Roxb. (Guangdong mulberry) (Huo 2000). In Japan, 24 Morus species and one subspecies were differentiated by Koidzumi (1917) based upon various morphological characteristics of the shoot, bud, leaf shape and size, flower, and fruit. There are three main mulberry species in Japan viz., M. alba, M. latifolia, and M. bombycis origin. The mulberry varieties of M. bombycis type

2

Cultivation, Utilization, and Economic Benefits of Mulberry

19

Table 2.4 Agro-climatic zones of Eastern and North Eastern region of India Sl. No.

States

Agro-climatic zones

1

West Bengal

Eastern Himalayan, Eastern plateau and Hills

Mulberry acreage (ha)

2

Odisha

Eastern plateau and hills

457

3

Bihar

Eastern Himalayan, Middle Gangetic plain

598

4

Madhya Pradesh

Central plateau and hills

2018

5

Chhattisgarh

Eastern plateau and hills

242

6

Jharkhand

7

Arunachal Pradesh

15,734

552 Eastern Himalayan region

278

8

Assam

2095

9

Manipur

3291

10

Meghalaya

3289

11

Mizoram

1679

12

Nagaland

13

Tripura

2064

14

Sikkim

300

694

Table 2.5 Agro-climatic zones of South India Sl. No.

State

Agro-climatic zones

1

Karnataka

Southern plateau and hills, West coast plains and ghats

106,384

2

Andhra Pradesh

Southern plateau and hills, East coast plains

44,607

3

Tamil Nadu

Southern plateau and hills, East coast plains, West coast plains and ghats

23,268

4

Kerala

West coast plain and ghats

5

Telangana

Southern plateau and hills, East coast plains

4770

6

Maharashtra

Western plateau and hills, Western coast plains, and ghats

7154

7

Madhya Pradesh

Western plateau and hills (some parts)

are generally cultivated in the colder area (Tohoku district), M. latifolia in warmer places (Kyushu district), and of M. alba in a wide range of geographic areas. Japan maintained a good collection of mulberry genetic resources more than 1300 accessions (Machii et al. 2000). In India, two cultivated (M. alba, M. indica) and two wild forms (Moris serrata, Moris laevigata) grow in the Himalayan region and the majority of the cultivated varieties are of M. indica type. In addition to these, other mulberry species, viz. M. nigra (black mulberry), Moris rubra, M. bombycis, and Moris kayayama, also

Mulberry acreage (ha)

144

reported from the Jammu and Kashmir region of India (Shabnam et al. 2016). Before independence, sericulture was promoted by Indian rulers of different dynasties in the country. At that time, most of the cultivated varieties are of the bush type in Bengal (Var. Bush Malda, Takda, Bombaiya, Kajli, Bishnupur, and Berhampore Local), Madras/Mysore (Mysore Local, Yenne ranginakaddi, Boodhukaddi, and Sultan Kaddi), and tree types in Kashmir (Shahtul, Botatul, Chahtatul) and Punjab (Sujanpur Local) (Chauhan et al. 2018). There is an increasing demand for location-specific mulberry cultivars as mulberry

20

P. Saini et al.

Table 2.6 Mulberry varieties in developed in India suitable for cultivation in different agro-climatic zones Agro-climatic zone

Mulberry cultivar

Pedigree

Leaf yield (MT/ha/yr)

Year of development/ release/ recommendation

Recommended condition

2000

Temperate, good rooting ability, well respond to input application and intercultural operations

Developer Institute: CSR&TI, Pampore North and North West

Chinese White

Clonal Selection

15–20

Goshoerami

Introduction from Japan

15–20

PPR-1

Goshoerami  Chinese White

16–20

2016 Presently under trial in AICEM IV (2019)

Temperate, high rooting ability, early sprouting after winter dormancy, moderately tolerant to frost damage

Chak Majra

Selection from natural population

25–30

2000

Sub-Tropical

Sujanpur

Selection from natural population

20–25

–

Temperate, Ruling cultivar

Developer Institute: CSR&TI, Berhampore East and North-East

S-1

Selection from OP seeds of Mandalaya

Irrigated: 28–29 Rainfed: 11–12

2000

S-1635 (triploid)

Selection from OP seeds of CSRS-1

Irrigated: 40–45 Rainfed: 8–14

2000

Irrigated and rainfed, high rooting ability, short internodal distance

S-146

Open Pollinated Seeds

30–35

2000

Irrigated

S-799

OP seeds of M. indica

35–40

2000

SV1

Somaclonal variant from tissue culture

35–38

–

TR-10

T-4 (4x)  Philippines (2x)

Hills: 7–8 Foot hills: 12–14

2000

Rainfed hills of Eastern and Central India Himachal and Doon valley, high rooting ability, fast growth after pruning

BC259

Backcrossing of hybrid of Matigare local (M. indica)  Kosen(M. latifolia) with kosen twice

Hills: 9–10 Foot hills: 15–16

2000

Rainfed and hills

C-763

M. multicaulis  Black cherry

30–32

–

Laterite soil

C-776

English Black  M. multicaulis

28–30

2002

C-2016

Hosur local  S162

12–14

–

Rainfed, high temperature

35–39

–

Irrigated

C-2017

(continued)

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Cultivation, Utilization, and Economic Benefits of Mulberry

21

Table 2.6 (continued) Agro-climatic zone

Mulberry cultivar

Pedigree

Leaf yield (MT/ha/yr)

Year of development/ release/ recommendation

Recommended condition

C-2028

Chinese white  S1532

36–37

2012

Flood prone areas in Eastern and NE India, tolerant to flood/water logging/stagnation of 4– 6 weeks, high membrane stability, high abscisic acid and low ethylene content

C-1730 (triploid)

T25 (4x)  S162 (2x)

15–16

2012

Rainfed red laterite soils of Eastern and Central India, tolerant to drought/moisture stress

S-1608

Selection from OPH (CSRS-1)

30–35

–

Irrigated

C-2038

CF1-10  C763

Irrigated: 53–54 Rainfed: 17– 21

2017

Irrigated and Rainfed

TR-4

Triploid from OP seeds

4–5

–

Hills and foot hills

TR-23 (triploid)

T-20 (4x)  S162 (2x)

Hills: 11–12 Foot hills: 24–25

2017

Rainfed hills of Eastern and NE India, fast growth after pruning, very early defoliation

Kosen

Introduction from Japan

Hills: 4–5 Foot hills: 10–12

1965

Hills and foot hills, quick sprouting after pruning

C-2058 (C-9)

Berhampore A  Shrim 2

34–35 (irrigated, under 50% NPK)

2020

Low input soils or 50% RDF in Eastern and NE India, low leaf senescence, quick sprouting, high survival rate, low internodal distance, early vigor after pruning

C-1360 (Ganga)

Philippines  Vietnam-2

Irrigated: 57

AICEM IV (2019)

Irrigated, high leaf thickness, high regeneration

C-2060 (Gen-1)

Kajli OP  V-1

58–60

2020

Irrigated, quick sprouting and early maturity, short internodal distance, high survival rate, low leaf senescence, tolerant to low temperature stress, high leaf yield under winter

C2020

CW  S1532

–

–

Water logging areas (continued)

22

P. Saini et al.

Table 2.6 (continued) Agro-climatic zone

Mulberry cultivar

Pedigree

Leaf yield (MT/ha/yr)

Year of development/ release/ recommendation

Recommended condition

Irrigated

Developer institute: CSR&TI, Mysore South

Kanva-2 or M5

OPH Selection from the seedling population of Mysore local

30–35

1968

S-30

EMS treatment of Berhampore Local

30–35

–

35–40

1986

Irrigated, Suitable for young age silkworm rearing, grow well in red laterite soils

S-54

40–45

1984

Irrigated

S-41

30–35

S-36

Irrigated

Victory-1

S-30  Ber. C-776

65–70

G-2

M. multicaulis  S-34

38–40

2003

Irrigated (Chawkie rearing)

60–65

2003

Irrigated (Late age rearing)

15–17

1990

Rainfed

S-34

Selection from polycross progeny

13–17

1990

Rainfed conditions with alkaline soils

MSG-2

BR-4  S-13

22–23

2015

Rainfed

RFS-135

OPH from Kanva-2

10–12

1986

RFS-175

10–12

1990

MR-2 (Mildew Resistant-2)

Selection from OPH

Irrigated: 30–35 Rainfed: 10–12

–

Irrigated and rainfed, high rooting ability, resistant to powdery mildew

AR-11 (Alkaline Resistant-11)

Open pollinated selection from K2

15–18

1999

Rainfed and semi-arid

AR-12 (Alkaline Resistant -12)

S-41 (4x)  Ber.C-776

23

2000

Irrigated, alkaline soil with pH > 8.5

Sahana

K2  Kosen

25

2000

Irrigated, grows well under shade in a coconut garden in peninsular states of India

RC-1 and RC-2 (Resource Constraint)

Punjab Local (M. indica)  Kosen (M. latifolia)

23–25

2002

Limited irrigation, suitable for late age rearing

G-4 S-13

1997

Irrigated, Suitable for young age and late age silkworm rearing, popular in Karnataka, Tamil Nadu, Andhra Pradesh, Kerala, Maharashtra

(continued)

2

Cultivation, Utilization, and Economic Benefits of Mulberry

23

Table 2.6 (continued) Agro-climatic zone

Mulberry cultivar

Pedigree

Leaf yield (MT/ha/yr)

Year of development/ release/ recommendation

Recommended condition

AGB-8 (Advanced Generation Breeding -8)

(Sujanpur 5x Philippines)  (K2  Black cherry)

45–47

AICEM IV (2019)

Sub-optimal

Anantha

Selection from RFS135 (OPH of K2)

2010

Irrigated

Irrigated: 35–40 Rainfed: 15

2000

Irrigated (high temperature and red soils) and rainfed, 75% water content, 80% moisture retention capacity

Vishala (triploid)

Irrigated: 45–50 Rainfed: 16– 18

2010

Irrigated (southern red soil and north black soil) and rainfed, water content – 76%, MRC – 85%

Thalaghattapura (TG)

35–40

–

Irrigated (Maland region and southern red soils)

Leaf yield potential about 5–8% higher than V-1 variety

–

Southern red soil regions, Northern black soil regions

Developer institute: KSSRDI, Karnataka South

DD (Viswa)

Clonal selection

Suvarna-1

M5  Mysore local

Suverna-2

M5  Viswa

Suverna-3

–

cultivation spreads rapidly across various agroclimatic zones. Previously, the cultivars Mysore Local in Karnataka and Berhampore Local in West Bengal were very common in traditional Sericultural areas, with yield potentials of about 20–25 tons/ha/year when irrigated. Two open pollinated hybrids (OPH) selections, K2 or M5 (Kanva-2) and Ber. S-1 (Mandalaya), was developed and released in Karnataka and West Bengal, respectively, in the mid-1960s, with a yield potential of 30–35 tons/ha/year under irrigated conditions. (Mallikarjunappa 2001). The mulberry leaf productivity has been hampered by several abiotic stresses (drought, cold, heat), soil stresses (acidity, alkalinity, salinity, toxicity), groundwater depletion, low soil organic content, soil nutrient deficiency (Vijayan et al. 2022a; Vijayan and Gnanesh 2022), and biotic stresses (diseases, pests, and

– –

weeds) in a varied range of agro-climatic conditions across the country (Table 2.9). Through various breeding methods, several mulberry varieties have been developed in India and out of which 18 mulberry varieties have been recommended for the different agro-climatic conditions in India for cultivation (Table 2.10).

2.3.3 Mulberry Cultivation Practices The success of high-quality cocoon yields is entirely dependent on proper mulberry garden planning and maintenance, as well as good silkworm rearing monitoring. Before plantation selection of mulberry variety, preparation of land, identifying the planting season, preparation of seed, spacing, and irrigation practices should be taken care of. Fertilizer application is another

24

P. Saini et al.

Table 2.7 Mulberry varieties cultivated in China Variety

Species

Ploidy level

Breeding method

Leaf yield (Kg/ha/Yr)

Recommended areas for cultivation

Tong Xiang Qing

M. multicaulis

2n = 2x = 28

–

34,500

Zhejiang Province

Selection from a natural population

33,750

Hu Sang 197 Huo Sang (Fire mulberry)

M. mizuho

2n = 3x = 42

Selection from a natural population

34,050

Nong Sang 8

M. alba

2n = 2x = 28

Selection from a natural population

45,000

Zhejiang province, cultivation in the river and hill side as medium low bush

Hong Cang Sang

M. multicaulis

Selection from Tong Xiang Qing

33,900

Zhejiang and Jiangsu Provinces

Hu Sang 199

M. alba

Selection from a natural population

29,250

Yu 2

M. alba

Selection from hybridization of Hu-sang 39  Guangdong Jing-sang

31,200

–

Zhong Sang 5801

M. atropurpurea

Selection from the hybridization of Hu-sang 38  Guangdong Jing-sang

23,400

–

Heyebei (Hu Sang 32)

M. multicaulis

Selection from a natural population

40,000

Zhejiang and Jiangsu Provinces

Hei You Sang

M. alba

Sichuan Province, Recommended for cultivation in the foothill areas as medium bush

2n = 3x = 42

Natural triploid

20,700

Natural triploid

18,000

2n = 2x = 28

–

18,750

Da Hua Sang Xiao Guan Sang Jia Ling 16

–

2n = 3x = 42

Xiqing (4x)  Yu 2(2x)

High productivity

Sichuan, Xinjiang, Guizhou, Henan and Shaanxi

Guandong Jing Sang

M. atropurpurea

2n = 2x = 28

Heterogeneous variety

33,750

South China

Lun 40

2n = 3x = 42

Selection

46,500

Lun 109

2n = 2x = 28

Selection from a natural population

39,450

Selection

39,000

Sha 2

South China, tolerance to drought but week tolerance to cold

Da 10

2n = 3x = 42

Natural triploid

15,000

South China

Kang Qing 10

2n = 2x = 28

Selection from a natural population

37,050

South China, early sprouting and early maturity (continued)

2

Cultivation, Utilization, and Economic Benefits of Mulberry

25

Table 2.7 (continued) Variety

Species

Hei Lu Cai Sang

M. multicaulis

Breeding method

Leaf yield (Kg/ha/Yr)

Recommended areas for cultivation

–

19,500

Shandong China

Da ji Guan Sang

–

20,000

Shandong China, strong tolerance to cold and wind, recommended for cultivation in yellow river area

Xuan 792

Selection

30,000

Shandong and North China

Niu Gen Sang

–

15,000

Hebei province

Hong Pi Wa Sang

–

18,000

Hubei China

–

20,250

Shanxi China

Natural triploid

17,700

South Xinjiang, Strong resistance to drought and cold

HeiGe Lu

Ploidy level

M. alba

He TianBai Sang

2n = 2x = 42

Yun Sang 2

2n = 2x = 28

Dao Zhen Sang TuantouHeyebai

M. multicaulis

2n = 2x = 28

Selection

27,000

Yunnan

Selection

22,500

Guizhou

Selection from natural population

35,680

Yangtze river basin of China

Huo (2000)

factor that influences quality production and high yields. Mulberry is grown as a bush in tropical and dwarf and trees in temperate climates.

2.3.3.1 Climate and Soil Type In temperate region conditions, February–March and in tropical/sub-tropical regions monsoon season (June–July) is the most suitable time for plantations, respectively. The survival of cuttings will be low under hot summer conditions; therefore, it will be better to take plantation under the scheduled time. Mulberry can be cultivated under diverse edaphic conditions such as red loamy soil, alluvial soil, and black cotton soil. The mulberry can be even grown on slopes of hilly areas which are not prone to waterlogging. In the case of slightly slope lands, proper drainage must be provided. Mulberry can be planted on both gentle and steep slopes. Contour plantation can be undertaken when the slope is less than 15% and for 15–3-percent slope,

terraces can be formed for mulberry plantation. Mulberry cultivation is undertaken under a varied range of soil types in India. For a good and healthy plantation healthy, fertile loam to clay soil with 6.5–6.8 pH is most suitable (Datta 2000). Soil with low salt and acid concentration is ideal for mulberry cultivation. Problematic soils (saline and alkaline) are not good for mulberry growth and development. Gypsum, sulfur, or green manuring are the way for the correction of For good growth of mulberry, the saline and alkali soils can be improved by the application of gypsum, sulfur, or green manuring, while acid soils can be improved by the application of lime and green manure (Table 2.11). A pH range of 5.5 (acidic) to 9.0 (alkaline) can be tolerated and for soil correction amendments like gypsum and lime could be used. In West Bengal and Kashmir, it is grown in alluvial soils. These soils are generally gray, light brown to brown, sandy loam to loam in soil texture, slightly alkaline to neutral in reaction,

26

P. Saini et al.

Table 2.8 Major mulberry varieties cultivated in Japan Variety

Species

Ploidy level

Breeding method

Year of recommendation

Remarks

Ichinose

M. alba

2n = 2x = 28

Selection from Shirodashi saplings of Nezumigaeshi

1898

Suitable for young and late age silkworm rearing, medium resistance to bacterial wilt, powdery mildew and rust disease, dwarf disease

Kairyo Nezumigaeshi (KNG)

M. alba

2n = 2x = 28

Selection from Shirodashi saplings of Nezumigaeshi

1907

Suitable for young and late age silkworm rearing, resistant to bacterial wilt, powdery mildew and rust disease

Roso

M. latifolia

2n = 2x = 28

Selection from the natural population

–

Drought tolerant, resistant to bacterial wilt, powdery mildew and rust disease, susceptible to dieback

Akagi

M. bombycis

2n = 3x = 42

Selection from the natural population

–

Suitable for young and late age silkworm rearing during spring and winter, resistant to bacterial wilt, powdery mildew and rust disease

Kenmochi

M. bombycis

2n = 2x = 28

Selection from natural population

1910

Suitable for young and late age silkworm rearing, resistant to bacterial wilt, powdery mildew and rust disease

Ichihei

M. bombycis

2n = 3x = 42

–

–

Resistant to bacterial wilt, powdery mildew and rust disease

KairyoRoso

M. latofolia

2n = 2x = 28

–

–

Drought tolerant, resistant to bacterial wilt, powdery mildew and rust disease, gall midge

Kokuso13

M. latifolia

2n = 2x = 28

–

1922

Resistant to bacterial wilt, powdery mildew and rust disease, medium resistance to dwarf disease

Kokuso 70

M. latifolia

2n = 2x = 28

–

1922

Resistant to bacterial wilt, powdery mildew and rust disease

Kokuso 21

M. latifolia

2n = 2x = 28

Selection from cross between Nganuma  Shiso

1949

Resistant to bacterial wilt and medium resistant to rust disease

Kokuso 27

M. alba

2n = 2x = 28

Selection from cross between Nganuma  KNG

1949

Resistant to bacterial wilt and powdery mildew, medium resistant to rust and dwarf disease

Kairyo Ichinose

M. alba

2n = 2x = 28

Selection from cross between Ichinose  Shirame-roso

–

Resistant to bacterial blight and powdery mildew, medium resistant to rust disease

Sarkar (2009)

2

Cultivation, Utilization, and Economic Benefits of Mulberry

27

Table 2.9 Agro-climatic conditions and issues to be addressed Sl. No.

Area

Zones/states

Problems/constraints

1

High altitude hilly regions

North and West, North-East, Parts of West Bengal, Karnataka

• • • •

Acidic Soil Cold and high dormancy Higher incidence of powdery mildew More acceptability of other cash crops

2

Plain-irrigated

South, Central, East, Maharashtra, Madhya Pradesh, Uttar Pradesh

• • • •

Medium yield Competition with horticultural crops High incidence of fungal diseases Pest and disease infestation

3

Semi-arid & rainfed regions

North-East, North West, Parts of Karnataka, Tamil Nadu, Andhra Pradesh, Maharashtra, MP, Odisha, Jharkhand, Bihar, Chhattisgarh

• Scarcity of water • Erratic and deficit rainfall • High atmospheric temperature, low humidity and high wind velocity • Lower yields • Soil alkalinity

4

Regions with Saline soil

Coastal West Bengal, Karnataka, Tamil Nadu, Andhra Pradesh

• Saline soils • Saline irrigation water

5

Regions with alkaline soils

Karnataka, Tamil Nadu, Andhra Pradesh

• Alkaline soils • Hardy irrigation water

Sivaprasad et al. (2021)

Table 2.10 Mulberry varieties authorized in India Sl. No.

Variety

Leaf yield potential (Mt/ha/yr)

Year of authorization

Suitable zone

1

G-4

55–60

2017

South India irrigated

2

C-2038

50–55

2017

Eastern and NE India Irrigated

3

TR-23

15–20

2017

Hills of Eastern India

4

Victory-1

55–60

2010

South India Irrigated

5

Vishala

55–60

2010

All India under Irrigated condition

6

Anantha

50–55

2010

South India Irrigated

7

DD

50–55

2000

South India Irrigated

8

S-13

12–15

2000

South India Rainfed

9

S-34

12–15

2000

South India Rainfed

10

S-1

35–40

2000

Eastern and NE India Irrigated

11

S-7999

35–40

2000

Eastern and NE India Irrigated

12

S-1635

40–45

2000

Eastern and NE India Irrigated

13

S-36

40–45

2000

South India Irrigated

14

S-146

14–16

2000

N. India and Hills of J&K Irrigated

15

TR-10

11–15

2000

Hills of Eastern India

16

BC259

11–15

2000

Hills of Eastern India

17

ChakMajra

25–30

2000

Sub-temperate

18

Chinese White

25–30

2000

Temperate

Source AICEM, Central Silk Board

28 Table 2.11 Soil amendments to be followed to bring soil pH to 6.8

P. Saini et al. pH range

Quantity of gypsum (MT/ha)

7.4–7.8

2.0

7.9–8.4

5.0

8.5–9.0

9.0

9.1 and above

14.0

pH range

Quantity of lime/ha Plains (MT)

Hilly areas (MT)

Soil type

5.5–6.5

1.25

2.5

Sandy

2.50

5.0

Sandy loam

5.0

7.5

Loamy

7.5

8.75

Clay loamy

Adopted from Datta et al. (2000)

and non-calcareous. They are generally deficient in nitrogen, humus, and occasionally in phosphorous. In Karnataka, the mulberry soils are predominantly red loam derived from granites and gneisses. The main characteristics of red sandy loam are shallow to medium in depth, well-drained, color bright red to pale brown, poor in water-holding capacity and bases, sandy to sandy loam in texture, alkaline to neutral in reaction, and low in organic matter. Most of the soils are deficient in available phosphorous and contain variable amounts of potash (Rangaswami et al. 1995).

2.3.3.2 Planting Material, Propagation, and Planting Schedule Being a perennial tree plant, both sexual and asexual mode of propagation is available in mulberry. It is amenable to propagation through the sowing of seeds, raising of seedlings, saplings through cutting, grafting, budding, and layering methods. Among the various propagation methods, raising mulberry sapling through stem cutting is the most common and obvious method followed in almost all the mulberry growing countries. About six to eight-month-old healthy, well-developed shoot with active buds is suitable for preparation of cuttings. The cuttings of 15–20 cm long having 3–4 active buds and 22–25 cm long with 5–6 healthy buds are best for raising mulberry under irrigated and rain fed conditions, respectively. The rooting ability of

stem cuttings depends on genotype (regenerative ability), environmental factors (temperature, humidity), physiological state (root primordial development, stored nutrient), and management practices (intercultural operations, i.e., fertilization, irrigation, weeding, etc.). The tropical mulberry varieties have better rooting ability than the temperate mulberry varieties. The rooting ability of the temperate genotype could be improved by treatment of cuttings with growth regulators (Indole Acetic Acid, Indole Butyric Acid, and Naphthalene Acetic Acid) and by grafting a poor rooter as scion on a good rooter genotype as stock (Ravindran and Rajanna 2005). The land should be plowed properly and irrigated adequately before planting. To avoid field mortality, the cutting should be treated with appropriate fungicides and planted in a straight position with active buds above the soil surface. For improving survival, the cuttings can be treated with Vesicular–Arbuscular Mycorrhiza (VAM). For the proper establishment of mulberry plantations, early spring and late autumn seasons are best suited. Planting in the winter and summer seasons is to be avoided. When planting is made in spring, care is taken not to delay the planting; otherwise, the sprouted buds fall off and the plants do not grow well. In India, the planting season varies in different parts (Ravindran and Rajanna, 2005). In Karnataka, mulberry is planted during July–August, with the onset of the

2

Cultivation, Utilization, and Economic Benefits of Mulberry

South-Western monsoon. Subsequent rains help the proper establishment of the crop. In West Bengal, cuttings are planted during November (late autumn) after the cessation of North-East monsoon rains. Planting in the rainy season will result in the rotting of cuttings. In Kashmir, before the onset of spring, planting activities are initiated, and before sprouting, cuttings/saplings are planted during the February–March season.

2.3.3.3 Planting Distance The planting distance depends upon the agroclimatic conditions (sunshine, rainfall, temperature, etc.), soil fertility status, the intensity of cultivation practices adopted including the pruning and harvesting methods, and also the mulberry variety planted. For optimum plant density of mulberry variety, training systems and harvesting methods should be taken into consideration. Regarding inter-plant spacing, plenty of considerations like plant habit, branching pattern, canopy structure of variety, intercultural operations for maintaining proper growth, and soil fertility status should be taken care of. In general, mulberry varieties that have profuse branching habits must be planted with wider spacing than those having less branching. In mulberry plantations raised under rainfed conditions, where moisture is a limiting factor wider spacing should be followed (Rangaswami et al. 1995). Cutting of 6–8 months old or sapling of 3–4 months can be used for plantation under different spacing, viz. 60  60 cm (27,778 plants/ha), 90  90 cm (12,346 plants/ha), 90 + 150  60 cm (13,888 plants/ha), and 90 + 120  60 cm (15,873 plants/ha). In the case of pit system, the sapling is planted at a distance of 22 cm with a row to row distance of 60 cm covering 76,923 plants/ha. In the southern region of India, mulberry is also planted in ridges and furrows under the Kolar system where 0.30– 0.45 m ridges and furrows are made and mulberry is planted at a distance of 0.10–0.15 m. A Strip system of planting is followed in West Bengal where the spacing of 0.6 m is kept in between strips. In Kashmir, a tree-type plantation is raised in a block with a 2.7  2.7 m distance between row to row and plant to plant. The dwarf

29

and bush-type plantations are also available in Kashmir with 72  36 cm and 36  16 cm, respectively (Kamili and Masoodi 2000). Under irrigated and rainfed situations, 36  36 cm and 24  24 cm spacing is used for bush-type plantation and 72  36 cm and 72  48 cm for dwarf plantation in the Jammu division (Table 2.12) (Ravindran and Rajanna 2005).

2.3.3.4 Training and Pruning System After planting, the saplings are cut to a uniform height. In the low cut and medium cut forms, the saplings are cut 15–30 cm above the ground level. In high cut form, which sets the length of the main stem at one meter or more, the saplings are cut to a height of about 10 cm from which a stout main stem is allowed to develop. During the following year, the main stem is cut to the desired height. In this case, the completion of the plant form is delayed by one year. But the merit is that since a stout main stem is formed, the branches develop luxuriantly. When the main stem is one meter or more in height support is provided to prevent the sapling from lodging on the ground due to wind. The inter-cultivation such as weeding and mulching is necessary for the maintenance of a good mulberry plantation. Weeding helps in removing unwanted plants which compete with mulberry for water and nutrition and hampered mulberry growth. Weeding not only removes the weeds but also loosens the soil to allow rainwater to soak deep into the soil and for better aeration and nitrification. Mulching helps in conserving soil moisture, keeps the soil loose and friable, protects the plants from winter injury, and keeps down the weeds. Pruned mulberry twigs are spread in between rows as mulches. Also, paddy straw, leaf mold, autumn leaves, stubbles, etc., serve as natural mulches (Rangaswami et al. 1995). Pruning is an important process in agricultural and horticultural crops for maintaining proper canopy shape, plant structure, and enhancement in leaf yield and nutritive value. In mulberry, it is practiced solely to improve the foliage yield, plant shape, and for an early harvest of leaves for silkworm rearing. It is beneficial in adjusting the leaf production period to synchronize with the

30

P. Saini et al.

Table 2.12 Mulberry cultivation under different Sericultural areas Regions

Variety

Spacing (cm)

Pruning (cm)

Manure and fertilizer NPK (kg/ha/yr)

South India (1) Irrigated

Kanva-2 S-36 S-54 V-1 DD MR-2

Pit system 60  60 90  90 Kolar system 45  30 Paired row (150 + 90)  60

15–20

20 T & 300:120:120 20 T & 350:140:140

(2) Rainfed

Mysore Local S-13 S-34

90  90

15–20

10 MT & 100:50:50

Eastern and North-East India

S-1 S-799 S-1635 C-2038

Strip system 60  15 120  90 45  23

15–20

Irrigated: 20 MT & 336:180:112 Rainfed: 10 MT & 150:50:50

Hills of Eastern India

TR-10 BC259 Kosen

North India Temperate

Goshoerami Chinese White

72  36 72  48

15–30 50–60 100

10 MT & 300:150:150

Sub-tropical

S-146 S-1635 ChakMajra

90  90 180  90 270  270

15 60–70 1.2–1.5 m

Irrigated: 20 MT & 300:120:120 Rainfed: 10 MT & 150:75:75

Adopted from Ravindran and Rajanna (2005)

silkworm rearing. It is also useful in extending mulberry leaf production in all the seasons, viz. spring, summer, and autumn. It also helps in practicing the intercultural operations for the maintenance of plantation, i.e., aeration and sunlight for luxuriant and healthy growth, inducing more vegetative growth than reproductive growth, removing dead fists and damaged stems, and controlling pest incidence. By proper pruning time and method, it is possible to get two to three harvests in temperate regions and up to five to six harvests per year in tropical regions. According to agro-climate, geographical conditions, leaf harvest, and silkworm rearing mulberry plants are pruned in different ways, i.e., low cut (15–30 cm), mid-trunk (60–70 cm), and high trunk (120–150 cm), and different pruning schedule, viz. bottom pruning (15–30 cm), middle pruning (60–70 cm), top pruning (green softwood portion), and Kolar system (branches

cut to ground level with both pruning and shoot harvest). Recurrent pruning over the years leads to damage to the branches which created dead fists at the stump top and weakens the plants. Therefore, rejuvenation pruning at the height of 2–3 cm below the crown height or first forking helps in restoring plant vigor, increasing the mulberry life span, and improvement in leaf yield and quality (Ravindran and Rajanna 2005; Bogesha and Jayaram 2014).

2.3.3.5 Irrigation Of all the input factors in mulberry cultivation, irrigation has a direct correlation with leaf productivity. Water is an essential component of every living system for all the necessary physiological processes such as photosynthesis, respiration, transpiration, growth, and development. Mulberry plants with an abundant supply of water are characterized by luxuriant growth, the

2

Cultivation, Utilization, and Economic Benefits of Mulberry

foliage well developed, and the leaves succulent and shiny, whereas mulberry plants of dry regions with limitation of soil moisture tend to be stunted with reduced leaf size. A light green color with succulence and a glossy surface is generally indicative of adequate soil moisture and satisfactory plant growth. Stunted mulberry growth, with small dark green puckered and over and early matured leaves that become powdery and fragile, are indicative of lack of soil moisture. The practice of withholding irrigation until the plants show wilting symptoms is not desirable since it would involve additional energy, water, and nutrients for the plant to pick up normal growth. Therefore, it becomes indispensable to maintain soil moisture for mulberry growth and leaf production. The frequency of irrigation in mulberry depends upon several factors such as growth phase, soil type, and agro-climatic conditions. When the mulberry plant is in the active growth stage, i.e., sprouting and foliar development, the crop needs to be irrigated frequently in the tropics like India where plant growth takes place around the year. In tropical areas, the most critical period of irrigation is from November to April whereas, in temperate regions, the active growth stage is from March to October. In mulberry, different methods of irrigation have been followed such as furrow, flat bad, basin, sprinkler, and drip methods (Ravindran and Rajanna 2005; Bogesha and Jayaram 2014).

2.3.3.6 Nutrients and Fertilizers Nutrients are very much important for proper growth and development of every living system, and it is provided in the form of nutrition. There are 17 nutrient elements as per the essentiality criteria of Arnon and Stout (1939). Of these elements, nitrogen (N), phosphorous (P), and potassium (K) are the primary macro-nutrients, essential for growth, development, reproduction, biosynthesis of biomolecules such as carbohydrates, proteins, fats, synthesis of phytohormones involves in various metabolic processes, and enzymes. The deficiency of these nutrient elements causes several abnormalities in plants

31

which ultimately affect all the biological processes essential for better functioning. In mulberry, nitrogen deficiency leads to stunted vegetative growth, drying of leaves/ shed prematurely, and reduces protein and moisture content which directly influence the mulberry leaf nutritive value. The supply of optimum nitrogen quantity to mulberry plants speeded the vegetative growth vigorously, enlargement of leaf size, succulent, and deep dark green with high chlorophyll content making leaf suitable for silkworm feeding. Phosphorous is important for cell division and meristematic activities, its deficiency resulted in stunted growth with reduced leaf size, and older leaves turn into reddish-purple. Among all the nutrient elements, potassium is considered a quality element. The potassium deficiency showed in older leaves with chlorosis and mottled leaf, necrosis along the leaf margins. Potassium deficiency is not seen in most mulberry plantations. These three major nutrient elements are available in the form of urea, diammonium phosphate (DAP) and murate of potash (MOP), and other combinations and are applied in mulberry plantations as per the recommended dose (Table 2.12).

2.3.3.7 Management of Disease and Pest Mulberry is affected by several diseases and insect pests which significantly affects not only the leaf productivity, leaf quality but also hampered silkworm rearing which directly impacts silk quality. Leaf spot, powdery mildew, leaf blight, leaf rust, root rot, and root-knot nematodes are the major problems (Arunakumar et al. 2019a, b, 2021; Gnanesh et al. 2021, 2022; Manojkumar et al. 2022). Besides diseases, there are more than 20 insect pests that severely affect leaf productivity as well as quality. Dissemination of diseases and pests is favored by environmental factors like temperature, humidity, rainfall, etc. Feeding of infected mulberry leaves by silkworms affects cocoon production (Vijayan et al. 2022b). Therefore, it is necessary to take control measures for the management of diseases and pests.

32

P. Saini et al.

2.3.3.8 Leaf Harvest Leaf production is not only important but utilization of mulberry leaf is also another important factor. The harvesting of leaves depends upon the type of rearing practices. There are three methods of mulberry leaf harvesting, viz. leaf picking, branch cutting, and whole shoot harvest. The leaf yield varied as per the mulberry variety, planting system, and agro-climatic conditions. The leaf picking method is very much cumbersome and labor intensive whereas the mulberry leaf is picked up individually. This leads removal of terminal buds and promotes auxiliary buds growth. The first leaf harvest starts after ten weeks of bottom pruning and subsequent picking for about seven to eight weeks; in this way, about six to seven leaf harvests are possible. The entire branch is fed to silkworms in branch picking. Branch cutting is a simple method, minimizes the labor cost and less wastage of leaf, and helps maintain hygienic conditions in silkworm rearing. The whole shoot harvest method involves cutting of shoot from near to the ground and fed to 4th instar worms. This system is practiced in the Kolar region of Karnataka and the Malda district of West Bengal. This method is suitable where the sprouting takes place around the year (Ravindran and Rajanna 2005; Bogesha and Jayaram 2014).

2.4

Factors Affecting Mulberry Leaf Productivity

Leaf productivity is the prime breeding objective in all crop plants. In mulberry also, leaf yield is an important trait that is targeted through different breeding projects. The leaf yield is a polygenic trait, and it is resultant of multiple traits like plant height, a number of branches, length of branch and number of active buds per meter length, internodal distance, leaf area, leaf weight, and biomass (Bindroo et al. 1990; Sahu et al. 1995; Tikader and Kamble, 2008; Vijayan et al. 1997). No doubt, mulberry can be cultivated under a diverse range of edaphic conditions, but leaf yield is an important factor. For the

enhancement in the leaf productivity per hectare, a region wise recommended package of practices needs to be followed because the mulberry leaf is the sole food of silkworms. Quality mulberry leaf is essential for successful silkworm rearing and high cocoon production. Several edaphic, physical, seasonal, and cultural factors influence both quantities as well as the quality of mulberry leaf. Mulberry has an extensive deep root system and for sustaining high leaf productivity and quality; soil plays a major role as it provides the essential nutrient elements which are indispensable for mulberry growth and development, oxygen for root respiration, mechanical support, and moisture. Feeding silkworms on the quality mulberry leaf will improve both the quantity of cocoon as well as the quality of silk, and this will be possible only when mulberry is grown under good soil conditions. It is found that under loamy soil conditions the content of crude protein, moisture, and crude fat is increased and carbohydrate and ash content decreased whereas, under gravelly soils, the leaves become converse. Therefore, cultivation of mulberry under fertile and healthy soil not only improves mulberry productivity and quality but also enhances the raw silk quality. The nutritive value of the mulberry leaf is also influenced by seasonal variation in different mulberry genotypes. Biochemical analysis of different temperate mulberry varieties showed that during the spring season the chlorophyll, protein, and carbohydrate content is on the higher side whereas, during autumn rearing the amount of chlorophyll, protein, and carbohydrate reduced significantly (Table 2.13). It is suggested that the application of fertilizers and manures in mulberry is necessary for the enhancement of the nutritive value of mulberry leaf which subsequently increases the quality of cocoon production (Rohela et al. 2020b). This difference in the mulberry leaf nutrition content is due to the difference in weather conditions from the spring to the autumn season. Under temperate conditions, the average sunshine varied from 5.0 to 10.0 h than the 9.0 to 13.0 h in the tropics. The lesser hours of

2

Cultivation, Utilization, and Economic Benefits of Mulberry

Table 2.13 Biochemical analysis of mulberry leaves during spring and autumn 2019

Mulberry variety

33

Total chlorophyll (mg/g)

Total carbohydrate (mg/g of dry weight)

Total protein (mg/g of dry weight)

Spring

Autumn

Spring

Autumn

Spring

Autumn

Goshoerami

6.20

4.20

262.00

226.48

49.62

40.20

KNG

6.70

4.18

265.00

160.22

53.44

40.02

Ichinose

6.80

6.28

270.00

161.20

50.80

41.28

Chinese White

6.50

5.82

248.00

187.42

40.96

36.24

PPR-1

5.70

4.86

268.00

192.48

47.92

38.22

TR-10

7.10

3.80

310.00

280.00

46.88

40.16

Rokokuyaso

5.40

3.64

270.00

244.12

45.14

37.78

Kariyoroso

5.20

3.42

280.00

212.32

40.30

34.33

Adopted from Annual Report 2019–20 of CSR&TI, Pampore

sunshine lead to a decrease in carbohydrate and protein content. The effect of climatic factors differs season, species, and cultivar wise (Ho et al. 1985; Sakai and Larcher 1987; Kumar 1990; Bari et al. 1990, Wisniewski et al. 2003; Petkov 2012). Reduction in temperature and relative humidity drastically reduced the growth and developmental processes whereas an increase in the temperature fastens the growth and yielded more leaf harvest and biomass (Tzenov 2017). The nutritive value of the leaves is determined by chemical estimation and feed value and rearing performance. The leaves harvested from irrigated conditions possess high moisture status and protein content and are more nutritious than the leaves under rain fed conditions. Feeding the silkworm with leaves grown under supplemental irrigation generally improves the silkworm health, larval weight, cocoon weight, shell weight, and denier. Also, the high moisture content of leaves attracts the silkworm which increases the rate of digestibility and nutrient assimilation (Rahmathulla et al. 2006). Thus, irrigation plays a pivotal role in enhancing leaf productivity as well as cocoon productivity. Among the nutrient elements, nitrogen played a major role in improving leaf quality. The nitrogen is supplied to mulberry in the form of chemical fertilizers, and it increased the crude protein content in mulberry leaves. The leaves become more succulent, and their maturity is delayed.

2.5

Utilization of Leaf

Mulberry (Morus spp.,) is a well-known and highly recognized plant species for better utilization of its leaf foliage in diversified ways (Thaipitakwonga et al. 2018). The Mulberry plant which is a perennial and woody natured tree species is widely distributed across the globe due to its nature of wide plasticity to survive under favorable as well as extremely harsh and disruptive environmental conditions (Ercisli and Orhan 2007). As it can be survived from below 0 °C to above 45 °C, the leaf foliage can be found in all regions including tropical, subtropical, temperate, and cold arid zones of most countries (Zhao 2009; Khan et al. 2013). Across the world, mulberry leaf has been in use since ancient days and it has wider acceptance regarding its leaf foliage as an ayurvedic source, as feeding material for monophagous silkworm larvae (Bombyxmori L.), as a source of high carbon sequestration in clearing air pollution, as green fodder for livestock’s and as a source of nutrients and phytopharmaceutical compounds for human beings (Rohela et al. 2020b). Because of the abovementioned applications and utilization in diversified fields; now in the present decade, mulberry is widely cultivated for revenue generation, rural empowerment, health benefits for humans, and also for providing a sustainable environment for future generations

34

P. Saini et al.

ANIMAL HUSBANDRY AYURDVDIC & TEA

SERICULTURE PHARMACEUTICAL & COSMETIC INDUSTRY

Fig. 2.1 Diversified uses of mulberry leaf across sericulture, animal husbandry, pharmaceutical, cosmetic, ayurvedic, and tea industries

(Srivastava et al. 2006). In the present era, mulberry leaf foliage is widely utilized across the various industrial sectors such as sericulture, animal husbandry, food industry, tea industry, cosmetic industry, and pharmaceutical industry (Gupta et al. 2005; Iqbal et al. 2012) (Fig. 2.1). Mulberry leaf foliage has various superior characteristics as compared to other plant species in considering it as a wonderful plant; as it is a rich source of essential biochemical compounds (vitamins, organic acids, minerals, and amino acids) required by humans, as a source of high palatability and digestibility natured leaf foliage with dietary proteins to livestock animals; as a

sole source of food material for producing commercial raw silk by the silkworms, well recognized as a good source of phytopharmaceutical compounds and also regarded as one of the best plants in providing sustainable environment (Asano et al. 2000; Srivastava et al. 2006; Yigit et al. 2010; Younus et al. 2017). The mulberry plants with broader leaves were also found to be good in the removal of carbon-based air pollutants (Lu et al. 2004); hence, mulberry plantation drives were also recommended and carried out in many European countries for the removal of carbon-based air pollutants, especially in the urban areas (Jian et al. 2012).

2

Cultivation, Utilization, and Economic Benefits of Mulberry

In this part of the book chapter, the utilization of mulberry leaves for medicinal, Sericultural, environmental, and industrial based applications was discussed in detail with a thorough literature review.

2.5.1 Utilization in Sericulture Since the old days, the mulberry leaf is mainly recognized as a sole source of feeding material for rearing the silkworm larvae of B. mori L., as these commercial insect larvae are monophagous (Rohela et al. 2018a). As compared to other continents, it is widely cultivated as a food plant for silkworm rearing in the countries of the Asian continent: majorly in China, India, Japan, Thailand, Malaysia, Indonesia, Korea, Vietnam, etc., (Sanchez 2000). Because of its economic importance in producing commercial cocoons, a lot of progress has been achieved concerning its cultivation, disease and pest management, agronomical practices, and also in developing and evolving new mulberry varieties which are suitable for specific ecological zones (Qin et al. 2012). As stated above even though the mulberry leaf was mainly utilized for silkworm rearing, the utilization of leaf is not limited only to silkworm rearing but also for other purposes (i.e., food, fiber, fodder, and fuel) which will be discussed in the later part of this book chapter. By seeing the economic progress, the countries of continents other than Asia have also started utilization of mulberry leaf in sericulture practice for revenue generation and employing rural people (Rohela et al. 2018b). Hence, this tree species is now cultivated widely in many countries of America, Africa, Australia, and Europe (Ercisli and Orhan 2007). Silkworm larvae of B. mori after it emerges from eggs; it passes through five instars stages to become an adult moth, during these stages the larvae feed on mulberry leaves and grows robustly to gain the weight of 10,000 times in comparison with their original weight of right after it emerges from the eggs (Rahmathulla 2012). Among the various species of the

35

mulberry, M. alba and M. indica are mainly utilized in India for feeding the silkworm larvae (Rohela et al. 2020a). During the initial stages, i.e., during the first and second instar larval stages priority is given to those varieties which possess a high content of moisture (> 70%) and mulberry leaves were fed by chopping into some sized pieces. During the third, fourth, and fifth instar stages of larval development, priority is also given to mulberry varieties with high contents of biochemical compounds such as proteins, carbohydrates, and chlorophyll (Das and Sikandar 1999). The nutrient quality of mulberry leaves plays a pivotal role in the active growth of larvae, timely molting, and subsequent production of quality cocoons in terms of shell weight (shell ratio), unbreakable filament length, denier, and renditta (Dandin et al. 2003). Freshly harvested mulberry leaves are highly succulent and palatable in nature to silkworms and ruminant animals (Ba et al. 2005; Simbaya et al. 2020). It contains approximately 71–77% moisture content, 8–13% of carbohydrate, 8–11% of natural detergent fiber, 5–9% of crude protein, 0.6–1.5% of crude fat, 4–10 mg of iron/100 g of fresh leaves, 0.16– 0.28 g of ascorbic acid/100 g of fresh leaves, 10– 14 µg of b-carotene/100 g of fresh leaves, 0.38– 0.78 g of calcium/100 g of fresh leaves, and 0.2– 1.1 mg of zinc/100 g of fresh leaves (Srivastava et al. 2006). Along with the above constituents, mulberry leaves also possess several other phytochemical compounds such as anthocyanins, alkaloids, coumarins, flavonoids, simple phenols, polyphenols, phenolic acids, saponins, stilbenes, sterols, and terpenes (Ahmad et al. 2013; Ramesh et al. 2014). Among the various constituents of mulberry leaf, the protein is the major biochemical compound that is responsible for boosting the metabolic activities of silkworm larvae resulting in active growth of larvae, cocoon formation (silk production) by larvae, and egg production by the adult moths (Zuhua 1994). Among the various proteins of plants, nitrate reductase is a key enzyme that catalyzes the reaction of converting nitrate to nitrite and providing the nitrogen source to mulberry plants (Ghosh et al. 1994). So

36

mulberry varieties with high NR activity and with higher soluble protein contents result in higher yield and good quality of mulberry leaf production (Ghosh et al. 2006), and it will reflect in the quality and quantity of cocoon and raw silk production. Therefore, the demand for greater raw silk production in India and other countries is mostly fulfilled by having mulberry varieties with higher leaf yield and quality leaf production. Higher quantity and quality leaf production is a polygenic character that is further influenced by the physiological and biochemical processes of the mulberry plant (Vijayan et al. 1997). The higher yield of leaf and biomass production is majorly dependent on the photosynthetic assimilation of CO2 (Menon and Srivastava 1984).

2.5.2 Mulberry Leaf for Livestock In most parts of the world including tropical and temperate regions, cereal crops, weeds, shrubs, grasses, and agricultural by-products are majorly fed as calorified and proteinaceous food material to ruminant animals (Leng 2002). But these dietary products which are used as animal fodder possess fewer calories, low organic matter, lower protein content, and deficiency in essential mineral elements (Garg and Gupta 1992). Further, the unpalatable and indigestible nature of these fodder results in lower intake of food material voluntarily by animals which often results in the undergrowth of domesticated animals/livestock and ultimately reflects in low production of livestock-based products such as meat and milk (Shayo 1997). Among the various alternative sources of fodder that can provide a balanced diet with regard to its protein content (14–30%), palatability due to its succulent nature and in vivo digestibility nature (75–855) in ruminants, the mulberry tree species was used as an alternative source and it is in practice since a long time in using its leaves in both fresh as well as in dry form as an animal feed (Datta 2000; Ba et al. 2005). Mulberry leaf due to its wide distribution in every part of the world, due to leaf quality

P. Saini et al.

parameters, due to palatability, and digestibility nature of leaves; the mulberry plants are now considered one of the sources for feeding domesticated livestock animals such as cows, buffaloes, sheep, goats, poultry, and rabbits (Sanchez 2000). Mulberry leaf possesses several phytochemical constituents which include flavonoids, alkaloids, anthocyanins, stilbenes, saponins, etc. (Chen et al. 2015a, b). There are several studies conducted regarding the phytochemical contents of mulberry leaves which can induce higher and quality production of milk and meat (Li et al. 2020a, b). Among the various phytochemicals, flavonoids (which are found abundantly in mulberry leaves) are a type of polyphenolic compounds that possess diversified biological (antioxidant potential) and pharmacological activities by which it enhances the growth and development of livestock animals and result in the quality production of animal products (Olagaray and Bradford 2019). Several studies were conducted on different livestock animals by utilizing mulberry leaf as a feeding material (Table 2.14). In a study conducted by Li et al. (2020a, b), it was found that mulberry leaf flavonoids by feeding at 45 g per day can alleviate heat stress tolerance as well as enhance milk production in buffaloes. Mulberry leaf flavonoids are also reported to regulate fat metabolism by mediating the transcriptional signaling of genes involved in fat metabolism (Zheng et al. 2014). Mulberry leaf flavonoids up-regulate the uncoupling protein-1 (UCP-1) of brown adipose tissue in animals and increase its body temperature by releasing the dietary energy; thus, this mechanism makes the animals fed with mulberry leaf flavonoids alleviate their heat stress tolerance capacity (Sheng et al. 2019). Further, improved glucose metabolism and insulin activity were achieved by upregulation of UCP-1 in activating the adipose tissue and increasing the body temperature (thermogenesis) of animals (Ravussin and Galgani 2011; Chondronikola et al. 2014). In another study, mulberry leaf flavonoids were shown to improve glucose metabolism and restoring of ATP homeostasis by inducing the

2

Cultivation, Utilization, and Economic Benefits of Mulberry

37

Table 2.14 Studies on mulberry leaf as a feed material for livestock animals #

Mulberry

Part

As a source of

Animal

Major finding

References

1

Morus alba

Leaves

Proteins

Sheep (Ovisaries)

Rapeseed meal can be replaced by mulberry leaf as a protein supplement from the feed material of sheep

Liu et al. (2001)

2

Morus alba

Leaves

Flavonoids

Buffalo (Bubalusbubalis)

Mulberry leaf flavonoids by feeding at 45 g per day can alleviate the heat stress tolerance as well as enhance the milk production in buffaloes

Li et al. (2020a, b)

3

Morus spp.

Leaves

Quercetin

Cow (Bostaurus)

Supplementation of quercetin (36 mg/day) in cows has resulted in release of increased insulin, showed insulin sensitivity, and increased glucose metabolism

Gohlke et al. (2013)

4

Morus spp.

Leaf Pellets

Protein

Cow (Bostaurus)

The mulberry leaf pellet supplementation (as a protein source) of 600 g/day along with rice straw as feed for beef cattle has improved digestibility and efficiency of rumen fermentation process

Huyen et al. (2012)

5

Morus alba

Leaves

Flavonoids

Bull (Bosspp.)

The calf fed with 3 g of mulberry leaf flavonoids has showed increased feed efficiency than control

Wang et al. (2018)

6

Morus alba

Leaves

Flavonoids

Calves (Bosspp.)

The calves fed with mulberry leaf flavonoids have showed increased metabolic rates and efficient rumen fermentation process

Yang et al. (2016)

7

Morus alba

Leaves

Flavonoids

Calves (Bosspp.)

The mulberry leaf flavonoids as feed have increased the body weight, digestibility and increase in serum growth hormone levels and insulin-like growth factors (ILG-1) in calves after the age of 56 days

Zhang et al. (2017a, b)

(continued)

38

P. Saini et al.

Table 2.14 (continued) #

Mulberry

Part

As a source of

Animal

Major finding

References

8

Morus alba

Leaves

Leaf extract

Rats (Rattusspp.)

The highest dose of mulberry leaf extract of 100 mg/kg of diet was lowering psychotic behavior in rats by significantly inhibiting serotonin activity

Rayam et al. (2019)

9

Morus alba

Leaves

Whole leaf

Goats (Capra hircus)

The mulberry leaf foliage is of high nutritive value and the nutrient digestibility of leaves was improved by feeding in hanging the leaves as compared to feed supplied in trough

Kouch et al. (2003)

10

Morus alba

Leaves

Proteins

Sheep (Ovisaries)

The mulberry leaves have high content of crude protein (20%) and low crude fiber (12%) which has resulted in better digestibility when fed as a mixed forage diet to sheep

Kandylis et al. (2009)

11

Morus alba

Leaves

Whole content as dry matter

Sheep (Ovisaries) & Goats (Capra hircus)

The mulberry leaves fed as dry matter to sheep and goats has increased the digestibility and weight in compared with control ones

Prasad and Reddy (1991)

12

Morus alba

Leaves

Leaf meal for nitrogen balance

Pigs (Susscrofadomesticus)

The study confirmed mulberry leaf meal as the high nutritive value and can be given as a nitrogen balance to pigs for easy digestibility and faster growth

Ly et al. (2014)

13

Morus alba

Leaves

Whole leaf

Lambs (Ovisaries)

The ruminal dry matter digestibility of feed is increased in lambs when the diet was mixed with mulberry leaves. The mulberry leaves may reduce the cost of conventional protein supplements in the diet of lambs

Chavira et al. (2011)

(continued)

2

Cultivation, Utilization, and Economic Benefits of Mulberry

39

Table 2.14 (continued) #

Mulberry

Part

As a source of

Animal

Major finding

References

14

Morus alba

Leaves

Leaf meal as protein source

Pigs (Susscrofadomesticus)

The growth and weight of pigs has increased by feeding with sweet potatoes as energy source and mulberry leaf as protein source

Araque et al. (2005)

15

Morus spp.

Leaves

Protein

Cows (Bostaurus)

The partial replacement of corn grain and cotton seed meal with ensiled mulberry leaf had no substantial effects on the ruminal microflora composition

Niu et al. (2016)

16

Morus spp.

Leaves

Protein

Goats (Capra hircus)

Feeding of fresh mulberry leaves for 60 days of duration has enhanced the digestive ability, better utilization of nutrients and subsequently leading to enhanced lactation performance in goats

Kumar et al. (2015)

17

Broussonetiapapyrifera

Leaves

Leaf meal

Cows (Bostaurus)

Paper mulberry leaves when fed to dairy cows, it has increased the total antioxidant capacity, superoxide dismutase, antibodies, and immune responses

Hao et al. (2020)

18

Morus alba

Leaves

Proteins, Digestible Nutrients and as Energy source

Cattle (Bostaurus)

Cattle fed with mulberry leaves has showed improved digestibility of dry matter, organic matter, and metabolize energy and net energy of mulberry leaves is higher than the different grasses used in the study

Vu et al. (2011)

19

Morus spp.

Leaves

Flavonoids

Sheep (Ovisaries)

The mulberry leaf flavonoids has improved the digestibility of organic matter, reduced the count of methanogenic microbes from the sheep micro biota, and also reduced the methane formation

Ma et al. (2017)

(continued)

40

P. Saini et al.

Table 2.14 (continued) #

Mulberry

Part

As a source of

Animal

Major finding

References

20

Morus alba

Leaves

Mulberry leaf extract

Salamander Fish (Andriasdavidianus)

Mulberry leaf extract improved the growth performance, gastric acid secretion, digestive capacity, and increased immune parameters in Salamander

Li et al. (2020a, b)

21

Morus alba

Leaves

Leaf Meal

Sting fish (Heteropneustesfossilis)

Mulberry leaf meal as diet has increased the survival rate, feed conversion ratio, and also disease prevention rate in sting fish

Bag et al. (2012)

22

Morus alba

Leaves

Proteins and as a source of other Nutrients

Goats (Capra hircus)

Leaves of mulberry when fed to goat bucks, it showed higher intake of dry matter with better digestion and efficient utilization of nutrients

Bakshi and Wadhwa (2007)

23

Morus alba

Leaves

Carbohydrates, Proteins and Minerals

Rabbits (Oryctolaguscuniculus)

Commercial diet replaced with 50% of wilted mulberry leaves has shown higher weight gain in rabbits with feed to gain ratio of 1:4.5

Khan et al. (2020)

AMPK-PGC-1 signaling pathway in skeletal muscles (Meng et al. 2019). Several studies were conducted on utilizing mulberry leaf as feed material in sheep. In a study, it was found that rapeseed meals can be replaced by mulberry leaf as a protein supplement from the feed material of sheep (Liu et al. 2001). The mulberry leaves have a high content of crude protein (20%) and low crude fiber (12%) which has resulted in better digestibility when fed as a mixed forage diet to sheep (Kandylis et al. 2009). The ruminal dry matter digestibility of the feed is increased in lambs when the diet was mixed with mulberry leaves (Chavira et al. 2011). The mulberry leaves may reduce the cost of conventional protein supplements in the diet of lambs (Chavira et al. 2011). The mulberry leaf flavonoids have improved the digestibility of organic matter and reduced the count of methanogenic microbes from the sheep

microbiota and also reduced methane formation (Ma et al. 2017). The mulberry leaf foliage is of high nutritive value, and the nutrient digestibility of leaves was improved by feeding in hanging the leaves as compared to feed supplied in a trough to goats (Kouch et al. 2003). Leaves of mulberry when fed to goat bucks showed a higher intake of dry matter with better digestion and efficient utilization of nutrients (Bakshi and Wadhwa 2007). Feeding fresh mulberry leaves for 60 days of duration has enhanced the digestive ability and better utilization of nutrients and subsequently led to enhanced lactation performance in goats (Kumar et al. 2015). The mulberry leaves fed as dry matter to sheep and goats have increased the digestibility and weight compared with control ones (Prasad and Reddy 1991). Similarly, research was also carried out on cows regarding the feeding of mulberry leaf

2

Cultivation, Utilization, and Economic Benefits of Mulberry

material as a sole food material or as a supplement to the normal diet of cattle. Cattle fed with mulberry leaves as sole food material have shown improved digestibility of dry matter, organic matter, and metabolize energy, and the net energy of mulberry leaves is higher than the different grasses used in the study (Vu et al. 2011). The mulberry leaf pellet supplementation (as a protein source) of 600 g/day along with rice straw as feed for beef cattle has improved digestibility and efficiency of the rumen fermentation process (Huyen et al. 2012). Supplementation of quercetin (36 mg/day) in cows has resulted in the release of increased insulin, showed insulin sensitivity, and increased glucose metabolism (Gohlke et al. 2013). The calves fed with mulberry leaf flavonoids have shown increased metabolic rates and an efficient rumen fermentation process (Yang et al. 2016). The partial replacement of corn grain and cotton seed meal with ensiled mulberry leaf had no substantial effects on the ruminal microflora composition (Niu et al. 2016). The mulberry leaf flavonoids as feed have increased the body weight, digestibility and increase serum growth hormone levels, and insulin-like growth factors (ILG-1) in calves after the age of 56 days (Zhang et al. 2017a, b). The calf fed with 3 g of mulberry leaf flavonoids has shown increased feed efficiency than control (Wang et al. 2018). Paper mulberry leaves when fed to dairy cows has increased the total antioxidant capacity, superoxide dismutase, antibodies, and immune responses (Hao et al. 2020). Apart from sheep, goats, buffaloes and cows, experimentation with a mulberry leaf as feed material is also carried in other animals. The growth and weight of pigs have increased by feeding mulberry leaf as a protein source along with sweet potatoes as an energy source (Araque et al. 2005). A study confirmed mulberry leaf meal has a high nutritive value and can be given as a nitrogen balance to pigs for easy digestibility and faster growth (Ly et al. 2014). The highest dose of mulberry leaf extract of 100 mg/kg of diet was lowering psychotic behavior in rats by significantly inhibiting serotonin activity (Rayam et al. 2019). Mulberry leaf extract improved the

41

growth performance, gastric acid secretion, digestive capacity and increased immune parameters in Salamander (Li et al. 2020a, b). Mulberry leaf meal as a diet has increased the survival rate, feed conversion ratio and also disease prevention rate in sting fish (Bag et al. 2012). Commercial diet replaced with 50% wilted mulberry leaves has shown higher weight gain in rabbits with feed to gain ratio of 1:4.5 (Khan et al. 2020).

2.5.3 Mulberry in Food and Tea Industry Mulberry leaf is also utilized in the food and tea industry as it is composed of several nutraceutical compounds which include antioxidants, carbohydrates, vitamins (B complex and C), and proteins as major components of interest (Chan et al. 2016). Mulberry leaves also possess vital mineral elements such as calcium, iron, potassium, phosphorous, and Zinc (Lown et al. 2017). Due to its role in promoting the health benefits of humans through the active ingredients of leaves, mulberry is compared as a life enhancer (Kumar and Chauhan 2008). Mulberry leaves are highly palatable in nature and hence used to make herbal teas and tinctures as a common practice of health benefitting hot drinks across several Asian countries. In Korea, tea is made by using the mulberry leaves either in fresh or dried form Mulberry tea has the potential to make the liver function in a normal way, it affects improving the eye sight and mulberry tea is recommended as a hot drink to all ages (Wang et al. 2011). Along with the leaves, young shoot tips and buds were also used in making the mulberry tea (Wani et al. 2017). Mulberry leaves are highly nutritious and contain several antioxidant molecules, vitamins, anthocyanins, flavonoids, and other phenolic compounds (Alfrey2017); hence, mulberry tea gives potential health benefits to humans. Mulberry leaf is also included as a nutritional supplement in preparations of different traditional Indian food items like pakoda, paratha, saag, fried bajji, dhokla, steamed products, and cakes which has received wide acceptability

42

(Srivastavaet al. 1997). As the leaves are composed of several phytochemical compounds which can lower cholesterol and blood sugar levels along with relieving inflammations, leaves were commonly included as an ingredient in the preparation of teas and food items to fight against diabetes and heart diseases and allergies (Chan et al. 2016). Mulberry leaf extract of one gram was given along with meals thrice a day to type 2 diabetes patients has shown significant results in terms of lowering blood sugar levels compared to control ones (Riche et al. 2017). Hence, mulberry leaf extract was suggested to be taken as a complementary meal for type 2 diabetes patients for normal maintenance of sugar levels after having meals (Riche et al. 2017). In a study, people suffering from higher cholesterol levels were given mulberry leaf extract (280 mg) as a supplement to their meals thrice a day, after 12 weeks of giving mulberry leaf extract as a supplementary diet; the bad cholesterol (LDL) levels were reduced by 5.6 and 19.7% of increase of good cholesterol (HDL) levels in those patients (Aramwit et al. 2011). Similarly, in another study supplement of the mulberry leaf containing 1-deoxynojirimycin for 12 weeks reduced the triglyceride levels in patients suffering from high triglyceride levels (Kojima et al. 2010). Mulberry leaf extracts as supplements to food/meals were also reported to prevent the diseases like hypertension, atherosclerosis, heart diseases, and cellular damage (Lim and Choi (2019)). Some studies indicated that mulberry leaves as an additional supplement to a natural diet will result in weight loss and loss of obesity by causing extra fat burning (Sheng et al. 2019). Similarly, hyperpigmentation can also be prevented by using mulberry leaf extract as a part of meals (de Freitas et al. 2016). Anti-cancer activity of mulberry leaf extracts against liver cells was also reported in some test tube based research (Naowaratwattana et al. 2010). The effects of mulberry leaf extracts as supplementation to meal or diet of humans and its potential health benefits are summarized in Table 2.15.

P. Saini et al.

2.5.4 Mulberry Leaf for Medical Use All the parts of mulberry (i.e., leaf, stem, bark, and root) have high medicinal values as it is composed of several pharmaceutically important compounds (Zhu and Lu 2001). In Indian traditional ayurvedic medicinal practice, mulberry tree was described as “Kalpa Vruksha” (Datta 2000). Here in this book chapter, the medicinal values of mulberry are elaborated mainly by focusing on the ayurvedic applications of leaves instead of root and stem parts. The crude extracts of mulberry leaves (Fig. 2.2) were used for curing illnesses of humans, curing insomnia, curing burns, preventing chronic hepatitis, overcoming vitamin deficiencies like beriberi, treating nose bleeding, for treatment of muscular swellings, reducing cholesterol levels, boosting the immunity, anti-aging, snake-bites, heart strokes, curing of constipation, for treatment of night sweating, diarrhea, cold, lowering blood glucose levels, controlling the weight of humans, facilitating discharge of urine, anti-neoplastic activity, eye illness, insect-bites, acne, wound healing, and increasing the life span of humans (Yi et al. 1997; Chu et al. 2001; Ye and Ye 2001; Lea and Lee 2001; Bajpai et al. 2012). Mulberry leaf juice as a moisturizer can be applied over the skin for keeping the skin fresh and moist during the winter seasons (Shiva Kumar et al. 1996). Mulberry leaf and barks contain several pharmaceutically important phytochemical compounds which help in combating many serious human diseases such as hyperlipidemia, protection from cardiovascular diseases, treatment of sore throat, atherosclerosis, strengthening the joints, delay in the aging process, prevent liver and kidney damage, antioxidant, neuroprotective, anti-fatigue, anxiolytic, vasoactive action, macrophage activation, anti-helminthic, anti-fungal, anti-bacterial, anti-viral, prevent premature white hair formation in humans, anti-thrombotic, antiobesity activity, hypertension, filariasis, anticancer, fever, cold, cough, diabetes mellitus, improve eye sight, inflammation, and promote the increased synthesis of progesterone and

2

Cultivation, Utilization, and Economic Benefits of Mulberry

43

Table 2.15 Potential health benefits of mulberry leaf extracts (MLE) when used as a food supplement to human beings #

Supplement

Function

Treatment

References

1

MLE

Anti-cancer activity

Liver Cancer

Naowaratwattana et al. (2010)

2

MLE

Fat burning

Obesity

Sheng et al. (2019)

3

MLE

Prevention of excess pigment formation

Hyperpigmentation

de Freitas et al. (2016)

4

MLE

Reduces the blood pressure and cholesterol levels

Hypertension, Atherosclerosis, Heart diseases, and cellular damages

Lim and Choi (2019)

5

MLE with 1deoxynojirimycin

1-deoxynojirimycin supplementation with meal reduces the triglyceride levels in patients

Hyperlipidemia

Kojima et al. (2010)

6

MLE

Mulberry leaf extract as a supplementary to diet reduces the bad cholesterol (LDL) levels increases the good cholesterol (HDL) levels

Hypercholesterolemia

Aramwit et al. (2011)

7

MLE

Lowering of blood sugar levels

Diabetes

Riche et al. (2017)

8

MLE

Decreases the inflammation

Allergic

Chan et al. (2016)

9

MLE

Combats inflammation and oxidative stress

Chronic disease

Liguori et al. (2018)

10

MLE

Antimicrobial activity against dental caries causing microbial pathogens

Dental caries

Tahir et al. (2017)

11

MLE

Protects against cardiometabolic risks

Heart diseases

Thaipitakwonga et al. (2018)

12

Dry MLE

Mulberry leaf extract can cure filariasis

Filariasis

Peiyi and Zhenhai (1990)

estrogen hormones, rheumatism, and arthritis (Chen et al. 2016a; Zhang et al. 2017a, b; Zhou et al. 2017). Important pharmaceutical biomolecules are reported from the mulberry leaf by various researchers (Enkhmaa et al. 2005; Kiran et al. 2019; Rohela et al. 2020b). Among all the identified compounds, pharmaceutically important are 1-deoxynojirimycin,2-arylbenzofuran, cyanidin-3-O-beta-D glucopyranoside, quercetin, quercetin-3-(6-malonylglucoside), artoindonesianin O, carotenoids, vitamin A, vitamin C, vitamin E, fagomine, moracin-c, morin, chlorogenic acid, caffeic acid, 4-O-caffeoylquinic acid, kaempferol-3,7-glucopyranoside, c-amino butyric acid, coumaric acid, p-hydroxybenzoic acids, 3,8-diprenyl-4,5,7-trihydroxyflavone, etc. (Andallu and Varadacharyulu 2002; Kang et al.

2005; Chen and Li 2007; Ansari et al. 2009; Dat et al. 2010; Park et al. 2013; Eva et al. 2015; Qiao et al. 2015; Chen et al. 2016b; Zeni et al. 2017; Ionica et al. 2017; Ganzon et al. 2017; Kiran et al. 2019). The pharmacological action of the listed pharmaceutical compounds was also reported (Fig. 2.3). Among the different compounds, 1deoxynojirimycin (DNJ) has wide potential applications for improving human health, and this compound can be extracted from leaves, stem bark and root bark of mulberry (Kiran et al. 2019). Among the different parts, DNJ content was reported to be extracted in higher concentration from mulberry stem bark followed by leaves and roots (Liu et al. 2015). In one study; DNJ content was estimated and compared in leaves of 132

44

P. Saini et al.

IMPROVING EYE SIGHT

CONSTIPATION

WOUND HEALING

BURNS

INSECT BITES

IMMUNITY

COLD

ILLNESS

DIARRHOEA

INSOMNIA

ALLERGIES INCREASING LIFE SPAN OF HUMANS

Fig. 2.2 Ayurvedic applications of mulberry leaf in traditional medicinal practice

mulberry varieties belonging to 9 mulberry species (Hu et al. 2013). And it was reported that DNJ content was higher in younger and delicate leaves than the old and fully matured leaves, it was also found that DNJ content in leaves is independent of the type of mulberry variety, species and place of existence of mulberry plantation (Hu et al. 2013). 1-deoxynojirimycin was reported to possess pharmacological actions of anti-diabetic (Chung et al. 2013), anti-hyperlipidemia (Chen and Li 2007), antioxidant (Katsube et al. 2006), antiinflammatory (Park et al. 2013), anti-hypertensive (Kojima et al. 2010), anti-cancer (Shuang et al. 2017), anti-obesity (Kim et al. 2017), and delaying of the aging process (Chen et al. 2015a, b). Apart from DNJ; mulberry leaves also possess several phytochemical compounds which provide potential health benefits to Humans. Vitamin B complex and vitamin C of mulberry leaves boost the immune system, provide antioxidant properties, and help in metabolisms of carbohydrates and fats. Similarly, c-amino butyric acid (GABA) found in mulberry leaves lowers the blood pressure and rutin found in the leaves

maintains normal blood circulation and improves the functioning of the heart. Higher content of superoxide dismutase and flavonoids found in mulberry leaves can eliminate free radicals (Fujun 1989). Volatile glycosides, various inorganic ions, fagomine and hydrophobic flavonoids isolated from mulberry leaves were reported to possess anti-diabetic activity (Fallon et al. 2008; Hunyadi et al. 2014; Bazylak et al. 2014; Kiran et al. 2019). Similarly; quercetin-3-(6-malonylglucoside) phytochemical isolated from leaves were found to exhibit anti-atherogenic properties (Enkhmaa et al. 2005). Moracin-C, chlorogenic acid, and flavonoids were reported for anti-hyperlipidemia activity (Andallu and Varadacharyulu 2002; Li et al. 2005; Zeni et al. 2017). Carotenoids, vitamins A, E, and C, caffeic acid, kaempferol-3,7glucopyranoside, coumaric acid, p-hydroxybenzoic acids, 2-aryl benzofuran, and 4-Ocaffeoylquinic acid compounds isolated from mulberry leaves were reported for antioxidant activity (Butt et al. 2008; Andallu et al. 2009; Memon et al. 2010; Yang et al. 2010a, b; Eva

2

Cultivation, Utilization, and Economic Benefits of Mulberry

Anti-Bacterial

45

Anti-Obesity Anti-aging effects

Anti-Allergic

Anti-Oxidant

Anti-pyretic

Hepatoprotective

Anti-Diabetic Hypolipidemic Anti-hypertensive Anti-Schistosomal

Anti-Inflammatory

Cardiovascular Protectant

PHARMACOLOGICAL ACTIVITIES OF IDENTIFIED PHARMACEUTICALS FROM MULBERRY LEAVES

Neuroprotective

Anti-Viral

Vasoactive

Anti-atherogenic

Anti-Cancer Controls Skin Infections

Fig. 2.3 Pharmacological activities of pharmaceuticals identified from mulberry leaves

et al. 2015; Ionica et al. 2017; Ganzon et al. 2017). The anti-cancer activity was reported from the compound 3, 8-diprenyl-4,5,7-trihydroxy flavone which was isolated from mulberry leaves (Dat et al. 2010). The neuroprotective activity was reported from the mulberry leaf extracted compounds of artoindonesianin O; cyanidin-3-O-beta-D glucopyranoside,-amino butyric acid, and quercetin (Kang et al. 2005; Ansari et al. 2009; Qiao et al. 2015). Similarly, morin for vasoactive action (Fang et al. 2005), polyphenols for anti-aging (Zheng et al. 2014), and c-amino butyric acid for anti-fatigue (Chen et al. 2016a) properties were reported from mulberry leaves (Fig. 2.4).

2.5.5 For Environmental Remediation There is an essential need to address the increasing global concern about environmental issues such as removal of pollutants (from air, water and soil), afforestation to increase the green

cover, control over the release of harmful chemical pollutants from industries, restoration of polluted and degraded environmental sites, and self-purification approach of the environment to purify the atmosphere. Among the various strategies needed to overcome environmental issues, phytoremediation is considered an ecofriendly and safe approach to providing a sustainable environment for future generations. Phytoremediation is a promising process of removing environmental pollutants by using various plant species which absorb, transfer, uptake, degrade, or detoxify harmful and toxic pollutants (Susarla et al. 2002). Among the various plant species, mulberry being a tree species has various advantages for being utilized as a phytoremediation plant species (Rohela et al. 2020b). Mulberry has broad sized leaves which can nullify the air pollutants; due to its deep rooted system, it can withstand the adverse conditions (drought, salinity, heavy metals, wind currents, flooding) posed by the environment and can absorb, degrade, or

46

P. Saini et al.

PHYTOPHARMACEUTICALS OF MULBERRY LEAF

Rutin

Quercetin

Flavonoids

Moracin-C & Chlorogenic Acid

Artoindonesianin O; Cyanidin-3-O-beta-D glucopyranoside

3,8-diprenyl-4,5,7trihydroxyflavone

γ-amino butyric acid (GABA)

Neuroprotecve

An-Atherogenic

An-Fague

1-deoxynojirimycin (DNJ) An-Cancer

Improves Funconing of Heart

An-inflammatory

Carotenoids, Caffeic acid, Kaempferol-3, 7glucopyranoside, Coumaric acid; 2Arylbenzofuran and 4-OCaffeoylquinic acid

An-Oxidant

An-hyperlipidemia

An-Diabec

An-hypertensive

An-Obesity

An-Aging

Fig. 2.4 Pharmacological activities of phytopharmaceuticals identified from mulberry leaves

detoxify the harmful pollutants such as heavy metals and other chemicals (Ghosh et al. 2017). Hence, mulberry plants and their leaves can be utilized for environmental safety and protection. In this book chapter, the main focus will be on the utilization of mulberry leaves in addressing environmental issues (Fig. 2.5). Mulberry is a fast growing tree species with high biomass production through its strong photosynthetic activity. Therefore, it was suggested that mulberry can be planted in urban areas to address the air pollutants. Mulberry leaves due to its stronger photosynthetic rate was reported that one hectare of mulberry plantation can absorb 1000 kg of carbon dioxide from the air in a single day, and in turn release approximately 730 kg of oxygen in a single day, this amount of oxygen could be utilized for the breathing of nearly 1000 people for a day (Tan et al. 2010). Similarly, it was reported that one hectare of mulberry plantation can absorb 49,290 kg of carbon dioxide from the air in a year and release approximately 35,850 kg of oxygen in a year as per the preliminary estimates (Qin et al. 2010). One hectare of mulberry trees was reported to absorb about 6.24  104 kg of

carbon dioxide in a year and in turn, release 4.60  104 kg of oxygen in a year (Jian et al. 2012). Hence, mulberry plants and their leaves are compared as a good source of carbon sink. Along with purifying the air, mulberry plants can also adsorb the dust particles and retain them on the surface of broader sized leaves (Zhang et al. 1997). In a single year, one hectare of mulberry plantations can retain 2720 kgs of dust on the leaves (Tan et al. 2010). Mulberry plants and their broader sized leaves have high resistibility and hence could be able to withstand wind currents and sand storms (Lin et al. 2008). Mulberry leaves have high resistance and absorbing ability toward environmental pollutants. It has a wide absorption ability of carbonbased atmospheric gaseous pollutants (carbon monoxide and carbon dioxide) through the process of carbon sequestration (Lu et al. 2004). They can absorb harmful industrial gasses such as fluoride, chlorine, sulfide, and nitrogen gases (Qin et al. 2010; Liu 2011). Leaves of mulberry in mulberry forestry can absorb large quantities of sulfur dioxide gases on daily basis (approximately 5.7 g of sulfur dioxide per kg of mulberry leaves/day) from the atmospheric gaseous

2

Cultivation, Utilization, and Economic Benefits of Mulberry

47

PURIFICATION OF AIR

ADSORBS THE DUST PARTICLES

CARBON SEQUESTRATION

ACCUMULATIONOF HEAVY METALS IN LEAVES

REMOVAL OF AIR POLLUTANTS

WITHSTANDS THE SAND STORMS

WITHSTANDS THE WIND CURRENTS

ABSORBS THE HARMFUL INDUSTRIAL GASES SUCH AS FLUORIDE, CHLORINE, SULPHIDE &NITROGEN GASES FROM ATMOSPHERE

Fig. 2.5 Role of mulberry leaves in environmental safety and protection

pollutants (Lu and Jiang 2003). Hence, mulberry plants are regarded as sulfur dioxide pollutionresistant tree species (Qin et al. 2012). Similarly, mulberry leaves were reported for stronger absorption and retention capabilities against harmful industrial pollutant metals such as lead and copper from the atmosphere of industrial areas (Lu and Li 2002). Lu and Li (2002) also reported on the high content of fluorine absorption by mulberry leaves (0.45 mg/g of leaves) from the fumigation testing conducted by the research team. Mercury heavy metal present in the soil can be absorbed and up taken in every part of mulberry (Hashemi and Tabibian 2018). Mulberry plants were noticed with undamaged leaves in the presence of high levels of chlorine gas in the atmosphere in the industrial areas (Lu et al. 2004). Due to these properties, mulberry tree species are recommended for phytoremediation of air pollutants in urban areas, chemical factories, and industrial areas for the removal of carbon-based gaseous pollutants, chlorine, fluorine, and other harmful gaseous pollutants (Olson and Fletcher 1999).

Further mulberry plants on urban plantation drive along the road sides, in public gardens, parks, along the banks of river channels, like street trees, as carbon sequestrating plants in the industrial areas, for effective utilization as city landscapes and also mulberry is regarded as green afforestation tree species (Wang et al. 2010; Qin et al. 2012; Jian et al. 2012). As mulberry is having the ability to remove air pollutants from the atmosphere with the help of its leaves and remove heavy metals and other types of soil pollutants through its roots, this plant species is considered ideal for providing a sustainable environment for future generations and helps in the sustainable development of nature (Jian et al. 2012).

2.6

Economics of Mulberry Leaf Production

Production of quality mulberry leaves is the basic requirement for a successful bivoltine cocoon crop. Stem cutting is the easiest method

48

P. Saini et al.

of obtaining a healthy and vigorous mulberry sapling. Saplings are raised in the flat or raised nursery beds. Development of a vigorous rooting system is essential for raising saplings of a high yielding mulberry variety. The temperate mulberry varieties have low rooting ability and for their propagation grafting is the best approach, while the sub-tropical or tropical mulberry varieties have good rooting ability. The cost of production of mulberry leaf varies with respect to yearly fluctuating rates of labor, inputs (vermi compost, fertilizers), and agroclimatic conditions. The production of a number of mulberry saplings in one acre of land also varies with respect to spacing between cuttings. In general, for one acre of land, about 1.60 lakh mulberry cuttings can be planted with 20 cm 10 cm spacing and of these with an 80% survival rate 1.28 lakhs saplings can be raised with the production cost of Rs. 0.60 per sapling (Bogesha and Jayaram 2014). Maintenance of nursery is very much important for the production of healthy and vigorous saplings. Under temperate and sub-tropical climatic conditions

year old saplings and tropical climatic conditions, six months old saplings are suitable for transplanting. For one acre, 4840 mulberry plants can be planted with 3  3 ft spacing. Following the recommended package of practices, it is possible to obtain 30–35 metric tons of chawkie leaf per hectare per year from S-36 and V-1 mulberry varieties in south Indian conditions whereas, 1500–1900 kg of chawkie leaf under north Indian conditions can be harvested for two crops and remaining leaf used for late age rearing (Dhar 2014). With this quantity of mulberry leaf, it is possible to conduct rearing of approximately 1.66–1.94 lakh DFLs of chawkie worms per hectare per year or 20,000– 24,000 DFLs per crop in south India; whereas, about 7500–9500 DFLs of silkworm seed could be chawkie reared from one hectare of mulberry garden under north Indian conditions during each season (Dhar 2014). The cost of production of one kilogram of chawkie leaf will be near about Rs. 3.00 (Dhar 2014). The detailed cost of production of mulberry leaf for one hectare per year is given in Table 2.16.

Table 2.16 Economics of mulberry leaf production kg/ha/yr #

Particulars

Rs/ha/yr

A

Operational cost

1

Tractor tilling and harrowing (12 h @ Rs. 1100/hr)

13,200.00

2

Farmyard manure (20 MT/ha @ Rs. 1402/MT)

28,044.00

3

Chemical fertilizers (Doses, rates and man-days varied region to region) Southern conditions: N:P:K:: 260:140:140 kg/ha/yr in eight split doses in eight crops harvest Northern conditions: N:P:K:: 250:150:150 kg/ha/yr (Temperate areas) N:P:K:: 200:100:100 kg/ha/yr (Sub-tropics areas)

33,723.00

4

Irrigation (40 man-days @ Rs. 350.00 per man-days)

14,000.00

5

Hoeing and weeding (45 man-days @ Rs. 350.00 per man-days—2 times)

31,500.00

6

Miscellaneous

2000.00

7

Interest on working capital

11,000.00

Total variable cost

133,467.00

B

Fixed costs

1

Apportion cost of establishment of mulberry garden

11,546.00

C

Total leaf production cost

145,013.00

D

Total leaf production (kg)

44,000

E

Total cost/kg of leaf

3.29

Source Central Silk Board (2020)

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Cultivation, Utilization, and Economic Benefits of Mulberry

2.7

Conclusion and Future Prospective

Mulberry is one of the fast growing perennial crops and the only food source of the B. mori. It can be cultivated in different environmental conditions such as temperate, tropical, and subtropical environments. The yield-scaled parameters are highly dependent on the soil types, nutrient inputs, and crop management. Apart from the feed material for silkworms, mulberry is having high potential to generate additional income utilizing in food and tea industry, ayurvedic medicine, pharmaceutical industry, and also environmental safety and protection and fodder for animals. Mulberry wastes find their major route of reutilization in biogas and composting. Most of the research findings reported to date are involved in the exploration and other attributes of Mulberry crop. Additionally, due to the high growth nature, mulberry crops can also be substantially used to enhance the terrestrial C sink. The real dynamics of the rhizodeposition of the different germplasm also need to be estimated along with the long-term C storage potential. Being aerobic in nature, the mulberry fields can also act as a methane sink. Therefore, the population dynamics of the methanotrophs and their community analysis also need to be assessed along with their controlling factors. This will support enhancing the futuristic methane sink if the proper community analysis and their methane entrapment potential are evaluated. Considering the potential of mulberry genetic resources, future research should focus more on utilizing them to create new and valuable products that can benefit small-scale farmers in their social and economic upliftment. The economics for the utilization of mulberry by-products also needs to be evaluated for ensuring profitable returns through new ventures. Thus, based on the information available to date, it can be suggested that mulberry crop is having diverse applications to generate income as well as environmental protection, especially in reference to climate change and phytoremediation.

49

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54 Organization of UN and Oxford & IBH Publishing Co. Pvt. Ltd., pp 1–150 Ravindran S, Rajanna L (2005) Mulberry production management. In: Rajanna L, Das PK, Ravindran S, Bhogesha K, Mishra RK, Singhvi NR, Katiyar RS, Jayaram H (eds) A text book on mulberry cultivation and physiology. Central Silk Board, Bangalore, pp 1– 54 Ravussin E, Galgani JE (2011) The implication of brown adipose tissue for humans. Annu Rev Nutr 31:33–47. https://doi.org/10.1146/annurev-nutr-072610-145209 Rayam S, Kudagi BL, Buchineni M, Pathapati RM, Immidisetty MR (2019) Assessment of Morus alba (mulberry) leaves extract for antipsychotic effect in rats. Int J Basic Clin Pharmacol 8:2130–2133 Riche DM, Riche KD, East HE, Barrett EK, May WL (2017) Impact of mulberry leaf extract on type 2 diabetes (Mul-DM): a randomized, placebo-controlled pilot study. Complement Ther Med. 32:105–108. https://doi.org/10.1016/j.ctim.2017.04.006.Epub. PMID: 28619294 Rohela GK, Phanikanth J, Shabnam AA, Shukla P, Sadanandam A, Ghosh MK (2018a) In vitro regeneration and assessment of genetic fidelity of acclimated plantlets by using ISSR markers in PPR-1 (Morus sp.): an economically important plant. Sci Hortic 241:313– 321. https://doi.org/10.1016/j.scienta.2018.07.012 Rohela GK, Shabnam AA, Shukla P, Aurade R, Gani M, Srinivasulu Y, Sharma SP, Kamil AN (2018b) In vitro clonal propagation of PPR-1, a superior temperate mulberry variety. Ind J Biotech 17:619–625 Rohela GK, Phanikanth J, Mir MY, Shabnam AA, Shukla P, Sadanandam A, Kamil AN (2020a) Indirect regeneration and genetic fidelity analysis of acclimated plantlets through SCoT and ISSR markers in Morus alba L. cv. Chinese white. Biotech Rep 25:313–321. https://doi.org/10.1016/j.btre.2020. e00417 Rohela GK, Shukla P, Muttanna RK, Chowdhury SR (2020b) Mulberry (Morus spp.): an ideal plant for sustainable development. Trees Forests People 2:100011. https://doi.org/10.1016/j.tfp.2020.100011 Sahu PK, Yadav BRD, Sarat Chandra B (1995) Evaluation of yield components in mulberry germplasm varieties. Acta Botanica 23:191–197 Sakai A, Larcher W (1987) Frost survival of plants. Springer, Berlin, p 321 Sanchez MD (2000) Mulberry: an exceptional forage available almost worldwide! World Anim Rev 93:1–21 Sarkar A (2009) Characteristics features of cultivated mulberry varieties. In: Mulberry breeding. Kalyani Publishers, pp 30–42 Sarkar T, Mogili T, Doss SG, Sivaprasad V (2018) Tissue culture in mulberry (Morus spp.) intending genetic improvement, micropropagation and secondary metaboloite production: a review on current status and future prospects. In: Kumar N (ed) Biotechnological approaches for medicinal and aromatic plants. Springer Nature Singapore Pte. Ltd, pp 467–487. https://doi.org/10.1007/978-981-13-0535-1

P. Saini et al. Shabnam AA, Rathore MS, Dhar A, Srinivasulu Y, Chauhan SS, Sharma SP (2016) Mulberry (Morus) diversity in Jammu and Kashmir. Ind Hortic J 6 (1):48–52. 202-15-IHJ-0510-2015-11 Shayo CM (1997) Uses, yield and nutritive value of mulberry (Morusalba) trees for ruminants in the semiarid areas of central Tanzania. Trop Grassl 31:559– 604 Sheng Y, Liu J, Zheng S, Liang F, Luo Y, Huang K, Xu W, He X (2019) Mulberry leaves ameliorate obesity through enhancing brown adipose tissue activity and modulating gut microbiota. Food Funct 10(8):4771–4781. https://doi.org/10.1039/ c9fo00883g.Epub. PMID: 31312821 Shiva Kumar GR, Anantha Raman KV, Magadum SB, Datta RK (1996) Medicinal values of mulberry. Indian Silk 34(7):15–16 Shuang E, Yamamoto K, Sakamoto Y, Mizowaki Y, Iwagaki Y, Kimura T et al (2017) Intake of mulberry 1-Deoxynojirimycin prevents colorectal cancer in mice. J Clin Biochem Nutr 61:47–52 Simbaya J, Chibinga O, Salem AZ (2020) Nutritional evaluation of selected fodder trees: mulberry (Morus alba Lam.), Leucaena (Leucaena luecocephala Lam de Wit.) and Moringa (Moringa oleifera Lam.) as dry season protein supplements for grazing animals. Agrofor Syst 94:1189–1197 Sivaprasad V, Chattopadhyay S, Suresh K (2021) Host plant improvement—mulberry: status, challenges and perspective. In: Brainstorming on host plant improvement organized by CSR&TI, Berhampore on 5th January 2021, pp 1–5 Srivastava S, Kapoor R, Srivastava RP (1997) Delicious delicacies from nutritious mulberry. Indian Silk 36 (1):39–40 Srivastava S, Kapoor R, Thathola A, Srivastava RP (2006) Nutritional quality of leaves of some genotypes of mulberry (Morusalba). Int J Food Sci Nutr 57:305– 313 Susarla S, Medina VF, McCutcheon SC (2002) Phytoremediation: an ecological solution to organic chemical contamination. Ecol Eng 18: 647–658 Tahir L, Aslam A, Ahmed S (2017) Antibacterial activities of Diospyrosblancoi, Phoenix dactylifera and Morusnigra against dental caries causing pathogens: an in vitro study. Pak J Pharm Sci 30:163–169 Tan C, Feng Y, Long H (2010) The important role of mulberry in low carbon and ecological economy of china. Sichuan Canye 1:12–15 (In Chinese) Thaipitakwonga T, Numhomb S, Aramwit P (2018) Mulberry leaves and their potential effects against cardiometabolic risks: a review of chemical compositions, biological properties and clinical efficacy. Pharmaceutical Biol 56(1):109–111. https://doi.org/ 10.1080/13880209.2018.1424210 Tikader A, Kamble CK (2008) Studies on variability of indigenous mulberry germplasm on growth and leaf yield. Pertanika J Trop Agric Sci 31:163–170 Tzenov P (2017) Climate changes effect on sericulture in Europa Caucasus and Central Asia. In: 8th Bacsa

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international conference “climate changes and chemicals—the new sericulture challenges”, Azerbaijan, pp 8–15 Vijayan K (2009) Approaches for enhancing salt tolerance in mulberry (Morus L.)—a review. Plant Omics 2 (1):41–59. http://www.pomics.com/Vijayan_ January2009_2_1_41_59.pdf Vijayan K, Gnanesh BN (2022) Genomic research in mulberry for higher silk productivity. In: Seritech, The new concepts in sericulture, The 26th International sericultural commission congress, 7–11th Sept 2022, Cluj-Napoca, Romania, pp 49–74 Vijayan K, Tikader A, Das KK, Chakraborti SP, Roy BN (1997) Correlation studies in mulberry (Morus spp.). Indian J Genet Breed 57:455–460 Vijayan K, Doss SG, Chakraborti SP, Ghosh PD (2009) Breeding for salinity resistance in mulberry (Morus spp.). Euphytica 169(3):403–411. https://doi.org/10. 1007/s10681-009-9972-x Vijayan K, Ravikumar G, Tikader A (2018) Mulberry (Morus spp.) breeding for higher fruit production. In: Al-Khayri JM, Jain S, Johnson D (eds) Advances in plant breeding strategies: fruits. Springer, Cham, pp 89–130. https://doi.org/10.1007/978-3-319-919447_3 Vijayan K, Gnanesh BN, Shabnam AA, Sangannavar PA, Sarkar T, Zhao W (2022a) Genomic designing for abiotic stress resistance in mulberry (Morus spp.) In: Genomic designing for abiotic stress resistant technical crops. Springer Nature https://doi.org/10.1007/ 978-3-031-05706-9_7 Vijayan K, Arunakumar GS, Gnanesh BN, Sangannavar PA, Ramesha A, Zhao W (2022b) Genomic designing for biotic stress resistance in mulberry (Morus spp.) In: Genomic designing for biotic stress resistant technical crops. Springer Nature https://doi. org/10.1007/978-3-031-09293-0_8 Vu CC, Verstegen M, Hendriks W, Pham K (2011) The nutritive value of mulberry leaves (Morus alba) and partial replacement of cottonseed in rations on the performance of growing Vietnamese cattle. AsianAustralas J Anim Sci 24:1233–1242 Wang B, Yang CT, Diao QY, Tu Y (2018) The influence of mulberry leaf flavonoids and Candida tropicalis on antioxidant function and gastrointestinal development of preweaning calves challenged with Escherichia coli O141: K99. J Dairy Sci 101:6098–6108. https://doi. org/10.3168/jds.2017-13957 Wang H, Meng B, Han H (2010) The discussion on mulberry as a green afforestation tree species. North Seric 31(1):45–47 Wang ZH, Wu Y, Zhang YZ (2011) Analysis on organoleptic quality and nutrient active ingredients of mulberry-leaf teas made by different processing techniques. Sci Seric 37(2):272–277 Wani MY, Mir MR, Baqual MF, Ganie NA, Bhat ZA, Ganie QA (2017) Roles of mulberry tree. The Pharma Inno J 6(9):143–147

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Wisniewski M, Bassett C, Gusta L (2003) An overview of cold hardiness in woody plants: seeing the forest through the trees. Hort Sci 38:952–959 Yang X, Yang L, Zheng H (2010a) Hypolipidemic and antioxidant effects of mulberry (Morus alba L.) fruit in hyperlipidaemia rats. J Food Chem Toxicol 48:2374– 2379. https://doi.org/10.1016/j.fct.2010.05.074 Yang Y, Gong T, Liu CH, Chen RY (2010b) Four new 2arylbenzofuran derivatives from leaves of Morus alba L. Chem Pharm Bull 58:257–260 Yang CT, Diao QY, Qu PB, Si B (2016) Effects of Candida tropicalis and mulberry leaf flavonoids on nutrient metabolism and rumen fermentation of calves. Chinese J Anim Nutr 28:224–234. https://doi.org/10. 3969/j.issn.1006-267x.2016.01.029 Ye W, Ye C (2001) Nutritional value of mulberry leaves and perspectives as feed. In: Mulberry for animal feeding in China, proceeding of a workshop, May 14– 17, 2001, Hangzhou, P.R. China, pp 29–35 Yi Y, Chun L, Xiaoqing Z (1997) Research on purity and features of Superoxide dismutase of mulberry leaf. J. Anhui Agri Univ 24(3):296–203 Yigit D, Akar F, Baydas A, Buyukyildiz M (2010) Elemental composition of various mulberry species. Asian J Chem 22:3554–3560 Yokoyama T (1962) Synthesized science of sericulture. Central Silk Board, Bangalore, 20p Younus WM, Mir MR, Baqual MF, Ganie NA, Bhat ZA, Ganie QA (2017) Roles of mulberry tree. The Pharma Inno J 6(9):143–147 Zeni ALB, Moreira TD, Dalmagro AP, Camargo A, Bini LA, Simionatto EL, Scharf DR (2017) Evaluation of phenolic compounds and lipid-lowering effect of Morusnigra leaves extract. An Acad Bras Ciênc 89 (4):2805–2815 Zhang G, Yang J, Zhao X, Feng K, Gao X (1997) Study on the root system distribution mulberry and its characteristics of soil and water conservation. Sci Seric 23:59–60 Zhang LY, Qu PB, Tu Y, Yang CT (2017a) Effects of flavonoids from mulberry leaves and candida tropicalis on performance and nutrient digestibility in calves. Kafkas Univ Vet Fak Derg 23:473–479. https://doi.org/10.9775/kvfd.2016.17111 Zhang Q, Zhang F, Thakur K, Wang J, Wang H, Hu F (2017b) Insights into morin from mulberry derived cell cycle arrest and apoptosis in human cervical carcinoma HeLacells. Food Chem Toxicol 112:466– 475 Zhao Q (2009) Dikaner: the peculiar village at edge of desert. Xinjiang Humanit. Geogr 4:078 Zheng S, Liao S, Zou Y, Qu Z, Shen W, Shi Y (2014) Mulberry leaf polyphenols delay ageing and regulate fat metabolism via the germline signalling pathway in Caenorhabditis Elegans. Agarose Gel Electrophor 36:9719. https://doi.org/10.1007/s11357-014-9719-z Zhou M, Chen QQ, Bi JF, Wang YX, Wu XY (2017) Degradation kinetics of cyanidin 3-O-glucoside and

56 cyanidin 3-Orutinoside during hot air and vacuum drying in mulberry (Morus alba L.) fruit: a comparative study based on the solid food system. Food Chem 229:574–579 Zhu X, Lu H (2001) Composition and medicinal value of mulberry leaves In: Mulberry for animal feeding in China, proceeding of a workshop, May 14–17, 2001, Hangzhou, P.R. China, pp 58–62

P. Saini et al. Zuhua S (1994) Evolution and life history of silkworm. In: Silkworm Physiology. Zunlinang, X., Shulin, L., Yungen, M., Chuanxi, Z., Xiaofeng, W., Zuhua, S. and Fangye, Z. (Eds.), China, pp 1–24

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Mulberry Breeding for Higher Leaf Productivity Thallapally Mogili, Tanmoy Sarkar, and Belaghihalli N. Gnanesh

3.1

Introduction

Mulberry (Morus spp.) is one of the fastest growing perennial hardy plants in the world and the sole food for silkworm Bombyx mori L. Mulberry is also a unique plant enabled due to its wider geological distribution across countries, ability to grow under different conditions and multiple uses (Rangaswami et al. 1976; Dandin 1986; Rohela et al. 2020), The quantity and quality of foliage produced in a unit area over a specific time have a direct bearing on silkworm cocoon harvest, farm economy and in turn has great economic importance in sericulture industry. It is a heterozygous, open-pollinated species and exhibits a wide range of variability at genotypic, phenotypic and ploidy (2n = 28–308) levels. Hence, a wide range of ploidy is available for their utilization in crop improvement programs. Genetic improvement in a crop is considered a permanent resolution, and it is very

T. Mogili (&)
T. Sarkar Central Sericultural Research and Training Institute, Manandawadi Road, Srirampura, Mysuru, Karnataka 570008, India e-mail: [email protected] B. N. Gnanesh Molecular Biology Laboratory—1, Central Sericultural Research and Training Institute, Manandawadi Road, Srirampura, Mysore, Karnataka 570008, India

much essential to increase productivity (Allard 1960; Noleppa 2016). The major objectives of mulberry genetic improvement through conventional breeding are aimed at enhancing their leaf yield per unit area of cultivation, both in terms of quantity and quality, which in turn enhances the silkworm rearing capacity and production at farmers’ level (Vijayan and Gnanesh 2022). As the cost of mulberry leaf production contributes > 50% of the total cost of cocoon production, much attention is being paid to increasing its leaf productivity (Dandin 1986; Sarkar 2000). Under temperate conditions, the growth of the mulberry is ceased due to cold conditions and winter dormancy, thereby restricting the silkworm rearing only to warmer seasons. On the contrary, under tropical conditions, mulberry can be grown around the year enabling 5–6 shoot harvests and cocoon crops per year. The important criteria for genetic improvement of mulberry are high rooting ability, quick sprouting, fast growth, high leaf yield and its quality and tolerance/resistance to abiotic and biotic stresses. It is imperative to develop mulberry varieties specific to different situations prevailing in the chosen areas/zones for horizontal expansion of sericulture in traditional and non-traditional states/countries with heterogeneous climatic and soil conditions. Systematic breeding work for increasing productivity and quality was initiated by Japan at the beginning of the twentieth century. Research on mulberry

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_3

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started in 1916 and varieties Kokuso-13 and Kokuso-70 were developed and released in 1922 (Machii et al. 1999b). Perennial nature, long generation time and heterozygous nature of mulberry are some of the inherent problems which do not permit the use of various conventional breeding methods employed in annual cross-pollinated crops. The non-availability of pure lines limits the knowledge on the inheritance pattern of important economic characters for improvement programs. Mulberry is propagated vegetatively through stem cuttings and grafting. The superior hybrid identified in the breeding process can be easily conserved through vegetative propagation without any segregation of genes/characters. The open pollination and heterozygous nature of mulberry parents results in generating wider variable hybrid progeny and thus promises a higher rate of success in the development of superior varieties. Mulberry also exhibits higher plasticity with differential response under various cultivation conditions/prentices (Gray 1990; Sarkar 2009; Urs et al. 2011).

3.2

Breeding Objectives for Evolving Superior Mulberry Varieties

The ultimate goal of mulberry genetic improvement is to develop productive varieties with high-quality leaves in the shortest possible time at a reasonable cost. Improved mulberry varieties should have the characteristics such as high yield, good quality of the leaf, tolerant to adverse conditions/adaptable to chosen environmental conditions and resistant to diseases and pests. Based on necessities, mulberry genetic improvement strategies can be broadly directed toward the following approaches: 1. Breeding for improvement of leaf productivity and quality in targeted areas 2. Breeding for development of abiotic stresstolerant mulberry varieties 3. Breeding for development of varieties resistant to diseases and pest.

3.3

Prerequisites for Mulberry Improvement Breeding Programs

Prerequisites for the evolution of a mulberry variety with desirable characteristics are welldefined breeding objectives, detailed knowledge of available genetic variability in the crop, knowledge of reproductive system and breeding behavior, the genetic information of inheritance on the traits involved in the breeding, selection of suitable parents for desirable traits in line with breeding objectives and methods and procedures of evaluation and selection. The above information is essential for a successful breeding program. The general breeding program for mulberry improvement is multidirectional integrated approaches (Fig. 3.1) consisting of evaluation of existing germplasm/local progeny for specific traits and selection of desirable parents, breeding methods for creating variability, screening and selection and isolation of superior varieties for desirable conditions.

3.3.1 Germplasm and Genetic Diversity A well-characterized and documented genetic resource (germplasm) is one of the most important prerequisites for the mulberry crop improvement program. The information on the genetic diversity in Morus species indicates the level of divergence among the accessions. This could help breeders to understand and predict which parental combination would produce the best offspring. Many mulberry germplasm accessions are being collected and conserved by all sericultural important countries, namely China (2600), Japan (1375), South Korea (208), Bulgaria (140), France (70), Italy (50), USA (23), Indonesia, Taiwan (5) and Mexico(5) (Vijayan et al. 2019). Jolly and Dandin (1986) prepared guidelines for the collection, conservation and evaluation of mulberry germplasm for making the breeding programs much directional. Machii et al. (1997; 1999a, b) have published an exhaustive catalogue for characterization and

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Mulberry Breeding for Higher Leaf Productivity

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General method of mulberry evoluon

Exisng Variability Introducons

Germplasm

Pre-breeding

Creaon of variability by employing breeding methods Local races

Cross breeding

Polyploidy breeding

Mutaon breeding

In-vitro methods

Inducon of ploidy

Irradiaon of genotypes

Inducon of variability

Inter-mang Selecon of parents

Raising of hybrid progenies in nurseries

Hybridisaon

Hybridisaon

Raising hybrid progenies

Raising triploid progenies

Isolaon of Mutants

Inducon of somac hybrids, haploids etc.

Transplantaon to field, Isolaon of promising hybrids by individual trait assessment and selecon of promising hybrids Primary yield evaluaon in comparison to local popular variees Final yield evaluaon

Mul-locaonal /Regional test

Naonal level Evaluaon test (AICEM-India)

Recommendaon of new variety

Recommendaon and Disseminaon of new variety

Mulplicaon –Breeders, foundaon and cerfied seed

Fig. 3.1 Mulberry crop improvement program for evolution of superior mulberry varieties

evaluation of mulberry genetic resources. In India, 1291 mulberry accessions (1006 indigenous, 285 exotic) are being maintained at Central Sericultural Germplasm Resources Centre, Hosur, Tamil Nadu. Mulberry accessions 1125 have been systematically characterized with more than 100 descriptors and published in 5 catalogues (Thangavelu et al. 1997, 2000; Tikader et al. 2006; Borpuzari et al. 2013). Morphological variability in mulberry germplasm is reported by various researchers in India and abroad, and standard descriptors were adopted for the recording of data on morphological characters and documentation (Dandin and Jolly 1986; Cappellozza et al. 1995, 1996; Machii et al. 1997, 2001; Tikader and Ananda Rao 2002a; Ananda Rao 2002; Tikader and Roy

2003; Kazutoshi et al. 2004). The germplasm accession can be utilized as one of the parents of the breeding program, while adapted/cultivated varieties could be used as another parent for cross-breeding to develop a superior variety in which the desired characteristics of the selected germplasm accessions could have been transmitted. Several studies revealed that there are considerable genetic variations among indigenous and exotic mulberry germplasm accessions. These studies grouped the accessions into many clusters to select suitable parental combinations to be utilized in breeding programs (Fotedar and Dandin 1998; Mala et al. 1997; Rajan and Sarkar 1998; Rajan et al. 1997; Tikader et al. 1999a, b; Vijayan et al. 1999a, b; Tikader and Roy 2003; Huang et al. 2014).

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3.3.2 Pre-breeding Study As indigenous, exotic, wild and landraces are being maintained in main germplasm stations, pre-breeding assessment is the first step in utilizing their diversity. The naturally occurring variations which exist among the wild relatives, primitive varieties/landraces and exotic genotypes are still underexploited in breeding programs due to various constraints and barriers. Wild relatives which possess varying levels of resistance/tolerance to multiple stresses provide important sources of genetic diversity for the crop improvement program. Wild mulberry (Morus laevigata) possesses unique features of a large leaf, long inflorescence, long fruit and high timber value. This species is also adapted to wide agro-climatic conditions right from sea level up to the elevation of 1500 m including humid tropical regions to semitemperate regions. Similarly, M. serrata grew in higher altitudes (700–2200 m) supposed to be tolerant to drought and other biotic/abiotic stresses (Tikader and Thangavelu 2003, 2005; Tikader and Dandin 2007; Tikader and Kamble 2008; Li et al. 2017a, b). The exploitation of such diverse sources of variation is of prime importance to circumvent problems related to genetic vulnerability and genetic erosion. In addition to crossability studies, pre-breeding activities adopting micro-plot or hot spot techniques helped in the identification of a group of promising germplasm accessions for their utilization in genetic improvement programs for specific conditions, viz. temperate conditions (18 exotic accessions), tropical and sub-tropical conditions (31 accessions), region-specific conditions and biotic/abiotic stress conditions (Jhansilakshmi and Gargi 2018).

3.3.3 Floral Biology and Anthesis Studies on the floral characters, anthesis, receptivity of stigma, pollen fertility, synchronization of flowering of parents and crossability barriers are very much essential for initiating breeding programs. Flowers of mulberry are

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predominantly dioecious, sometimes monoecious and bisexual flowers are also observed. Flowers are small and lightweight and do not contain any nectar or emit any smell. The major pollinating agent is wind. Hence, the reproductive biology of mulberry germplasm accessions was studied in detail for the selection of parents (Lev 1970; Ogurtsov 1978; Jolly et al. 1987; Tikader 1999; Tikader et al. 1995, 1999a, b, 2000; Vijayan et al. 1997a, b). Under tropical conditions, the peak period of dehiscence of anthers was observed between 8 AM to 12 Noon during March–April 8.00 AM to 10 AM and 2 PM to 4 PM during November–January (Das et al. 1970a; Balakrishna et al. 2002b). Mulberry pollen can be successfully stored for one month under the refrigerated condition at 2–4 °C (Das and Katagiri 1968; Das and Sarkar 1971). However, the viability of pollen declined gradually with the increase of storage period. Stigma receptivity varies from 7 to 12 days.

3.3.4 Crossability Study Crossability among the different Morus species and their inheritance pattern was studied extensively (Das and Krishnaswami 1965; Dandin et al. 1987; Dwivedi et al. 1989b; Tikader and Dandin 2001). The intra- and inter-species crossability studies were carried out in mulberry and the results proved the possibilities of getting F1 hybrids (Tikader and Ananda Rao 2002b; Govindaraju and Basavaiah 2010). Among the intraspecific hybridization, a maximum seed set was observed in M. indica and M. alba (80%) followed by M. multicaulis (50%), M. bombycis (47%). Seed set and seed germination percentage were low (3.58%) in intraspecific M. laevigata crosses (Tikader and Ananda Rao 2002a, b). Seed setting percent was moderate to high when cultivated species (M. indica) was crossed with different wild species M. cathayana (39.5%), M. tiliaefolia (41.11%), M. serrata (47.6%) and M. pendula (75%). In general, sexual compatibility in mulberry was good and most of the interspecific hybridization showed appreciable seed germination

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Mulberry Breeding for Higher Leaf Productivity

percentage (Dwivedi et al. 1989a, b, c; Tikader and Ananda Rao 2002b).

3.3.5 Study of Genetics of Mulberry for Designing a Breeding Scheme Assessment of genotypic and phenotypic variations, heritability, combining ability, genetic advance, selection index, correlation and path coefficient enhanced selection efficiency of mulberry breeding programs (Das and Krishnaswami 1969; Sarkar et al. 1997a, b, 2003; Susheelamma et al. 1988; Goel et al. 1998; Banerjee et al. 2007, 2011; Masilamani et al. 2000; Murthy et al. 2010; Mamrutha et al. 2010, 2017; Doss et al. 2012; Huang et al. 2014; Biradar et al. 2015; Suresh et al. 2017, 2018; Rahman and Islam 2020). The indirect selection method indicated that the total length of all shoots, the total weight of above-ground biological yield and the weight of 100 leaves had a significant positive association and have high impacts on yield (Sarkar et al. 1992; Rahman et al. 1994, 2006). In mulberry, a line  Tester analysis was carried out using 3 females (lines) and 5 male (testers) genotypes to determine genetic interaction in the expression of various quantitative characters including leaf yield. This study indicated broad genetic variability among the progenies (Vijayan et al. 1997a, b). The ratio of general combining ability (GCA) and specific combing ability (SCA) indicated the predominance of nonadditive genes in mulberry. The genotype, namely China White (female) and MS-1 (male), was the best general combiners among the tested parents and the cross (Berhampore-1  Kajli) was found the best cross for leaf yield. In another study, the female, viz. Sujanpur-5, MR-2 and M. multicaulis, and the male, viz. S-523, were found to be good combiners among the genotypes tested (Balakrishna et al. 2005). Results suggest that selective crossing followed by proper screening may be the best approach for breeding for the development of high yielding mulberry varieties. To find out important yield-contributing parameters for the selection of desirable

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genotypes, interrelationships of leaf yield and yield components were studied by Susheelamma et al. (1988). Rahman et al. (1994) and found that the total length of all shoots, the total weight of all aerial branches and 100 leaf weights had significant positive association and also high direct effects on leaf yield. Mulberry accessions showed variability in various photosynthetic traits (Das et al. 1999; Singhal et al. 2000; Sengupta et al. 2012; Setua et al. 2012), the photosynthetic efficiency could be used as an important parameter in the selection and development of mulberry varieties (Sitarami Reddy et al. 2018) Mulberry accessions also exhibited variability in physiological parameters, and earlier studies indicated that leaf area duration (LAD), biomass duration (BMD) at any stage of growth, leaf area index (LAI) and crop growth rate (CGR) at a later stage of growth could be utilized for predicting leaf yield while screening and selection of mulberry (Mogili et al. 1992; Jalaja et al. 2010; Rukmangada et al. 2020).

3.4

Conventional Breeding Approaches

For improvement of mulberry leaf productivity under specific agro-climatic regions, several breeding approaches could be adopted, viz. (1) importation or introduction of varieties, (2) screening and selection from natural populations or landraces/cultivars, (3) cross-breeding (4) polyploid breeding, (5) mutation breeding, (6) utilization of heterosis through F1 hybrid seed complex.

3.4.1 Importation or Introduction of Mulberry Varieties Introduction of promising varieties is the shortest and simple way to improve the mulberry productivity in a particular agro-climatic area, and it helps in enriching pre-breeding resources for a breeding program as well as direct utilization in the field. The most important consideration in this approach is the collection of genotypes from

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the country of similar climatic conditions. Hence, most of the earlier mulberry varieties of temperate origin, which had been introduced to the Southern part of India, did not perform well. Mulberry varieties, viz. Goshoerami, KNG, Tsukasaguwa, Ichinose, Rokokuyose and Kokuso-20, were introduced from Japan to the Kashmir valley. These introduced varieties acclimatized in this region; subsequently, they replaced low yielding varieties and they were under cultivation till the year 2020. However, these varieties had less rooting ability and were not amenable for propagation through stem cuttings (Chauhan et al. 2018). A mulberry variety collected from Mandalaya, Myanmar, was introduced in India in 1970, renamed as S-1 and recommended for cultivation in the Eastern and North-Eastern parts of India. This variety was again introduced to Bangladesh and found superior in comparison with the local varieties. Similarly, a popular South Indian variety Kanva-2 was introduced in Egypt and Uganda successfully. Mulberry variety Yunsang59 of the former Soviet Union was introduced to China, and it is suitable for both young and late age silkworm rearing. Calabrese and Catania-1 and Catania-2 were introduced to Brazil from Italy, and Kokuso-21, Kokuso-27, KNG of Japan were introduced to Brazil (Sarkar 2009). While the introduction of exotic mulberry genotype, all the quarantine measures and genetic resource transfer rules must be followed to avoid the entry of pests/pathogens and unauthorized exploitation of genetic resources across the countries. The genetic resources exchange programs should comply with the breeders of introduced varieties.

3.4.2 Screening and Selection from Natural OpenPollinated Populations or Varieties Owing to open pollinating and heterozygous nature, mulberry has a great number of natural hybrids, polyploids and spontaneous mutants. Through a long period of continuous natural selection, large number of good genotypes exist

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in nature which is a good source of germplasm for screening and selection and breeding resources. Since the natural hybrids are extensively distributed in various regions of China, the Regional Research Institutes have initially focused on the collection, evaluation and identification of superior mulberry varieties for the respective sericultural areas. Mulberry varieties, viz. Heybai, Tuantuheyebai, Tongxiangsing, Husang-197, were primarily recommended for Zhejiang Province. Similarly, Lunjiao-40 and Xiaoguansang were recommended for Guangdong Province while Husang, Dajiguan and Helusang were recommended for Sichuan and Shandong province, respectively, for commercial exploitation. Waktole and Wosene (2016) screened locally available genotypes and identified Kumbi and M4 as superior for field cultivation. In this method, the desirable hybrids were directly selected from the open-pollinated hybrid populations that arose due to natural pollination and there was no crossing barrier among the genotypes of the species. In India, mulberry crop improvement program was started during the early 1960s, and the varieties like Mysore local and other local landraces (e.g., Yenneranginakaddi, Boodhu kaddi and Sultan kaddi) of M. indica L. were identified in South India (Dandin 1997). In the early phases, Kanva-2 mulberry variety with entire leaves was isolated from lobed leaf Mysuru local variety from a cultivated farm in Southern India. This variety ruled the entire south of India for about four decades and was found suitable for both irrigated and rainfed water stress conditions and also performed well in Uganda and Bangladesh when introduced. Similarly, broadleaf mulberry variety “Bombai” with high leaf yield was isolated from a lobed leafy Kajli in West Bengal. As an alternate strategy to the earlier method, the open-pollinated hybrid selection method was adapted in India. In this method, seeds were collected from selected female parents after random mating. The segregated hybrid population raised in the experimental plot and for hybrids with desirable traits were screened. The shortlisted hybrids were evaluated under field conditions for growth, yield, yield-contributing attributes and leaf quality by adopting standard

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Mulberry Breeding for Higher Leaf Productivity

evaluation procedures. Though many attempts were made, very few mulberry varieties were developed in India. Varieties MR-2 and S-146 were developed through an open-pollinated hybrid selection method. These varieties did not exhibit higher leaf yield compared to checks but exhibited improvement concerning disease resistance (mildew) and adaptability, respectively. Continuous efforts have been made to develop and select mulberry varieties with desirable traits and mulberry varieties, viz. RFS135, RFS-175, S-13, AR-10 and AR-11, have been found suitable for soil moisture stress and sub-optimal irrigated conditions (Susheelamma 1987; Susheelamma et al. 1992b; Mogili et al. 2017a, b). Damasco et al. (2011, 2014) reported the development of 5 best performing mulberry varieties (Alf-004, S61-019, S54-019, S61-011 and Alf-025) based on growth, leaf yield, and quality and propagation traits. However, the development of a large hybrid population is required for obtaining hybrids with desirable traits. Subsequently, the breeder’s choice has been shifted toward cross-breeding strategy for achieving hybrids with desirable traits using traitbased cross-breeding approaches.

3.4.3 Cross-Breeding/Controlled Pollination by Hybridization Hybridization or cross-breeding is the most effective, popular conventional breeding method, wherein desirable traits of two or more genetically dissimilar parents are combined into one. The generation of new populations with high variability through cross-breeding approach is the stepping stone for the development of new hybrid varieties. In mulberry, the F1 population is exploited for commercial utilization using the advantage of vegetative propagation. The hybridization process involves various steps like (a) selection of parents, (b) synchronization of flowering, (c) bagging of female and male inflorescences, (d) collection of pollen and crossing, (e) collection of fruits and harvesting of seeds, (f) raising of seedlings, screening of

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individual hybrids, selection of superior ones and its multiplication followed by evaluation along with control varieties in experimental plots. The selection of parents is the foundation of success in obtaining a desirable hybrid as per the breeder’s goal. The breeder should analyze the variability in different germplasm accessions, heredity of parents and expression pattern of particular characters in the progeny, viz. dominance/recessive. Ideally, both parents should have superior traits. If any of the parents have poor character, the other parent should compensate for the particular character. Parents selected for yield improvement experiments should be relatively high yielding genotypes. Many factors influence the yield potential, and it is necessary to select the parents possessing various yield-contributing characters. It is better if the parents are distantly related, for example, crosses between two varieties of two different species or different origins (geographically distant) or crosses between exotic  indigenous genotypes. Further, crosses may be made between parents from the same latitude but not from the same longitude. When exotic genotypes are utilized, the breeder should have enough information on their habitat, dormancy and rooting ability. According to the breeding experience in mulberry, the heredity of some economic characters shows that (i) growth vigor is a dominant character, (ii) the length of internode is usually intermediate between both the parents, (iii) the thickness of leaf blade of progeny is less than both parents or some time intermediate, (iv) the sprouting rate of hybrid always tends to that of the early budding parent, (v) mulberry blight and dogare blight are recessive in heredity (Yang 1994).

3.4.3.1 Method of Pollination After the selection of parents as per breeding objectives, selected parents are raised in pots or maintained in the field (Breeders’ Plot). It is essential to avoid pollination with undesirable pollen. Hence, two methods are commonly used either by isolating the parents by space or by mechanical means in which the female parent is covered with bags. The objective of controlling

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cross-pollination in mulberry is to pollinate the desired female parent with the functional pollen of the selected/desired male plant at an appropriate time when stigma is fully receptive. As the mulberry plant is mostly dioecious, controlling open pollination is easy. The cross-pollination of mulberry could be carried out in glass houses (Indoor crossing) and in a field where both the parents are grown (outdoor crossing). In the indoor method, crossing is done under entirely controlled condition which ensures complete exclusion of foreign pollen. In this method, at the initial stage, the selected female and male plants are raised in bigger pots using one-year-old saplings/grafts and maintained under normal conditions outside the glasshouse. Before the initiation of the crossing program, the female parent (4–5 plants per cross) is transferred to a separate crossing chamber inside the glasshouse and pruned in spring and maintained at 28 °C with humidity of 70–80%. The male plants are also pruned simultaneously and maintained separately in the glasshouse for synchronizing the pollination (March/April). Before the pollination, the pollen is collected from the male flowers in folded butter paper bags and transferred to a glass vial. Pollination is carried out by applying pollen to the stigma of the mature female flower using a soft paint brush, and this step is repeated for 4–5 days. After completion of controlled pollination, the male plants are removed from the glasshouse and the female plants are maintained in a crossing chamber till the fruits are matured and ripened. Ripened fruits are collected, and the seeds are extracted, dried and preserved with proper labels. In Japan, seeds are sown either in pot or continuous peat ban pans/in peat pellets (Jiffy-7) for germination in a glasshouse. After approximately 40 days of growth, the seedlings are transferred to nursery beds. While transplanting, seedlings with small and lobed leaves, lateral and lodging shoots, and less vigor are rejected. In each nursery bed, approximately 250 seedlings are transplanted at a distance of 25 cm between rows and 15 cm between seedlings. Finally, from these nursery beds, less than 15% of the seedlings are transplanted to the individual selection field. In

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contrast, the outdoor crossing method is adopted in India and China, where the entire crossing program is conducted outdoor in the field either in Breeding or in germplasm plots. For this purpose, long butter paper bags are used to cover the entire branches bearing female inflorescences. The bags are tied as soon as the female flowers come out of the buds. Before bagging the branches, the tip of the branch is cut to prevent linear growth of the branch. When both male and female flowers are matured, the pollen is collected from male flowers and is kept in a Petri dish with the help of a hairbrush. These pollen thus collected are dusted on the stigmatic surface of the female flowers after removing the bags from the branches and again the branches are bagged just after completion of pollination. The paper bag is removed from the branches once the stigma becomes brown and not receptive. The pollinated inflorescences are again covered with a cloth bag to protect the fruits from birds and animals. This outdoor crossing method cannot avoid the possibility of contamination with unwanted pollen fully. Fruits are collected carefully, and seeds are extracted, dried and preserved. Seeds remain viable for more than a year but it is desirable to sow the seeds within six months of collection. In India, seeds collected are sown in either earthen pots, plastic cups, or nursery beds under glasshouse conditions or artificial shade conditions. Seedlings are maintained for 3–4 months by regular watering, application of fertilizers and adopting plant protection measures. While transplanting the seedlings in the plot, those with deep lobation, lateral shoots, lodging, coarseness and small size leaves and poor vigor are discarded.

3.4.3.2 Types of Hybridization/ Controlled Pollination There are many ways of sexual hybridization based on the type of parent materials used. The parents may belong to different species of the same genus. Hybridization is broadly classified as intervarietal and distant hybridization based on the taxonomic relationships of the parents. In crop improvement programs, intervarietal hybridization is the most commonly used

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Mulberry Breeding for Higher Leaf Productivity

method, where parents involved in hybridization belongs to the same species; may be two varieties, races of the same species. In general, intervarietal crosses may be simple or complex depending on the number of parents. For example, (a) single cross (use of two parents to produce the F1), complex cross (use of more than two parents to produce hybrids), (b) advanced generation cross (crossing of advanced generation of A  B with an advanced generation of C  D elite hybrids–three to four hybrid parents) (Kesavacharyulu et al 2006; Sarkar 2009), (c) mixed pollen cross (mixed pollen of elite parents to produce hybrids) and (d) backcross. The backcross method is mainly employed to transfer one or two economic characters from a local variety to a superior variety that lacks those traits. The variety A is crossed with variety B, and the resultant F1 is backcrossed with A (recurrent parent). Repetition of the process is necessary to develop genotypes with desired traits. For example, the Kosen variety is suitable for hilly areas but lacks good rooting ability. Hence, Kosen was crossed with a good rooting genotype, Matigara local. The resultant hybrids of Kosen with introduced high rooting ability were backcrossed twice with Kosen. From the resultant progenies, a genotype, viz. BC259 with improved rooting potential, has been identified and recommended for hilly areas.

3.4.3.3 Synchronization of Flowering Time and Pollen Collection Many times, both the parents do not flower at the same time. In such a case, male flowers mature earlier than females. Hence, it is necessary to collect pollen from the male parents and preserved them in desiccators at 5–10 °C. The vitality of preserved pollen grains can last for approximately 20 days. The other method of synchronization is the storage of stem/shoot cuttings. Before sprouting of branches of male parent grown in field, its branches are cut, wrapped in rice straw and stored at 5–10 °C. These sticks (stems) are taken out just 10– 15 days before the female parent flower in the field. The cuttings of the male plant are planted

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in moist sand beds or pots and maintained at 20– 25 °C until they flower. Then the pollen of male flowers is collected for controlled pollination.

3.4.3.4 Hybridization Through Cuttings When synchronization of both the parents becomes very difficult, cuttings of both the parents can be collected, wrapped in rice straw and stored at 2.5–5 °C. These cuttings when needed can be taken and forced to flower in a pot or seedbed. 3.4.3.5 Modification of Flowering Studies have indicated that sex can be modified by altering environmental conditions such as temperature, humidity, photoperiod, light intensity and chemical as indicated in (Table 3.1). Through controlled pollination, a large number of mulberry varieties were developed in various countries using the genotypes belonging to different Morus species, viz. M. alba, M. indica, M. bombycis, M. latifolia, M. multicaulis and M. atropurpurea (Datta 2000; Yong 2000; Machii et al. 2000; Pan 2000 and Kazutoshi et al. 2004).

3.4.4 Polyploidy Breeding Polyploidy is a condition in which the cells of an organism have more than two paired (homologous) sets of chromosomes. In Genus Morus, the basic chromosome number (x) is 14 and diploids have two sets (28) of chromosomes. However, mulberry (Morus) exhibits a high degree of polyploidy ranging from diploid (2n = 2x = 28) to decosoploidy (2n = 22x = 308). Natural polyploids are common in mulberry though diploids with 28 chromosomes or triploids with 42 chromosomes (2n = 3x = 42) are more frequent (Kundu and Sharma 1976; Basavaiah et al. 1989, 1990; Maode et al. 1996; Venkatesh 2015). Tetraploids with 56 chromosomes (2n = 4x = 56), hexaploids with 84 chromosomes (2n = 6x = 84), octoploids with 112 chromosomes (2n = 8x = 112) and decosoploidy (2n = 22x = 308) are also found in nature (Table 3.2). A spontaneous haploid of M.

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Table 3.1 Sex modifying factors and sex expression Factors

Sex expression Male

Female

Temperature

High

Low

Photoperiod

Long

Short

Light intensity

High

Low

Relative humidity

Normal

High

Leaf harvesting

No harvesting

Over harvesting

NAA and Ethrel and Colchicine

No treatment

Treatment

Table 3.2 Chromosome numbers in some species of genus Morus and their level of ploidy Species

Somatic number (2n)

Ploidy level (multiples of basic number x = 14)

M. indica

28

2x (Dilpoid)

M. alba

28

2x (Diploid)

42

3x (Triploid)

M. australis

42

3x (Triploid)

M. atropurpurea

42

3x (Triploid)

M. mongolica

56

4x (Tetraploid)

M. laevigata

28

2x (Diploid)

56

4x (Tetraploid)

M. tiliefolia

84

6x (Hexaploid)

M. cathayana

56

4x (Tetraploid)

84

6x (Hexaploid)

112

8x (Octoploid)

308

22x (Decosoploid)

M. nigra

notabilis was found in China and has been exploited for whole genome sequencing (He et al. 2013). Recently, some Chinese scientists are considering 14 as diploid chromosome number (2n = 2x = 14), i.e., considering 7 as the basic chromosome number and classified the genotypes with 28 chromosomes as tetraploid (2n = 4x = 28), with 35 chromosomes as pentaploid (2n = 5x = 35) in M. mongolica (Li et al. 2014) with 42 chromosomes as hectaploid (2n = 7x = 49) in M. wittiorum (Li et al. 2017a, b) and, with 98 chromosomes (2n = 14x = 98) in Yun 7 wild mulberry (Li et al. 2017a, b). However, based on chromosome level genomics and genomic synteny analysis coupled with

karyotype analysis, Jiao et al. (2020) considered that M. alba with 28 chromosomes to be diploid (2n = 2x = 28). The flow cytometry method is being used for quick identification of ploidy levels in mulberry (Han et al. 2013; Yang et al. 2017; Gnanesh et al. 2023). In general, polyploids have considerable value in crop plants particularly crops that are grown for their vegetative parts and they play an important role in the natural selection and better adaptability of species in the new ecological niches (Udall and Wendel 2006). Some of the polyploids of mulberry possess better economic characteristics and are also expected to offer a wide range of genetic

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variation at breeders’ disposal enabling them to make more effective selections. The reasons for the existence of a large number of polyploids are natural crossing between diploid and tetraploid plants mostly by the failure of meiosis that results in the formation of unreduced chromosomes in gametes. Sometimes the failure of mitosis results in the doubling of the chromosome set and thus results in repetition of the whole basic chromosome set. Fertilization of unreduced gametes by normal gamete of the same species produces triploids with three sets (Sugiyama 1959; Dwivedi et al. 1989b). Majority of mulberry polyploids are superior to diploids as they have thick and big leaves with more weight, rich in nutrients and moisture content. Despite their superior leaf quality, the leaf productivity and rooting behavior of most of the tetraploids are usually lower than that of their putative diploids (Susheelamma et al. 1991). Both induced and naturally occurring tetraploids and triploids were reported in Morus by Seki (1951), Tojyo (1954, 1963, 1966, 1985), Elakobarova (1973), Alekperova (1979), Abdullaev (1962), Dzhafurov and Alekperova (1979), Dzhafurov and Nadzhafov (1984), Huang (1981), Chu and Sun (1986), He and Zhou (1989), Zhu and Guo (1989), Guo and Wu (1989, 1990) and Yang and Yang (1989, 1991), Yang et al. (2006), Lu et al. (2012), Zhang et al. (2015). However, productive tetraploids, namely Qiangsang 2 (Lu et al. 2012) Tuansang 11 (Zhang et al. 2015) and Yusang 5 (Li et al. 2018a, b), have been recommended for commercial utilization. Hybridization followed by polyploidization has played a significant role in the development of many superior triploid/polyploid mulberry varieties. A large number of triploids with many desirable characters were reported from Japan, China and India. Tojyo (1966, 1985) and Tojyo et al. (1986, 1992) have developed three superior triploids, namely Shinkenmochi, Aobanezumi and Mitsugeri, which exhibited higher yield compared to KNG and Ichinose, and these varieties showed resistance to cold and diseases. Dzhafurov et al. (1985) have reported a triploid variety AZNISH-9 which is resistant to Pseudomonas syringe. Development of tetraploid and

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triploid varieties with good nutritive quality has been reported by Seki and Oshikane (1959), Dzhafurov and Alekperov (1979). Superior triploid variety, viz. Jialing-16, has a 20% higher yield over Husang-32 (Maode et al. 1997) and another variety, viz. Dhazhonghua with a 25% higher yield over Heybei, was reported from China. New tetraploids Qiangsang 2 (Lu et al. 2012) and Tuangsang 11 (Zhang et al. 2015), a good number of reports are available on induction of auto-tetraploids and their utilization in the development of triploids in India. Both induced and naturally occurring tetraploids and triploids were reported in Morus by Datta (1954), Das and Krishnaswami (1965), Kedaranath and Lakshmikanthan (1965), Das et al. (1970a, b). Autotetraploids have been induced by colchicine treatment of mulberry varieties Kanva-2 (Sastry et al. 1968), RFS-135 (Dwivedi et al. 1986), S30, S-36, S-41 and K-2 mutant (Dwivedi 1994; Dwivedi et al. 1988, 1989b; Sikdar and Jolly 1994, 1995), V-1, S-13, S-34 (Jhansilakshmi and Sarkar 1995). All these productive tetraploids have been crossed with promising diploids like Ber.C-776 and diploid genotypes (Dwivedi et al. 1989b; Sikdar and Jolly, 1995) for the development of promising triploids. AR-12, a triploid genotype has been identified as tolerant to alkalinity (Mogili et al. 2008; Sathyanarayana and Mogili 2013; Sathyanarayana et al. 2008; Jalaja et al. 2013). High yielding triploid varieties, viz. Tr-4, Tr-8, Tr-10, TR-23, S-1635, and Vishala exhibited higher yield and wider adaptability across various agro-climatic areas (Verma et al. 1986; Chakravarty et al. 2018).

3.4.4.1 Artificial Induction of Polyploidy Many chemicals, like acenaphthene, chloral hydrate and sulphanilamide, are found to induce polyploidy, but colchicine is the most effective one and it can be used as an aqueous solution or as a paste with lanoline. The concentration of colchicine treatment ranges from 0.01 to 1.0%. Based on the type of the plant material, the concentration varies and the commonly used dosage is 0.1 to 0.4%. Cold or heat shock treatment or radiation treatment (Gamma rays, X-

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rays) of germinal tissue for a short duration was found to be effective in inducing polyploidy. The detailed procedure has been given below. Colchicine Treatment for Seeds Dry seeds of selected variety are soaked in 0.1 to 0.4% colchicine solutions for different durations. The seeds are washed in running water and sown on a wet thick filter paper kept in a Petri dish for germination. Seedlings with thick, dark green leaves are transferred to soil and screened for polyploidy. Abdullaev (1967) developed tetraploids of seven local and two introduced mulberry varieties through this seed treatment method. Application of 0.05–0.2% colchicine solution once a day on the growing portion of seedlings at the cotyledon expanding stage for several days is another method of using seedlings of selected parents. Colchicine Treatment for Bud This is the most common method in developing tetraploid in mulberry. Colchicine (0.1–0.4%) aqueous solution is applied on the buds using a cotton swab (Das et al. 1970a, b) or through injecting colchicine solution into the buds. As colchicine treatment affects only active tissues, treatment should be given to the tissues that will develop into vegetative, sexual or both types of organs. Hence, mulberry plants need to be pruned for allowing their buds to bulge and sprout before exposure to colchicine. Colchicine treatment through the cotton swab: For induction of tetraploidy in a selected diploid variety, the saplings are raised in pots and pruned after establishment. At the bud-bulging stage, a small incision is made on the apical portion of the bud and it is covered with a cotton swab dipped in 0.01–0.1% colchicine solution. The bud is maintained wet by dropping colchicine solution using a dropper at regular intervals. Care needs to be taken to keep the cotton swab wet throughout the treatment period and the cotton swab on the apical position only. In the second method, remove the dormant/unwanted buds from the lower portion before the treatment and dip the apical portion of the sapling in 0.2 to 0.4% colchicine solutions for 24–48 h. In the

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third method, excised vegetative buds are grown in vitro adding colchicine to the culture media (Chakraborti et al. 1998). Colchicine treatment through injection: inject 0.2% aqueous solution of colchicine to the swelling vegetative buds once a day for three consecutive days or inject 0.1% colchicine once in a day for 4–6 days using a micro-syringe (Fig. 3.2). Induction of Tetraploidy by Radiation The tetraploidy in mulberry can also be induced by gamma irradiation. Irradiation of the bud is carried out at its swelling stage by acute gamma rays with a dosage of about 5000 rads, at the rate of 1500– 2000 rad/hour. Then repeated cutting-back treatments are carried out to isolate tetraploids (Katagiri and Nakajima 1982; Yang et al. 1997). Development of Tetraploids Through Hybridization Tetraploids can also be produced by crossing a hexaploid with a diploid or vice versa. A promising allotetraploid was developed through a cross of Zhongsang 5801 (2x)  Tianmumosang (6x) in China. Verma et al. (1986) successfully developed amphiploids (4x = 56) from a cross between M. alba var. China White and M. indica var. Ber S799 followed by colchicine treatment to the hybrid seedlings (Guo and Wu 1989). They observed that out of treatments, 0.4% colchicine was most effective and 0.6% was lethal.

3.4.4.2 Cutting Back Method When the chemical/radiation treatment is successful, a few malformed leaves appear at the bottom of the shoot. A small leafless portion with short internodes and various types of leaves, viz. diploid, tetraploid and mixoploid, also appear. Very rarely complete tetraploid shoots develop immediately after mutagenic treatment. Hence, cutting-back treatment is applied on the region, just above the fully expanded tetraploid leaf (Fig. 3.2) where a complete tetraploid shoot develops from that bud. If a complete tetraploid shoot does not appear after the first cutting-back treatment, repetition of the same is done for

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Fig. 3.2 Method of induction of tetraploidy in mulberry by injection method and cutback treatment for complete tetraploidy

obtaining a complete tetraploid shoot (Yang et al. 1997). Once the complete tetraploid shoot is obtained, the shoot is allowed to mature and then the shoot is used for multiplication through cuttings to get several complete tetraploid plants.

3.4.4.3 Induction of Triploids Through Hybridization Through a crossing of a tetraploid female with a diploid male or vice versa, triploid can be developed (Yang et al. 1997; Sikdar and Jolly 1995; Maode et al. 1997). In addition, triploids can also be obtained by crossing normal haploid male gamete and diploidized female gamete by colchicine treatment (Dwivedi et al. 1989b; Sugiyama 1962). Triploids are also produced through endosperm culture (Thomas et al. 2000) and hypocotyl explants (Wang et al. 2011). 3.4.4.4 Salient Features of Polyploids In mulberry, majority of induced polyploids are superior to diploids as they have thicker and larger leaves. Their leaves are rich in nutrients and moisture content with a reduced stomatal number. The polyploid mulberry plants are inferior to their diploid counterparts in terms of vegetative growth and rooting ability (Das and Prasad 1974; Dwivedi et al. 1986, 1988; Susheelamma et al. 1991). In tetraploids, the size of palisade and spongy parenchyma cells are increased by 38 to 73% with an increase of 23 to 50% in the number of chloroplasts per cell. Further the stems of tetraploid exhibit thick

phloem, thin xylem, large pith, more vascular bundles in comparison with diploids. Vessels are big, but less in number in tetraploid. The ratio of cortex plus xylem to pith in the stem is approximately 20, 23 and 15, respectively, for the diploid, triploid and tetraploid. Root tissues also show a similar tendency. However, reduction in the height of the plant, number of branches, intermodal distance and number of stomata per unit area indicated that the duplication of the gene does not always increase in size but may also reduce it (Zhang et al. 2018). Differences in morphological, molecular and physiological traits at varied ploidy levels have been studied in mulberry (Mathi Thumilan and Dandia 2009; Mogili et al. 1990).

3.4.4.5 Identification of Mulberry Polyploids For identification of polyploids, the following methods can be used. 1. Morphological observations: Tetraploids are generally characterized by enlarged organs, thick large and dark green leaves, and soft shoots. The characters of triploids are in between tetraploids and diploids (Eswar Rao 1996; Sikdar 1990; Susheelamma et al. 1991; Yang and Yang 1991; Vijayan et al. 1998, 1999a, b). 2. Chloroplast count technique: Fixing of young leaf samples in Carnoy's solution for 4–8 h and transfer to 95% alcohol until the leaves

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become pale. Hydrate by passing through 85, (g) Induced mutants in mulberry can be utilized in three ways, viz. directly as new varieties 75, 50 and 35% ethanol for five minutes in for commercial exploitation in the field, as each solution. After washing with water, keep pre-breeding resources and as basic material the leaf piece on a slide, the lower side of the for re-irradiation for further improvement. leaf blade facing air and mount in a drop of 1:2 iodine-potassium iodide solutions (lg. Iodine and 2 g. potassium iodide dissolved in 3.4.5.2 Common Mutagens Used 100 ml of distilled water). 50–100 stomata in Mulberry should be observed for ploidy confirmation The agents capable of inducing mutation are (Sikdar 1990; Sikdar et al. 1986; Yang 1994). called mutagens. Broadly, mutagens are of two types, i.e., physical mutagens and chemical mutagens. Physical mutagens include ionizing radiations like gamma rays, X-rays, neutrons and 3.4.5 Mutation Breeding in Mulberry non-ionizing radiations like UV rays and visible lights. Chemical mutagens include alkylating Mulberry is considered the most suitable material agents such as ethyl methanesulfonate (EMS), for experimental mutagenesis, as once a desirable methyl methanesulfonate (MMS), N-nitroso-Nmutant is identified, it can be propagated suc- methylurea (NMU), N-nitroso N-ethyl Urea cessfully either by cuttings or grafts. Mutation (NEU), diethyl sulfate (dES) and base analogues breeding in mulberry paved the way for devel- like hydrazine and hydroxyl amine, 5oping varieties with better leaf quality, resistance Bromouracil (BU). Most of the mutagens are to diseases and abiotic stress, high adaptability, carcinogenic in nature and care should be taken improved rooting ability and enhanced dry mat- while handling those mutagens. Gamma rays are ter per unit area of the leaf (Hazama 1967, 1968a, widely used physical mutagens for mulberry. It is b; Nakajima 1973; Kukimura et al. 1975, 1976; imposed under gamma chambers to treat plant Yang 1986; Li et al. 2018a, b; Wang et al. 2017; parts such as stem cuttings, saplings, buds, pollen Vijayan et al. 2022a). grains and somatic tissues raised through tissue culture. The plant materials are irradiated by 3.4.5.1 Advantages of Mutation keeping them inside a shield and sent down for Breeding: exposure to the irradiation source (60Co) which is situated underground. Gamma fields are also (a) Increased mutation rate, extended mutation used for whole plant irradiation and are normally spectrum and creation of new genotypes circular fields with powerful gamma source (b) Improving one or two characters in otherwise cobalt (60Co) in the center. The treatment intenwell-adapted varieties. The main advantages sity varies based on the placement of are that the basic genotype of the variety is plants/saplings at various distances from the usually only slightly altered while the radiation source and is generally used for chronic improved character(s) is added and the time treatment with a lower dose. Pollen grains are required to breed the improved variety can be also irradiated with ionizing and UV rays. shorter than the hybridization to achieve the Radiation breeding in mulberry was initiated same results. first in Japan in 1957 after the establishment of (c) Break down of character linkages and re- gamma radiation facility as well as gamma field arrangement of gene order facility. The radiosensitivity, mutation frequency (d) Shortens breeding program and spectrum are quite different among varieties (e) Facilitates distant hybridization and over- and species of mulberry. However, it is reported comes incompatibility that tetraploids and triploids seem to be less (f) Production of haploids sensitive to irradiation than does diploids.

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Table 3.3 Irradiation dosages recommended for different parts of mulberry Radiation source

Material

Dose rate (kR/hour)

Lethal dose (kR)

Optimum dose (kR)

Gamma rays (60Cobalt) or

Sapling buds

2–5

10–11

5–6

Scion buds

2–5

10

4.5–6

Growing saplings

2–5

Dry seeds

5–6

35

15–25

137

Cesmium source

X-rays

4.5–6.0

Wet seeds

5–6

30

15–20

Germinating seeds

5–6

25

10–20

Fresh pollen

2–4

>4

Dormant buds

5–7

32

Sprouting buds

> 12.5

5–10

Thermo-neutrons (neutrons/cm2

Scion buds

5  1011

1  1011

Dry seeds

1  1012

5  1011

P Beta-rays

Source Yang (1994), kR—kilo roentgen

A variety with a higher mutation rate is likely to give a wider range of mutation spectrum. Ideal radiation doses for different parts of mulberry are given in Table 3.3. Fujita and Wada (1982) opined that mutation frequency under acute irradiation is higher than in chronic irradiation. Nakajima (1973), Katagiri and Nakajima (1982) opined that cutting-back treatment of irradiated primary bud induces a high frequency of mutations and rate of whole mutated plants. Reirradiation of mutants is also practiced to increase the mutation frequency and mutation spectrum in mulberry (Fujita and Wada 1982). Chemical mutagens are generally used to treat seeds but rarely for vegetative parts like buds, cuttings and scions. Chemical mutagens are known to produce a higher rate of gene mutation. Among the chemical mutagens, EMS was found effective in mulberry and resulted in promising hybrids like S-30, S-36, S-41 and S-54 (Sastry et al. 1974). Gatin and Ogurtsov (1981) developed mutants by treating mulberry stem cuttings with NMU. Colchicine is also used in combination with dimethyl sulfoxide (Wang et al. 2017) and combination with 6 benzylaminopurine (Liu et al. 2014) for the production of tetraploid mulberry varieties Wangsangyou 1 and Shushen 1, respectively.

3.4.5.3 Handling of Mutated Plant Materials After irradiation in the vegetative tissues (Vegetative generation), various effects of mutation such as inhibition of growth, fasciation of shoots, development of deformed leaves, short internodes and dark greenness of leaves could be observed on an irradiated shoot. As meristem of the buds composes many undifferentiated cells, mutations may not be induced in all these cells. For isolation of wholly mutated shoot, the irradiated plants should be pruned/cut repeatedly. This procedure is called cutting back which is presented in Fig. 3.3 handling of mutated plant materials in different generations is described in Table 3.4. After irradiation of bud, it sprouts and grows as a new shoot (M1V1). The shoot consists of deformed leaves in the bottom portion, followed by a portion that is leafless or may consist of small leaves. The upper portion of the shoot possesses normal or mixed/chimeric leaves. After 35–45 days of growth, the shoot is pruned just below the leafless portion. If there is no leafless portion just above the deformed leaf on the shoot, the shoot is pruned just after the 10th leaf from the base of the shoot. If the numbers of leaves are less than 10 on the shoot, the apical

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normal shoot

young leaves

mutated shoot

normal part

irradiaon mutated bud

scales

pruned leafless part malformed leaf part

Sapling

Apical Poron

M1V1

M1V2

Fig. 3.3 Isolation and Identification of somatic mutants at M1V2 Generation and establishment of clones Table 3.4 Mutated plant materials in different generations Material

Stem cuttings/grafts/whole plant of mulberry variety lacking one or two desired character (s)

Treatment

Acute/Chronic Radiation (Cobalt-60) or Chemical mutagen treatment with appropriate dose/Concentration

M1V1 generation

Pruning of shoot grown from irradiated buds (M1V1) and initial selection from lateral shoots for mutant shoots

M1V2 generation

Identification and isolation of somatic mutants and establishment of clones. Cutting back of non-mutant shoots from chimeric shoots

M1V3 generation

Further isolation of somatic mutants. Vegetative propagation and preliminary evaluation of mutants

M1V4 to M1V7 generations

Evaluation of mutant clones. Assessment of the characters decided for improvement. Release as a new variety

Source Sarkar (2009)

portion of the shoot is pruned for allowing its lateral buds to sprout and grow. The second cutting-back treatment is applied on the lateral shoots (M1V2) developed from the buds located on the basal part of the irradiated shoot. If the newly sprouted/developed lateral shoots again exhibit malformed leaves at the base, leafless portion in the middle and normal leaves at the top, the shoot is pruned just after the 10th or 5th leaf from the base (Katagiri 1970; Katagiri and Nakajima 1982). Thus, the lateral buds are allowed to sprout and develop shoots (M1V3).

The wholly mutated shoots thus developed at any stage of cutting-back treatment are propagated through grafting or stem cuttings. Nakajima (1973) and Katagiri (1970) reported the isolation of 17–23% whole mutants after first cutting-back treatment itself. In Japan and China, the studies indicated that applying cutting back method directly above the leafless portion is most effective in isolating numerous “wholly mutated” shoots. However, in the M1V2, most of the radiation injuries disappear from the shoots. The important vegetative

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generation for selecting bud mutant is M1V2. In case of re-irradiation of induced mutants, the same process may be repeated and it may give higher mutation frequencies and wider mutation spectra.

3.4.5.4 Mutation in Cell and Tissue Culture Somaclonal variations are a source of variability. But they are of low rate and narrow spectrum. The combination of in vitro culture (somaclonal variation) and mutagenesis have proved to increase mutation rate and mutation spectrum. In wheat, Hezhu-8 is the first variety developed through somaclonal variation. Somatic hybrids like SV-1 were developed at CSRTI, Berhampore. 3.4.5.5 Mutation as a Tool in Cell Fusion Gamma fusion is another method, where mutation technique can be used for inactivation or fractionization of the genetic material of donor cells before fusion with another recipient or species. 3.4.5.6 Application of Mutation Breeding Mutation breeding in mulberry was initiated in Japan in 1957, and about 50 mutants were isolated in 1967 and out of which two varieties were exploited in the field. Hazama (1968a) induced various mutants of KNG using acute gamma irradiation; most of them were chlorophyll and morphological mutants without any practical utility. However, two mutants found beneficial to have 7% more leaf thickness and 20–25% shorter intermodal distance and 15% higher leaf yield over control. In China, few mutant lines, viz. Fu sang-10, R81-1 and R81-2, were developed from Ichinose variety through gamma irradiation. R81-1 is characterized by chromosomal alteration from diploid to tetraploid with a 23% and 20% increase in width and size of the leaves, respectively. Another mutant line L7681 was obtained

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through N2 laser ray exposure of Fl hybrid seeds of Cangxi 49  Yu 2 cross. This mutant line is not only superior in leaf yield but also quality. A few popular mutant varieties such as Xiansang-305, Xinyiyuan, Singu 11-6 have been introduced for cultivation in the farmers’ field in China. Nakajima (1973) has attempted to induce die-back disease resistance caused by Diaporthe nomurai Hara in mulberry through mutation breeding and but the resultant mutant line showed various degrees of susceptibility, while Kukimura et al. (1975, 1976) failed to induce resistance. Fujita and Wada (1982) isolated two resistant lines after gamma irradiation and also opined that it is possible to develop useful mutant lines of mulberry with high rooting ability.

3.4.6 Utilization of Heterosis through F1 Hybrid Seed Complex For effective utilization of heterosis in F1 progeny which has vigorous growth, good leaf quality and resistance to adverse conditions, Japanese Scientists put forward the sexual propagation scheme for commercial exploitation in the field. This technology was also adapted by Russian and Chinese scientists. In the early days of research, hybrid seed propagation was not effective because of more heterogeneity and inferior F1 plants. Chinese scientists have made efforts to standardize the hybrid seed complex method, and in the year 1962, this technology was introduced as mulberry variety in the name of “Jinsang.”

3.4.6.1 Advantages of Hybrid Seed Complex Varieties Hybrid seed complex varieties are cultivated through seeds, and it can be stored in a market for sale (market commodity). It shortens the commercialization period of newly developed varieties by eliminating the long-drawn and costly step of grafting/sapling preparation in the nursery. It also helps in the selection of superior genotypes from the hybrid progeny.

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3.4.6.2 Disadvantages The heterogeneity appears to be high in the progeny raised through hybrid seed complex as compared to the clones propagated through vegetative means like grafts/stem cuttings. There is a decrease in the vigor of plantations in subsequent years. There is also a reduction in leaf size in subsequent years. However, these have been rectified by better parental selection. 3.4.6.3 The Important Parameters for Parental Selection (a) Parental plants should have higher leaf yield with a bigger leaf size (b) Unisexual (c) Synchronized flowering period (d) Good affinity and high seed setting capacity (e) High heritability of desirable characters (f) Both the parents should be relatively pure in nature.

3.4.6.4 Breeding Program After selection of suitable parental genotypes, the breeding scheme is prepared and breeding experiments are conducted which include the following activities, viz. cross-breeding (50–100 cross combinations), separation of hybrid (F1) seeds, sowing of hybrid seeds in experimental plot and comparative yield evaluation of the hybrid progenies in comparison with local check variety. The selection of hybrid progenies (F1) could be made based on breeding objectives and percentage of homozygosis of the F1 population. The important criteria for the selection of F1 progenies are as given below. 1. In the F1 population, the progenies with less vigor or poor growth (undesirable characters, viz. lateral branches, small leaf, susceptibility to diseases, etc.) should be less than 15%. 2. F1 progenies should have erect branching patterns for easy inter-cultivation. 3. The average shoot length of Fl progeny (per plant) should be more than 1.2 m with a coefficient of variance (CV) < 35%. 4. Number branches/F1 plant should be 2 to 5 during 1st year and 2.5 to 3 from 2nd year onwards (CV < 30%).

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5. Leaf size (in China) of F1 hybrid should be 18 cm (length)  14 cm (width) with CV < 15%. 6. Leaf yield of F1 progenies should be 30 MT/ha (average of first five years) (CV < 15%) 7. The optimum leaf thickness of F1 hybrid is 125 µm (CV < 15%). 8. Relative resistance to diseases should be on par with cv. Husang 199. 9. If the breeding objective is the development of disease-resistant variety, then the F1 progenies should be significantly more resistant than the National or Provincial check variety. If the objective is the development of leaf yield and quality, mulberry variety Tongxiangqing should be considered as a check for evaluation of F1 progenies. The cross combination of high yielding/ disease-resistant F1 progenies has to be certified by the Provincial or National Mulberry and Silkworm Variety Evaluation Commission. Then, the parents of the particular cross are planted for “hybrid seed” production for commercial purposes.

3.4.6.5 Production of Hybrid Seeds in the Garden The garden is prepared for the production of mulberry hybrid seeds for commercial purposes. The steps followed in this method are given below. (a) The male and female plants must be planted in the garden, and their ratio should be 1:5. In one Mu (Mu = l/15th of a hectare) area of plantation, about 771 female and 154 male plants are accommodated in Zhejiang Province and 2000–5000 plants in Guangdong Province. (b) The spacing of the plantation should be 120  60 cm. (c) Mulberry garden should be highly fertile. Application of more organic manure, P and K application is advisable. For mulberry seed garden, the fertilizer dosage of N: P: K is 5:3:4 and soil moisture should be high during the blooming period.

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(d) For higher pollen production and effective fertilization, the height of the male plant should be 10 cm higher than the female parent. (e) If any bisexual flowers are present in the garden, they have to be removed. (f) The seed garden should be 500 m away from other mulberry gardens. (g) Contrary to the foliage/cocoon production garden, more branches have to be maintained for better fruit production. (h) Care should be taken to prevent mulberry pests and diseases. (i) After fruit collection, the seeds are harvested from the fruits and the seeds are utilized for the establishment of a foliage/cocoon production garden. Following the above-mentioned method, it is possible to produce 350–450 kg of mulberry seeds during the first two years and 500–800 kg of seeds from the third year onwards from one hectare garden.

3.4.6.6 Cultivation of Hybrid Seed Variety for Leaf Production and Silkworm Rearing The mulberry plantation is established either by sowing of F1 seeds at the rate of 45,000 seeds/ha or by raising F1 seedlings in elevated nursery beds, followed by transplantation of these seedlings in the garden. While raising F1 seedlings in nursery beds, the seeds are sown in rows on elevated nursery beds (25–30 cm between rows and 2–3 cm between the seeds in a row). The cultural operation such as irrigation, application of fertilizers, providing of shade, maintaining of soil temperature needs to be carried out at the seedling stage. The seedlings with poor growth need to be removed. Seeds that are sown in June will exhibit their inherent characters in August. During this period, the seedlings with poor growth are removed from the nursery beds. Transplantation of the seedlings is carried out in January (dormant stage) with a spacing of 100  33 cm (Density of about 30,000 plants/ha). Immediately after transplantation, the

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tip, viz. 1/3 or 2/5 of the seedling, is cut. Firstyear harvesting could be rational; thereafter, the same cultural operations need to be followed for leaf harvest (Liu et al. 2014). By adopting the hybrid seed complex method in China, varieties such as Sa 2  Lun 109, Tang 10  Lun 109, Zhongsang 5801  Yue 2 (Fengchi), Lunjiao No. 408  Yue 82, Lunjiao No. 408  Husang 7, M1  9104, M2  93146, M2 x 93133, M1  94126, Tasang  Ji 681 were developed (Yang 1994; Zhu et al. 2013; Liu et al. 2014; Tang et al. 2016).

3.4.7 Breeding for Stress-Tolerant Mulberry Varieties The productivity and quality of mulberry leaves, very often, are affected by environmental constraints such as cold (low temperature), drought/soil moisture deficit (due to scarcity of rainfall and irrigation water), salinity and alkalinity. The reduction in leaf yield is also dependent on the mulberry varieties under cultivation. In general, it has been reported that the realized yield of a variety reduced to 1/3 under stress conditions as compared to that of ideal conditions (Sarkar 2009). Tolerance is a complex phenomenon and associated with many morphological, physiological, anatomical and biochemical characteristics. The tolerant varieties are identified primarily through indirect breeding method, viz. selection, where the genotypes developed elsewhere are evaluated under stress conditions/hot spot areas for foliage yield and stress tolerance. In the direct breeding method, a cross is made between two suitable parents. Here, one parent may have stress-tolerant traits/it may be a local variety selected from a hot spot area, while other parents may be a high yielding genotype.

3.4.7.1 Breeding for Drought/Soil Moisture Stress Tolerance Drought stress is the most widespread limiting factor for mulberry affecting leaf productivity in many countries where sericulture is a source of livelihood. Droughts are expected to be more

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frequent in some areas due to changing climate. Some key steps for the development of droughttolerant genotypes are given below: (1) Characterization of pre-breeding resources (germplasm accessions/genotypes) for identification of tolerant genotypes (under simulated drought) and evaluating these pre-breeding materials in various agroclimatic conditions (hot spots) with variable precipitation levels (2) Strategic crossing (Cross-breeding) among the parents having different but complementary traits for a given environment followed by screening, selection and field evaluation. Susheelamma et al. (1990) have evaluated 92 mulberry germplasm accessions for ten morphophysiological traits like stomatal frequency, stomatal size, leaf thickness, cuticle thickness, palisade parenchyma-to-spongy parenchyma ratio, moisture retention capacity, length of root, dry weight of the root, root-to-shoot ratio (length and weight basis) and 10 yield-contributing parameters like number of primary and secondary shoots per plant, length of shoot, intermodal distance, number of leaves per meter length of shoot, leaf area, the weight of 100 leaves, moisture percentage and percentage of sprouting and rooting. Finally, two genotypes ACC142 and ACC109 have been identified as drought-tolerant genotypes out of 23 accessions evaluated under rainfed/natural stress conditions. Two drought-tolerant high yielding varieties, viz. S-13 and S-34, were developed and recommended for red loamy soils and block cotton soil, respectively (Susheelamma 1987; Susheelamma and Jolly 1986; Susheelamma et al. 1989, 1992b). Dorcus and Vivekanandan (1997) have screened 8 mulberry genotypes by withholding water and found S-13 and S-34 tolerant to water stress. Jhansilakshmi et al. (2014) have screened 121 diverse mulberry genetic resources collected from different arid and semiarid geographical regions under progressive water deficit stress at CSGRC, Hosur, for 20 physiological parameters. They have reported that early vigor and low leaf

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senescence could be used as indicators for drought tolerance, and these can be used for rapid selection from large populations. Mulberry varieties, C1730, suitable for non-irrigated conditions of Eastern and North-Eastern India and MSG-2 for South India have been recently evolved (Sivaprasad et al. 2021). Zhang et al. (2018) evaluated eight mulberry varieties based on anatomical studies of stem and leaf and reported Wubusang and Baisang varieties as drought-tolerant.

3.4.7.2 Breeding for Cold Tolerance Cold-tolerant mulberry varieties were developed in various temperate countries mostly by the cross-breeding method. The experiments for the development and evaluation of cold-tolerant varieties were conducted in hot spot areas/winter seasons. For example mulberry varieties, viz. Yukishinogi, Yukishirazu, Yukimasari (Japan), Hongcang sang, Huosang, Longsang 1, Liaolu 11, Poluosang (China, Wang et al. 2009), Harkov-8 (Former USSR), BC2-59, PPR-1 (India), were developed for cultivation in temperate regions. Doss et al. (2011) have identified two high yielding varieties, viz. CT-44 and CT-11, which are found suitable for Indian temperate conditions. 3.4.7.3 Breeding for Salt Tolerance Studies have been conducted to identify salinitytolerant mulberry genotypes through screening and selection methods under coastal saline soils (Anas and Vivekanandan 1994a, b; Agastian and Vivekanandan, 1997) and under simulated saline conditions (Anas and Vivekanandan 1999; Mogili et al. 2002, 2008; Prakash et al. 1998; Sarkar et al. 2017; Yin et al. 2018). Based on the screening in coastal hot spot areas, two genotypes, viz. Tr-4 and Tr-10, were identified as salttolerant varieties. Mogili et al. (2002) conducted a detailed study under various levels of simulated stress conditions wherein Victory-1 and AR-10 exhibited higher survival rates and higher leaf yields under higher salinity levels (EC 8.0 dS m−1). These two varieties had a higher K: Na ratio and lower Na and Cl in all plant parts, viz. root, shoot and leaves. Ber. C-776 has been

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Mulberry Breeding for Higher Leaf Productivity

identified as a salinity-tolerant variety as it can tolerate salinity levels up to EC 7.8 mhos/cm under in vitro screening experiments followed by field study (Chakraborti et al. 2000). The variety has shown a 28.1% higher leaf yield over S1635. Approaches for developing saline/abiotic stress-tolerant mulberry varieties have been reviewed by various researchers (Vijayan 2009; Vijayan et al. 2008, 2009; Sarkar et al. 2017, 2018; Aruna et al. 2019). Lin et al. (2018) studied osmatic regulative substances and antioxidants in different tissues in three mulberry varieties and found that Da 10 was tolerant to salt stress.

3.4.7.4 Breeding for Alkalinity Tolerance In India, attempts have been made to identify variety tolerant to alkalinity which led to the identification of a triploid genotype viz. AR-12 (Mogili et al. 2008; Sathyanarayana and Mogili 2013; Sathyanarayana et al. 2008). AR-12 exhibited comparatively lower Na: K indicating selective uptake, transport and preferential partitioning of minerals (Jalaja et al. 2013). Urs et al. (2011) reported that mulberry, viz. RC-2, showed a high degree of plasticity when exposed to soil moisture, alkalinity and salinity stresses. Tr-23 is recommended for acidic soils in hilly regions of Eastern and North-Eastern India (Sivaprasad et al. 2021). 3.4.7.5 Development of Varieties Suitable for Sub-optimal Irrigation and Inputs Among various agronomic inputs to which mulberry responds well and quickly are irrigation and fertilizers. Irrigation helps in better utilization of manures and fertilizers has a major role in increasing the productivity in a unit area of mulberry plantation. Sometimes irrigated areas are turning to be semi-irrigated due to irregular and less annual precipitation and depletion of the groundwater source. Scarcity of availability of irrigation water decreases leaf yield and quality of mulberry. Hence, there is a requirement to develop varieties that can respond reasonably well under limited or sub-

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optimal irrigation and efforts of breeding resulted in the development of mulberry variety AGB 8 for sub-optimal irrigated conditions with 47 tons of leaf yield/ha/year. The small and marginal farmers may not have the capacity to purchase and apply required quantity of fertilizers at the proper time. The yield of a high yielding variety reduces drastically when the quantities of fertilizers inputs are reduced. Hence, there is a need to develop a variety that will produce a reasonable leaf yield at a reasonable level of fertilizer application. The genotypes should have the capacity to yield higher when the input constraints are removed. Mulberry varieties RC1 and RC2 have been recommended for sub-optimal irrigation and fertilizers [50% of recommended irrigation and fertilizers (Urs et al. 2011)].

3.4.7.6 In Vitro Screening for Abiotic Stress Tolerance In vitro screening method has been applied for the identification of genotypes tolerant to various abiotic stresses like drought, salinity and alkalinity. This is an indirect method for preliminary screening of a large number of genotypes. In many crop plants, salinity and drought-tolerant lines have been isolated using this in vitro technique. Mulberry genotypes showed response to simulated abiotic stresses such as drought (polyethene glycol induced), salinity (NaCl and Na2CO3 + NaHCO3 induced) under in vitro conditions (Rama Rao et al. 1997; Tewari et al. 2000; Vijayan et al. 2003, 2004, 2022a).

3.4.8 Breeding for Disease and Pest Resistance Mulberry is affected by several diseases caused by fungus, bacteria, mycoplasma, nematode and virus and cause severe damage to crops and productivity (Vijayan et al. 2022b). The dwarf disease caused by mycoplasma-like organisms that mostly occur in temperate countries like Japan and China is considered to be the most dreaded disease. The severity of diseases varies from region to region. Development of disease-

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resistant varieties by harnessing genetic resistance (Arunakumar et al. 2019a, b, 2021; Gnanesh et al. 2021, 2022) and introgression of high yielding varieties are the most sought after method to leaf vis-a-vis cocoon productivity. The common mulberry diseases and resistant varieties (Sarkar 2009) are presented below:

3.4.8.1 Mulberry Dwarf Disease This is caused by mycoplasma-like organism and is mostly found in temperate areas. The dwarf disease-resistant mulberry varieties like Tokiyutaka, Oshima (Japan), Tongqiangshing, Hongkong sang, Hu sang-199, Nongsang-8 and Yu-2 (China) are in cultivation. 3.4.8.2 Bacterial Blight This is caused by Pseudomonas mori and is widely distributed in various cultivation regions. Mulberry varieties like Ukranian-1, Harkov 3 (erstwhile USSR), Hayatesakari, Kenmochi, Sinichinose, Minamisakari, Kosen (Japan) Husang-199, Nongsang-8, Yu-2, Jiantol heybaitao (Chun 1996), S-36 and S-41 (India) were found to be relatively resistant to bacterial blight. 3.4.8.3 Bacterial Wilt Mostly prevalent in China, caused by Bacterium mori and the mulberry varieties, viz. Nongsang-9, Yu-2, Zhongsang-5801, Jingling-16, were found to be resistant to bacterial wilt. Recently, 76 mulberry hybrid combinations were evaluated for resistance to bacterial wilt disease and resistant hybrid seed varieties were identified (Zhu et al. 2014). 3.4.8.4 Powdery Mildew This disease is caused by Phyllactinia guttata (earlier P. corylea) and is prevalent in all the mulberry growing areas. Mulberry varieties, namely Akagi, Aobanezumi, Fukayuki, Fukushima, Oha, Ichinose, KNG, Kairyoroso, Kokuso20, Kosen (Japan), Dahuasang, Xiaoguan sang, Kang Qing-10 (China), MR-2, BC259 (India), have exhibited medium to high resistance to powdery mildew disease. Jhansilakshmi et al. (2016) screened 144 germplasm accessions for resistance to powdery mildew and tukra

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(Maconellicoccus hirsutus) and reported that 21 accessions exhibited resistance reaction to powdery mildew and 9 accessions to tukra disease. Chattopadhyay et al. (2010, 2011) evaluated germplasm accessions for resistance to mildew under field and glasshouse conditions, and they found the relationship between leaf micromorphological traits and mildew resistance.

3.4.8.5 Dieback Disease The disease occurs during snowfall in temperate areas. This disease incidence is routinely measured before the recommendation of new varieties. Mulberry varieties, viz. Yukishiraju, Fukayuki, Shinso-2, were found to be resistant to dieback disease in Japan. 3.4.8.6 Root rot Diseases of the root system are a major problem in mulberry cultivation, as managing them is quite challenging in comparison to foliar diseases. Of late, outbreaks of root rot disease have become a serious threat in the Southern part of India, often resulting in the devastation of entire plantations. Various types of root rots such as dry rot caused by Fusarium solani and Fusarium oxysporum, black rot caused by Botryodiplodia theobromae (=Lasiodiplodia theobromae), Rhizopus rot caused by Rhizopus oryzae and charcoal rot caused by Macrophomina phaseolina (= Rhizoctonia bataticola) have been reported in mulberry from India (Philip et al. 1995; Radhakrishnan et al. 1995; Chowdary 2006; Arunakumar and Gnanesh 2023; Gnanesh et al. 2021, 2022; Vijayan et al. 2022b). Most of the mulberry cultivars are prone to charcoal rot (Chowdary 2006). Thirty-five promising germplasm accessions and popular varieties were screened for resistance to root rot and root-knot. Several crosses were made using medium to resistant genotypes with high yielding varieties. Promising 6 hybrids exhibiting resistance reaction to root rot and root-knot (Meloidogyne incognita, Kofoid and White) and higher leaf yield over population means were shortlisted for further evaluation (Doss et al. 2018). Eight promising resistant accessions (G2, ME-0006, ME-0011, ME-0093, MI-0006, MI-0291, MI-

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0489 and MI-050) were found to be resistant to black root rot (Gnanesh et al. 2022).

3.4.8.7 Root-Knot Nematodes (RKN) Meloidogyne incognita causes the most devastating soil-borne menace in mulberry (Manojkumar et al. 2022). Its infection leads to the development of root-knot disease complex with root rot pathogens and results in deterioration of leaf quality and yield loss of up to 20% (Govindaiah et al. 1996). Recently, 415 diverse germplasm accessions were screened for RKN resistance under glasshouse conditions. Among them, 69 accessions were identified as immune/resistant and the shortlisted 20 accessions were further evaluated under field conditions of RKN hot spots. Eight germplasm accessions, viz. BR-8, Karanjtoli-1, MI0437  MI-0364 (P-2), Nagalur Estate, Tippu, Calabrese, Thai Pecah and SRDC-3, were identified as resistant to Meloidogyne incognita under field condition. These germplasm accessions can be utilized as either rootstocks or for further RKN resistance breeding programs (Arunakumar et al. 2021).

3.5

Mulberry Variety Development: Screening, Selection and Evaluation

In general, the breeding process starts from the selection of parents, mating, screening, selection and the recommendation of varieties. As mentioned earlier, as per the breeding objectives, a large number of hybrid seedlings ranging from 10,000–20,000 are raised from 15 to 20 cross combinations. Mulberry variety development process followed in India, Japan and China is similar with minor variations and takes 11– 13 years for regional release and inclusion in Multi locational evaluation (Sarkar and Fujita 1993a, b, c, d; Mogili 1994, 2000; Sarkar et al. 1997a, b; Sarkar 2009). The comparative steps adopted in major sericulture countries are presented below.

79 Indian system

Japanese system

Chinese system

1. Selection of parents and crossing— 1 year

Selection of parents and crossing— 1 year

Selection of parents and crossing— 1 year

2. Raising of seedlings and establishment in field—1 year

Raising of seedlings— 1 year

Raising of seedlings— 1 year

3. Selection (a) Screening— 3 years (b) Primary yield evaluation —4 years

3. Selection (a) First selection— 5 years (b) Multiplication by grafting— 1 year

3. Selection (a) First selection— 5 years (b) Multiplication by grafting— 1 year

4. Evaluation Final yield evaluation—4 to 5 years

4. Evaluation (Comparison test) Second selection—4 to 5 years

4. Evaluation (Comparison test) Second selection—4 to 5 years

Duration: 13– 14 years

Duration: 12– 13 years

Duration: 12– 13 years

At the Regional Testing Centers/All India Coordinated Evaluation of Mulberry—4 to 5 years

Local trial (Mulberry variety authorization program)— 6 years

Local trial (Mulberry variety authorization program)— 6 years

Registration and Mass multiplication and Variety release—1 to 2 years

Registration and Mass multiplication and Variety release—1 to 2 years

Registration and Mass multiplication and Variety release—1 to 2 years

Total duration: 19–20 years

Total duration: 19–20 years

Total duration: 19–20 years

3.5.1 Transplantation of Seedlings and the First Selection The selection process begins at the seedling stage before transplantation to the field by the rejection of weak and seedlings with undesirable traits. Generally, seedlings are planted in close spacing to accommodate a large number of seedlings in a

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unit area. In Japan, the plantation method used is paired row system, i.e., (40 + 120)  20 cm. The hybrids with undesirable traits are removed from the second year onwards. In India, a slightly modified spacing of (60 + 120)  40 cm is suggested by Sarkar et al. (1997a, b) and spacing of 60  60 cm is also followed. 1. Planting pattern of seedling of the crosses: Seedlings from one cross are planted in both rows of the paired row to make a block in the field. The plantation is maintained by applying 20 tons of farmyard manure and NPK @ 300:120:120 kg per hectare per year from the second year onwards. 2. Method of numbering: In Japan, the progeny of a particular cross is numbered indicating the year of crossing, the code number of cross and the plant number. For example, 21-A-1 indicates that in the crossing year 2021, the cross detail (S-36  V-1) code is “A” and the plant number is “1”. The same number is continued till the end of the second selection or the final yield trial and specific names are given in addition to the experiment number. There is no specific numbering system in India; the above similar system can be adopted in India.

3.5.2 The First Selection/First Stage Observations Observations are recorded after approximately one year of establishment of the seedling plantation. The plants are pruned in the following year and the data collection starts. In the early screening, visual observations are made mostly on morphological traits such as leaf lobation, presence of lateral shoots, leaf color, leaf surface, appearance, the posture of the shoots, petiole length and disease resistance, followed by rejection of undesirable seedling in the second year. Though the total leaf area of a plant contributes to the yield of the plant, in India hybrids with unlobed leaves are preferred while selecting. But in Japan, 4–5 lobed leaves are preferred. Hence in the selection process, plants with more

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lobes and thin leaves are rejected. The presence of lateral shoots is one of the most undesirable traits as it creates thin and small leaves. Generally, dark green leaves with a smooth surface are preferred and the pale green and rough surface ones are rejected (selection-1). Observations are recorded for disease resistance of the hybrids across various seasons. Leaving the rejected hybrids, the remaining hybrids are screened for important phenotypic characteristics, viz. sprouting rate, total and average shoot length; longest shoot length, intermodal distance, above-ground biological yield (Joso), shoot weight and number of leaves per meter length of shoots and leaf yield are collected from late second year and 3rd year covering all the seasons (Selection-2) (Dandin and Kumar 1989). Studies made to find out a few parameters, viz. contributing to the selection of desirable hybrids in mulberry indicated that total length of all shoots, the total weight of all shoots and 100 leaf weights showed highly significant correlations with leaf yield (Sarkar et al. 1986, 1988; Rahman et al. 1994; Wakatole and Wosene 2016). Further analysis of data indicated that these parameters have a high correlation with leaf yield both at genotypic and environmental levels (Sarkar et al. 1997a, b) and can be used to shortlist the promising hybrids. However, a study on juvenile-mature correlations does not give a reliable estimate in mulberry (Prasad et al. 1995). In India, during the third year, promising 100– 150 hybrids are subjected to rooting efficiency and hybrids with > 60% rooting and leaf yield values higher than the population mean are shortlisted for inclusion in primary yield trials in tropical conditions (selection-3).

3.5.3 First Selection/Second Stage Observations/Primary Yield Evaluation In tropical countries like India, the selected hybrids are multiplied by stem cuttings and planted an experimental plot along with check varieties for conducting primary yield trial either in the augmented design or lattice design of 6  6, 8  8

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and even more depending on the number of hybrids selected. Due to complexity, the lattice design was replaced with augmented design or randomized block design (RBD) along with check varieties. The primary yield trial provides more reliable data than that of the first stage observations (first selection) which is obtained from a single plant. In this trial, more hybrids are tested in a smaller plot along with ruling variety as check-in replication under similar micro-climatic conditions. The trial is conducted for 3 years after the establishment of the plantation. Data on leaf yield, yield-contributing parameters are recorded for 3 years. During the 3rd year, the quality of the leaf is assessed through silkworm moulting tests and biochemical assays. In Japan, under temperate conditions, the late observations are continued in the same selection plot and selection is based on a single plant only. The inferior undesirable hybrids are rejected based on comparison of pair or comparison with standard control without statistical methods.

3.5.4 Second Selection or Final Yield Evaluation The final yield evaluation (India) or the second selection (Japan/China) consists of the assessment of 8–14 superior hybrids which have been identified from the first selection/primary yield evaluation (Sarkar and Fujita 1993c, d). In this trial, the hybrids are evaluated in a larger experimental plot with an appropriate statistical design. Data is recorded for 3 years after the establishment of the plantation. During the 3rd year of data collection, the leaf quality is assessed through silkworm bio-assay and chemo-assay. The superior genotypes which outperform the check variety are recommended either for field release or inclusion in the multilocational trial/All India Coordinated Evaluation of Mulberry (AICEM).

3.5.5 Experimental Design for Mulberry Breeding Experiments Varietal comparison tests are mainly carried out in the field as a part of the selection process for

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good varieties. These field tests can be affected by environmental conditions, reducing the accuracy of the results. To eliminate environmentcaused errors and to increase the accuracy of the experiment, the following methods are adopted. The field plot test should be carried out under uniform soil conditions and similar microclimatic conditions, i.e., sunshine and ventilation. The test varieties should be of the same type, i.e., high yielding varieties should be compared with high yielding varieties and tolerant varieties with other tolerant varieties. Relatively uniform planting measures should be adopted, i.e., size of saplings, pruning, manuring and other management methods. 1. Layout of the experiment: The randomized block design (RBD) is the most common technique and is used for testing a few genotypes. The test genotypes are planted in random order in the blocks. The number of replications required and the size of each plot depend upon the soil heterogeneity. Chaturvedi and Sarkar (2000) have recommended a nearly square type of plot with 22.68 m2 which accommodate 63 plants under 60  60 cm spacing and a minimum of 48 plants per test genotype per replication. Narrow or rectangular plots increase none effective plantation area due to more number of border-covering plants. 2. Spacing: Generally, the plantation is carried out at the onset of monsoon (June–July; Sept– Oct). Plantation could be done under paired row system (90 + 150) x 60 cm or 90  90 cm or 60  60 cm. Paired row system facilitates partial mechanization and saves manpower requirements. Trenches or pits are made and the saplings are planted deep in the soil. After the plantation is over, the saplings are cut at 15–20 cm above the ground level and maintained as per recommended package of practices (Sarkar et al. 1997a, b). 3. Duration of the trial: The final yield evaluation/second selection is conducted for four years in tropical countries like India and six years in temperate countries like Japan

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and China. The reason behind the increased (b) Silkworm Bioassay: Comprehensive evaluation of leaf quality is done through moulting duration in the temperate areas is that in test, grown silkworm appraisal by feeding of tropical countries about 5 harvests are colmulberry leaves to silkworms. lected in a year and only 3 harvests in case of temperate areas due to winter dormancy and Moulting test: It is an easy and fast method to the number of harvests remains 15 for both cases. Growth and yield data are collected assess the leaf quality through feeding the young from the second year onwards (Sarkar 2009). larvae with young leaves of tested genotypes and 4. Record of data on growth yield and quality: check varieties (Benchamin and Anantharaman Growth data is collected from five plants per 1990; Mala et al. 1992; Rahman et al. 2000). The genotype per replication, while yield data is larvae of the 1st and 2nd instar stages are used recorded from the whole plot. Parameters for this test. Feeding for 60% of the feeding considered at this stage are similar to the late duration out of total time required for I and II instar larvae is followed. The percentage of observation of the first selection (PYE). 5. Appraisal of leaf quality: Leaf quality iden- worms settled for moult and larval weight are tification is usually performed during the last considered for calculating the index. Whole instar appraisal: In this method, the stage of selection under similar field management conditions by rearing silkworms and larvae (1st to 5th instar stages) are fed with both through chemical analysis (Das and Prasad standard check varieties and mulberry genotypes 1974; Katagiri and Machii 1988; Rajabov to appraise. The parameters considered are larval duration, cocoon weight/10,000 larvae, number et al. 2021). (a) Chemical assay: The most important leaf of cocoons/10,000 larvae (Effective rate of rearconstituents considered are moisture percent- ing) shell weight/10,000 larvae and cocoon age and its retention capacity, proteins, car- weight/100 kg of leaves and finally reeling bohydrate, fat and vitamin contents. parameters. Fifth instar rearing test: In this method, I to IV According to Chinese observations, nitrogen, soluble sugar and carbohydrates have a sig- instar worms are fed with leaves of one standard nificant effect on cocoon quality. The ratio of variety while V instar worms are fed with mulcarbohydrates to crude proteins (C/N ratio) is berry genotypes to be appraised. For preliminary closely related to silkworm rearing. It is screening, a minimum of 50 larvae per replicareported that the suitable C/N ratio is 3.5–5.0 tion and for formal appraisal, 400–500 for grown larvae, 0.98–1.0 in 3rd to 5th larvae/replication covering a minimum of 3–4 leaves in spring (Lou Cheng Fu 1994). crops are considered. The parameters considered Fukuda et al. (1961) reported that silkworms are the same as for the whole instar appraisal. ate more mulberry leaves with 3.14% nitro- Identification of genotype for high fecundity, gen than the leaves with 2.67% nitrogen. The whole instar silkworm appraisal and fifth instar average levels of amino acid content in mul- appraisal is followed with an additional paramberry leaves were estimated by Machii and eter of number of eggs laid by moths. Productive efficiency of cocoon shell (PECS) Katagiri (1991). Generally, higher the conappraisal: Assessment of productive efficiency of tents of proteins and soluble sugars in leaves, better the leaf quality, as they are the main cocoon shell is considered as an accurate critenutrients of silkworms (Lou Cheng Fu 1994). rion for evaluating the quality of test genotypes However, the young larvae need leaves with a (Katagiri and Machii 1988; Machii and Katagiri high content of soluble sugar and moderate 1990; Sarkar and Fujita 1994; Jalaja and Suresh content of proteins, while grown larvae Kumar 2011). The weight of the larvae, amount demand the leaves with high content of pro- of leaves supplied, amount of leaves consumed teins and moderate content of soluble sugars. are recorded on daily basis. Then data is recorded

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Mulberry Breeding for Higher Leaf Productivity

on the quantum of leaves consumed, amount of leaves digested, cocoon weight and cocoon shell weight. It was reported that the PECS has significantly correlated with the amount of the firstday feces of the fifth instar larvae, which, therefore, can be used to appraise the quality of mulberry leaves to be tested. Sometimes, final yield evaluation is also conducted in different locations of the region simultaneously, particularly for the development of tolerant varieties with sustainable yield (Susheelamma et al. 1992a, b). Based on the results obtained during the final yield evaluation and analysis of data, one or two promising hybrids are finally selected and recommended for the region. The top-performing hybrids are multiplied from the third year onwards, and sufficient seed material is maintained for further multiplication and supply.

3.5.6 Multi Locational Trial and Variety Authorization Multilocational trial helps in assessing the adaptability of the genotypes in different environments/regions, other than the regions where they have been developed. The multilocational trial is also called as mulberry variety Authorization program/All India Coordinated Experiment on Mulberry in India, local trial in Japan and National examination Commission for mulberry and silkworm in China. There are 18 locations in India, 16 locations in Japan and 8 locations in China for the mulberry variety authorization program. In Japan, the varieties are also tested under dense plantation of 0.8  0.5 m (25,000 plants/ha). The genotypes, which are to be tested in different agro-climatic conditions, are collected from different breeding institutes. The trial is conducted in the larger plots under randomized block design with 3–5 replications in wider spacing and national and regional popular varieties as controls. The trial is conducted for three years after establishment. In this trial, data is recorded on leaf yield, important growth parameters, rooting ability, feeding quality and

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disease resistance. In temperate countries, special care is taken to find out resistance to dwarf and dieback diseases. In Japan and China, the experiments on disease resistance are conducted separately in 4–5 locations/hot spots where natural disease incidence is high. The committee organizing the evaluation collects the performance data from all the test centers and subjects to location-wise pooled and stability analysis (Eberhart and Russel 1966). The committee recommends the varieties based on performance and adaptability and gives a registration number apart from the commercial name suggested by the breeder. In India, the first All India Coordinated Experiment for Mulberry (AICEM) was initiated in 1993, and 3 trials have been completed and a total of 19 varieties were recommended/ authorized for cultivation in different agroclimatic conditions of India (Saratchandra et al. 2011; Mogili et al. 2017a, b) and presented in Tables 3.5 and 3.6. Similarly, about 19 varieties have been recommended in China (Tables 3.7 and 3.8) and in Japan, through these trials, 19 varieties have been so far authorized and released for field exploitation by the Ministry of Agriculture, Forestry and Fisheries (Tables 3.9 and 3.10).

3.6

Mulberry varieties developed through Conventional Breeding Approaches

Morus species are naturally distributed in 49 countries, and out of which in 29 countries, it is cultivated for silkworm rearing and silk production. Though a large number of mulberry varieties are maintained across various countries, a few of them are used for commercial cultivation.

3.6.1 Major Mulberry Varieties Cultivated in India In India, the mulberry improvement program was initiated during the early 1960s, and spearheaded by Central Silk Board (CSB) constituted research

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Table 3.5 Major mulberry varieties of India and their salient features Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

Mysore local

Native to South India

South India under both rainfed and irrigated conditions

Diploid belongs to M. indica L. Leaves heterophyllous, ovate; Poor leaf yield; Tolerant to drought, fast sprouting; Female and male plants

Kanva-2 (K2/M-5)

Selection from natural population of Mysore local variety, recommended in 1968

South India under irrigated and rainfed conditions

Diploid belongs to M. indica L. Slightly spreading with many branches; Leaves entire, medium size, cordate, green; Leaf yield about 30 tons/ha/yr under irrigated condition, Good rooting ability; widely adaptable; Flowers female

S-30

Chemical mutagenesis-EMS treated Berhampore local seeds progeny

South India under irrigated conditions

Diploid belongs to M. indica L. Erect branches with short internodes; leaves entire, cordate, Leaf yield about 35–40 tons/ha/yr under irrigated condition, good leaf quality; Moderate rooting ability; Flowers female

S-36

Chemical mutagenesis-EMS treated Berhampore local progeny, recommended in 1984

South India under irrigated conditions

Diploid belongs to M. indica L. Slightly spreading with many branches; Leaves entire, cordate, large, golden green; Leaf yield about 40 tons/ha/yr under irrigated condition, good leaf quality; Moderate rooting ability; Flowers female. Suitable for young age silkworms

S-41

Chemical mutagenesis-EMS treated Berhampore local progeny

South India under irrigated conditions

Triploid belongs to M. indica L. Fast growing with many branches; Leaves entire, cordate, dark green with coarse surface; Leaf yield about 40 tons/ha/yr under irrigated condition, medium leaf quality; good rooting ability; fast sprouting and early maturity; flowers male

S-54

Chemical mutagenesis-EMS treated Berhampore local progeny, recommended in 1984

South India under irrigated conditions

Diploid belongs to M. indica L. Slightly spreading with many branches; Leaves entire, cordate, large, green; Leaf yield about 45 tons/ha/yr under irrigated condition, moderate leaf quality with low leaf moisture retaining ability; Good rooting ability; Flowers female

RFS-135

Open-pollinated hybrid from Kanva-2

South India under protective irrigation

Diploid belongs to M. indica L. Slightly spreading with many branches; Leaves entire, cordate, large, green; Leaf yield about 40 tons/ha/yr under irrigated condition and 30–35 under protective irrigation; moderate leaf quality; Good rooting ability; Flowers predominantly male (continued)

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Table 3.5 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

RFS-175

Open-pollinated hybrid from Kanva-2

South India under protective irrigation

Diploid belongs to M. indica L. Slightly spreading with many branches; Leaves entire, cordate, large, green; Leaf yield about 50– 55 tons/ha/yr under irrigated condition and 30–35 under protective irrigation; moderate leaf quality; Good rooting ability; Flowers predominantly male

S-13

Selection from poly-cross progeny of Kanva-2. Recommended during 1990

South India Under Rainfed conditions in sandy soils

Diploid belongs to M. indica L.; many erect branches with short internodes; fast growing with extensive root system; leaves entire, ovate, medium size, glossy; leaf yield 13–16 tons/ha/yr under rainfed conditions; good leaf quality; male flowers

S-34

Selection from poly-cross progeny of Kanva-2 and recommended in 1990

South India- Rainfed and moderate alkaline soils

Diploid belongs to M. indica L. many erect branches with short internodes; fast growing with extensive root system; leaves entire, cordate, medium size, wavy surface, dark green; Leaf yield 13– 16 tons/ha/yr under rainfed conditions; good leaf quality; Male flowers

MR-2

Selection from open-pollinated hybrids

South India hills and plains of Tamil Nadu

Diploid belongs to M. sinensis; many erect branches with short internodes; fast growing with extensive root system; leaves heterophyllous 3–4 lobed, ovate, wrinkled, dark green; leaf yield about 30–35 tons/ha/yr; resistant to powdery mildew; good leaf quality; flowers monoecious

Victory-1 (V-1)

Cross-breeding S-30  Ber.C776. Recommended in 1997 for irrigated areas

South India For irrigated areas, popular variety

Diploid belongs to M. Indica L; many erect branches with long internodes; fast growth, extensive root system; leaves ovate, glossy, dark green; leaf yield about 60 tons/ha/yr under irrigated conditions; Good leaf quality and high moisture retaining capacity; many male flowers

G-2

Cross-breeding M. multicaulis  S-34 (M. indica)

South India, exclusively for commercial chawki rearing centers

Diploid, slightly spreading, early sprouting; short internodes; leaves cordate, medium size, dark green and smooth; Good leaf quality Chawki leaf yield 38 tons/ha/yr; many female flowers, profuse fruiting (continued)

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Table 3.5 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

G-4

Cross-breeding M. multicaulis  S-13 (M. indica). Recommended in 2017 through AICEM for irrigated areas

South India—irrigated areas

Diploid, erect fast growing branches with short internodes; early sprouting; leaves cordate, large dark green, wavy surface; Good leaf quality; Leaf yield about 65 tons/ha/yr; high rooting ability and water use efficiency; Flowers female and many

AR-10

Open-pollinated hybrid of Kanva-2

South India- rainfed conditions

Diploid belongs to M. indica L.; many erect branches with short internodes; fast growing with extensive root system; leaves entire, ovate, medium size, glossy; Leaf yield 13–16 tons/ha/yr. Male flowers

AR-11

Open-pollinated hybrid of Kanva-2

South India- rainfed areas

Diploid belongs to M. Indica L; erect, many branches with short internodes; leaves entire, small, thick, semi-glossy, cordate; yields 9–10 tons per ha/yr; profuse root system; predominantly male

AR-12

Polyploid breeding S-41 (4x) (♀)  Ber.C776 (♂) followed by evaluation under alkaline soils hot spot area

South Indiarecommended for alkali soils where pH ranges from 8.5 to 9.4

Triploid belongs to M. multicaulis. Semi-erect, medium branches; leaves entire, large, wide ovate; deep green, slightly rough, glossy; leaf yield 25–30 tons/ha/yr under alkaline soil; Good quality, profuse root system; Predominantly male

Sahana

Cross-breeding Kanva-2  Kosen evaluation under coconut shade

South Indiarecommended as intercrop in coconut plantations

Diploid belongs to M. indica L. Semi-erect medium branches; leaves large, cordate, glossy, deep green; Yields 30–38 tons/ha/yr under coconut shade (40,000 lux intensity); moderate rooting, Female flowers

Anantha

Clonal selection from RFS175 evaluation under irrigated conditions

South India- irrigated areas

Diploid belongs to M. indica L. Semi-erect many branches; leaves cordate, large, glossy smooth; yields about 60tons/ha/yr; medium keeping quality

RC-1

Cross-Breeding Punjab local  Kosen and screening under sub-optimal conditions

South India - resource constraints, i.e., 50% of recommended fertilizers and irrigation

Diploid belongs to M. indica L; semi-erect, medium branches; leaves 2–3 lobed, cordate, glossy, large thick; yields 22–25 tons per ha/yr under 50% irrigation and fertilizers; good quality; high plasticity; female flowers (continued)

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Table 3.5 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

RC-2

Cross-Breeding Punjab local  Kosen and screening under sub-optimal conditions

South India—resource constraint areas

Diploid belongs to M. indica L; erect, many branches; leaves entire, medium size and ovate to cordate, deep green; yields 25–35 tons per ha/yr under 50% irrigation and Fertilizers; good quality; high plasticity; female flowers

MSG-2

Cross-breeding BR-4  S-13-multilocational evaluations

South India—rainfed areas

Diploid, erect, many branches, short internodes; leaves entire, medium to large, cordate, smooth surface and dark green; yields 22– 23 tons/ha/yr under rainfed conditions; male flowers

AGB-8

Advanced generation breeding (Sujapur5  Philippines)  (K2  Black cherry)

South India—under AICEM IV

Diploid, erect, many branches, long internodes; leaves entire, large, cordate, smooth and glossy and dark green; Yields 45–47 tons/ha/yr under 60% of irrigation; male flowers

Viswa

Clonal selection from DD variety by KSSRDI, India

South India—high temperature black and red soil areas

Diploid, Branches simple, erect to spreading; leaves entire, ovate to wide ovate and medium to large in size, good quality; yields 40–45 tons/ha/yr; predominantly male, bisexual

Vishala

Clonal selection from local plantations of farmers garden of Sidlaghatta, Kolar, Karnataka

South India- Irrigated southern red soil and North black soil of Karnataka

Triploid, spreading nature, strong branches, many; leaves entire, large, cordate, slightly coarse surface; early sprouting and fast maturity of leaves; medium quality; yields 45–50 tons/ha/yr under irrigated conditions

Thalaghattapura

Clonal selection

South India- irrigated areas of malnad region and southern red soils of Karnataka

Diploid, branches simple, erect to spreading; Leaves entire medium to large in size, good quality; Yields 35–40 tons/ha/year

Kajli

Native to West Bengal

West Bengal, in early stage, widely distributed in West Bengal

Diploid belong to M. indica L. branches very thin, short; highly lobed leaves; very poor leaf yield; strongly resistant to drought and diseases

Bombai

Clonal selection by farmers from local variety in Malda

West Bengal

Diploid belong to M. indica L.; slightly spreading, more branches with fast growth; leaves large, thin

Ber.S-1

Introduction from BurmaMandalaya

Eastern and North-Eastern India

Diploid belongs to M. indica L.; cultivated under rainfed and irrigated conditions; erect branches, short inter nodes; leaves are entire, ovate, small size, thick; good quality; yields about 12 under rainfed and 30–35 tons/ha/year under irrigated conditions; predominantly male (continued)

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Table 3.5 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

Ber.C-776

Cross-breeding M. multicaulis  Black cherry

Eastern India

Diploid belong to M. indica L.; erect nature, many fast growing branches, long inter nodes; leaves entire, medium size, green; yields 30–35 tons/ha/yr; medium leaf quality; flowers male and also recommended for saline soils

Ber.S-799

Selection from open-pollinated progeny of M. indica L.

Eastern and North-Eastern India

Diploid belongs to M. indica L., erect profuse branches, short inter nodes; leaves entire, ovate, medium size, thick; medium quality; yields about 35 tons/ha/yr under irrigated conditions; predominantly male

S-1635

Open-pollinated hybrid selected from CSRS-I seedlings and evaluated by AICEM, CSB, Bengaluru

Eastern and North-Eastern India and recommended for entire India

Triploid belongs to M. indica L.; suitable to both irrigated and rainfed areas; early sprouting semierect, stout branches; leaves entire, large, deep green, thick, slightly rough surface, fast maturity; yields 40–45 tons/ha/yr; Predominantly male

TR-4

Polyploidy breeding Triploid from open-pollinated progeny

Hills of Eastern India

Hills and foothills and yields 4–5 tons/ha/yr

TR-10

Polyploidy breeding, Induced tetraploid of Ber.S1x Phillippine (diploid), Selection

Recommended for Hills and foothills of Eastern India

Triploid belongs to M. indica L.; erect with medium branches, fast growth and quick maturity; leaves cordate, entire, large, thick and deep green, glossy; yields 12–15 tons/ha/yr in hilly areas; good quality; sterile and occasionally male flowers

TR-23

Polyploidy breeding, T-20 (4x)  S-162 (2x)

Hills and foothills of Eastern India

Recommended for acidic soils and hilly soils, yields 11–12 tons/ha/yr

SV-1

Somaclonal variant through tissue culture

Eastern India

Yields 35–38 tons/ha/yr under irrigated conditions of Eastern India

BC259

Backcross method, Matigara black  Kosen (Twice)

Hills and foothills of Eastern India

Yields about 7–8 tons/ha/yr

C-2028

Cross-breeding China white  S-1532

Eastern India

Recommended for waterlogged areas, yields 35–36 tons/ha/yr

C-1730

Cross-breeding, T25 (4x)  S-162; Triploid

Eastern India

Rainfed areas of Odisha, yields 13– 14 tons/ha/yr

C-2038

Cross-breeding CF1-10  C-763

Irrigated conditions of North-East India

Recommended for irrigated and rainfed areas; yields 54–55 tons/ha/yr

C-1360

Cross-breeding Philippines  Viethnam-2

East and North-East India

Yields 57–58 tons/ha/yr under irrigated conditions

S-146

Selection from unknown openpollinated source

North India—Foothills of J and K

Yields 8–10 tons/ha/yr under subtemperate conditions (continued)

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Table 3.5 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

China white

Clonal selection from exotic variety (China)

North India

Suitable for temperate conditions and yields 15–20 tons/ha/yr; good rooting, predominantly female

Chak majra

Selection from natural variability

Sub-temperate

Suitable for sub-temperate conditions of Jammu area; yields 25–30 tons/ha/yr. Predominantly male

Goshoerami

Introduction from Japan

North and North West India

Suitable for low-temperature areas and yields 11–15 tons/ha/yr under hilly areas

PPR1

Polyploidy—Cross-breeding Goshoerami  Chinese white

North and North West India

Temperate hills, yields 16–20 tons/ha/yr

Table 3.6 Authorized mulberry varieties of India and their salient features Name

Station and breeding method

Year (AICEM)

Recommended to

S-36

CSRTI, Mysuru Mutation Breeding

2000

Irrigated conditions of South India

S-13

CSRTI, Mysuru Selection from OPH of Kanva-2

2000

Rainfed areas (red loamy soils) of South India

S-34

CSRTI, Mysuru Selection from OPH of Kanva-2

2000

Rainfed areas (Black cotton soils) of South India

Vishala (DD)

KSSRDI, Thalagattapura Clonal selection

2000

Irrigated areas of South India

S-1

CSRTI, Berhampore, Introduction

2000

Eastern and North-East India

S-799

CSRTI, Berhampore, Selection from OPH

2000

Eastern and North-East India

S-1635

CSRTI, Berhampore, Polyploidy breeding-Triploid

2000

Eastern and North-East India National Check

C-776

CSRTI, Berhampore Cross-breeding

2000

Saline soils

S-146

CSRTI, Berhampore Selection from OPH

2000

Irrigated conditions of North India and Hills of Jammu and Kashmir

Tr-10

CSRTI, Berhampore, Polyploid breeding- Triploid

2000

Hills of Eastern India

BC2-59

CSRTI, Berhampore, Backcross Matigara local  Kosen (twice)

2000

Hills of Eastern India

Chak Majra

CSRTI, Pampore (RSRS Jammu) Selection from Natural variability

2000

Sub-temperate

China white

CSRTI, Pampore Clonal selection

2000

Temperate

Vishala

CSRTI, Berhampore Clonal selection?

2010

All India under irrigated conditions

Victory-1 (V-1)

CSRTI, Mysuru, Cross-breeding S-30  Ber.C-776

2010

Irrigated conditions of South India (continued)

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Table 3.6 (continued) Name

Station and breeding method

Year (AICEM)

Recommended to

Anantha

CSRTI, Mysuru Clonal selection

2010

Irrigated conditions of South India

G-4

CSRTI, Mysuru, Cross-breeding M. multicaulis  S-34

2017

Irrigated conditions of South India

C-2038

CSRTI, Berhampore, Cross-breeding, CF1  C-76

2017

Eastern and North-Eastern India

Tr-23

CSRTI, Berhampore, Polyploid breeding, T-20  S-162

2017

Hills of Eastern India

AICEM All India Coordinated Experiment for Mulberry

institutions (CSRTI-Mysore; CSRTIBerhampore; CSRTI-Pampore) and later by Karnataka State Sericulture Research and Development Institute (KSSRDI). The mulberry varieties, namely Kanva-2, S-135, S-175, S-13, AR-11, MR-2, S-1, S-799, S-146, S-1635, were developed through selection from openpollinated hybrids (OPH). Few mulberry varieties such as S-30, S-36, S-41 and S-54 were evolved by chemical mutagenesis at CSRTIMysuru. The mulberry varieties developed through cross-breeding include S-34, V-1, G-4, G-2, RC-1, RC-2, Sahana, MSG-2, AGB-8, C2028, C-2038, BC259, C-1360, PPR-1, etc. (Das 1986; Sastry 1984; Dandin et al. 1994; Sarkar et al. 1999, 2000; Balakrishna and Susheelamma 2004; Balakrishna et al. 2002a; Yadav 2004; Mogili et al. 2005, 2013, 2017a, b; Vijayan et al. 2012, 2017, 2019; Sivaprasad 2016, 2019; Sivaprasad and Mogili 2018; Shivaprakash et al. 2018; Sivaprasad et al. 2021) Some of these varieties are most popular with the farmers for their productivity and adaptability. Clonal selection, a common breeding practice, has led to the evolution of high yielding variety, viz. Vishala (45–50 MT/ha/year) is recommended for irrigated conditions in Karnataka. A triploid variety (AR12) with tolerance to soil alkalinity was recommended for cultivation in alkaline soils of the Southern part of India and Tr-23 is recommended for acidic soil in hilly regions and C1730 for drought tolerance of Eastern and North-Eastern India (Tikader and Kamble 2007; Chouhan et al. 2017). Due to the advent of these

superior varieties, cultivation package and practices resulted in the improvement of mulberry leaf productivity (MT/ha/year) by leaps and bounds (South: 30–35 to 60–65; East and NE: 20–30 to 55; North and NW: 10–15 to > 20) leading to the sustainable cocoon production under assured irrigation/input conditions. The major mulberry varieties with salient features cultivated in different parts of India are presented in Tables 3.5 and 3.6. The Distinctness, Uniformity and Stability (DUS) test guidelines have been developed for mulberry (Morus spp.) with 35 DUS descriptors and 34 example varieties which facilitate registration and protection of new/extant mulberry varieties (Girish Naik et al. 2016).

3.6.2 Major Mulberry Varieties Cultivated in China There are more than 1000 cultivated varieties of mulberry in China (Huang et al. 1997; Yong 2000; Pan 2000). Most of them were developed from the four species, viz. Lu mulberry (M. multicaulis Perr), white mulberry (M. alba L.), mountain mulberry (M. bombycis Koidz) and Guangdong mulberry (M. atropurpurea Roxb.). In China, more systematic breeding work for mulberry improvement was initiated during 1950 by adopting 12 words principle “Select locally, Propagate locally, Popularize locally for leaf yield, quality and vigor” (Yang 1994). Natural superior hybrids and spontaneous mutants were

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Table 3.7 Mulberry varieties cultivated in China and their salient features Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

Husang-197

Selection from natural population

Irrigated areas of Yangtze river basinZhejiang Province

Diploid, belongs to M. multicaulis. Leaf yield potential 33.75 tons/ha/yr, Suitable for both young and late age silkworm rearing, Moderately resistant to dwarf disease and brown spot, susceptible to bacterial wilt

Husang-199

Selection from Natural population from Zhejiang province

Zhejiang and Jiangsu provinces

Diploid and belongs to M. alba, branches many, slightly spreading; yields about 30 tons/ha/yr; poor leaf quality; good rooting ability; moderately tolerant to drought, low resources

Husang-32 (Heyebai)

Selection from Natural population from Zhejiang province

Widely distributed in Zhejiang and Jiangsu provinces

Diploid belongs to M. multicaulis.; wider adaptability, slightly spreading canopy; large leaves, good quality; yields about 40 tons/ha/yr; moderately resistant to all diseases

Tongxiang Qing

Indigenous

Widely distributed in Zhejiang Province

Diploid belongs to M. multicaulis; wider adaptability, spreading canopy; large leaves. Good quality; yields about 35 tons/ha/yr; moderately resistant to dwarf and brown spot diseases

Hong Cang sang

Selection from openpollinated hybrids of Tongxiang Qing

Widely distributed in Zhejiang and Jiangsu provinces

Diploid belongs to M. multicaulis. wider adaptability, Straight canopy; large leaves, good quality, yields about 34 tons/ha/yr; moderately resistant to dwarf and brown spot diseases but susceptible to bacterial wilt

Huo sang

Indigenous and natural triploid

Widely distributed in Zhejiang Province

Triploid belongs to M. mizuho.; canopy spreading; tender leaves red in color, large leaves, medium leaf quality; yields about 34 tons/ha/yr; moderately resistant to brown spot but susceptible to bacterial wilt, dwarf and powdery mildew diseases

Nong sang-8

Selection from natural population in Zhejiang Province

In river and hillside as medium bush in Zhejiang Province

Diploid belongs to M. alba L.; canopy straight; large leaves, good quality; yields about 45 tons/ha/yr; resistant to dwarf disease, brown spot and dirty leaf diseases; High rooting ability and propagated through cuttings

Xiao Guan sang

Native of Sichuan Province

Widely distributed in east and north of Sichuan Province on foothills

Diploid belongs to M. alba L.; straight canopy; ovate dark green leaves, medium leaf quality; yields about 18 tons/ha/yr, medium resistant to powdery mildew but susceptible to dwarf disease, bacterial disease and brown spot, Tolerant to drought and cold (continued)

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Table 3.7 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

Yu-2

Cross-breeding, Hu sang39  Guangdong Jing sang

Widely cultivated in Jiangsu and Zhejiang Province

Diploid belongs to M. alba L.; semierect tree; cordate light green leaves with luster surface, medium leaf quality; yields about 32 tons/ha/yr; resistant to dwarf disease, black wilt and bacterial diseases, tolerant to drought and cold and highly responsive to higher dose fertilizers

Guangdong Jing Sang

Native of pearl river delta of Guangdong province

Widely distributed in Guangdong province and South China

Diploid belongs to M. atropurpurea Roxb.; recommended for high-density plantations, trees erect; dull entire leaves, ovate or cordate, medium leaf quality; yields about 34 tons/ha/yr; strongly resistant to dwarf disease but sensitive to powdery mildew; tolerant to repeated pruning and high humidity but sensitive to drought and cold

Lun 109

Selection from natural population from pearl river delta of Guangdong Province

Widely distributed in Guangdong Province

Diploid, belongs to M. atropurpurea Roxb.; strong shoots with profuse side branches, grows fast; leaves cordate and thick, medium leaf quality; yields 40 tons/ha/yr; weak resistance to all diseases, medium tolerant to drought weak to cold

KangQing-10

Selection from natural population from west of Guangdong province

Widely distributed in Jiangsu and Zhejiang provinces

Diploid, belongs to M. atropurpurea Roxb.; early sprouting and early maturing type, spreading tree nature, branches strong and many; leaves slightly downwards, cordate and thick, medium leaf quality; yields about 37 tons/ha/yr, strong resistance to bacterial wilt, powdery mildew, susceptible to pest infestation, weak tolerance to cold, high rooting ability

Xuan 792

Cross-breeding, Husang39  Guangdong jing sang

Widely cultivated in Jiangsu and Zhejiang provinces

Diploid belongs to M. alba L.; semierect trees, many branches; leaves cordate, light green with good luster, medium leaf quality; yields about 32 tons/ha/yr; strong resistance to dwarf disease and black wilt bacterial diseases; susceptible to pest infestation; tolerant to drought and cold and highly responsive to higher dose fertilizers

Hei Lu Cai sang

Indigenous to Shandong province

Cultivated in yellow river area

Diploid belongs to M. multicaulis Perr.; straight trees, many thin branches; leaves ovate, dark green; medium quality; yields about 20 tons/ha/yr; moderately resistant to brown spot disease (continued)

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Table 3.7 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

Tuantou Heyebai

Natural selection from Heyebai

Widely cultivated in Yangtze river basin in China

Diploid belongs to M. multicaulis Perr.; spreading trees with drooping branches; leaves cordate, light green, medium luster, medium quality; yields about 36 tons/ha/yr; resistant to dwarf but susceptible to blight

Hei Yu sang

Natural triploid

Widely cultivated in South China

Diploid belongs to M. alba.; erect trees, slightly spreading, medium branches; leaves ovate, dark green, with good luster, good leaf quality; yields about 21 tons/ha/yr; medium resistance to dwarf and black wilt and resistant to powdery mildew and brown spot diseases; good rooting ability

Lun-40

Natural triploid

Widely distributed in South China

Triploid belongs to M. atropurpurea Roxb.; fast growing, slightly spreading, many branches; leaves cordate, thick, medium quality; yields 47 tons/ha/yr; weak resistance to bacterial blight and red rust diseases; suitable for dense plantation

Hey Tian Bai sang

Selection from natural populations from South Xinjiang

South Xinjiang

Triploid belongs to M. alba L.; trees spreading, medium strong branches; leaves ovate, dark green, slightly spreading, with dull luster, good leaf quality; yields about 18 tons/ha/yr; resistant to drought and cold

Yun Sang-2

Selection from natural populations at Yunnan province

Yunnan province

Diploid belongs to M. alba L.; tree slightly spreading many branches; leaves cordate, thick, medium quality; 27 tons/ha/yr

Sha-2

Selection from natural populations from Pearl river delta

Pearl river delta

Diploid belongs to M. atropurpurea Roxb.; slightly spreading canopy, strong branches many; leaves ovate or long cordate, thick, medium quality, yields 40 tons/ha/yr; weak resistance to bacterial wilt and strong resistance to thrips and mites

Hong Pi Wa sang

Native variety of Hubei

Hubei, China

Diploid belongs to M. multicaulis Perr.; slightly spreading trees with many branches; leaves cordate, light green, good luster, medium quality; yields about 18 tons/ha/yr; medium resistance to bacterial blight, powdery mildew and dirty leaf diseases

Da Ji Guan

Indigenous to Shandong

Shandong and Hebei provinces

Diploid belonging to the species of M. multicaulis Perr.; yields 20 tons/ha/yr, good leaf quality (continued)

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Table 3.7 (continued) Mulberry variety

Breeding method and parents used

Area of cultivation

Salient features

Hei Lu Cai Sang

Indigenous to Shandong, China

Shandong and Hebei provinces

Diploid belonging to the species of M. multicaulis Perr; yields 19.5 tons/ha/yr, good leaf quality

Xuan 792

Selected from the variants of native mulberry in Shandong

Shandong and Hebei provinces

Diploid belonging to the species of M. multicaulis Perr; yields 30 tons/ha/yr, good leaf quality

Niu Gen Sang

Native variety of Hebei

Shandong and Hebei provinces

Diploid belonging to the species of M. multicaulis Perr; yields 15 tons/ha/yr, medium leaf quality

Hei Ge Lu

Native variety of Shaanxi, middle of Yellow river

Shaanxi province

Diploid belongs to M. alba L.; yields 20.25 tons/ha/yr, medium leaf quality

Zhong Sang 5801

Selected from the hybridization of “Hu-sang 38  Guangdong Jingsang”

Shaanxi Zhejiang and Jiangsu provinces

Diploid belongs to M. atropurpurea Roxb.; yields 23.4 tons/ha/yr, medium quality leaves

Da Hua Sang

Indigenous to Sichuan

Anhui, Hubei Hunan, Sichuan Provinces

Natural triploid (2n = 3x = 42), belonging to the species M. alba L.; yields 18.7 tons/ha/yr, Good quality

Xiao Guan Sang

Indigenous to Sichuan

Anhui, Hubei Hunan, Sichuan Provinces

Diploid belonging to the species M. alba L.; yields 18.75 tons/ha/yr, medium quality

Jia Ling 16

A triploid bred by crossing “Xiqing” (tetraploid) with “Yu 2” (diploid)

Anhui, Hubei, Hunan, Sichuan Provinces

Early sprouting and middle maturity type; Highly productive, good leaf quality

Da 10

Natural triploid (2n = 3x = 42) Selected from Pearl River Delta of Guangdong

Guangdong and Guangxi Provinces

Triploid belonging to the species M. atropurpurea Roxb.; yields 15 tons/ha/yr

Dao Zhen Sang

A native variety selected from Guizhou

Yunnan and Guizhou Provinces

Diploid (2n = 2x = 28) belongs to M. alba L.; yields 22.5 tons/ha/yr, medium leaf quality

Tong 10  Lunjiao 109 Seed complex

Seed complex variety— Cross-breeding

Guangdong Province (Pearl river delta)

Diploid belongs to M. atropurpurea; High yield and easy multiplication and establishment; suitable for dense plantations, early sprouting

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Table 3.8 Authorized mulberry varieties of China Mulberry variety

Breeding method

Year

Recommended for

Yu 2

Cross-breeding

1989

The Changjiang River valley

Xuan 792

Selection from natural population

1989

The Huanghe River valley

Yu 151

Cross-breeding

1989

The Changjiang River valley

Lunjiao 40

Selection from local variety

1989

The Zhujiang River valley

Huangsang 14

Selected from local seedling mulberry

1989

The Changjiang River valley

Shi 11

Selection from local variety

1989

The Zhujiang River valley

Tang10  Lun 109

Hybrid mulberry seed

1989

The Zhujiang River valley

Jihu 4

Cross-breeding

1989

Northeast zone

Yu 237

Cross-breeding

1989

The Changjiang River valley

Xuanqiu 1

selected from local seedling mulberry

1989

Northeast zone

7307

selected from local seedling mulberry

1989

The Changjiang River valley

Huamingsang

Selected from local seedlings mulberry

1994

Chizhou Xuanzhou Anqing in Anhui Province and Linyi in Shandong Province

Wan 7707

Selected from local seedling mulberry

1994

Chizhou Xuanzhou Anqing in Anhui Province and Linyi in Shandong Province

Xiang 7920

Cross-breeding

1995

The Changjiang River valley

Shigu 116

Mutation breeding

1995

The Changjiang River valley, The middle and lower reaches of the Huanghe River

Yu 711

Cross-breeding

1995

The Changjiang River valley, The middle and lower reaches of the Huanghe River

Hongxin 5

Cross-breeding

1995

The Changjiang River valley, The middle and lower reaches of the Huanghe River

Xinyizhilan

Introduced variety

1995

The Changjiang River valley; The Hanghe River valley

Xinyiyuan

Mutation breeding

1995

The Changjiang River valley, The middle and lower reaches of the Huanghe River

Beisangyihao

Selected from local seedling

1995

The Changjiang River valley, mulberry middle and lower reaches of the Huanghe River

Dazhonghua

Plyploidy breeding

1996

The Changjiang River valley

Huangluxuan

Selection from local variety

1998

The Huanghe River valley

7946

Cross-breeding

1998

The Huanghe River valley

Jialing 16

Polyploid breeding

1998

The Changjiang River valley

Nongsang 8

Cross-breeding

2000

The Changjiang River valley

Nongsang 14

Cross-breeding

2000

The Changjiang River valley

Nongsang 12

Cross-breeding

2000

The Changjiang River valley

Xiansang 305

Mutation breeding

2001

The Huanghe River valley

Canzhuan 4

Selected from local seedling mulberry

2001

The Changjiang River valley (continued)

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Table 3.8 (continued) Mulberry variety

Breeding method

Year

Recommended for

Wangsangyou 1

Chemical mutagenesis (0.4% colchicine + 3% dimethyl sulfoxide mixture on 7707) Tetraploid

2014

Anhui, Hefei Province. Approved by Forest variety Evaluation Committee of Anhui in 2014

Sha-2  Lun 109

Seed complex variety- Cross-breeding

2014

Guangdong Province (Pearl river delta)

Yusang 5

Mutation and Cross-breeding, Tetraploid

2018

Mengzi Yunan Province

collected and evaluated at their natural growing areas through screening and selection for direct utilization in sericulture. By adopting this approach, breeders have identified superior varieties, viz. Heyebai (national check), Tuantauheyebai, Tongxiangqing, Lunjiao 40, Husang, which have been released for cultivation. Interspecific and intervarietal cross-breeding of genotypes of various regions resulted in the development of varieties, viz. Yu 2, Yu 117, Nonsang 14 and Nonsang 8, with further improvement in yield (30–80%) and quality (15– 30%). Mutation and polyploid breeding methods have also shown encouraging improvement of quality and disease resistance (Yang 1986). For example, R81-1, a tetraploid superior variety both in terms of quality and leaf productivity, was evolved by doubling the genome of Ichinose (polyploidization) using gamma irradiation (Yang 1986). The utilization of “Commercial seed complex varieties” was found to perform well at the farmer’s level, mainly in Guangdong province. Improved mulberry varieties like

Longsang 1 and Wangsangyou 1(Wang et al. 1993, 2016), Jialing 16 (Li et al. 1994), Xuan 792 (Song et al. 1995), Jiantol heyebaito (Chun 1996), Nongsang-10 (Dongfeng et al, 1997) Shengtong 1 (Ye et al. 2000), Suhu-16 (Shi et al. 2000), Shansang 402, Hangguo 2 (Su et al. 2006, 2012), Chuansang_98-1 (Liu et al. 2009), Jinsang 4 (Gao et al. 2012), Lucha-1 (Chen et al. 2012), Shushen 1 (Liu et al. 2014), Palchung (Gyoo et al. 2014), Yusang-3 (Fu et al. 2015), Chuansang 48-3 (Yang et al. 2016), Yuesang-11 (Tang et al. 2016), 8033, 9204, Jinsang 1. The salient features of major varieties of China are presented in Tables 3.7 and 3.8.

3.6.3 Major Mulberry Varieties Cultivated in Japan In Japan, most of the cultivated varieties developed from four species, viz. M. bombysis, M. alba, M. latifolia and M. acidosa (Machii et al. 1999a, b). Mulberry varieties, viz. Hinichinose,

Table 3.9 Major varieties cultivated in Japan and their salient features Mulberry variety

Breeding method and parents used

Salient features

Jumonji

Selection from natural population

Diploid belongs to M. alba L. Selection from natural population. Tree form procumbent; Leaves yellowish green

Akagi

Selection from natural population

Triploid belongs to M. bombycis Koidz.; tree slightly spreading with many long branches; leaves 0–2 lobed, green thick, medium coarse with good luster, medium quality; resistant to bacterial wilt, powdery mildew but susceptible to die back; suitable for young and late age silkworms during spring and winter; 10% rooting ability (continued)

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Table 3.9 (continued) Mulberry variety

Breeding method and parents used

Salient features

Ichinose

Selection from “Shirodashi” saplings of Nezumigaeshi and recommended in 1898

Diploid belongs to M. alba L.; suitable for both young and late age silkworms; tree slightly spreading with many branches; leaves 4 lobed, green thick and medium coarse; medium quality; medium resistance to bacterial wilt, powdery mildew and rust diseases and medium resistance to dwarf disease

Kairyo Nezumigaeshi (KNG)

Selection from “Shirodashi” saplings of Nezumigaeshi in 1907

Diploid belongs to M. alba L.; suitable for both young and late age silkworms; tree slightly spreading with many long branches; leaves 4 lobed, green thick, smooth and good luster; medium quality; resistant to bacterial wilt, powdery mildew; 30% rooting ability

Kenmochi

Selection from natural population during 1910

Triploid belongs to M. bombycis Koidz.; tree spreading with many long branches; leaves 2–4 lobed, dark green thick, medium coarse with poor luster; medium quality; resistant to bacterial wilt, powdery mildew, die back but susceptible to dwarf disease; suitable for young and late age silkworms; > 90% rooting ability

Roso

Selection from natural population

Diploid belongs to M. latifolia Poir; tree slightly spreading with many long branches; leaves entire, green thick, smooth with good luster; medium quality; resistant to bacterial wilt, powdery mildew but susceptible to die back; tolerant to drought; poor rooting ability

Kokuso-13

Selection and released in 1922

Diploid belongs to M. latifolia Poir; Tree is slightly spreading with many branches; leaves unlobed, green thick, smooth surface with good luster; medium leaf quality; resistant to bacterial wilt, powdery mildew and rust diseases and susceptible to dwarf disease; about 60% rooting ability

Kokuso-70

Selection and released in 1922

Diploid belongs to M. latifolia Poir; Tree is slightly spreading with many branches; leaves unlobed, green thick, smooth surface with good luster; medium leaf quality; resistant to bacterial wilt, powdery mildew and rust diseases and susceptible to dwarf disease; about 60% rooting ability

Kokuso-20

Cross-breeding Naganuma  Shiso and released in 1949

Diploid belongs to M. latifolia Poir; tree is erect type with medium number of branches; leaves entire, large, green thick, smooth surface with good luster; medium leaf quality; susceptible to dwarf disease hence not popularized

Kokuso-21

Cross-breeding, Naganuma  Shiso and released in 1949

Diploid belongs to M. latifolia Poir; Tree is erect type with medium number of branches; leaves unlobed, large, green thick, smooth surface with good luster; good leaf quality; resistant to bacterial wilt, medium resistant to rust, susceptible to dwarf disease and die back; poor rooting (20%) ability (continued)

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Table 3.9 (continued) Mulberry variety

Breeding method and parents used

Salient features

Kokuso-27

Cross-breeding, Naganuma  Kairyo Nezumigashi and released in 1949

Diploid belongs to M. alba L.; Tree is slightly spreading with medium number of branches; leaves 4 lobed, large, green, thick, smooth surface with good luster; medium leaf quality; resistant to bacterial wilt and powdery mildew and medium resistant to rust and dwarf diseases; very poor rooting ability

Kairyo Ichinose

Cross-Breeding Naganuma  Kairyo Nezumigashi and released in 1949

Diploid belongs to M. alba L.; Tree is spreading type with medium number of branches; leaves 4 lobed, large, green, thick, smooth surface with good luster; medium leaf quality; resistant to bacterial blight and powdery mildew and medium resistant to rust and susceptible to dwarf diseases; medium rooting ability

Kanmasar

Selection from natural population 1966

Cold-resistant variety popular in northern Japan; leaves 2–3 lobed, fast growth, responds well to fertilizers

Asayuki

Natural tetraploid 1966

High yielding variety cultivated in snow area (100 cm); resistant to die back and dwarf diseases

Fukayuki

Selection from natural population 1966

Under cultivation in northern Japan where snow fall ranges up to 150 cm; large, lobed leaves, dark green; excellent shoot growth in spring, high yield

Wasemidori

Selection from natural population 1966

Suitable for mild climatic areas of Japan; early sprouting and late hardening- suitable for young, late age silkworms

Atsubamidori

Selection from natural population 1966

Very high yielding in fertile fields with mild climate; branches resistant to lodging by wind, large and very thick leaves

Ichihei

Selection from natural population

Triploid belongs to M. bombycis Koidz.; tree slightly spreading with medium long branches; leaves 0–2 lobed, green thick, coarse with good luster; medium quality; resistant to rust, medium resistant to bacterial wilt and powdery mildew but susceptible to dwarf disease; about 40% rooting ability

Kairyoroso

Selection from natural population

Diploid belongs to M. latifolia Poir; tree spreading with many long branches; leaves entire, green thick, smooth surface with good luster; good quality; resistant to bacterial wilt, powdery mildew and rust and gall midge; 30% rooting ability

Kokuso-13

Selection

Diploid belongs to M. latifolia Poir; tree erect with medium number branches; leaves entire, green thick, smooth surface with good luster; good quality; resistant to bacterial wilt, powdery mildew and medium resistant to dwarf disease

Ichibei

Natural triploids

Distributed in northern part of Japan

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Table 3.10 Authorized mulberry varieties of Japan and their salient features Name

Norin number

Station and year

Salient features

Shin-ichinose

Norin-1

NISES, Tsukuba, 1970

Suitable to warm areas, best quality, resistant to bacterial blight

Yukishinogi

Norin-2

NISES, Ojiya, 1972

Suitable to medium snow areas, high yield, resistant to die back

Yukishirazu

Norin-3

NISES, Ojiya, 1976

Suitable to high snow areas, spreading type, high yield, resistant to die back

Minamisakari

Norin-4

NISES, Kyushu, 1976

Suitable to warm areas, moderate yield and quality, resistant to dwarf disease

Shinkenmochi

Norin-5

NISES, Tohoku, 1981

Suitable to cold areas, good rooter, high yield and quality, susceptible to dwarf disease

Tokiyutaka

Norin-6

NISES, Tsukuba, 1981

Suitable to temperate areas, low yield and moderate quality, resistant to dwarf disease

Hayatesakari

Norin-7

NISES, Kyushu, 1981

Suitable for both hot and cold areas, moderate yield and quality

Aobanezumi

Norin-8

NISES, Tohoku, 1982

Suitable for both warm and cold areas, high yield but low quality, susceptible to dwarf disease

Ooyutaka

Norin-9

NISES, Tsukuba, 1986

Suitable for warm areas, high yield and moderate quality, resistant to dwarf disease

Mitsushigeri

Norin-10

NAES, Tohoku 1988

Suitable for cold areas and dense planting, High yield and moderate quality, High rooting ability

Yukimasari

Norin-11

NAES, Ojiya 1989

Suitable for snow areas, high yield and high quality, High rooting ability, resistant to die back but susceptible to dwarf disease

Mitsuminami

Norin-12

NAES, Kyushu 1990

Suitable for hot areas and mechanical pruning, moderate yield and quality, resistant leaf blight

Tachimidori

Norin-13

NISES, Tsukuba 1990

Suitable for temperate areas, moderate yield, quality and rooting, resistant to dwarf disease

Yukiasahi

Norin-14

NAES, Ojia, 1991

Suitable for snow areas, high yield, quality and rooting ability, moderate resistant to dwarf disease

Hinosakari

Norin-15

NAES, Kyushu, 1992

Suitable for mechanical harvest, high rooting ability, moderate quality and resistant to diseases

Hachinose

Norin-16

NAES, Matsumoto, 1992

Suitable for cold areas, moderate yield and high quality, high rooting ability, resistant to powdery mildew

Waseyutaka

Norin-17

NISES, Matsumoto, 1994

Suitable for cold areas, high yield, moderate resistance to diseases

Mitsusakari

Norin-18

NAES, Kyushu, 1996

Suitable for warm areas, resistant to bacterial blight and powdery mildew, susceptible to dwarf disease

Senshin

Norin-19

NISES, Tsukuba, 1998

Suitable for warm areas, resistant to dwarf disease

Natsunobori

Norin-20

NISES, Tsukuba, 2000

Suitable for temperate areas, vigorous growth and high yield

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T. Mogili et al.

Yukishinogi, Minamisakari, Shinkenmochi, Hayatesakari, Aobanezumi, Mitsuminami and Senshin, were developed through hybridization based on a selective breeding approach. Shinichinose (F1 hybrids) was derived from the cross of Ichinose (M. alba)  Kokuso 21 (M. latifolia) and is considered as one of the best hybrid varieties (Machii et al. 1999a, b). Varieties of M. bombycis are cultivated in a cold region, whereas M. latifolia varieties are cultivated in warm places. The salient features of major varieties of Japan are presented in Tables 3.9 and 3.10.

3.6.4 Major Mulberry Varieties Cultivated in Thailand In Thailand, at the initial stage, low yielding local varieties were cultivated. Introductions of a few varieties, viz. Buriram (BR-60), Buriram

4/2 and Sakolnakhon, have replaced the local varieties. These varieties have a leaf yield potential of 18.75 to 25 tons/ha/year. While other varieties, viz. Noi and Khun Pai, have a leaf yield potential of 16 tons/ha/year, the salient features of major varieties of Thailand are presented in Table 3.11.

3.6.5 Major Mulberry Varieties Cultivated in Brazil In Brazil, all the cultivated mulberry varieties belong to M. alba. Commercial companies have disseminated the Miura, Korin clones and Calabrese varieties. Genetically modified clones (IZ and FM) are very productive and with more nutritious leaves (Fonseca et al. 1985a, b, c, 1986, 1987a, b, c; Almeida and Fonseca, 2002). The salient features of major varieties of Brazil are presented in Table 3.12.

Table 3.11 Major varieties cultivated in Thailand and their salient features Mulberry variety

Breeding method and parents used

Salient features

Noi

Native local variety

Diploid, erect plant; leaves ovate, large, 0–2 lobed and dark green; medium leaf yielder with moderate quality

Kun Pai

Native Thai variety

Diploid, erect plant; leaves ovate, large, 0–2 lobed and green in color; medium leaf yielder with moderate quality

Thai pecha

Natural triploids

Triploid, erect plant with many branches with fast growth; leaves ovate, medium size, entire and dark green, thick blade; medium leaf yielder with moderate quality

Thai beelad

Natural triploids

Triploid, erect plant; leaves ovate, small size, entire and green with thick leaf blade; medium leaf yielder with moderate quality

BR-10

Diploid, erect plant; leaves ovate, large, entire and green; medium leaf yielder with moderate quality

Buriram 60 (BR-60)

Cross-breeding (Luin Jio 44  Noi)

Diploid, erect plant; leaves ovate, large, 0–2 lobed and green; medium leaf yielder with moderate quality; moderately resistant to powdery mildew and thrips infestation

Sakonnakhon (SK)

Cross-breeding (Luin Jio 40  Khunphai)

Diploid

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Table 3.12 Major varieties cultivated in Brazil and their salient features Mulberry variety

Breeding method and Parents used

Salient features

Branca da Espanha (Spanish white)

Introduction from Spain

Bears both lobed and unlobed leaves with smooth and glossy texture; good adaptation; good yield; good rooting ability and propagated through cuttings

Calabresa

Introduction from Italy

Bears 5 lobed leaves with smooth and glossy texture; medium adaptation; good yield (5 t/ha/year); good rooting ability and propagated through cuttings

Catania-1

Introduction from Italy

Well-adapted, vigorous, productive, low rooting ability

Catania-2

Introduction from Italy

Bears un-lobed, deep green, glossy leaves; well-adapted with fast growth and high yield; poor rooting ability and propagated through grafts only

Catania paulista

Native of Limeira, Paulo state

Characteristics resemble to Catania varieties. Precocious, productive and vigorous, low rooting ability

Contadini

Introduction from Italy

Bears un-lobed, green, glossy leaves; well-adapted with fast growth and high yield; good rooting ability and propagated through cuttings

Formosa

Introduction from Thaiwan

Bears both lobed and un-lobed, ovate, green, glossy leaves; well-adapted with fast growth and high yield (8.6 t/ha/year); good rooting ability and propagated through cuttings

Fernao Dias

Selection from natural population from Sao Paulo state

Bears both lobed and un-lobed, ovate, green, glossy leaves; well-adapted with fast growth and high yield (8.6 t/ha/year); good rooting ability and propagated through cuttings

Florio

Italian origin

Not well-adapted; precocious but not productive; not well propagated through cuttings

Galiana

Native of Limeira, Paulo state

Medium, tardy, vigorous and rustic; not well-propagated through cuttings

Iamada

Originally from Promissao county, Sao Paolo State

It is precocious; but little productive; good propagation through cuttings

Kokuso-21

Introduction from Japan

Not well-adapted; Tardy, produces few branches that grow slowly; no propagation through cuttings

Kokuso-27

Introduction from Japan

Not well-adapted; Tardy, produces few branches that grow slowly; no propagation through cuttings

Lopes lins

Selection from natural population from Sao Paulo state

Bears both lobed and un-lobed, green, glossy leaves; welladapted with fast growth and high yield (8.6 t/ha/year); good rooting ability and propagated through cuttings

Miura

Selection from natural population from Batose county

Bears both lobed and un-lobed, green, glossy leaves; welladapted with fast growth and high yield; good rooting ability and propagated through cuttings

Moretiana

Introduction from Italy

Very good adaptation; productive and rustic; little tardy; no propagation through cuttings

Moscatela

Originated from Italy

Good adaptation; precocious; rustic and productive; good propagation through cuttings

Nezumigaeshi

Introduction from Japan

Good adaptation; vigorous, rustic and productive; No propagation through cuttings (continued)

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T. Mogili et al.

Table 3.12 (continued) Mulberry variety

Breeding method and Parents used

Salient features

Nostrana

From Limeira, Sao Paulo

It is rustic; vigorous, precocious; easy propagation through cuttings

Paduana

Native from Borborema, Sao Paulo

It is precocious; rustic and productive; good propagation through cuttings

Pendula

Originated from Rio de Janeiro state

It is precocious; rustic but little productive

Rosa

Originated from Italy

Good adaptation but low productivity; slow development; good propagation through cuttings

Rosa da Lombardia

Originated from Italy

Bad adaptation; medium, tardy; low productivity; slow development and precocious leaf maturation; poor rooting ability

Rosol

Originated from Registro county, Sao Paulo

It is precacious, rustic, vigorous and productive; Leaves wrinkled, coarse: Low adaptability and low feeding ability

Selvagem

Native of Campinas, Sao Paolo state

It is rustic, vigorous and precocious; low productive; excessive lobed leaves

Serra-das-Araras

Originated from Araras mountain range, Rio de Janeiro state

It is very precocious, rustic, low productive; very intense blossoming; easy propagation through cuttings

Siciliana

From Barbacena county, Minas Gerais State

It is precocious, rustic,; low productive; easily propagated through cuttings

Talo Roxo

From Compinas, Sao Popaolo

Precocious, low production; easy propagation through cuttings

Tiete

From Tiete county, Sao Paolo

Precocious, rustic, productive; leaves are coarse, less palatability to silkworms

Ungaresa

From Limeira, Sao Paolo

Precocious, rustic, low productive; good propagation through cuttings

Korin

From Fiacoes de Seda Bratac collection

Very vigorous and productive; good propagation through cuttings

Issaokina (IZ 3/2)

Cross-breeding Contadini  Catania Paulista

Bears both lobed and un-lobed, cordate, light green, glossy leaves; well-adapted with fast growth and high yield (8.6 t/ha/year); good rooting ability and propagated through cuttings

Capucho (IZ 5/2)

Cross-breeding Branca da Espanha  Catania

Bears both lobed and un-lobed, cordate, greenish, glossy leaves; well-adapted with fast growth and high yield (7.9 t/ha/year

Campinas (IZ 10/1)

Cross-breeding Lopes Lins  Catania Paulista

Bears un-lobed, cordate, light green, glossy leaves; welladapted with fast growth and high yield (8.0 t/ha/year

Luiz Paolieri (IZ 13/6)

Cross-breeding Fernao Dias  Kokuso-27

Bears both lobed and un-lobed, cordate, light green, glossy leaves; well-adapted with fast growth and high yield (11.8 t/ha/year

Rio da Pedras (IZ 15/7)

Cross-breeding Calabresa  Nezumigaeshi

Bears lobed and un-lobed, cordate, small, light green, glossy leaves; well-adapted with fast growth and medium yield (6.5 t/ha/year); flowers small and female

Rosa da Fonseca (IZ 19/13)

Cross-breeding Talo Roxo  Kokuso-27

Bears un-lobed, green, glossy leaves; well-adapted with fast growth and high yield (10 tons/ha/year) (continued)

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Mulberry Breeding for Higher Leaf Productivity

103

Table 3.12 (continued) Mulberry variety

Breeding method and Parents used

Salient features

Sempre Verde (IZ 29/1)

Cross-breeding Capinas  Nezumigaeshi

Bears heterophyllous, cordate, light green, glossy leaves; well-adapted with fast growth and high yield (8.2 tons/ha/year)

Tamarina (IZ 56/4)

Cross-breeding Formosa  Catania Paulista

Bears un-lobed, cordate, green, glossy leaves with more shrivel texture; well-adapted with fast growth and high yield (12 tons/ha/year). Flowers female and many

Javanesa (IZ 56/4)

Cross-breeding Formosa  Kokuso-27

Bears un-lobed leaves with truncate base, glossy and smooth texture; productive variety (9.35 tons/ha/year) with fast growth. Flowers female and medium size

3.7

Conclusion and Future Prospects

At present, conventional breeding approaches have shown significant achievements in developing superior mulberry varieties with higher leaf yield, better leaf quality, wider adaptability,

tolerance to cold/drought, soil alkalinity, salinity, resistance to diseases and pests. Nowadays, there is a paradigm shift in research focus on mulberry breeding from the development of high leaf yielding varieties for silkworm rearing, its multipurpose exploitation for pharmaceutical, cosmetic, food and beverage industries and its positive impact on environmental safety approach

MULBERRY Breeding Approaches to Improve producvity, Quality & Adaptability

Seri-composng & Byproduct ulizaon

Culvated Mulberry

Silkworm Rearing

Intercropping – Nitrogen enrichment

By product Ulizaon

Cocoon Producon

Poultry, cale feed, cosmecs, Amino acids, pharmaceucals, pupal oil etc,

Improved Producvity Employment Generaon Boost to Rural Economy

Animal husbandry - Fodder

Agro-forestry

Environmental Conservaon

Fodder, tea, fuel, jam, mber, sport goods, paper, wine, vitamins, carotene, amino acids, chlorophyll, alkaloids etc.

Conservaon of soil,, water & degraded lands

Sustainable Sericulture

Fig. 3.4 Schematic model for sustainable mulberry sericulture through different organic linkages for upliftment of rural economy

104

(Bhattacharya et al. 2019; Rohela et al. 2020; Centhyea et al. 2021). It is appropriate to call it the most suitable plant for sustainable development (Fig. 3.4). Hence, future strategies toward mulberry improvement may be decided depending on the future needs. In respect of mulberry leaf productivity and quality, to sustain productivity under climate change scenario, it is essential to bring greater diversity into the gene pool, introduction and utilization of un-adapted and productive exotic and wild species. The following points may be considered for future crop improvement programs for higher leaf productivity coupled with quality and adaptability: (1) mulberry varieties with improved nutrient uptake efficiency, higher biomass production, insensitive to micro-climatic conditions, resistance to pest and diseases, (2) development of mulberry varieties productive and resistant to root rot, root-knot and mildew diseases, (3) mulberry varieties with a higher yield, quality and adaptability to support highly productive silkworm races, (4) development of mulberry varieties resistant to mulberry pest such as thrips, mites, whitefly and sucking pest, (5) development of mulberry variety for temperate areas by utilizing accessions including exotic ones with good rooting and low dormancy through hybridization and selection, (6) development of quicker, a robust evaluation system for rapid outreach of new mulberry varieties to farmers. Further, integrated approaches utilizing advanced genomic tools including transgenesis and marker-assisted selection in tandem with conventional breeding methods could be necessary toward mulberry genetic improvement programs.

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T. Mogili et al. of the 20th Congress of the International commission held at Bangalore, India, 15–18th Dec, pp 10–17 Tikader A, Vijayan K, Raghunath MK, Chakraborti SP, Roy BN, Pavankumar T (1995) Studies on sexual variation in mulberry (Morus spp.). Euphytica 84:115–120 Tikader A, Rao AA, Ravindran S, Naik VG, Mukherjee P, Thangavelu K (1999a) Divergence analysis in different mulberry species. Indian J Genet 59:87–93 Tikader A, Vijayan K, Chakroborti SP, Roy BN (1999b) Sexual behaviour in mulberry germplasm (Morus spp.). Bull Ind Acad Seric 3:33–37 Tikader A, Rao AA, Mukherjee P (2000) Pollen morphology studies in mulberry (Morus spp.). Indian J Seric 39:160–162 Tikader A, Chandrasekhar M, Borpuzari MM, Saraswat RP, Ananda Rao A, Sekar S (2006) Catalogue on mulberry (Morus spp.) germplasm-Volume III and IV. Central Sericultural Germplasm Research station, Hosur, Tamilnadu Tojyo I (1954) Tetraploid mulberry tree induced by the colchicines method. J Seric Sci Jpn 23:278 Tojyo I (1963) Autotetraploid induced by colchicine treatments in mulberry seedling. J Seric Sci Jpn 32:34 Tojyo I (1966) Studies on the polyploidy in mulberry tree I. breeding of artificial autotetraploids. Bull Sericult Expt Sta Jpn 20:187 Tojyo I (1985) Research of polyploidy and its application in Morus. JARQ 18:222–227 Tojyo I, Watanabe Y, Hayasaka S (1986) Breeding of new triploid mulberry cultivars Shinkenmochi and Aobanezumi. Bull Tohoku Natl Agric Expt StnYatabe 30:151–250 Tojyo I, Watanabe Y, Hayasaka S, Ohwada K (1992) A new triploid mulberry, Morus alba variety, Mitsugeri. Bull Tohoku Natl Agric Expt Stn 34:1–27 Udall JA, Wendel JF (2006) Polyploidy and crop improvement. Crop Sci 46:S3–S14 Urs MKP, Rajashekar K, Sarkar A (2011) Evaluation of mulberry (Morus spp.) genotypes for tolerance to major abiotic stresses. J Ornam Horticult Plants 1 (3):167–173 Venkatesh KJ (2015) Studies on micromorphology and karyotype analysis of three mulberry genotypes (Morus spp.). AJPCT 3(2):192–198 Verma RC, Sarkar A, Sarkar S (1986) Induced amphiploids in mulberry. Curr Sci 55:1203 Vijayan K (2009) Approaches for enhancing salt tolerance in mulberry (Morus L)—a review. Plant Omics 2 (1):41–59 Vijayan K, Gnanesh BN (2022) Genomic research in mulberry for higher silk productivity. In: Seritech, The new concepts in sericulture, The 26th international sericultural commission congress. 7–11th September 2022, Cluj-Napoca, Romania, pp 49–74 Vijayan K, Tikader A, Roy BN, Qadri SMH, Pavankumar T (1997a) Studies on stigma receptivity in mulberry (Morus spp.). Sericologia 37:343–346

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Vijayan K, Chauhan S, Das NK, Chakraborti SP, Roy BN (1997b) Leaf yield component combining ability in mulberry (Morus spp.). Euphytica 98:47–52 Vijayan K, Chakraborti SK, Doss SG, Tikadar A, Roy BN (1998) Evaluation of triploid mulberry genotypes, morphological and anatomical studies. Indian J Seric 37:64–67 Vijayan K, Das KK, Doss SG, Chakraborti SP, Roy BN (1999a) Genetic divergence in indigenous mulberry (Morus spp.) genotypes. Ind J Agric Sci 69:851–853 Vijayan K, Sahu PK, Chakrabortti SP, Roy BN (1999b) Evaluation of tetraploids for triploid breeding in mulberry. Indian J Genet 59(4):515–522 Vijayan K, Chakraborti SP, Ghosh PD (2003) In vitro screening of mulberry (Morus L) for salinity tolerance. Plant Cell Rep 22:350–357 Vijayan K, Chakraborti SP, Ghosh PD (2004) Screening of mulberry (Morus spp.) for salinity tolerance through in vitro seed germination. Indian J of Biotechnol 3:47–51 Vijayan K, Chakraborti SP, Ercisli S, Ghosh PD (2008) NaCl induced morpho-biochemical and anatomical changes in mulberry (Morus spp.). Plant Growth Regul 56:61–69 Vijayan K, Doss SG, Chakraborti SP, Ghosh PD (2009) Breeding for salinity resistance in mulberry (Morus spp.). Euphytica 169(3):403–411 Vijayan K, Srivastava PP, Jayarama Raju P, Saratchandra B (2012) Breeding for higher productivity in mulberry. Czech J Genet Plant Breed 48(4):147–156 Vijayan K, Jayaramaraju P, Singhvi NR, Ravikumar G (2017) Genetic improvement of mulberry in India: challenges and prospects. Sericologia 57(4):185–197 Vijayan K, Reddy RA, Mishra RK (2019) Contribution of breeding and biotechnology to crop improvement in mulberry (Morus spp.). In: VI Asian Pacific congress of sericulture and insect biotechnology, 2–4 March Mysore, India, pp 25–32 Vijayan K, Gnanesh BN, Shabnam AA, Sangannavar PA, Sarkar T, Zhao W (2022a) Genomic designing for abiotic stress resistance in mulberry (Morus spp.) In: Genomic designing for abiotic stress resistant technical crops. Springer Nature. https://doi.org/10.1007/ 978-3-031-05706-9_7 Vijayan K, Arunakumar GS, Gnanesh BN, Sangannavar PA, Ramesha A, Zhao W (2022b) Genomic designing for biotic stress resistance in mulberry (Morus spp.) In: Genomic designing for biotic stress resistant technical crops. Springer Nature. https://doi. org/10.1007/978-3-031-09293-0_8 Waktole S, Wosene G (2016) Evaluation of mulberry (Morus spp.) genotypes for growth, leaf yield and quality traits under Southwest Ethiopian condition. J Agron 15:173–178 Wang JE, Yang DM, Yang YI (1993) Breeding of coldresistant mulberry variety “Longsang 1.” Canye Kexue 19(1):1–5 Wang JK, Liu ZY, Wang Y, Jiang W, Feng YG, Wang DF (2009) Identification trials on mulberry

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Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective Raju Mondal, Gulab Khan Rohela, Prosanta Saha, Prashanth A. Sangannavar, and Belaghihalli N. Gnanesh

4.1

Introduction

Mulberry is perennial, fast-growing, deciduous woody plant that belongs to the order Rosales, family Moraceae and genus Morus (Zeng et al. 2015); it grows well in tropical, sub-tropical, and temperate conditions. About 24 species and one sub-species of this plant are identified (Yuan and Zhao 2017). Because of adequate environmental adaptability, tree mulberry plants are being cultivated in different parts of the world for silkworm rearing. It is also reported that the nutrition of mulberry leaves greatly influences growth and

R. Mondal (&) Mulberry Tissue Culture Lab, Central Sericultural Germplasm Resources Centre (CSGRC), Hosur 635109, India e-mail: [email protected] G. K. Rohela Biotechnology Section, Central Sericultural Research and Training Institute, Pampore, Jammu & Kashmir 192121, India P. Saha Department of Botany, Durgapur Government College, Durgapur, West Bengal 713214, India P. A. Sangannavar RCS, Central Office, Central Silk Board, Bengaluru, Karnataka 560068, India B. N. Gnanesh Molecular Biology Laboratory-1, Central Sericultural Research & Training Institute, Mysore 570008, India

development of silkworm larva, and subsequent cocoon quality (Sudhakar et al. 2021). Now-adays, mulberry leaves are also used as a raw material for some products including mulberry herbal tea, wine, and beverage (Tchabo et al. 2017). Additionally, the leaves are rich in alkaloids, flavonoids steroids, tannins terpenoids, and saponins (Hussain et al. 2017). Because of abundance of a range of pharmacological compositions, mulberry plant parts are used in Indian and Chinese medicines (Yang et al. 2010). Recent frontier researchers also illustrated mulberry trees as rich in bioactive compounds which result in relieving many potential diseases (Yang et al. 2010; Mondal et al. 2022). The genus Morus comprised of several species, among which viz., M. alba, M. nigra, M. rubra, M. indica. M. tartarica, M. papyrifera, and M. tinctoria (Venkatesh 2021) have gained much attention. Though, a number of 217 plant name (as accepted species and/or synonym and/or unresolved) were recorded in the repository database which was discovered by various workers across the world (http://www. theplantlist.org). Hence, taxonomic nomenclature has not been resolved to date. Besides that, this diverse Morus spp. comprised chromosome numbers vary from 2n = 28 to 22n = 308 (diploid to decosoploid) with ploidy level x to 22x, and based on meiotic behaviors, basic chromosome numbers of the genus have been recognized as x = 14 (Venkatesh 2021). Therefore, complex taxonomic identity and divers

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_4

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genome duplication hinder the improvement of the mulberry crop improvement. Besides that, conventional breeding is also hindered because of the nature of its heterozygosity, crosspollination behavior and lack of mutant lines. In the last two decades, traditional breeding methods contributed immensely to developing promising/superior genotypes for commercial exploitation on the basis of cytogenetical data and important functional traits (especially related to stress, and yield) (Banerjee et al. 2008; Sarkar et al. 2017, 2021). In mulberry, the use of genomic methods like genetic engineering (cloning, and overexpression) and marker-assisted selection for the generation of climate smart varieties as well as improved productivity that led to expansion for first-rate silk production was also illustrated (Vijayan et al. 2006, 2009, 2014, 2022a; Gulyani and Khurana 2011; Sarkar et al. 2017). Recently nuclear genome of M. notabilis (He et al. 2013), M. alba (Jiao et al. 2020), and M. indica (Jain et al. 2022) were also sequenced. The recent consequence of climate change affecting the global productivity of mulberry (Sarkar et al. 2021). Hence, development of climate smart mulberry is a prerequisite for future sustainability and current climatic condition insisting to explore the publicly available potential genomic resources for the improvement of mulberry production by implementation of genomic and frontier molecular biology tools. The enormous pool of genes in Morus spp. can be a significant resource for future crop improvement. In the present discussion, we have focused on the current status, challenges, and outlook of mulberry genome analysis through the implementation of integrated omics study, advanced breeding program, genetic engineering/biotechnological techniques and bioinformatics for mulberry genetic improvement toward stress tolerance.

4.2

Current Status and Challenges

The genetics of mulberry still remains challenging as its nature of immense heterozygosity as well as the long period to develop a variety of about 15–20 years (Vijayan et al. 2012). A long

juvenile period (2–3 years) is the major setback for conventional breeding in mulberry. Further, high heterozygosity and lack of inbred lines related to the accessions make the genetic improvement through conventional breeding highly laborious and time consuming. Evaluation of germplasm and categorizing accessions based on diversity that exists among them is required for selecting suitable parents for breeding. Previously, in absence of molecular markers, germplasm was evaluated using phenotypic and/or functional traits such as morpho-physiological traits (early sprouting, winter hardiness) and agronomical traits like tolerance to abiotic and biotic stress such as drought, saline, alkaline, and resistance to pests/diseases (Arunakumar et al. 2021; Gnanesh et al. 2021; 2022; Tikader and Kamble 2007; Vijayan et al. 2022a, b). The expression of most phenotypic traits is highly influenced by environmental factors and stage of development; the genetic base of all the characteristics of mulberry could not be clarified. Though the use of different molecular markers as well as biochemical markers such as isozymes was also implemented in mulberry to explain the genetic diversity among species (Ananda Rao et al. 2011). Plant tissue culture is the core of biotechnology-based breeding methods and helps mulberry plants in conservation and micro propagation pathways (Sarkar et al. 2018). It also adds to the improvement of the phytochemical profile of mulberry plants in direct and indirect ways. Somaclonal variation has an enormous advantage in mulberry (Sarkar et al. 2018); it enables to generation of desired traits. Additionally, developments of haploid and double-haploid (DH) lines have a greater advantage for genetic analysis of important traits, gene discovery, and its utilization in cross-breeding approaches. Hence, the development of somaclones, haploid, and doublehaploid (DH) lines is the possible mulberry crop improvement strategies in near future. Additionally, the contribution of polypoidy (whole genome duplication) and alternative splicing toward genetic adaptability was not studied in mulberry. Hence, isoforms involved in tree speciation should be recognized. Another important aspect of cross-kingdom interaction (interaction with

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Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective

microbes) and their impact on mulberry productivity should be undertaken to enhance productivity. Genomics is providing information on the identity, site, effect, and function of genes affecting traits—understanding that will increasingly drive the use of biotechnology in mulberry. Genomics sets the base for post-genomics actions, such as proteomics and metabolomics to produce information on gene and protein structure, as well as their interactions and functions. These will help to understand systematically the molecular biology of mulberry for their practical utilization. Genomics and proteomics offer a major help for rapid recognition of genes and pathways that are linked with agronomically and economically important traits. Molecular markers, on the other hand, are existed abundantly, stable across the growing conditions and developmental stages, and are free from pleiotropic and epistatic effects. Several researchers used DNA markers such as random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), amplified fragment length polymorphism (AFLP), and simple sequence repeats (SSR); single nucleotide polymorphism (SNP) used for assessing the genetic diversity/uniformity among the mulberry accessions/genotypes (Arunakumar et al. 2021; Gnanesh et al. 2023; Muhonja et al. 2020; Rohela et al. 2018, 2020; Shinde et al. 2021; Vijayan et al. 2014). Plant genome sequencing has several impacts such as(a) Whole genome sequencing (WGS) serves as an anchor genome for closely related species and speeds up gene discovery. For example, genomic information on corn, wheat, and rice helps to complement the understanding of phylogenetic relationships, evolutionary consequences, adaptability, etc. (Messing and Llaca 1998). (b) The complete genome sequence provides the foundations for a comprehensive understanding of comparative as well as specific gene functions (Kaul et al. 2000). (c) Receiving near-one-plant sequence information can also be useful for future studies. For

(d)

(e)

(f)

(g)

(h)

4.3

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example, Arabidopsis and rice are distant angiosperm taxa and serve as model systems in dicot and monocot respectively and provide platforms for the comparative study (Liu et al. 2001; Rensink and Buell 2004). WGS provides a platform for understanding genes and genetic variation associated with important complex traits. Additionally, the intervention of science especially genetic engineering enables manipulation of gene expression as per requirement. The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce desirable changes in the genome. Sequencing technology can give a better platform to a molecular taxon. For example, genotype/accessions can be tagged with DNA bar-coding. Moreover, the SNP markers can be the link point of NGS and genome-wide association studies (GWAS). The integration of high-throughput genome sequencing technology with aid of omics technologies (Fig. 4.1) can illustrate the genetic architecture of any complex metabolic pathway.

Comparative Study Between M. Notabilis, M. Alba, and M. Indica Genome

4.3.1 Morus Notabilis Among the various species of mulberry, genome sequencing was first reported successfully in M. notabilis by employing the whole genome shotgun sequencing method (He et al. 2013). M. notabilis is a wild species of mulberry that is mostly found in the high altitude ranges (1200– 2800 m) of Southwestern parts of China (Chen et al. 2014). In M. notabilis, the monoploid

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FISH, NGS, CRISPR, TALENs, ZFN

Genomics

Metabolomics

GC–MS, HPLC Fig. 4.1 Integrated omic approaches for mulberry improvement. The application of phenomics, genomics, transcriptomics, proteomics, metabolomics, and bioinformatics analysis will reveal performance, mutational landscape, gene expression profile, abundance of proteins,

the genetic architecture of metabolic pathways, and evolutionary relationship of mulberry, respectively. The integrated omic approaches will be solved fundamental queries regarding complex traits viz. yield, tolerance to stress, etc.

chromosome number was reported as 7, and the total number of chromosomes as 14 (He et al. 2013). The genome size of M. notabilis was reported as 330 Mb with approximately 29,000 genes; out of the whole genome size (*330 Mb), nearly one-third of it (*128 Mb) was found as repetitive sequences (He et al. 2013). As per the comparative genomic analysis reported by He et al. (2013), the gene sequences of mulberry have evolved three times faster than other sequenced plant species of Order-Rosales, and this faster evolution of genes in mulberry was predicted as a piece of molecular evidence that has facilitated wide spreading and survival nature of Morus spp. under varied climatic conditions

across the world. Hence, mulberry is widely spread from its native range of Asia and adapted well in other continents of Africa, USA, Europe, and Australia. The genomic sequencing of M. notabilis was carried by sequence assembling approach by using contig N50 length of 34,476 bp and scaffold N50 length of 390,115 bp, and out of all the scaffolds used, the largest scaffold comprised of 3,477,367 bp (He et al. 2013). Studies suggest that the total genome size of 330 Mb by sequence assembling approach, the length of gaps was found as 16,281,000 bp (4.9%), and the length of non-gapped continuous sequence was found as 314,510,000 bp (95.1%). The total GC

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Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective

content of M. notabilis was found to be 35.02% which is similar to other sequenced eudicots (He et al. 2013). Out of the whole genome size of 330 Mb, the repetitive sequences of 127.98 Mb were found from the assembled region, and the repetitive sequences of 18.48 Mb were estimated from the unassembled region (Table 4.1). Hence, it was estimated that *47% of genome size is comprised of repetitive sequences, and out of this, > 50% of repetitive sequences are of Copia like and Gypsy like long terminal retrotransposons type of transposable elements (He et al. 2013). By using RNA-seq data and expressed sequenced tags (ESTs)-based approaches, *29,338 genes were predicted out of which 27,085 genes were predicted as protein encoding genes and 2253 genes as partial genes (He et al. 2013). These results were further supported by de novo gene prediction (99.93%), homology-based gene prediction (69.94%), and RNA-seq/EST (58.38%)-based gene prediction approaches (He et al. 2013). Further, when all the 29,338 genes were annotated by using RNA-seq data and ESTsbased approaches, the average length of an mRNA was predicted as 2849 bp, an average coding region as 1156 bp and an average of 4.6 exons per gene (He et al. 2013). By using RNAseq data; 213,241, 285, 360, and 404 genes were found as tissue specific expressible genes in the tissues of bark, root, winter bud, male flower, and leaf, respectively (He et al. 2013). Further 1805 genes were identified as housekeeping genes which get expressed continuously in all the above five tissues of Morus; these 1086 genes include 116 genes encoding for ribosomal proteins and 26 genes encoding initiation factors of the translation process (He et al. 2013). Krishnan et al. (2014a) have developed the first online database ‘MulSatDB’ for mulberry microsatellites and identified 217,312 microsatellites or simple sequence repeats (SSR) from the whole genome sequence and 961 numbers of SSR from the EST sequences of M. notabilis. Krishnan et al. (2014a) have used identification tool of microsatellite (MISA) and searched for simple sequence repeats of hexa (with a minimum of 5 repeats), penta (5 repeats), tetra (5 repeats), tri (6

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repeats), di (10 repeats), and mono (10 repeats) with an interrupting gap sequence of 100 bp between the repeats. Among the identified microsatellites; 245 were hexanucleotide repeats, 741-pentanucleotide repeats, 2521-tetranucleotide repeats, 7485-trinucleotide repeats, 21,453dinucleotide repeats, and 151,152 are identified as mononucleotide repeats (Table 4.1). Also identified the average distribution length as 1522 bp per single SSR in the genomic sequence of Morus notabilis (Krishnan et al. 2014a). The developed site of MulSatDB (http://btismysore. in/mulsatdb) is freely accessible for browsing the SSR markers of mulberry which will be useful for designing the primers for screening the mulberry varieties with desired traits from a large number of mulberry accessions.

4.3.2 Morus Alba The whole genome size, chromosome number, and ploidy level of M. alba were recently reported by Jiao et al. (2020). The genome size of M. alba was elucidated as 346.39 Mb by using Oxford Nanopore, Illumina HiSeq, and highthroughput chromosome conformation capture (Hi-C) data-based molecular techniques (Jiao et al. 2020). From the whole genome of 346 Mb, the repetitive sequences were in the length of 180.11 Mb (Table 4.1), which is almost 535 of the whole genome (Jiao et al. 2020). By using ab initio and homology-based gene prediction methods, the total number of protein encoding genes in M. alba is identified as 22.767 genes with an average gene length of 3209 bp, coding length of 1148 bp with an average number of 5.09 exons per gene (Jiao et al. 2020). It was also predicted that as per phylogenomic analysis, the M. alba diverged from the M. notabilis approximately 10 million years ago (Jiao et al. 2020). There are also few reports about the identification of microsatellites, transcriptomic studies by carrying the sequencing of expressed sequence tags (EST’s), and sequencing of transcripts under different abiotic stress conditions (Vijayan et al 2022a). A total of 21,229 expressed genes were sequenced in M. alba by Dai et al. (2015).

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Dhanyalakshmi et al. (2016) generated 10,191 ESTs from leaf tissue under drought conditions, among which 5319 ESTs were used for annotation. Liu et al. (2017) have identified the 1,01,589 salinity stress responsive genes from the leaf, stem, and root region-based 180,694 transcripts with a total length of 72,058,872 bp and mean length of 709 bp under the salt stress conditions. Shang et al. (2017) have carried the transcriptomics work on floral development in mulberry and identified a total of 87,719 genes which get expressed during the development of flower buds at six different stages. Mathithumilan et al. (2013) analyzed 2391 EST sequences and 1094 genomic sequences (a totally 3485 sequences) for the presence of microsatellite regions from the ‘Dudhia white’ genotype of M. alba. After analysis, a total of 900 microsatellites were identified from the genomic sequences (Mathithumilan et al. 2013). Among the identified simple sequence repeats, 303 are dinucleotide repeats-DNR (33.67%), 167 are mononucleotideMNR repeats (18.56%), 155 are trinucleotide repeats-TNR (17.22%), and remaining repeats were identified as tetra, penta, hexa, and long nucleotide repeats in less frequency (Mathithumilan et al. 2013). Among the different types of DNR identified in M. alba, ‘TC’ type of dinucleotide repeats was most predominant (25.5%) followed by ‘CT,’ while in most of the plant species, ‘AT,’ ‘GA,’ and ‘AG’ were reported as the most frequent dinucleotide repeats (Lagercrantz et al. 1993; Edwards et al. 1996; He et al. 2003). In M. alba, ‘CG’ and ‘CA’ dinucleotide repeats were identified as the least abundant type (Mathithumilan et al. 2013). Among the trinucleotide repeats of microsatellites, ‘GAA’ was most abundant (15.9%); similarly, the tetranucleotide repeats of ‘AAAT,’ pentanucleotide repeats of ‘AAAAC,’ and hexanucleotide repeats of ‘AAAAAG’ were found as most abundant in M. alba (Mathithumilan et al. 2013).

4.3.3 Morus Indica Recently, a draft genome sequence of M. indica was reported by Jain et al. (2022) using

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cultivated Indian mulberry cv Kanva-2. With gene completeness of 96.5%, a set of highquality genome sequences was obtained by employing four distinct technologies, including Illumina (short-read), single-molecule real-time sequencing (long-read PacBio), chromosomal conformation capture (Hi-C), and optical mapping (Bionano). The draft de novo genome assembly with 365 scaffolds and a total size (bp) of 505,298,450 was illustrated (Table 4.1). Genome sequence analysis has revealed the presence of repetitive DNA (49.2%), proteincoding genes (27,435), genes with more than one transcript (1546), multi-exonic transcripts (23, 792, 86.72%), and > 90% genes are confirmed based on transcript evidence (Jain et al. 2022). In comparison, the repetitive DNA content (49.2%) of M. indica is in between the values of M. notabilis (38.8%) and M. alba (52.9%). As per the genome analysis, slightly higher number of genes was predicted in M. indica (27,435) as compared to M. notabilis (27,085) and M. alba (22,767). The comparative whole genome alignment has revealed high levels of similarity (> 50%) with M. alba (Jain et al. 2022). Study also reported the phylogenetic relationships, divergence time (*3.37 mya), GO assignment (13, 163), transcription factor (2036), and species-specific gene *4.8% of M. indica. At sets of 8293, 15,586, and 10,343 genes were identified which involved in the biological process, molecular function, and cellular process (GO terms), respectively. Interestingly, several GO terms were reported to be unique to M. indica, including response to hormone (abscisic acid and jasmonic acid) stimulation, secondary metabolic process, post-translational protein modification, phosphate metabolic process, peptide transport, and cell wall organization/ modification. Using seven different vegetative and reproductive tissues/organs (leaf, root, stem, winter bud, male inflorescence, female inflorescence, and fruit), transcriptomics analysis of numerous accessions was performed (Jain et al. 2022). In addition, 21 accessions were used for genome resequencing, which in turn revealed *2.5 million SNPs and *0.2 million insertions/deletions. Jain et al. (2022) reported

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Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective

the GC content in M. indica was 31.2% which is slightly lower compared to genome of the other two reported species (M. alba-33.2% and M. notabilis-33.1%), and 10.3% bases accounting to Ns (Table 4.1). Most of the portions in the M. indica genome had a GC content of 30–40%, which was comparable to that of Arabidopsis thaliana and Fragaria vesca genomes (Table 4.1). These findings and generated MindGP database (http://tgsbl.jnu.ac.in/MindGP) provide a complete resource for accelerating mulberry genomes research in order to better the species. Based on sequence similarity of 311 single copy genes, the evolutionary relationship of M. indica was compared with other sequenced plants (Cannabis sativa, Fragaria vesca, Malus domestica, M. notabilis, Prunuspersica, Pyrusbretschneideri) genomes (Jain et al. 2022). The phylogenetic analysis revealed M. indica as the closest relative of M. notabilis (Family: Rosaceae) and C. sativa (Family: Cannabaceae) based on the togetherness clustering under the constructed phylogenetic tree (Jain et al. 2022).

4.4

Chloroplast Genome

4.4.1 Importance of Study of Chloroplast Genome in Plant Biotechnology Information about chloroplast genome is an essential prerequisite for targeting varietal improvement by chloroplast genetic engineering approaches. Such approaches can prove to be very useful for tree species like Morus, where varietal improvement by conventional breeding approaches becomes highly challenging due to the perennial habit of the plant. Moreover, chloroplast genetic engineering can directly and specifically target alteration in leaf characteristics without directly affecting other traits of the plant. This is pertinent in the case of mulberry, as here only the leaf is of commercial value for the silk industry. Hence, chloroplast engineering approaches targeting an increase in moisture retention of the leaves and their nutritional

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improvement can have a boosting effect on the production of silk. Chloroplasts serve as biological factories, continuously synthesizing food for the plant and thereby sustaining life on this planet. The green tissues of a healthy plant contain multiple copies of chloroplast (  20) per cell which are inherited maternally. The chloroplast genome (cpDNA) is haploid in nature and has a prokaryotic architecture. The genes of the cpDNA are often organized into operons, and hence, multiple genes are driven by a single promoter. As the chloroplast genome is maternally inherited and there is a lack of genetic recombination, so conservation of its gene content and gene order is ensured. Thus, the study of the chloroplast genome is very important from the perspective of comparative phylogenetics among species and developing species-specific DNA barcode (Ravi et al. 2008). Alongside that, the study of cpDNA and chloroplast genetic engineering has gained prominence over the past decade due to several advantages of the process, such as: 1. The organization of genes of the cpDNA into operons ensures formation of polycistronic mRNA. Thus, multigene engineering can be achieved by chloroplast transformation. 2. As plant cell contains multiple copies of chloroplast, high level of amplification of the introduced foreign gene is possible. 3. Maternal inheritance of the chloroplast genome restricts the presence of genetically engineered cpDNA in the pollen and hence reduces the risk of transforming non-target species by pollen flow, ensuring an environment-friendly way of genetic engineering. 4. As the introgression of foreign DNA into the chloroplast is through homologous recombination, problems related to gene silencing and proper excision of selectable marker genes are obliterated. The above advantages have resulted in the utility of chloroplast transformation in diverse biotechnological applications from phytoremediation to molecular farming for antibiotic production, from biofuels to vaccines and industrial enzyme production, from biopharmaceuticals to

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agronomic-trait improvement across varied plant species like tobacco, Arabidopsis, carrot, potato, cotton, etc. (Adem et al. 2017). Such diverse application of chloroplast transformation necessitates extensive knowledge of the chloroplast genome of the target plant species. Knowledge about the chloroplast genome of the Morus species and quality improvement of its leaves become highly important as the leaves of these plants serve as the staple diet for the silkworm that runs the million dollar silk industries across the world.

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tRNA content of M. alba as reported by Luo et al. (2019) and He et al. (2020) was contradictory, which might be attributed due to their difference in location of sample collection. Chloroplast genome-based comparative phylogenetics revealed a close association of M. alba with M. cathayana, and M. notabilis with M. indica. The assemblages were connected with M. mongolica. The family Moraceae was in turn associated with the family Ulmaceae (Luo et al. 2019).

4.5 4.4.2 Studies on the Chloroplast Genome of Morus Species Mulberry (Morus sp.) belongs to the family Moraceae which consists of more than a thousand species including enormous trees like banyan and fig. The genus consists of about ten species, which grow across Asia, Africa, Europe, and the USA. Among these, to date, chloroplast genome sequencing has been achieved in eight species, the details of which are provided in Table 4.2. As a general pattern of the chloroplast genomes of the Morus species, all the reports indicated the presence of two identical inverted repeats (IR) regions and two single copy regions —large (LSC) and small (SSC). From Table 4.2, it is observed that the chloroplast genome size of the eight Morus species ranges between 158,000 and 160,000 bp, with the largest being that of M. alba (159,290 bp) and the smallest being that of M. mongolica (158,459 bp). All the species displayed similarity in their GC content and inverted repeat (IR) content. However, the large single copy region (LSC) of M. cathayana (88,143 bp) and M. alba (88,065 bp) was slightly greater than that of the other species. On the other hand, small single copy region (SSC) of M. multicaulis (20,035 bp) was the highest among the eight species. A similarity in protein-coding genes was exhibited by the Morus species, but the rRNA and tRNA genes of M. indica, M. notabilis and M. alba were conspicuously higher than the rest of the species. It is noteworthy that the rRNA and

Population Genomics and Diversity Analysis

Since the advent of genomics, population genetics has become dominated by complex statistical and computational methodologies. The discovery and development of molecular markers in mulberry have become progressively more rapid as NGS technologies have become increasingly cheaper and effective (Jain et al. 2022; Jiao et al. 2020; Vijayan and Gnanesh 2022). Among all types of molecular markers, available SSR commonly have been used in mulberry for population studies (Arunakumar et al. 2021; Gnanesh et al. 2023; Krishnan et al 2014b; Pinto et al. 2018; Yulianti et al. 2022). In Morus spp. even though studies on genetic distances, evolution, and kinship have been analyzed using DNA markers, the inferences are still controversial (Muhonja et al. 2019; Jiao et al. 2020; Gnanesh et al. 2023). To elucidate the genetic composition of the founding populations, Yulianti et al. (2022) based on the UPGMA clustering, principal coordinate analysis and structure analysis showed populations of M. australis in the Ogasawara Islands are genetically related to those in the Ryukyu Islands. Their study indicated that M. australis plants in the Ogasawara Islands are descendants of those introduced from the Ryukyu Islands. Pinto et al. (2018) used 214 germplasm accessions to identify markers associated with charcoal root rot resistance, the panel used in the study was weakly structured with two subpopulations, and most of the accessions were found to be

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Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective

123

Table 4.1 Comparative statement of genomic information of Morus spp Characteristics of genome

M. notabilis

M. alba

M. indica

Ploidy

2n = 2x = 14

2n = 2x = 28

2n = 2x = 28

Monoploid chromosome number

07

14

14

Total chromosomes in a diploid

14

28

28

Genome sequencing method and approaches

Whole genome shotgun sequencing method, RNAseq data, expressed sequenced tags (ESTs)based approaches

Oxford nanopore, illumina HiSeq, and high-throughput chromosome conformation capture (Hi-C-based approaches)

Illumina, PacBio, Hi-C sequencing, bio nano, and RNA-seq data

Genome size (bp)

330,378,613 (330.37 Mb)

346,393,484 (346.39 Mb)

505,298,450 (505.39 Mb)

Contig N50 (bp)

40,438

2,710,056

18,779,702

No. of contigs

842

398,46

18,139

Scaffold N50 (bp)

405,448

22,871,251

174,315

No. of scaffolds

31,301

16

365

GC content

0.3486

0.3429

0.3119

Chromosome length

–

98.38%

–

Complete BUSCOs (Benchmarking Universal SingleCopy Orthologs)

92.60%

94.30%

96.5%

Repetitive sequences (RS, in Mb)

147

180.11

248.6

Percentage (%) of RS in the Whole genome

47

52.85

49.2

Number of protein encoding genes

27,085

22,767

27,250

Average gene/mRNA length

2849 bp

3209 bp

1430

Coding sequence length

1156 bp

1148 bp

39

House keeping genes

1805

–

–

Reporting year

2013

2020

2022

References

He et al. (2013)

Jiao et al. (2020)

Jain et al. (2022)

admixtures. Similarly, Zhang et al. (2016) used a germplasm panel of 93 mulberry accessions of diverse origins to identify markers for a few important fruit traits. A total of 24 markers associated with fruit traits were identified. To explore the population structure and diversity of 132 mulberry cultivars from different areas, across China and Japan, Jiao et al. (2020) used whole genome resequencing data obtained from the mulberry cultivars to develop SNPs and examine whole genome genetic variations.

Population structure separated the cultivars into three groups. The phylogenetic tree based on the maximum-likelihood (ML) method clustered Japanese mulberry cultivars together and formed a basal lineage connected to the wild mulberry trees, whereas Chinese cultivars are separated into two groups. In their study, a clear genetic structure was observed, with samples from each geographical region. Every limited study has been conducted on linkage disequilibrium (LD) and association studies in mulberry.

158,484

158,459

159,113

159,154

158,680

159,103

159,265

159,050

159,290

Morus indica

M. mongolica

M. atropurpurea

M. multicaulis

M. notabilis

M. multicaulis

M. cathayana

M. alba

M. alba

NR Not reported

Genome size (bp)

Species

36.20

36.20

36.16

36.19

36.36

36.20

36.20

36.29

36.37

GC (%)

88,065

87,762

88,143

87,940

87,470

87,763

87,824

87,363

87,386

LSC (bp)

19,845

19,876

19,844

19,809

19,776

20,035

19,875

19,736

19,742

SSC (bp)

25,690

25,706

25,639

25,677

25,717

25,678

25,707

25,678

25,678

IR (bp)

77

82

78

78

84

78

78

80

83

Proteincoding genes

Table 4.2 Summary of the reported chloroplast genomes of Morus species

4

8

4

4

8

4

4

4

8

rRNA genes

30

36

30

30

37

30

30

30

37

tRNA genes

NR

NR

83

82

NR

81

83

78

NR*

SSR loci

Illumina sequencing

NR

cpDNA on Illumina Hiseq 2000 platform

cpDNA on Illumina Hiseq 2000 platform

Whole genome Illumina sequencing

Illumina Hiseq 2500 platform

Illumina Hiseq 2500 platform

cpDNA on Illumina Hiseq 2000 platform

Long PCR and shotgun sequencing

Method

He et al. (2020)

Luo et al. (2019)

Kong and Yang (2017)

Kong and Yang (2017)

Chen et al. (2016)

Li et al. (2016)

Li et al. (2016)

Kong and Yang (2016)

Ravi et al. (2006)

References

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Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective

4.6

Application of Genomics Resources for Mulberry Breeding

The development of inexpensive and easy-to-use allele-specific molecular markers is a critical step in the successful application of high-throughput MAS in breeding programs (Gnanesh et al. 2013). Relative to conventional linkage mapping, genome-wide association study (GWAS) is considered a precise high-resolution mapping tool for complicated quantitative features; GWAS inspects genetic distinction at whole genome level to locate candidate genes (Esvelt Klos et al. 2016; Zhang et al. 2016). In this connection, the identification of most abundant and suitable polymorphic SNPs in the genome of mulberry helps to improve biotic and abiotic stress tolerance mulberry genotypes through GWAS (Abdurakhmonov and Abdukarimov 2008; Vijayan 2010; Sarkar et al. 2017; Vijayan et al. 2022a, b). Additionally, the implementation of genotyping-by-sequencing (GBS) method is necessary to generate desirable SNPs for QTL and linkage disequilibrium (LD) mapping (Sarkar et al. 2017). Recently, a fast increase in available genome sequencing data has resulted in an intensification of computational-based pangenome analysis (Zekic et al. 2018). The pangenome refers to a species’ whole set of genes, which is made up of a core genome (which contains sequences shared by all members of the species) and effectively identifies causal genetic variants (e.g., SNPs) which can be useful for the improvement of many agronomically important genes in mulberry. Future strategies for mulberry improvement are presented in Fig. 4.2. With ongoing trends of high-throughput sequencing technology, plant scientists achieved significant attainment towards crop improvement. Though inconsistency was also reported specifically for the improvement of complex traits such as yield, drought tolerance because of inadequate understanding of (i) synchronization of nuclear to organelle protein expression; (ii) highly complex compartmentalized metabolic network system (Heinig et al. 2013);

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(iii) complication of the eukaryotic multilevel gene regulation system (Sweetlove et al. 2017); and (iv) expression of genes influences be diverse agro-climatic conditions induce an enormous challenge to develop climate smart crops (Mondal et al. 2021). Currently, a stupendous opportunity exists for data mining of wide-ranging online datasets and information available in the public domain. Bioinformatics tools and in silico data mining research have significantly contributed to generate a platform for computational biology (Swarbreck et al. 2011; Mondal et al. 2021). A range of repository databases, bioinformatics tools, and experimental datasets of different model and nonmodel crops are currently available and that will help in several aspects like understanding molecular relationships, divergence, duplication, cis-elements, microRNA mediated regulations, gene expression, co-expression network, interaction, post-transcriptional/translational mediated modifications, Gene Ontology (GO), etc. (Mondal et al. 2021). Though, to understand the genetic architecture of complex traits, information was found to be inadequate. Thus, further extensive study requires for the improvement of the mulberry crop as presented in Fig. 4.3.

4.7

Conclusion and Future Perspectives

Recent trends of research on mulberry are clearly soothing that mulberry is not only restricted to silkworm rearing but also used for the production of herbal tea, wine, beverage and is also considered an eminent source of pharmacological composites. Hence, the exploration of genomic research prerequisite to capitalizing on the benefit of mulberry genetic resources. Moreover, in the present chapter, we have focused on different aspects of information available in the public domain, challenges, and the future direction of mulberry genomic research. Moreover, in mulberry, there are no large-scale genome-wide association studies (GWAS), and this may be attributed to the very limited application of

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high-throughput SNP genotyping in mulberry. To address the needs of genes associated with abiotic/biotic stress, yield, and nutrient use efficiency, large-scale development of genomic resources and SNP detection is prerequisites for mulberry. Additionally in the future, cell-typespecific marker, good quality annotated genome

Collection

Species level diversity

M.australis M.bombycis M.indica M.latifolia M.alba M.nigra M.cathayana M.rotundiloba M.sinensis M.laevigata M.serrata M.tiliaefolia M. multicaulis M.rubra M. macroura

sequences, and information regarding ploidyassociated traits will be the most important research aspect for mulberry crop improvement. In this connection, we have also emphasized the application of integrated omic approaches through utilizing available genomics resources for future population genomic analysis.

Gene Bank

Core set

Crop wild relatives (CWR) Bi-parental population

Experimental Study

Trait Evaluation Phenomics, Genomics, Epigenomics, Transcriptomics, Proteomics, Metabolomics Bioinformatics

Anatomical, Physiological, Biochemical, Reproductive, Propagation, Growth and yield

Stress tolerant capacity, species specific variability, ecological behaviour, mutant & somaclonal development, DH-line generation 5%

GTCAATGAG 12%

GTCACTGAG

31%

15% 36%

GTCAATGAG

Marker based genotyping

Digital library

SSR/ SNPs Marker

Dissection of genetic resources

Low

High Genetic Gain

WGS, GBS, ABS

High

Low

QTLs/QTNs/allele mining/haplotypes Forward breeding

MAS/MABC

Genomic Selection

PAGE

Multi-stress tolerant genotype development

Transgenic

4

Mulberry Genome Analysis: Current Status, Challenges, and Future Perspective

b Fig. 4.2 Future strategies for mulberry improvement.

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will help to select genotype with specific traits including water (WUE) and nitrogen use efficiency (NUE), drought, saline and alkaline tolerance, etc. (ii) Pre-Breeding Program: Advancement of integrated screening strategies includes multi-omics approaches, multiple trait association study and evaluation of stress tolerance capacity, behavior, etc. (iii) Development of Digital Library: To understand the genetic architecture of complex traits, identification of gene (s) that associates with variation of a quantitative trait (QTL) should be emphasized through implementation of powerful strategies like genome-wide association study (GWAS), bi/multi-parental, and mutation/inbreed population mapping, integrated phenome (phQTL), metabolome (mQTL), proteome (pQTL) and expression (eQTL). (iv) Advancement of Breeding Approaches Forward breeding, MAS/MABC, promotion of alleles through genome editing (PAGE), transgenic approaches and can be used for simply inherited traits, while genomic selection can be used to integrate complex inherited traits like multiple stress tolerance capacity. The goal of global sustainable production of mulberry will be achieved by this comprehensive examination of genetic resources, which will save both time and money

Mulberry germplasm Central Sericultural Germplasm Resources Centre (CSGRC, http://csgrc.res.in), Hosur, India, comprised world-wide collections (a total of 1317 mulberry accessions) from different geographical regions (28 countries). Mulberry germplasm accessions belong to accepted eight species viz. M. alba L., M. auastralis Poir., M. cathayana Hemsl., M. indica L., M. macroura Miq., M. nigra L., M. rubra L., and M. serrata Roxb. were recognized according to the nomenclature of the Plant List (www.theplantlist.org) database. Other species like M. bombycis Koidz. (synonym of M. auastralis), M. laevigata Wall. ex Brandis (synonym of M. macroura Miq.), M. latifolia Poir. (synonym of M. alba L.), M. multicaulis Perri. (synonym of M. alba L.), M. rotundiloba Koidz. (unresolved), M. sinensis Loudon (unresolved), and M. tiliifolia Makino (synonym of M. cathayana Hemsl.) are also available. For future improvement, four major strategies are (i) Mulberry Genetic Resource Evaluation and Maintenance Program: Collections, conservation, and evaluation of germplasm for identification of elite germplasm and establishment of unique populations (coreset, bi-parental, crop wild relatives, reference breeds, mutants, inbreeds) that

Database •Genomics •Transcriptomics •Proteomics Data mining

Available resource for nuclear, chloroplast and mitochondrial genomics

Structural information Promoter structure

Gene structure

G1 G2 G3 G4 G5

t1 t2 t3 t4 t5

Protein structure

P1 P2 P3 P4 P5

3D structure

Phylogeny

Functional information In silico expression

PPI prediction

GO network

In silico mutation

Lab validation Gene expression 1 .8

Protein expression

Bio-assay

1 .6 Relative expressiuon

Lines development •Overexpressed •Mutant •Homozygous

1 .4 1 .2 1 .0 0 .8 0 .6 0 .4 0 .2 0 .0 C

2H

6H

12H

Fig. 4.3 Roadmap of the utilization of genetic information for genetic improvement in mulberry. Exploration and extensive data mining of repository genomic, and transcriptomics data from publically available databases like MorusDB, MindGP, and others (Table 4.3) will help prediction of structural (cis-elements in promoter, exon– intron arrangement structure, conserve motif, homology

modeling, and phylogenetic relationship, etc.), functional information (gene expression profile, protein–protein interactions, GO enrichment, gene mutations, etc.). Further, wet-lab validation through the development of transgenic, mutant lines, in turn, will help for a precise understanding of gene functions toward mulberry improvement

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Table 4.3 Some important repository database and bioinformatics tools for illustration of molecular information Bioinformatic resources

Database/tools

Web link

References

Genomics and transcriptomics

MindGP

http://tgsbl.jnu.ac.in/MindGP

Jain et al. (2022)

Genomics, transcriptomics and proteomics

MorusDB

https://morus.swu.edu.cn

Li et al. (2014)

Transposable element database

MnTEdb

https://morus.swu.edu.cn/ mntedb

Ma et al. (2015)

Plant transcriptional regulatory map

PlantRegMap

http://plantregmap.gao-lab.org

Tian et al. (2020)

Mulberry metabolome database

MMHub

https://mmdb.biodb.org

Li et al. (2020)

Molecular interactions, reactions, and relations

KEGG Pathway

https://www.kegg.jp/kegg/ pathway.html

Kanehisa and Goto (2000)

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R. Mondal et al. Vijayan K (2010) The emerging role of genomic tools in mulberry (Morus) genetic improvement. Tree Genet Genomes 6(4):613–625 Vijayan K, Gnanesh BN (2022) Genomic research in mulberry for higher silk productivity. In: The 26th international sericultural commission congress on Seritech, the new concepts in sericulture, ClujNapoca, Romania, 7–11 Sept 2022, pp 49–74 Vijayan K, Srivastava PP, Raju PJ, Saratchandra B (2012) Breeding for higher productivity in mulberry. Czech J Genet Plant Breed 48(4):147–156 Vijayan K, Srivatsava PP, Nair CV, Awasthi AK, Tikader A, Sreenivasa B, Urs SR (2006) Molecular characterization and identification of markers associated with yield traits in mulberry using ISSR markers. Plant Breed 125(3):298–301 Vijayan K, Raju PJ, Tikader A, Saratchnadra B (2014) Biotechnology of mulberry (Morus L.)—a review. Emirates J Food Agric 26(6):472 Vijayan K, Doss SG, Chakraborti SP, Ghosh PD (2009) Breeding for salinity resistance in mulberry (Morus spp.). Euphytica 169(3):403–411 Vijayan K, Gnanesh BN, Shabnam AA, Sangannavar PA, Sarkar T, Zhao W (2022a) Genomic designing for abiotic stress resistance in mulberry (Morus spp.) In: Genomic designing for abiotic stress resistant technical crops. Springer Nature https://doi.org/10.1007/ 978-3-031-05706-9_7 Vijayan K, Arunakumar GS, Gnanesh BN, Sangannavar PA, Ramesha A, Zhao W (2022b) Genomic designing for biotic stress resistance in mulberry (Morus spp.) In: Genomic designing for biotic stress resistant technical crops. Springer Nature https://doi. org/10.1007/978-3-031-09293-0_8 Yang X, Yang L, Zheng H (2010) Hypolipidemic and antioxidant effects of mulberry (Morus alba L.) fruit in hyperlipidaemia rats. Food Chem Toxicol 48(8– 9):2374–2379 Yuan Q, Zhao L (2017) The mulberry (Morus alba L.) fruit a review of characteristic components and health benefits. J Agric Food Chem 65(48):10383–10394 Yulianti W, Katoh S, Sugita N, Kokubugata G, Kato H, Murakami N (2022) Microsatellite markers reveal genetic differentiation of an invasive mulberry, Morus australis Poir. (Moraceae), among the Island Groups in Japan and its introduction to the Ogasawara Islands. Acta Phytotaxonomica et Geobotanica 73(1):1–8 Zekic T, Holley G, Stoye J (2018) Pan-genome storage and analysis techniques. Comp Genom 29–53 Zeng Q, Chen H, Zhang C, Han M, Li T, Qi X, Xiang Z, He N (2015) Definition of eight mulberry species in the genus Morus by internal transcribed spacer-based phylogeny. PLoS ONE 10(8):e0135411 Zhang J, Yang T, Li RF, Zhou Y, Pang YL, Liu L, Fang RJ, Zhao QL, Li L, Zhao WG (2016) Association analysis of fruit traits in mulberry species (Morus L.). J Hortic Sci Biotechnol 91(6):645–655

5

Relationship Between Genome Size and Ploidy Level in Mulberry Belaghihalli N. Gnanesh , Raju Mondal , H. B. Manojkumar, M. R. Bhavya, Pradeep Singh , G. S. Arunakumar, and Thallapally Mogili

Abbreviations

GC GS FCM SSR WGD PI

Genomic content Genome size Flow cytometry Simple Sequence Repeat Whole genome duplication Propidium iodide

B. N. Gnanesh (&)
H. B. Manojkumar
M. R. Bhavya
G. S. Arunakumar
T. Mogili Molecular Biology Laboratory-1, Central Sericultural Research and Training Institute, Mysuru, Karnataka 570008, India e-mail: [email protected] R. Mondal Mulberry Tissue Culture Lab, Central Sericultural Germplasm Resources Centre (CSGRC), Ministry of Textile, Government of India, Hosur 635109, India P. Singh Agri-Biotechnology Division, National Agri-Food Biotechnology Institute (NABI), Mohali, Punjab 140306, India

5.1

Introduction

Mulberry (Morus spp.) belongs to the Moraceae family consisting of 37 genera having approximately 1100 species including banyan, breadfruit, fig, and upas. Across the world, over 10 species along with more than 1000 cultivated varieties have been found (Dhanyalakshmi and Nataraja 2018); however, in India, six species are generally found (Dandin et al. 1987; Basavaiah and Rajan 1989). Mulberry has been commercially utilized as the sole food source of silkworms (Bombyx mori) in sericulture (Arunakumar and Gnanesh 2023; Gnanesh et al. 2021, 2022). Mulberry plants were described as a truly wild species in the Himalayan terrain of tropical and subtropical regions of India (Watt 2014). A comprehensive level of ploidy has been observed in Morus genus, ranging from haploid (2n = 2x = 14) to docosaploid (2n = 22x = 308). However, in sericulture, most of the diploid species are being used as an exclusive food source for the silkworms. Since 1920, numerous studies have attempted to comprehend meiotic behavior, which is regarded as a critical part of the breeding program. The ploidy status, size, and structure of chromosomes, as well as behavior in pairing and segregation during meiosis, have been systematically explored for the parents of popular mulberry cultivars and a few triploids (Basavaiah et al. 1990a, b; Chakraborti et al. 1999; Shafiei and Basavaiah 2018; Sarkar

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_5

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et al. 2021). Details of the reported ploidy level in different accessions of mulberry based on the cytology work, including their origin, parentage, special characters, and their institute involved in the development, are described in Table 5.1. However, studies are subjected to be critical because of intense chromosome size variation in mulberry (Datta 1954a, b). As a result, estimating genome size using flow cytometry (FCM) may be advantageous for analyzing inter-specific ploidy level variation and ploidy-associated traits to develop knowledge of mulberry genomes (Gnanesh et al. 2023). FCM using a DNA-selective fluorochrome is the most widely used technique for estimating plant genome size, since the invention of a quick approach for isolating nuclei from healthy plant tissues (Galbraith et al. 1983). Despite the fact that various types of plant tissue may be used for FCM analysis, many researchers choose to estimate 2C nuclear DNA concentration in rapidly developing young leaves to get high-resolution histograms (Bennett and Leitch 1995a). Since its inception, the number of people using FCM has expanded dramatically in plant biology. FCM has proven to be an effective and quick method of determining nuclear DNA content as well as polyploidy levels (Kron and Husband 2012; Hoang et al. 2019; Eeckhaut et al. 2005; Ochatt, 2008; Bourge et al. 2018). FCM testing is beneficial in differentiating mulberry germplasm at various ploidy levels, as well as providing insight into the extent of GC variability among some of the various ploidy levels of mulberry germplasm (Mondal et al. 2023a; Yamanouchi et al. 2010). The ploidy level and chromosome number of known accessions of mulberry by chromosome counting or flow cytometry (FCM) analysis are described in Table 5.2. In many breeding efforts, the FCM has been used to depict the GS and ploidy level of somatic tissue in numerous crops (Dutt et al. 2010; Kamiri et al. 2011). Moreover, estimation of GS and ploidy level diversity analysis remain to be elucidated in the mulberry population study. Additionally, different limitations hinder mulberry crop improvement: (i) due to the obvious everlasting growing habit and

B. N. Gnanesh et al.

complicated inheritance pattern, traditional breeding is excruciatingly time taking (Mathithumilan et al. 2013); (ii) mulberry lacks adequate molecular markers, preventing contemporary genomic techniques to crop enhancement (Mathithumilan et al. 2016); and (iii) marker-assisted breeding for specific trait improvement has been hampered by the shortage of co-dominant markers to be used in a genetic mapping (Pinto et al. 2018). Although many marker systems have been used to study genetic variation because mulberry is a highly crosspollinated heterozygous genome, Simple Sequence Repeats (SSRs) are often acceptable (Zhang et al. 2006; Tan et al. 2014; Mathithumilan et al. 2016; Arunakumar et al. 2021; Shinde et al. 2021; Vijayan et al. 2022a, b). Moreover, based on morphological and DNA marker technology, it is difficult to estimate and classify their ploidy level variation, and further chromosome study in Morus is quite challenging due to the size variation of the chromosome.

5.1.1 Estimation of Nuclear DNA Content Genome size is associated with an organism’s life history, development, physiology, ecology, genome dynamics, and evolution (Van’t Hof and Sparrow 1963; Beaulieu et al. 2008; Šímová and Herben 2012; Greilhuber and Leitch 2013; Bilinski et al. 2018; Simonin and Roddy 2018; Novák et al. 2020; Roddy et al. 2020; Gnanesh et al. 2023). It is defined as the amount of DNA in an individual’s unreplicated gametophyte nucleus. Estimation of nuclear DNA content in plants through flow cytometry (FCM) via DNAselective fluorochrome is now the prevailing method, and the execution of flow cytometry methodology for estimating genome size and ploidy level in mulberry is illustrated in Fig. 5.1. For estimating the genome content using FCM, selecting a suitable reference standard is a must, and there are two types of standardization processes: (1) internal standardization and (2) external standardization. For accurate measurements of genome size (GS), aneuploidy, and DNA base

Diploid

Triploid

Triploid

ME-0065

MI-0012

MI-0013

MI-0025

–

–

S-1

S-13

S-36

MR-2

Suvarna-1

Suvarna-2

Promising variety under rainfed conditions with yield potential of 10–12 MT/ha/year

Mathithumilan and Dandin (2009)

Rao (1996)

Rao (1996)

Shafiei and Basavaiah (2018), Gnanesh et al. (2023)

Kumara and Basavaiah (2016), Gnanesh et al. (2023)

Venkatesh (2017)

Mathithumilan and Dandin (2009)

Venkatesh (2015)

Rao et al. (2013), Gnanesh et al. (2023)

Venkatesh (2015), Gnanesh et al. (2023)

References

Venkateswarlu et al. (2004), Gnanesh et al. (2023) (continued)

CSRTI, Mysore

Improved high leaf-yielding triploid in South India

Fast-growing high-yielding variety with unlobed, deep green, succulent leaves, and good root proliferation

Mildew-resistant variety

Nutritionally superior used for silkworm rearing

Selection for rain-fed conditions

Superior agronomic attributes

Special features

Triploid

MI-0079

Birds Foot

OPH of K-2

KSSRDI, Bangalore

KSSRDI, Bangalore

KSSRDI, Bangalore

Evolved at Coonoor Sericulture Farm, Tamil Nadu

CSRTI, Mysore

CSRTI, Mysore

CSRTI, Berhampore

CSRTI, Mysore

Developed institute

Kumara and Basavaiah (2016)

Diploid

–

RFS-135

Clonal selection made in the collection from Dehradun

Colchicine treatment

M-5 (4x) as female and Viswa (2x) as male parent

M-5 (4x) as female and Viswa (2x) as male parent

Open-pollinated hybrid

Mutant—EMS treatment of Berhmpore local

OPH selection; RFS-135 and RFS-175

Clonal selection, seedling belongs to Mandalaya introduced from Burma

S-30 X Ber C-776

Origin

Diploid

Tetraploid

Diploid

–

–

M-5

Vishwa

Diploid

Diploid

Diploid

Diploid

MI-0308

V-1

Reported ploidy

Accession number

Germplasm

Table 5.1 Ploidy level based on the chromosome count and predicted ploidy level using flow cytometry (FCM) analysis

5 Relationship Between Genome Size and Ploidy Level in Mulberry 133

Diploid

Diploid

Diploid

Hexaploid

ME-0220

–

–

MI-0160

MI-0046

ME-0036

ME-0066

–

M. macroura

RC-1

RC-2

S-34

S-30

Goshoerami

Kosen

M. serrata

Diploid, triploid, and tetraploid

Diploid

Triploid

Diploid

Diploid

Uneuploid

Diploid

Tetraploid

Diploid

ME-0035

KNG

Reported ploidy

Accession number

Germplasm

Table 5.1 (continued)

Himalayan mulberry

Introduction from Japan

CSRTI, Pampore

CSRTI, Mysore

CSRTI, Mysore

S-30  Ber 776 Mutation breeding

CSRTI, Mysore

CSRTI, Mysore

Developed institute

Punjab local  Kosen

Punjab local  Kosen

Origin

Different ploidy level of M. serrata reported for the first time

Ruling variety in Northwest India

Developed for irrigation condition

Drought-tolerant variety

(Resource-constraint variety) showed better leaf yield under low in put conditions (50% less fertilizer and irrigation)

(Resource-constraint variety) showed better leaf yield under low input conditions (50% less fertilizer and irrigation)

Tetraploid male variety maintained in the germplasm bank attached to Bangalore University

Recommended for temperate region (16 to 17 MT/ ha/yr leaf yield)

Special features

(continued)

Tikader and Kamble (2008)

Basavaiah and Rajan (1989), Gnanesh et al. (2023)

Rao et al. (2013), Gnanesh et al. (2023)

Gnanesh et al. (2023)

Shah et al. (2019)

Venkatesh et al. (2014)

Venkatesh et al. (2014)

Venkatesh (2020)

Rao et al. (2013)

Rao et al. (2013)

Gnanesh et al. (2023)

Venkatesh (2017), Venkatesh and Munirajappa (2013)

Rao et al. (2013), Gnanesh et al. (2023)

References

134 B. N. Gnanesh et al.

Diploid

Diploid

Diploid

MI-0014

–

–

ME-0008

ME-0018

MI-0026

MI-0158

MI-0524

MI-0902

MI-0052

K-2

G-2

G-4

M. nigra

M. cathayana

Punjab local

C-776

Sahana

Vishala

Mysore local

Diploid

Triploid

Diploid

Diploid

Diploid CSRTI, Berhampore

Local variety of South Karnataka

Clonal selection

K-2  Kosen

CSRTI, Mysore

KSSRDI, Bangalore

Recommended for subtropical region (14 to 19 MT/ha/yr leaf yield under rain-fed condition)

Coconut shade-tolerant variety, and it has yield potential of 45–50 mt/ha/year under optimal conditions

Venkatesh (2015) Mallikarjunappa et al. (2008), Gnanesh et al. (2023) (continued)

Saratchandra et al. (2011), Sujathamma and Priya (2021), Gnanesh et al. (2023)

Rao et al. (2013), Gnanesh et al. (2023)

Venkatesh et al. (2014)

Rao et al. (2013)

Gnanesh et al. (2023)

Diploid

Ploidy level identification through flow cytometry

Venkatesh and Munirajappa (2013)

Tetraploid

Black cherry X Morus multicaulis

Gnanesh et al. (2023)

Gnanesh et al. (2023)

Mathithumilan and Dandin (2009)

Razdan and Thomas (2021)

References

Tikader and Kamble (2008)

Possibility of diploid

Variety developed by two diploids

Variety developed by two diploids

Special features

Rao et al. (2013)

CSRTI, Mysore

CSRTI, Mysore

CSRTI, Mysore

Developed institute

Docosaploid

Morus multicaulis X S-13

Morus multicaulis X S-34

Originated from the OPH seeds of Mysore local

Origin

Diploid

Natural triploid

MI-0799

AR-12

Reported ploidy

Accession number

Germplasm

Table 5.1 (continued)

5 Relationship Between Genome Size and Ploidy Level in Mulberry 135

Tetraploid

Tetraploid

Triploid

Triploid

Natural triploid

MI-0173

ME-0149

–

MI-0014

MI-0172

–

–

S-1635

M. tiliaefolia

RFS-135

K-2

S-1708

S-41

Lun 40-2

The reference column provides the source of publication

Through mutation

Colchicine treatment

Colchicine treatment CSRTI, Mysore

CSRTI, Mysore

Venkatesh (2014), Gnanesh et al. (2023)

Venkatesh (2015), Gnanesh et al. (2023)

References

Yongkang (2000)

Venkatesh (2014)

Venkatesh (2020)

Mathithumilan and Dandin (2009)

Dwivedi et al. (1986)

Gnanesh et al. (2023)

Ploidy level identification through flow cytometry

Recommended for subtropical region, and its foliage is of excellent quality Ideal for silkworm rearing in all Seasons also ruling highyielding variety of West Bengal

Promising variety under rainfed conditions with yield potential of 10–12 MT/ha/year

Special features

Triploid

CSR&TI, Berhampore

CSRTI, Mysore

Developed institute

Tikader and Kamble (2008)

OPH selection from CSRS-1

Open-pollinated hybridization of Kanva-2

Origin

Hexaploid

Natural triploid

Diploid

MI-0066

RFS-175

Reported ploidy

Accession number

Germplasm

Table 5.1 (continued)

136 B. N. Gnanesh et al.

5

Relationship Between Genome Size and Ploidy Level in Mulberry

137

Table 5.2 Confirmed ploidy level and chromosome number of known accessions of mulberry by chromosome counting or flow cytometry (FCM) analysis Ploidy level (2n)

Name

Diploid 2n = 2x = 28

Triploid 2n = 3x = 42

Tetraploid 2n = 4x = 56

Hexaploid 2n = 6x = 84

Accession number

References

V-1

MI-0308

S-13

MI-0012

Mathithumilan and Dandin (2009), Venkatesh et al. (2013, 2014), Venkatesh (2015), Gnanesh et al. (2023)

S-34

MI-0160

RFS-175

MI-0066

S-36

MI-0013

K-2

MI-0014

Mysore local

MI-0052

Sujanpur-5

MI-0017

M. australis

ME-0001

UP-22

ME-0254

S-1635

MI-0173

Vishala

MI-0902

Venkatesh et al. (2014), Kumara et al. (2021), Gnanesh et al. (2023)

Suvarna-1

MI-0772

Kumara and Basavaiah (2016), Gnanesh et al. (2023)

Suvarna-2

–

Shafiei and Basavaiah (2018), Gnanesh et al. (2023) Sarkar et al. (2021), Gnanesh et al. (2023)

AR-12

MI-0799

Pouri-2

MI-0652

RFS-135 (4x)

–

Dwivedi et al. (1986)

K-2 (4x)

–

Sastry et al. (1968), Mathithumilan and Dandin (2009)

M. serrata

–

Basavaiah and Rajan (1989), Yamanouchi et al. (2017), Gnanesh et al. (2023)

The reference column provides the source of publication for each chromosome count (Gnanesh et al. 2023)

structure, internal standardization is followed, where unknown and reference samples are chopped together and measured at the same time, whereas in external standardization, the nuclei of reference and unknown samples are isolated and analyzed separately. However, this method is not acceptable for the precise evaluation of genome content. External standardization may be used for primary detection of genome content for selecting a suitable internal reference standard and for ploidy level estimation (Temsch et al. 2022; Sliwinska et al. 2022). In many fields of plant research, knowing information on plant genomes has been increasingly significant, especially in the areas of biosystematics, ecology, population biology, and taxonomy (Castro et al. 2012). Genomes signify

a distinctive and legitimate level of organization, through exclusive and specific evolutionary histories. Genome size is an important speciesspecific fundamental characteristic that helps to understand the evolutionary associations among related species (Gregory 2001). Hence, in mulberry, it is essential to explore the genetic differentiation and variation in genomic content for the exploitation of heterosis and understanding the genetic basis of complex traits. Morus spp. is mostly diploid but may also exist in the higher ploidy state. Our knowledge related to the genome size of the different species of Morus maintained in India is still unknown. Variation in nuclear DNA content is an essential tool to understand the structural, functional, and evolutionary concepts of genetic information

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B. N. Gnanesh et al.

Collection of young leaf tissue and chopping

Ploidy level

2n

Genome size (GS)

Chopping buffer

4n

6n

Trait estimation

Filtering debris

DNA fluorochrome (Propidium iodide)

Flow cytometry analysis

Filtered homogenate

Correlation GS-traits

Ploidy breeding

Fig. 5.1 Application of flow cytometry for estimating genome size and ploidy level in mulberry

stored in an organism. It also helps to know the biological and adaptive value of diversity in the context of evolutionary and taxonomic clarifications (Gregory 2005; Greilhuber et al. 2010). The method of flow cytometry has been used in many plant species for precise and fast assessment of nuclear DNA (Bennett and Leitch 1995a, 2005; Bennett et al. 2000, Doležel 1991; Doležel and Bartos 2005). Previously, different researchers determined complete information on genome size using flow cytometry for domesticated and cultivated mulberry. The 2C genome content of diploid Morus alba, M. bombycis, M. latifolia, and M. rotunbiloba was 2C = 0.79 pg using Ethidium bromide (EB) (Horjales et al. 2003). The 2C = 0.704–0.746 pg was found using DAPI (4′,6-diamidino-2-phenylindole) and PI (Yamanouchi et al. 2010). The 2C DNA content was 1.70 ± 0.02 pg using Feulgen densitometry

(FD) (Ohri and Kumar 1986). The genome size estimated for M. alba is 1C = 345–366 Mbp (Yamanouchi et al. 2010) or 1C = 386 Mbp (Horjales et al. 2003). Flow cytometry analysis is more useful in distinguishing the mulberry at different ploidy levels, which provides information about the variation level of genome content among the different ploidy levels of mulberry germplasm (Yamanouchi et al. 2010).

5.1.2 Cytogenetics Toward Estimation of Genome Size and Ploidy Level Mulberry exists in a range of polyploidy levels, from haploid (M. notabilis, n = 2x = 14) to docosaploidy (M. nigra, 2n = 22x = 308). The diploid chromosomal number 2n = 2x = 28 comprised Morus spp. such as M. alba,

5

Relationship Between Genome Size and Ploidy Level in Mulberry

M. atropurpurea, M. bombycis, M. indica, M. latifolia, and M. rotundiloba (Datta 1954a, b). Majority of triploids (2n = 3x = 42) and tetraploid (2n = 4x = 56) belong to M. laevigata (Das 1961), whereas hexaploids (2n = 6x = 84) were reported in M. serrata (Basavaiah and Rajan 1989) and M. tiliaefolia (Seki 1952). Earlier reports also recommend that polyploidy can range up to docosaploidy (2n = 22x = 308) as in M. nigra (Basavaiah et al. 1990a, b; Yamanouchi et al. 2017). The main species of mulberry available in India are M. alba, M. indica, M. atropurpurea, M. nigra, M. serrata, M. latifolia, and M. laevigata (Dandin et al. 1987; Basavaiah and Rajan 1989).

5.1.3 Flow Cytometry (FCM) Toward Estimation of Genome Size and Ploidy Level In 2013, for the first-time chromosome conformation, an estimated genome size of 330 Mb in natural haploid M. notabilis was reported by He et al. (2013). Though till 2020, chromosomallevel genome information was restricted in Morus spp. However, Jiao et al. (2020) insight chromosomal-level genome information and investigated population genomics for a better understanding of economically important traits in diploid M. alba (2n = 28, 346 Mb). Recently, Jain et al. (2022) complemented by revealing another high-quality genome sequence of Indian cultivar Kanava-2 belonging to M. indica (2n = 28, 505.39 Mb) that offers a source for functional and translational genomics. Besides the scaffold estimated, the genome size of different mulberry species, specifically for higher ploidy levels, was also assessed by implementing FCM, since it was more robust and cheaper. The present incarnation of accessible polyploids of mulberry is explained in this section to better understand polyploidy variation at the species level. To address how and to what extent WGD occurred in mulberry germplasm collections, Gnanesh et al. (2023) chose a total of 157 mulberry accessions from seven distinct species, including popular Indian varieties, and submitted

139

them to FCM genome content analysis (Table 5.3). Genome content (GC) data indicates that it ranged from 0.72 (M. indica, diploid) to 2.89 pg (M. serrata, hexaploid). Other genome content studies revealed the 2C value of mulberry species ranged between 0.70 and 0.73 pg for diploid, 1.04 and 1.17 pg for triploid, 1.42 and 1.63 pg for tetraploid, 2.02 and 2.48 for hexaploid, and 7.26 pg for docosaploid (Yamanouchi et al. 2017). However, these estimates differ from previous FCM assessments, possibly because of differences in the DNA reference standard, dyeing time, and fluorochrome difference (Doležel et al. 1992; Guo et al. 2015). Propidium iodide (PI) is frequently used for GS estimation through FCM (Gnanesh et al. 2023; Doležel et al. 1992; Yamanouchi et al. 2017). Nevertheless, the estimated GS of diploid M. alba by Ohri and Kumar (1986) was around 1.7 for the n = 14, whereas diploid M. alba was 1.7 pg/2C (Greilhuber 1998; Chang et al. 2018). Genome size variation happened by detection method or due to environmental factors, such as soil nutrition, water stress, and severe climate conditions. Genetic factors like duplications or loss of non-essential genes in an ongoing evolutionary process also contribute to the variation in genome size (Benett and Leitch 1995; Chang et al. 2018, Greilhuber 1998; Greilhuber and Leitch 2013; Laurie and Bennett 1985; Rayburn et al. 1985). Xuan et al. (2022) verified that chromosome fissions/fusions are important mechanisms to describe the evolutionary concept of basic chromosome number differences among M. notabilis and M. alba.

5.1.4 Intra- and Inter-specific Nuclear DNA Content Variation of Morus spp. FCM usage has significantly improved GS estimation as a taxonomic marker and also in assessing how it corresponds to ecological and environmental cues along with phenotypic variables (Al-Qurainy et al. 2021). The GS estimation of the diverse collections available at the Central Sericultural Germplasm Resources

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B. N. Gnanesh et al.

Table 5.3 Genome size estimations of different Morus species Morus spp.a

n

Ploidy level

Range (pg)

2C Mean ± SD

Min 2C (pg)

Max 2C (pg)

M. alba

52

2n = 2x = 28

0.73–0.98

0.92 ± 0.06a

ME-0160

ME-0108

02

2n = 3x = 42

1.16–1.21

1.18 ± 0.03a

MI-0050

ME-0092

01

2n = 4x = 56

02

2n = 2x = 28

M. australis

1.45 0.81–0.83

0.82 ± 0.02a

ME-0002

ME-0001

M. cathayana

02

2n = 2x = 28

0.80–0.85

0.83 ± 0.03

ME-0254

ME-0018

M. indica

53

2n = 2x = 28

0.72–0.92

0.85 ± 0.06a

MI-0046

MI-0529

b

M. macroura

a

03

2n = 3x = 42

1.24–1.27

1.26 ± 0.02

MI-0799

MI-0173

03

2n = 4x = 56

1.69–2.01

1.89 ± 0.15c

MI-0454

K-2 (4x)

a

11

2n = 2x = 28

0.83–0.97

0.89 ± 0.07

MI-0787

MI-0782

03

2n = 3x = 42

1.23–1.28

1.25 ± 0.03b

MI-0079

MI-0521

1.53–2.02

1.82 ± 0.19

MI-0247

MI-0387

c

03

2n = 4x = 56

M. rotundiloba

01

2n = 2x = 28

0.86

M. serrata

01

2n = 6x = 84

2.89

M. sinensis

01

2n = 2x = 28

0.83

M. species

15

2n = 2x = 28

0.80–0.96

0.87 ± 0.09a

AGB-8

UP-105 (Acc105)

4

2n = 3x = 42

1.20–1.29

1.25 ± 0.04a

Vishala

Suvarna-3

Total accessions

157

n Data regarding the species and number of individuals sampled n = 1 Range ansd SD not analyzed a Mulberry species are arranged as per the accepted eight species, viz., M. alba L., M. auastralis Poir., M. cathayana Hemsl., M. indica L., M. macroura Miq., M. nigra L., M. rubra L., and M. serrata Roxb. It was recognized according to the nomenclature of The Plant List (www.theplantlist.org) database. Other species like M. bombycis Koidz. (synonym of M. auastralis), M. laevigata Wall. ex Brandis (synonym of M. macroura Miq.), M. latifolia Poir. (synonym of M. alba L.), M. multicaulis Perri. (synonym of M. alba L.), M. rotundiloba Koidz. (unresolved), M. sinensis Loudon (unresolved), and M. tiliifolia Makino (synonym of M. cathayana Hemsl.) are grouped as per the accepted nomenclature

Centre (CSGRC), Hosur, encompassing different species of Morus collected from tropic, subtropical, and temperate areas all over the world, including popular varieties cultivated in India, was investigated (Gnanesh et al. 2023). Genome content of studied mulberry accessions ranged from 0.72 to 2.89 pg, the highest amount of DNA (2.89 pg) was found in M. serrata (Hexaploid genome), and the least value (0.72 pg) was recorded in S-30, which was a high-yielding female parent as like standard check varieties S34 and V-1 (Table 5.3). Triploids detected in 4 species (M. alba, M. indica, M. macroura, and Morus spp.) showed a slight range of GS variation, 1.16 to 1.24 ± 0.04 pg, whereas tetraploids

showed the highest variability (1.45 to 2.02 pg) as compared to diploids and triploids.

5.1.5 Relation Between Phenotypic Trait Plasticity and Genome Size (GS) Polyploidy has been considered a source of evolutionary development, species expansion, as well as an ecological dead-end. Especially in plants, polyploids are not restricted, which occurred as a consequence of WGD events and appear to be associated with environmental conditions. Hence, understanding the phenotypic plasticity among the

5

Relationship Between Genome Size and Ploidy Level in Mulberry

ploidy groups (2x, 3x, 4x, and 6x) in Morus spp. is imperative. The female floral features, notably style length, were well-recognized attributes used to classify mulberries in terms of female accessions (Koidzume 1917; Minamizawa 1976; Chang 2006). Though, recently quantitative vegetative traits were considered and shown to be beneficial for mulberry plants (Chang et al. 2014, 2018; Gnanesh et al. 2023). Moreover, we have to consider both vegetative and reproductive traits to recognize the effect of WGD on phenotypic plasticity. Morphological variation of reproductive traits like mature inflorescence length and mature fruit length among the representative accessions of mulberry with different ploidy levels is illustrated in Fig. 5.2. The correlation study conducted by Gnanesh et al. (2023) indicates a significant positive correlation between GS and traits, viz., mature inflorescence length and fruit length (Table 5.4). Additionally, multiple traits-based PCA is referred to genome size linked with leaf area (LA), which is the account of coordinate and leaf-lamina length (LLL), leaf-lamina width (LLW), petiole length (PL), petiole width (PW), and (X+, Y− coordinate). However, further research will contribute to mature inflorescence length (MIL), mature fruit length (MFL), and inter-nodal distance (InD) (positive requires investigating how and to what extent GS is intimate to physiological and/or anatomical traits in mulberry). Tetraploids in mulberry have higher trichomes and thick cuticles and thus making them not suitable for rearing silkworms, whereas they can be exploited in triploid production which has useful traits helping to increase mulberry production (Mathithumilan and Dandin 2009). Chang et al. (2018) examined the variations of vegetative traits among diploid and triploid mulberry accessions. The triploid genotype ‘Elongated Fruit No. 1,’ recorded larger leaf length, leaf width, the ratio of leaf length to width, petiole length, petiole width, leaf thickness, and internode length than that of other diploids studied. T-test showed a significant difference between these two ploidy levels for leaf width, petiole width, and internode length. The linear regression relationship between ploidy

141

level and leaf width, petiole width, and internode length accounted for 60.6%, 61.9%, and 85.7%, respectively. From their studies, it is concluded that internode length might be a suitable trait for differentiating triploid from diploid individuals. The variation between diploid and triploid was determined by the internode length better than the other factors studied. Similarly, diploid and autotetraploid mature mulberry trees were morphologically characterized by Dai et al. 2015, and they found that bigger plants and organs were witnessed in the autotetraploid than in diploid. The leaf area was larger and thicker in autotetraploid compared with those of diploids. Cross-section studies in leaves showed that the cell size of spongy tissue and palisade tissue was higher in leaves of autotetraploid, but there was no difference in the cell number. Similarly, as compared to the diploids, the fruits were larger in autotetraploid in different fruit stages, whereas the fruit maturing period had no major difference and the fruit mean weight of autotetraploid was heavier than diploids. Moreover, the fruit length was longer and greater in autotetraploid as compared with those of diploids. Mathithumilan and Dandin (2009) from their studies on genetic analysis of diploid and tetraploid mulberry concluded that the doubling of chromosomes in mulberry is associated with a decrease in stomatal frequency.

5.1.6 Molecular Analysis/Genetic Variation and Population Structure The survival potential, capability to mitigate confrontational ecological factors, resilience, and viability of species are influenced by genetic variation and population structure. Hence, knowledge of genetic variation and population structure is critical to assessing evolutionary processes, sustainable utilization, crop improvement programs, and conservation of plant genetic resources (Mondal et al. 2023b; Zhang et al. 2019). To understand the extent of genetic diversity and relationship in Morus species, different molecular markers have been utilized. For

142

B. N. Gnanesh et al.

Fig. 5.2 Morphological variation among the accessions of mulberry with different ploidy level. a Mature inflorescence length, b Mature fruit length

5

Relationship Between Genome Size and Ploidy Level in Mulberry

143

Table 5.4 Correlation coefficients among quantitative characters in different accessions of mulberry IND (cm) Ploidy InD LLL LLW

0.264*

LLL (cm)

LLW (cm)

LS (cm2)

PL (cm)

PW (cm)

MIL (cm)

MFL (cm)

MFW (cm)

0.203

0.283*

0.188

−0.04

0.293**

0.287*

0.413**

0.065

0.449**

0.468**

0.480**

0.260*

0.328**

0.437**

0.492**

0.122

0.910**

0.847**

0.632**

0.762**

0.291*

0.414**

0.093

0.825**

0.531**

0.755**

0.275*

0.393**

0.159

0.488**

0.742**

0.364**

0.506**

0.459**

0.109

0.073

0.241*

0.355*

LA PL PW MIL MFL

0.647**

0.214 −0.105 0.152 −0.002 0.067

**

Significance at p < 0.01, *significance at p < 0.05 Inter-nodal distance (InD), leaf-lamina length (LLL), leaf-lamina width (LLW), leaf area (LA), petiole length (PL), petiole width (PW), mature inflorescence length (MIL), mature fruit length (MFL), and mature fruit width (MFW) (Gnanesh et al. 2023)

a few decades, the PCR-based markers like random amplified polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), Inter-Simple Sequence Repeats (ISSRs), fluorescence-based amplified fragment length polymorphism (AFLP), sequence-related amplified polymorphism (SRAP), and Simple Sequence Repeat (SSR) markers have been utilized in studies of relatedness, phylogeny assessment, cross-transferability to closely related species, characterization of accessions and cultivars, and genetic diversity analysis among mulberry cultivars and wild accessions (Sharma et al. 2000; Bhattacharya and Ranade 2001; Awasthi et al. 2004; Vijayan et al. 2004, 2022b; Mathithumilan et al. 2013; Bajpai et al. 2014; Arunakumar et al. 2021). Among various marker systems used for mulberry breeding programs, SSRs are the marker of choice due to their multiple attributes such as multi-allelic with co-dominant inheritance, high genome-wide coverage, high reproducibility and polymorphism, locus, and species-specific. SSRs are small tandem repeats of 1–6 bp nucleotide motifs and are ubiquitously present in plant genomes. Considering these characteristics, SSRs are extensively used markers for DNA fingerprinting studies, high-density mapping, population diversity study, conservation genetics, paternity testing, marker-assisted breeding,

etc. (Kalia et al. 2011; Zhong et al. 2021; Vijayan et al. 2022b; Vijayan and Gnanesh, 2022). Further, to enhance our knowledge of evolutionary biology and improve breeding programs in mulberry species, Gnanesh et al. (2023) have utilized the SSR markers for understanding the associations between genetic diversity, population structure, and different ploidy levels. Twenty informative SSR markers have revealed relatively high polymorphic information content (PIC) and observed heterozygosity value in 82 accessions, clearly explaining their potential as a marker of choice in the future improvement of mulberry breeding programs. Further, this study revealed a high level of within-species genetic diversity, high gene flow, a high level of genetic admixture, and a weak population structure in the studied mulberry species. The SSR analysis also revealed that there is no association between genetic diversity and ploidy level in studied accessions, as SSRs are not able to discriminate between different ploidy levels. Although substantial improvements have been attained in the mulberry crop through conventional breeding, these efforts have been continuously constrained by the complex inheritance pattern and perennial growth habit of mulberry including the lack of adequate molecular markers and paucity of genomic information (Mathithumilan et al. 2016; Pinto et al. 2018; Vijayan et al.

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2022a). The ever-increasing application of NGS methods for several research purposes has expanded the genomic resources in many species, predominantly in non-reference plant species (Zalapa et al. 2012). Recently, the genomic resources in mulberry are enriched with the chromosome-level reference genome sequencing of the Chinese cultivar (M. alba ‘Heyebai’) and draft genome sequencing of the Indian cultivar (M. indica cv. Kanava-2) (Jiao et al. 2020; Jain et al. 2022). These sequence repositories in the future will expedite the functional, translation genomics, and modern breeding approaches for mulberry crop improvement.

5.2

Conclusion

In the past two decades, genomic research using DNA markers especially RAPD and SSRs has played a major role in understanding the phylogeny of mulberry. Based on morphological and DNA maker techniques it is difficult to classify the ploidy level of mulberry, and further chromosome study in Morus is challenging as the size of the chromosome is very smaller. In this context, we discussed the value of genome size, mostly as a taxonomic marker, and the role of genome size in polyploidy differentiation and genomic research as well as the maintenance of plant diversity between and within the species of Morus. Also, the information on genetic variation will be helpful for mulberry breeders to explore the genetic dissimilarity found in Morus species by crossbreeding using these resources. Acknowledgements Dr. Gnanesh B.N. acknowledges the Science and Engineering Research Board (SERB), New Delhi, for financial support (SERBSB/S2/RJN049/2015).

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6

Transcriptomics: Current Status and Future Prospects for Identifying Trait-Specific Genes in Mulberry K. H. Dhanyalakshmi, Shivasharanappa S. Patil, Tinu Thomas, H. V. Chaitra, Hari Singh Meena, M. Savitha, and Karaba N. Nataraja

MAPK

Abbreviations

PYL4 GPCR1 RLK PYL1 PP2C ATL CML 38 LIKE PBP1 CDPK

PYRABACTIN RESISTANCE1-LIKE 4 G protein-coupled receptor1 Receptor-like Kinase PYRABACTIN RESISTANCE1-LIKE 1 Protein Phosphatase 2C Arabidopsis Tóxicos en Levadura Calmodulin-LIKE 38 LIKE PID BINDING PROTEIN 1 (PBP1) (PID- PINOID) Calcium‐Dependent Protein Kinase

MAPKKK PAP GTP RLP Snf1-b AP2 NAM CUC HSF WRKY

TIFY K. H. Dhanyalakshmi
S. S. Patil
T. Thomas
H. V. Chaitra
H. Singh Meena
K. N. Nataraja (&) Plant Molecular Biology Laboratory, Department of Crop Physiology, University of Agricultural Sciences Bangalore, GKVK Campus, Bengaluru, India e-mail: [email protected] M. Savitha Department of Crop Physiology, College of Sericulture, University of Agricultural Sciences Bangalore, Chintamani Campus, Chintamani, India K. H. Dhanyalakshmi Department of Plant Breeding and Genetics, College of Agriculture, Padannakkad, Kerala Agricultural University, Kasaragod, India

IAA AIL SHN ERF RAP bZIP ARP GATA

Mitogen-Activated Protein Kinase Mitogen-Activated Protein Kinase Kinase Kinase Purple Acid Phosphatase Guanosine 5′-Triphosphate Receptor-Like Proteins Sucrose non-fermenting 1-beta APETALA2 No Apical Meristem Cup-Shaped Cotyledon Heat Shock Factor WRKY domain (W-Tryptophan, R-Arginine, K-Lysine, Y-Tyrosine) TFY domain (T-Threonine, I-Isoleucine, F-Phenylalanine, Y-Tyrosine) Indole-3-Acetic Acid AINTEGUMENTA-LIKE SHINE Ethylene-Responsive Factor RELATED TO AP2 Basic leucine zipper Auxin-responsive protein Recognize promoter elements with a G-A-T-A core sequence

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_6

149

150

AP2-EREBP

ERF bHLH MYB RING finger protein RAX

DREB LAF Sep 2 PCL1 AUX ARR TGA SPL MYC GBL EH2 GolS PER HSP TIP CAX ERO NRAMP

HSF UGT ERD NRT FADS PIP PEI

K. H. Dhanyalakshmi et al.

APETALA2/ethylene-responsive element binding protein Ethylene-Responsive Factor Basic Helix-Loop-Helix MYB DNA-binding domain Really Interesting New Gene finger protein REGULATOR OF AXILLARY MERISTEMS1 Dehydration-Responsive Element Binding Long After Far-Red Light Stress enhanced protein 2 PHYTOCLOCK 1 Auxin Arabidopsis Response Regulators TGACG motif-binding factor SQUAMOSA promoter binding protein-like MYC DNA-binding domain G-Box-Like-1 Epoxide hydrolase 2 Galactinol synthase Peroxidase Heat shock protein Tonoplast Intrinsic Protein Cation/proton exchangers Endoplasmic reticulum oxidoreductin-1 Natural Resistance-Associated Macrophage Protein Heat Stress Transcription Factors Uridine 5′-diphosphoglucuronosyltransferase Early Responsive to Dehydration Nitrate Transporter Fatty Acid Desaturase Plasma membrane Intrinsic Interacting Protein Pectin Esterase Inhibitor

CALS PAL

6.1

Callose synthase Phenylalanine ammonia-lyase

Introduction

Mulberry (Morus spp.) is a commercial crop cultivated for rearing domesticated silkworm (Bombyx mori L.). The tree is also known for its diverse nutritional and medicinal qualities, and different parts such as leaves, roots, bark and twigs are used in Chinese traditional medicine for the treatment of various ailments (Huang et al. 2013a; Yuan and Zhao 2017; Lim and Choi 2019; Jan et al. 2021). In addition to these, the tree is being used as feed and supplement for ruminants (Sanchez 2000), phytoremediation (Olson and Fletcher 1999) and biomass energy generation (Lu et al. 2009). Due to its wide array of applications across diverse sectors of life and industry, trees are proposed to play a remarkable role in sustainable development (Rohela et al. 2020). Besides these beneficial features, the perennial system is also known for its inherent traits such as rapid growth, deep root systems (Datta 2000) and adaptability to adverse environmental conditions (Liu and Willison 2013). The tree is fastgrowing with tremendous biomass production potential (Datta 2000) and is also known for several drought tolerance traits (Feng et al. 2016). With diverse unique features and traits adorning the system, along with the availability of omics information at multiple levels, and being amenable for genetic transformation, mulberry is proposed to be a promising tree model system (Dhanyalakshmi and Nataraja 2018). Recently, RNA sequencing (seq) or transcriptomic approaches are widely used in diverse systems to understand biological processes. Such attempts have also been made in mulberry, which is helping the researchers to prospect novel genes and pathways and also provides methods for both mapping and quantifying gene transcripts. In this chapter, the efforts made in understanding the molecular basis of two important traits in mulberry rapid growth and stress adaptation using transcriptomics are discussed.

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Transcriptomics: Current Status and Future Prospects for Identifying …

6.2

Transcriptomics in Mulberry

6.2.1 Growth-Linked Traits in mulberry—An Overview Mulberry trees are periodically pruned and maintained as a shrub for foliage production. Suitable short (3–4 months) to long-term (10– 12 months) rotation coppicing can be practised for foliage production depending on agroclimatic conditions, whereas intensive coppicing is practised under irrigated conditions (Suzuki et al. 1988; Dandin et al. 2003). Nevertheless, the trees are capable of generating harvestable biomass by six months of planting and established plantations yield up to 60–70 t foliage/ha depending on the variety (Datta 2000). Even in marginal lands, annual biomass production of 17–22.5 t/ha (dry matter) is reported which is higher than that of many fast-growing plant species (Lu et al. 2009). This rapid growth potential of mulberry is one of the most appreciated traits, as an ideal system to prospect growth-linked genes. The biomass accumulation is dependent on diverse morphophysiological aspects such as plant physiological status, pruning methods, bud break and dormancy, branching behaviour and leaf functional traits (Suzuki et al. 1988; Suzuki and Kitano 1989; Yamashitha 1990; Cao et al. 2019). Moreover, there exists large variation for growth traits among mulberry genotypes (Rukmangada et al. 2018). However, molecular mechanisms associated with the growth traits are poorly understood, and attempts to identify and characterize the key regulators and associated pathways from other related tree crops are required for manipulating growth traits in mulberry.

6.2.1.1 RNA-Seq to Understand Growth-Linked Traits Growth traits have a complex regulation due to their polygenic nature and their responsiveness to internal as well as external cues (Gonzalez et al. 2020). The temporal expression of specific or general function genes also adds to its

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complexity. Transcriptomic studies have been instrumental in revealing the molecular regulation of specific traits in a more targeted way enumerating key genes and linked pathways, on a global level. Studies in bamboo, a species with one of the fastest growth rates, reveal the prominent roles of genes encoding transcription factors (TFs), plant hormones, cell cycle regulation, cell wall metabolism and cell morphogenesis for rapid growth (Peng et al. 2013). Aquaporin PIP gene co-regulated by the hormones auxin and gibberellin is proposed to be a candidate gene behind fast growth (Peng et al. 2013). Transcriptomic studies in two Populus genotypes with contrasting stem growth rates show that 1542, 2295 and 2110 genes are differentially expressed during pre-growth, fast growth and post-growth stages, respectively. Genes linked with the regulation of the cell cycle and cell growth have a key role in determining stem growth. This includes cell wall- and membrane-linked genes, signalling molecules, genes associated with DNA replication and hormone transport. PeuBELL15, a BEL1-like TF is the regulator of growth which probably coordinates the expression of genes relevant for cellulose synthesis and lignin biosynthesis (Han et al. 2020). Transcriptomic information is available in other tree systems related to different aspects of growth such as growth heterosis in rubber (Yang et al. 2018), wood formation (Li et al. 2017a) and phase change in Larix kaempferi (Xiang et al. 2019) and cambial growth in Chinese fir (Wang et al. 2013). However, in mulberry, very few studies are available in this direction. In mulberry, the major growth-linked trait is rapid bud break and leaf biomass accumulation. Several mulberry genotypes have been classified as high-growth genotypes (HGG) and lowgrowth genotypes (LGG) based on the growth attributes including leaf biomass (Rukmangada et al. 2018). As per the transcriptomic study performed using the fully expanded leaves of contrasting mulberry genotypes for growth (HGGs-Jalalgarah-3 and M. laevigata (H), a hybrid, bred from M. laevigata (♀)  Kanva-2 (♂); LGGs-Harmutty and Vadagaraparai-2),

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photosynthesis and related metabolic genes are upregulated in fast-growing species, while the slow-growing species is characterized by the upregulation of genes involved in the biosynthesis of secondary metabolites (Rukmangada et al. 2019). The study generated 34,096 unique transcripts wherein 505 transcripts were significantly upregulated and 597 were significantly downregulated in HGG when compared to LGG. In both HGGs, genes related to cell, cell part, organelle and organelle part of gene ontology (GO) were higher as compared to Harmutty. In Vadagaraparai-2, genes related to GO terms membrane, membrane part, catalytic activity, binding, metabolic process and cellular process were higher. In both HGGs, pathways related to photosynthesis, photosynthesis protein and oxidative phosphorylation in the energy metabolism pathway were enriched. In LGGs, plant hormone signal transduction, transporter and transcription factors were over-represented. The better growth performance of the HGGs was correlated with the enrichment of genes linked to energy metabolism and better photosynthesis (Rukmangada et al. 2019). As per the study, genes linked with genetic information processing pathway (linked with transcription, translation, folding, sorting and degradation, replication and repair and RNA family) and active metabolic process (carbohydrate, energy, lipid, nucleotide, amino acid, metabolism of other amino acids, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides and biosynthesis of other secondary metabolites) are important for leaf growth and development in mulberry. In another study, a transcriptomic approach was used to examine the genetic basis of improved growth traits linked with autopolyploidization in mulberry. Over 600 transcripts (approximately 2.87 of the total genome sequences) were differentially expressed between autopolyploids and the natural diploids (Dai et al. 2015). Among these, 4.9% of the genes are linked with plant hormones, cytokinin, gibberellin and ethylene. Other differentially expressed and upregulated genes in autotetraploid mulberry include those specifically

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expressed in chloroplasts, cytochrome genes and photosystem-related genes. The leaf crosssection studies indicated an increase in cell size in autopolyploids, but not cell number. In nutshell, autotetraploid mulberry with better growth traits could be due to the upregulation of genes linked to the photosynthetic process, biosynthesis and signal transduction of plant hormones (Dai et al. 2015).

6.2.1.2 Prospecting Growth-Linked Genes—Leads from Other Systems While global studies in understanding growthlinked traits are still emerging in mulberry and other related systems, targeted studies have been intensive in model systems like Arabidopsis. A study using 710 natural accessions of Arabidopsis proposed many genes linked with cell proliferation (MCM4), growth signalling (REM1.2), epigenetic modification (ORC1), hormone signalling (PPD1), ABF3, etc., to be associated with growth traits (Gonzalez et al. 2020). AtHD2D, a histone deacetylase is a promising candidate to promote growth by enhanced vegetative growth period (Han et al. 2016). Several genes in Arabidopsis (transcriptional regulators, genes associated with protein synthesis and modification, hormonal regulation, cell wall extension and signalling pathways) classified as Intrinsic Yield Genes (IYGs) are known to enhance growth and produce more biomass, but require further validation (Gonzalez et al. 2009). In perennial systems, the growth processes are more complex. Unique growth-linked processes in trees like the activity-dormancy cycle and wood formation during the long-life cycle led to the identification of tree model systems like poplar (Taylor 2002), which are now helping us to understand new players in growth traits. EARLY BUD BREAK 1 (EBB1), one of the key regulators of apical bud break was identified in poplar (Yordanov et al. 2014). EBB1 is required to activate meristem activity after winter dormancy. EBB3 that activates the cell cycle after bud break is a positive regulator of bud break (Azeez et al. 2021). An endo‐b‐1,4‐glucanase

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(KORRIGAN (KOR1) gene) required for cellulose biosynthesis identified in poplar is also a modulator of growth (Maloney and Mansfield 2010). Tree growth and shoot architecture depend upon activation of apical as well as axillary buds. Many signals including hormonal signalling pathways converge on the transcription factors such as BRANCHED1 (BRC1), which controls branching by regulating axillary growth (Wang et al. 2019). However, the information on branching and apical bud break in perennials are fragmented, and the complete regulatory pathway is unknown. In trees like hybrid aspen, branching processes are mediated by mutually antagonistic action of the flowering regulators TERMINAL FLOWER 1 (TFL1) and APETALA1 (LIKE APETALA 1/LAP1). Through the grafting experiment, it was shown that LAP1 promotes branching through local action in axillary buds. Short photoperiod and low temperature suppress branching by simultaneous activation of TFL1 and repression of the LAP1 pathway (Maurya et al. 2020). Nevertheless, such studies are not reported in mulberry, and concerted efforts are required to prospect growth-related genes in mulberry.

6.2.2 Stress-Adaptive Traits— Prospecting Genes and Key Pathways As a perennial system, mulberry has a long lifespan and a crop cycle of more than 50 years (Vijayan et al. 2011) that expose the trees to a wide range of biotic and abiotic adversities. Abiotic stresses like drought, salinity and alkalinity can cause yield loss up to 50–60% in mulberry, while biotic stresses caused by fungi, bacteria, viruses and pests can reduce yields by 10–30% (Rao 2002). These stresses have a profound impact on leaf quality as well. However, many mulberry genotypes and varieties are known to perform better amid adverse conditions (Guha et al. 2010; Durgadevi and Vijayalakshmi 2020). Large variability has been observed in available germplasm resources for tolerance to biotic and abiotic stresses (Vijayan 2010;

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Banerjee et al. 2009). There is evidence to show that the crop has demonstrated excellent adaptability to harsh natural environments like drought (Huang et al. 2013b), flooding (Liu and Willison 2013), cold (Fujikawa et al. 2006) and heavy metals (Jiang et al. 2019). Mulberry leaf exudes are toxic to caterpillars other than B. mori (Konno et al. 2006). Transcriptomic studies are available on some of these aspects giving insights into the molecular regulation of these traits and options for prospecting trait-linked genes.

6.2.2.1 RNA-Seq to Understand Abiotic Stress Tolerance Initially, Expressed Sequence Tags (ESTs) were used for gene discovery in mulberry. Several stress-responsive genes were identified from an EST library generated from the mature leaf tissues of M. indica var. K2 and validated through expression analysis (Lal et al. 2009). These include early-responsive to dehydration proteinrelated (ERD3, ERD15), osmotin gene, halotolerance protein (HAL3), cold-regulated genes, glutamine synthetase, dehydrins, remorin, ABC transporters, dehydration-responsive protein (RD22), late embryogenesis abundant 3 family gene, cold acclimation-related WCOR413-like, drought-responsive family proteins, etc. Around 2400 ESTs were generated from the root tissues of the same variety, out of which 130 ESTs were common with that of the leaf, but with differential expression (Checker et al. 2012). The key root specific stress-responsive genes identified in this study are ethylene-forming enzyme (AT1G05010), pleiotropic drug-resistance protein 12 (AT1G15520), plant U box 22 (AT3G52450) and an MLP-like protein (AT1G24020). Subsequently, a Suppression Subtractive Hybridization (SSH) of contrasting mulberry genotypes (K2-drought sensitive; AR12-drought tolerant) provided a better understanding of the gene regulation behind drought tolerance (Gulyani and Khurana 2011). The stress-responsive ESTs identified in this study consisted of diverse families of TFs (zinc finger, NAM, bHLH, PHD finger, bZIP, MYB, AP2 and jumonji domain-containing proteins, with but

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zinc-finger proteins having more abundance); signal transduction pathway genes (calciumdependent protein kinase (CIPK16), armadillo/ beta-catenin repeat protein, SET domaincontaining protein, patatin-related gene), phosphorylation/dephosphorylation-linked genes (GHMP kinase, inositol 1, 3, 4 triphosphate5/6 kinase, leucine-rich transmembrane protein kinase, protein kinase family proteins), phosphatases (protein phosphatase 2C, dualspecificity protein phosphatase, trehalose 6 phosphate phosphatase), hormone responserelated genes (gibberellin-regulated family protein, auxin-responsive protein, IAA-alanine resistance protein 1 and ethylene-responsive protein 6), protein biosynthesis and regulationrelated genes (ribosomal proteins, genes homologous to elongation factors, translation initiation factors, splicing factors) and genes linked with modifying, sorting and degradation of proteins. The library was also enriched in genes linked with cellular detoxification, transport, growth and metabolism (Gulyani and Khurana 2011). Transcriptome sequencing strengthened the gene discovery programmes and assisted in revealing the molecular mechanisms governing abiotic stress tolerance in depth. Drought The first report on the transcriptomic study of mulberry (Morus multicaulis) under drought through de-novo sequencing and assembly of mulberry variety Yu71-1 using Illumina RNAseq technology generated 54,736 contigs (Wang et al. 2014). Out of this, 27,013 contigs were annotated against 10110 GO terms and 247 Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways. Metabolic pathways (carbohydrate metabolism, glycan biosynthesis and metabolism, amino acid metabolism) were most represented followed by secondary metabolites and microbial metabolic pathways, indicating active metabolic processes and synthesis of secondary metabolites in the leaf. The study identified 1051 differentially expressed genes although very few were validated (Dehydrin, EH2, AP2, NAC4, WRKY6, GBL, HSF30). All genes except WRKY6 showed upregulation

K. H. Dhanyalakshmi et al.

(Wang et al. 2014). The study paved the lead for understanding the important pathways operating in mulberry. In another drought-specific study using the leaf tissues of drought-tolerant mulberry (M. alba L.) genotype (Dudia white), a large number of stress-responsive upstream regulatory or downstream functional genes such as kinases, ribosomal proteins, membrane proteins, transporters and transcription factors (TFs) were identified (Dhanyalakshmi et al. 2016; Dhanyalakshmi 2018). However, this study focused more on proteins of unknown functions (PUFs), which will be discussed in the later session. The transcriptome data generated in this study was also used to identify several DNAmarkers (Thumilan et al. 2016). It is also demonstrated that drought stress induces extensive alternative splicing events in mulberry (Jeevitha 2022). The study by Li et al. (2017b) revealed that miRNAs have critical roles in regulating gene expression in mulberry under drought stress. The study performed transcriptome-wide high-throughput degradome sequencing and reported 30 conserved miRNA families with 409 targets, while 199 novel miRNAs targeted 990 mulberry genes under nonstress conditions. Under drought stress, the 30 conserved miRNA families targeted 373 genes, while 950 target genes were identified for 195 novel miRNAs. Conserved miRNA families such as mno-miR156, mno-miR172 and mno-miR396 target 54, 52 and 41 transcripts, respectively, under drought, and most of these were TF genes. And these targets were consistent with the information from other systems (Li et al. 2017a, b). In addition to these, there is targeted expression analysis to prospect genes linked to drought tolerance (Mamrutha et al. 2017; Sajeevan and Nataraja 2016). Many drought-responsive ESTs have been identified and deposited in the NCBI database such as basic helix-loop-helix 144-like (KP732520.1) (Anju 2016), ethylene-responsive transcription factor RAP 2.3-like (KR778884.1) (Sankranthi 2018), nuclear transcription factor Y subunit A-1-like (Accession- KR150685.1), DEAD-box ATP-dependent RNA helicase 3-like (KT004595.1), NAC domain-containing transcription factor 78-like (KR063110.1),

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WRKY21-like and MaUSP1-like (Dhanyalakshmi and Nataraja 2021) (https://www.ncbi.nlm. nih.gov/nuccore). Salinity Mulberry is considered to be moderately tolerant to salinity (Vijayan et al. 2008). In-vitro studies using auxiliary buds have revealed that mulberry genotypes have large variability in its response to salinity (Vijayan et al. 2003). A significant correlation was observed for their performance exvitro. There is little information available regarding the molecular mechanisms underlying its salinity tolerance. Transcriptome analysis performed in mulberry accessions contrasting in salt tolerance (high-salt-tolerant accessionsCS6H and CS12H; low-salt-tolerant accessionsCS6L and CS12L) and in different plant parts (root, stem and leaves) generated 101,589 unigenes from 180,694 transcripts, wherein 34,273 transcripts (34.72%) were annotated at least in one public database (Liu et al. 2017). The differentially expressed genes under salt stress include heat shock proteins, stress-related TFs, ROS scavenging system-related genes and several receptor-like protein kinases. Among these, a greater number of DEGs were downregulated (mostly genes related to stress response). Genes encoding auxin binding protein, acid betafructofuranosidase, aquaporin, 3-oxoacyl-[acyl-carrierprotein] synthase II, methylesterase 17, rhodanese-like domain-containing protein 10, protein SOMBRERO, receptor-like protein kinase FERONIA, polyphenol oxidase and beta1,3-glucanase) were most differentially expressed in low-salt-tolerant genotypes under salt stress. Downregulation of the genes linked with “sulphur metabolism” and “cysteine and methionine metabolism” indicates a probable adaptation strategy against long-term stress conditions. The DEGs of low-salt-tolerant genotypes were enriched with genes associated with signal transduction and transcription regulation, with decreased transcript abundance. Enrichment of DEGs with genes involved in “oxidation–reduction process”, “peroxidase activity”, “oxidoreductase activity”, “cysteine and methionine

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metabolism” and “plant hormone signal transduction” indicates a late but stronger stress response in the low-salt-tolerant genotypes. The low-salt tolerance is attributed to the lower expression levels of genes linked with signal transduction and transcription regulation (Liu et al. 2017). The study highlights the relevance of aquaporins and heat shock proteins under salt stress in mulberry and forms a base for further enrichment of the understanding of salt stress tolerance in mulberry. Cold The cold responsiveness is poorly studied in mulberry. Transcriptomic study in this direction is necessary for understanding the molecular regulation and identification of key genes and pathways for improving cold tolerance in mulberry. Cold stress-responsive transcriptome in a M. alba genotype (Yu711) identified 3593 significantly expressed genes, out of which 2337 were upregulated genes and 1256 were downregulated. These genes corresponded to plant hormone signal transduction, MAPK signalling pathway, ubiquinone and another terpenoidquinone biosynthesis, nitrogen metabolism and biosynthesis of secondary metabolites (Adolf et al. 2021). The important upregulated DEGs were dehydration-responsive element-binding protein, adagio protein 3, germin-like protein, linoleate 13S-lipoxygenase and uncharacterized LOC21395589, while BURP-domain protein RD22-like, ethylene-responsive transcription factor ERF017, licodione synthase and UDPglycosyltransferase were significantly downregulated. Genes linked to hormonal signalling (TIFY genes) and TFs (AP2/ERF, WRKY, bZIP, GATA, HSF families) are important in stress response. The study suggests that hydrolases like glucan endo-1,3 b-glucosidase and b-amylase have a significant function under cold stress. There was an upregulation genes linked with cell wall organization or biogenesis such as endochitinase, COBAS genes, encoding pectinesterase inhibitors, mulatexin and galactoside 2alpha-L fucosyltransferase to protect the cells intact under cold stress. The study suggests that

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the genes linked to hydrolase activity, cell wall biogenesis and plant hormone signalling network are important for cold stress response in mulberry. Heavy Metals Mulberry trees that exhibit tolerance to heavy metals like copper (Cu), cadmium (Cd), zinc (Zn) and lead (Pb) (Zhao et al. 2013; Pehluvan et al. 2012; Jiang et al. 2019) have immense potential for phytoremediation of soils contaminated with heavy metals (Olson and Fletcher 1999; Jiang et al. 2019), without significant impact on the silkworm (Si et al. 2021). In mulberry seedlings, Cd significantly inhibits growth in higher concentrations while inducing antioxidant enzymes in shoots and roots in lower concentrations (Dai et al. 2020). The majority of the accumulated Cd get compartmentalized in the soluble fraction in roots and the cell walls of leaves and stem. Cd also integrates with the proteins and pectates in tissues. Cd stress (5, 10, 20 µM respectively) induced 178 (53 upregulated, 125 downregulated), 307 (161 up, 146 down) and 510 (235 up, 275 down) differentially expressed genes, respectively, among which 27 were common under all concentrations (Dai et al. 2020). Stress-activated pathways for mRNA regulation, stress response and signalling while secondary metabolism, hormone regulation and cell wall-related pathways were negatively regulated. Upstream signalling genes (calmodulinbinding transcription activator, protein kinase family proteins and G-proteins) and transcription factors (MYB, bHLH, WRKY, AP2 and zincfinger proteins) are shown to have a significant role in stress response. Downstream functional proteins like heat shock proteins are important candidates to engineer stress tolerance as they can protect proteins from stress-induced damages like misfolding, aggregation and premature degradation. There was an increased activity of the enzymatic antioxidants (SOD, POD, CAT, PAL and PPO) in leaves and roots indicating mechanisms protecting the cell from toxicity. Genes associated with several pathways, mainly including phenylpropanoid, flavonoids and terpenoids metabolites, were differentially regulated

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in response to heavy metal stress indicating its role under stress. In short, Cd detoxification in mulberry was operated through signalling and transcriptional regulation events. However, candidate genes conferring tolerance need to be identified and validated. In another study using two varieties of mulberry contrasting for Cd accumulation (low-Cd Zhehulusang (zhls) and high-Cd mulberry Xianmianzao (xmz)), 2785 and 1211 differentially expressed genes (DEGs) were identified in zhls and xmz, respectively (Jiang et al. 2020). Pathways such as flavonoid biosynthesis, photosynthesis, carbon fixation, porphyrin and chlorophyll metabolism and biosynthesis of secondary metabolism enriched the DEGs of zhls, while xmz was enriched with pathways of flavonoid biosynthesis, plantpathogen interaction and phenylpropanoid biosynthesis and phenylalanine metabolism. The enrichment of flavonoid biosynthesis pathway genes in both varieties indicates a conserved mechanism for tolerating stress in mulberry (Jiang et al. 2020).

6.2.2.2 RNA-Seq to Understand Biotic Stress Tolerance One of the major diseases affecting mulberry is the bacterial wilt caused by Ralstonia solanacearum. The transcriptomic study using bacterial wilt-resistant commercial cultivars of mulberry (M. atropurpurea) (KQ10 and YS283) and the susceptible cultivar YSD10 gives an insight into the molecular mechanisms behind disease resistance. The infection of R. solanacearum caused a differential regulation of 798 genes in resistant cultivars wherein 502 were upregulated and 296 were downregulated (Dai et al. 2016). The resistant cultivars were characterized by the specific regulation of genes encoding protein kinases, TFs and genes associated with cell wall modifications. Protein kinases involved 32 genes encoding serine or threonine-protein kinases out of which 27 were upregulated and 5 were downregulated, 11 receptor kinases (4 upregulated and 7 downregulated), mitogen-activated protein kinase kinase kinase, calcium-dependent protein kinase and SNF1 protein kinase. Specifically regulated TFs in tolerant cultivars include

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zinc finger, AP2, auxin related, NAC, HSF and other minor TFs (TGA family, bHLH family, TRY, MADS-box protein, stress-enhanced protein 2 (Sep2) and PCL1-like). Most interestingly, the resistant cultivars were characterized by the differential regulation of a large number of genes associated with cell wall modifications (glucosyltransferase family, pectinesterase inhibitor, glucanase and glycoprotein families) indicating its role in disease resistance (Dai et al. 2016). In another study using a resistant (KQ10) and susceptible cultivar (YSD10), pathways like plantpathogen interactions, biosynthesis of secondary metabolites and starch and sucrose metabolismrelated genes were differentially expressed. The study also indicated that flavonoids have an important role in stress tolerance in tolerant genotypes (Dai et al. 2019a). Besides the leaves, mulberry fruits have gained considerable economic importance. Fruit production has been affected by several devastating diseases. Efforts to understand the molecular basis of disease resistance are still emerging in mulberry. Recently, omic platforms have elaborated the molecular regulation of some of the diseases. One of them is a sclerotial disease in mulberry, and most M. alba L. varieties are known to be susceptible to the disease (Lv et al. 2021). Three pathogens are causing sclerotial disease, namely Ciboria carunculoides, Scleromitrula shiraiana and Ciboria shiraiana (Lv et al. 2021). The fruits of the mulberry cultivar (M. atropurpurea), YSD10, are popular in China. The fruit transcriptome of YSD10 analysed by Dai et al. (2019b) in two different post-infection stages by C. carunculoides (stage1: diseased fruit showing partial protrusion and the individual achenes showed global swelling; stage 2: the achenes turned brown) revealed that 9% of the transcriptome was differentially regulated after infection (1801 genes). Post-infection, 792 (430 up and 362 downregulated) and 1496 (677 up and 819 downregulated) DEGs were identified at stage 1 and stage 2, respectively. About 487 genes were common at both stages. These DEGs corresponded to plant-pathogen interactions,

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plant hormone signal transduction, phenylpropanoid biosynthesis, starch and sucrose metabolism and flavonoid biosynthesis pathways. The plant hormone signal transduction pathway genes were associated with brassinosteroid (BR) (17 genes including BAK1 or BRI1), abscisic acid (ABA) (PYL/PYR, PP2C), gibberellic acid (GA) (GID1), ethylene, cytokinin and auxin (AUX/IAA) signalling. The infection triggered calcium-mediated defence signalling pathways evident with the upregulation of 19 genes coding for calmodulin. Other signalling pathway genes involve protein kinases (MAPKs, particularly MAPKKK and MAPKK), flagellinsensing 2-like protein kinase (FLS2), serine/threonine-protein kinase PBS1 and brassinosteroid insensitive 1-associated receptor kinase 1. TFs accounted for 5.7% (104 DEGs, 65upregulated and 39 downregulated) of the differentially regulated genes. These include TFs from AP2 family (23 DEGs), MYB (15 DEGs), zinc finger (12 DEGs), WRKY (8 DEGs) and bHLH (8 DEGs). TFs related to stress response (AP2, MYB, bHLH and C2H2 zinc-finger families) were upregulated, while most developmentrelated TFs (AUX/IAA and squamosa promoter binding protein (SPB)) were downregulated (Dai et al. 2019b). Most of the genes related to photosynthesis and cellular growth (histone and the cytoskeleton proteins tubulin and actin) were downregulated post-infection. In summary, C. carunculoides infection induced pathways like plant hormone signal transduction and calciummediated defence signalling, while suppressing photosynthesis and cellular growth-related metabolism (Dai et al. 2019b). Mulberry (M. atropurpurea) cultivar Hongguo2 was used to study the molecular-level responses to C. shiraiana infection. Transcriptome analysis of healthy (CK), early-stage diseased (HB1) and middle-stage diseased (HB2) mulberry fruits generated a total of 5109 transcripts (926 up and 3183 downregulated) and 6760 (2655 up to and 4105 downregulated) DEGs for HB1 and HB2, respectively, when compared to control (Bao et al. 2020), and 3315 genes were common for both stages. Among the

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DEGs, genes are related to plant hormone signal transduction, transcription factors and phenylpropanoid biosynthesis. The most enriched top three highest significantly enriched KEGG pathways were flavonoid biosynthesis, plant hormone signal transduction and phenylpropanoid biosynthesis. Phenylpropanoid biosynthesis is suggested to play important role in mulberry response against the pathogen (Bao et al. 2020).

6.2.3 Other Important RNA-Seq Studies There are a few other important studies in mulberry at the transcriptome level. These include the studies on mulberry latex, flavonoid biosynthetic pathway and female flower development. Mulberry produces latex as a defence system against microbes and herbivores. Latex generally contains a large number of proteins such as peptidases, peptidase inhibitors, chitinases and anti-oxidative enzymes and is associated with various biological functions including transcription, translation, protein degradation and response to environmental stimuli (Cho et al. 2014). Transcriptome analysis of the latex led to the identification of genes encoding several proteins including peptidases, lectins, annexin, pathogenesis-related proteins etc. (Kitajima et al. 2012). This study is a lead to a prospect and characterize latex-related genes from mulberry. The transcriptomic study on flavonoid biosynthetic pathway along with metabolome profiling revealed that the flavanol 3-O-glucoside-Orhamnosyltransferase (FGRT) is the key enzyme during rutin biosynthesis in mulberry (Li et al. 2020a). The transcriptomic study on female flower development in mulberry highlights the relevance of AP2/ERF and MADS-box genes in floral development. The co-expression analysis suggested that nutrient reservoir, phosphate signal and secondary metabolism may play a key role in floral development (Shang et al. 2017). This information on female flower development regulation would be promising for mulberry

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breeding and biotechnology. Table 6.1 presents an overview of the important transcriptomic studies in mulberry.

6.2.4 Targeted Studies in Prospecting Trait-Linked Genes Several attempts have been made in the last decade in understanding and exploring the stressadaptive mechanisms in mulberry (Mamrutha et al. 2017; Sajeevan and Nataraja, 2016). This has led to the discovery and characterization of diverse families of stress-responsive genes linked with specific traits. Such genes can be used in improving mulberry traits as well as in other related systems. Some of the case studies are summarized below. One of the important candidate genes identified from mulberry is MiREM, a member of plant-specific family of proteins called remorins linked with stress response in plants (Checker and Khurana 2013). MiREM has ubiquitous expression in mulberry, with higher expression levels in mature leaves and stems. Arabidopsis plants overexpressing MiREM are tolerant to dehydration and salt stress at germination and seedling stages (Checker and Khurana 2013). A Shaggy-like protein kinase (SK), MmSK, was identified from M. alba, and the gene was upregulated under various abiotic stresses (Li et al. 2018a). SKs are associated with diverse aspects of plant growth, development and its response to the external environment. Virusmediated MnSK-silenced mulberry plants became susceptible to drought indicating the relevance of the gene (Li et al. 2018a, b). Another candidate is heterotrimeric guaninenucleotide binding proteins (G-proteins), which impart drought and salinity tolerance by modulation of reactive oxygen species (Liu et al. 2019). Several candidate TFs have been characterized in mulberry. The novel basic helix–loop– helix (bHLH) transcription factors associated with abiotic stress response in mulberry have been cloned and partially characterized (Sajeevan

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Table 6.1 Summary of transcriptome analysis in mulberry Species and tissue

Platform

SRA No/BioID

Enriched gene or pathways

Reference

I. Growth-linked transcriptomes Morus indica

Illumina HiSeq2500

SRX5028920 SRX5028919 SRX5028918 SRX5028917

Energy metabolism (photosynthesis) and secondary metabolite pathway

Rukmangada et al. (2019)

Morus alba

Illumina HiSeq2000

GSE70428

Photosynthesis, hormone signalling, plant-pathogen interaction, secondary metabolites

Dai et al. (2015)

II. Abiotic stress-related transcriptomes Drought Morus multicaulis (Leaves)

Illummina HiSeq 2000

–

Biosynthesis of secondary metabolites; carbohydrate metabolism glycan biosynthesis and metabolism, amino acid metabolism

Wang et al. (2014)

M. alba (Young leaves)

Illumina HiSeq2000

GEO database: GSE84889

miRNA, TFs, metabolic transporters, signal transduction factors

Li et al. (2017a, b)

M. alba (Leaves)

454 Genome Sequencer FLX Titanium system

NCBI SRA: SRP047446

Upstream signalling pathway genes, TFs, genes encoding functional proteins, PUFs

Dhanyalakshmi et al. (2016)

Illumina HiSeq 2000

Morus DB http://morus.swu. edu.cn/bio/ morusdb/data/ transcriptome

TFs, Ca & ABA signalling, oxidation–reduction process, peroxidase activity and oxidoreductase activity

Liu et al. (2017)

Illumina HiSeq 2500

–

Plant hormone signal transduction, MAPK signalling pathway, ubiquinone and biosynthesis of secondary metabolites

Adolf et al. (2021)

Salinity M. alba (Root, leaves and stem)

Cold M. alba (Young leaves)

Heavy metal (cadmium) Morus alba (Leaves)

Illumina HiSeq

Database accessions: SRP057498

Flavonoid biosynthesis, plantpathogen interaction, phenylpropanoid biosynthesis, porphyrin and chlorophyll metabolism, phenylalanine metabolism

Jiang et al. (2020)

M. atropurpurea (Roots, stems and leaves)

BGISEQ-500

NCBI GEO submission number GSE152672

mRNA regulation, metabolism of flavonoids, phenylpropanoids and terpenoids

Dai et al. (2020)

TFs, protein kinases, cell wall modification

Dai et al. (2016)

III. Biotic stress-related transcriptomes Bacterial wilt (Ralstonia solanacearum) Morus atropurpurea (Roots)

Illumina HiSeq 2000

GEO database: GSE60030

(continued)

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Table 6.1 (continued) Species and tissue

Platform

SRA No/BioID

Enriched gene or pathways

Reference

Morus atropurpurea (Roots)

Illumina HiSeq 2000

GEO database: GSE60030

Flavonoid biosynthesis (chalcone synthase, chalcone isomerase and flavonoid 3’-hydroxylase), plantpathogen interactions, biosynthesis of secondary metabolites and starch and sucrose metabolism

Dai et al. (2019a)

Activation of plant hormone signal transduction, calcium-mediated defence signalling pathways, suppression of photosynthesis and cellular growth

Dai et al. (2019b)

–

Phenylpropanoid biosynthesis, TFs, plant hormone signal transduction, eugenol enrichment

Bao et al. (2020)

DDBJ Sequence Read Archive accession number DRA000412

Jacalin like lectin, peroxidase, class I chitinase, b-1,3-glucanase, galacturonase-inhibitor protein and thaumatin-like protein

Kitajima et al. (2012)

SRR2868676

Hormone signal transduction AP2/ERF and MADS Box gene families

Shang et al. (2017)

(MMHub; https://biodb.swu. edu.cn/mmdb/

Flavonoids biosynthesis mapping and biosynthesis of rutin

Li et al. (2020a)

Sclerotial disease (Ciboria carunculoides) Morus atropurpurea (Fruits)

Illumina HiSeq 2000

GEO database: GSE111319

Sclerotial disease (Ciboria shiraiana) Morus atropurpurea (Fruits)

BGISEQ-500

IV. Other studies Herbivores and microorganisms Morus alba L. cv. Minamisakari (Petiole latex)

GS-FLX Titanium sequencer

Flower development Morus alba (Floral bud)

Illumina HiSeq2500

Flavonoid biosynthesis M. notabilis (Leaves)

Illumina HiSseq 4000

and Nataraja 2016). The authors isolated 1512 bp of bHLH-like gene and its transcript variant of 1419 bp. The genomic clone of MabHLH144-like is 2304 bp consisting of a single 786 bp intron at the 5′ UTR region, and the expression analysis indicated differential expression of the transcript variants in diverse tissue types and also under different abiotic stresses (Sajeevan and Nataraja 2016). MnDRED4A is a member of dehydrationresponsive element binding (DREB) family TFs that enhances tolerance to heat, cold, drought and salt stress in tobacco (Liu et al. 2015).

LncRNAs are known to be important regulators of various biological processes and also regulate plant response to environmental cues. MuLnc1 identified from M. multicaulis is cleaved by mul-miR3954 to produce secondary siRNAs (si161579) that can silence the expression of a gene encoding calmodulin-like protein (CML27). Heterologous expression of the gene in Arabidopsis improved its resistance to diseases, salinity and drought. MuLnc1 is considered to be a potential target for the improvement of mulberry (Gai et al. 2018). Likewise, miR166f (M. alba) has a target of homeobox-leucine

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Transcriptomics: Current Status and Future Prospects for Identifying …

161

zipper (HD-Zip) transcription factors and histone arginine demethylase, which are involved in responses to heat, cold, drought and salt stresses. The transgenic mulberry expressing miR166f was susceptible to drought, indicating it to be a positive regulator of drought tolerance (Li et al. 2018b). Mulberry is known for its potential for phytoremediation of heavy metal-contaminated soils. But genes like phytochelatin synthase (PCS) associated with the detoxification of heavy metals in other plant species are not yet validated in mulberry. Two PCS genes (MnPCS1 and MnPCS2) were identified from M. notabilis through genome-wide analysis and induced in root, stem and root tissue on exposure to Zn2+ and Cd2+ stress. Overexpression of the genes in Arabidopsis and tobacco model systems enhanced its tolerance to Zn2+ and Cd2+ stress and accumulation, indicating their phytoremediation potential (Fan et al. 2018).

6.2.5 RNA-Seq Studies Indicated Many Proteins of Unknown Functions (PUFs) in Mulberry

Fig. 6.1 Representation of the important differentially expressed genes linked to stress response in mulberry (Deduced from transcriptomic studies of Dhanyalakshmi et al. 2016; Wang et al. 2014; Liu et al. 2017; Adolf et al.

2021; Dai et al. 2016; 2020; Bao et al. 2020). Red (font colour) indicates upregulated genes; Blue (font colour) indicates downregulated genes, and ? indicates unidentified genes

Transcriptomic studies in mulberry have revealed the presence of a large number of genes with unknown functions. Genes encoding uncharacterized, hypothetical and unknown proteins have been classified as proteins of unknown functions (PUFs) (Dhanyalakshmi et al. 2016). Universally, PUFs constitute a large proportion of the transcripts identified in transcriptomes. In mulberry, about 23% of the water stress-induced genes in the SSH library of generated from mulberry genotypes contrasting for drought tolerance coded for unknown genes (Gulyani and Khurana 2011). About 40% of the mulberry ESTs annotated using corresponding Arabidopsis gene in the three categories each in biological process, cellular component and

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molecular function was annotated to be unknown. There was a high representation of unknown genes in the EST library from root tissue (Checker et al. 2012). Many of these genes exhibited significant expression under different abiotic stresses. The Proteins of Unknown Functions Annotation Server (PUFAS) available at http://caps.ncbs.res.in/ pufas/ specifically annotates PUFs from sequencing platforms using a sequence and structure-based strategy and can give a lead for functional validation experiments (Dhanyalakshmi et al. 2016) (Fig. 6.1).

6.3

Conclusion and Perspectives

As an important cash crop with multiple unique traits and features, mulberry is an excellent system to prospect trait-specific genes. A few targeted studies have been attempted to identify novel genes (Checker et al. 2012; Sajeevan and Nataraja 2016; Gai et al. 2018). However, comprehensive investigations at multiple omics levels are important for the complete understanding of the molecular regulation of the desired trait. For example, the information generated on flavonoid biosynthesis in mulberry at the genomic level (He et al. 2013) was complemented with metabolomics and transcriptomics (Li et al. 2020a). Integrated transcriptomic and metabolomic analysis contributed to an in-depth understanding of phenylpropanoid biosynthesis in mulberry fruit in response to devastating sclerotiniose pathogen infection and the potential of ugenol/isoeugenol enrichment in the fruit as a novel method for its control (Bao et al. 2020). A comprehensive genomic and transcriptomic information is available at the Morus Genome Database (MorusDB) (Li et al. 2014) and metabolomic data available at MMHub; Li et al. 2020b). There have been success stories on mulberry transformation with the gene of interest (Sajeevan et al. 2017; Li et al. 2018a, b). Hence, there is scope for intensive studies to understand the molecular regulation of specific traits and prospect specific genes.

Acknowledgements NNK acknowledges the Department of Biotechnology, Government of India, for financial support (BT/TDS/121/SP20276/2016).

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164 Lal S, Ravi V, Khurana JP, Khurana P (2009) Repertoire of leaf expressed sequence tags (ESTs) and partial characterization of stress-related and membrane transporter genes from mulberry (Morus indica L.). Tree Genet Genomes 5(2):359–374 Li T, Qi X, Zeng Q, Xiang Z, He N (2014) MorusDB: a resource for mulberry genomics and genome biology. Database Li R, Chen D, Wang T, Wan Y, Li R et al (2017a) High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberry (Morus alba). PLoS ONE 12(2): e0172883 Li WF, Yang WH, Zhang SG, Han SY, Qi LW (2017b) Transcriptome analysis provides insights into wood formation during larch tree aging. Tree Genet Genomes 13(1):19 Li R, Fan T, Wang T, Dominic K, Hu F et al (2018a) Characterization and functional analysis of miR166f in drought stress tolerance in mulberry (Morus multicaulis). Mol Breed 38(11):1–4 Li R, Liu L, Dominic K, Wang T, Fan T et al (2018b) (2018a) Mulberry (Morus alba) MmSK gene enhances tolerance to drought stress in transgenic mulberry. Plant Physiol Biochem 132:603–611 Li D, Chen G, Ma B, Zhong C, He N (2020a) Metabolic profiling and transcriptome analysis of mulberry leaves provide insights into flavonoid biosynthesis. J Agricul Food Chem 68(5):1494–1504 Li D, Ma B, Xu X, Chen G, Li T et al (2020b) MMHub, a database for the mulberry metabolome. Database Lim SH, Choi CI (2019) Pharmacological properties of Morus nigra L. (black mulberry) as a promising nutraceutical resource. Nutrients 11(2):437 Liu Y, Willison JM (2013) Prospects for cultivating white mulberry (Morus alba) in the drawdown zone of the three gorges reservoir. China. Environ Sci Pollut Res 20(10):7142–7151 Liu XQ, Liu CY, Guo Q, Zhang M, Cao BN et al (2015) Mulberry transcription factor MnDREB4A confers tolerance to multiple abiotic stresses in transgenic tobacco. PLoS ONE 10(12):e0145619 Liu CY, Liu XQ, Long DP, Cao BN, Xiang ZH et al (2017) De novo assembly of mulberry (Morus alba L.) transcriptome and identification of candidate unigenes related to salt stress responses. Russ J Plant Physiol 64 (5):738–748 Liu C, Xu Y, Feng Y, Long D, Cao B et al (2019) Ectopic expression of mulberry G-Proteins alters drought and salt stress tolerance in tobacco. Int J Mol Sci 20(1):89 Lu L, Tang Y, Xie JS, Yuan YL (2009) The role of marginal agricultural land-based mulberry planting in biomass energy production. Renew Energy 34 (7):1789–1794 Lv Z, Hao L, Ma B, He Z, Luo Y et al (2021) Ciboria carunculoides suppresses mulberry immune responses through regulation of salicylic acid signalling. Front Plant Sci 12 Maloney VJ, Mansfield SD (2010) Characterization and varied expression of a membrane-bound endo-b-1, 4-

K. H. Dhanyalakshmi et al. glucanase in hybrid poplar. Plant Biotechnol J 8 (3):294–307 Mamrutha HM, Nataraja KN, Rama N, Kosma DK, Mogili T et al (2017) Leaf surface wax composition of genetically diverse mulberry (Morus sp.) genotypes and its close association with expression of genes involved in wax metabolism. Curr Sci 25:759–766 Maurya JP, Miskolczi PC, Mishra S, Singh RK, Bhalerao RP (2020) A genetic framework for regulation and seasonal adaptation of shoot architecture in hybrid aspen. Proc Natl Acad Sci U S A 117 (21):11523–11530 Olson PE, Fletcher JS (1999) Field evaluation of mulberry root structure with regard to phytoremediation. Bioremediat J 3(1):27–34 Pehluvan M, Karlidag HU, Turan M (2012) Heavy metal levels of mulberry (Morus alba L.) grown at different distances from the roadsides. J Anim Plant Sci 22 (3):665–670 Peng Z, Zhang C, Zhang Y, Hu T, Mu S et al (2013) Transcriptome sequencing and analysis of the fast growing shoots of moso bamboo (Phyllostachys edulis). PLoS ONE 8(11):e78944 Rao AA (2002) Conservation status of mulberry genetic resources in India. Paper contributed to expert consultation on promotion of global exchange of sericultural germplasm resources, Satellite session of 19th ISC Congress. Bangkok, Thailand, pp 21–25 Rohela GK, Muttanna PS, Kumar R, Chowdhury SR (2020) Mulberry (Morus spp.): an ideal plant for sustainable development. Trees For People 100011 Rukmangada MS, Ramasamy S, Sivaprasad V, Varkody GN (2018) Growth performance in contrasting sets of mulberry (Morus Spp.) genotypes explained by logistic and linear regression models using morphological and gas exchange parameters. Sci Hortic 235:53–61 Rukmangada MS, Sumathy R, Naik VG (2019) Functional annotation of mulberry (Morus spp.) transcriptome, differential expression of genes related to growth and identification of putative genic SSRs, SNPs and InDels. Mol Biol Rep 46(6):6421–6434 Sajeevan RS, Nataraja KN (2016) Molecular cloning and characterization of a novel basic helix–loop–helix-144 (bHLH144)-like transcription factor from Morus alba L. Plant Gene 5:109–117 Sajeevan RS, Nataraja KN, Shivashankara KS, Pallavi N, Gurumurthy DS et al (2017) Expression of Arabidopsis SHN1 in Indian mulberry (Morus indica L.) increases leaf surface wax content and reduces postharvest water loss. Front Plant Sci 8:418 Sanchez MD (2000) World distribution and utilization of mulberry, potential for animal feeding. In: FAO electronic conference on Mulberry animal production (Morus1-L), p 111 Sankranthi S (2018) Functional validation of RAP2. 3like, an Apetala2/Ethylene Responsive Factor (AP2/ERF) identified from Indian mulberry (Morus alba L.). Dissertation, University of Agricultural Sciences, Bengaluru

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7

Proteomics in Mulberry Liu Yan, Lin Tianbao, Zhang Cankui, and Lv Zhiqiang

DEPs

Abbreviations

2D PAGE

AKR2A BmNPV CAT Cd CEF COI1 DAPs

Two-dimensional polyacrylamide gel electrophoresis Ankyrin-repeat containing protein 2 A Bombyx mori nuclear polyhedrosis virus Catalase Cadmium Cyclic electron flow Coronatine insensitive1 Differentially accumulated proteins

L. Yan
L. Tianbao
L. Zhiqiang (&) Sericultural and Tea Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China e-mail: [email protected] L. Yan e-mail: [email protected] L. Tianbao e-mail: [email protected]

DNJ ER ET IEF /SDS-PAGE

iTRAQ JA JAZ MD PSII ROS RuBP SA SE SOD TMT TrxM4 Trx-Prx TrxX UV-B

Differentially expressed proteins 1-Deoxynojirimycin Endoplasmic reticulum Ethylene Isoelectric focusing-sodium dodecyl sulfate–polyacrylamide gel electrophoresis Isobaric tags for relative and absolute quantification Jasmonic acid (JA) JA ZIM-domain Mulberry yellow type dwarf disease Photosystem II Reactive oxygen species Ribulose-1,5-bisphosphate Salicylic acid (SA), Sieve element (SE) Dismutase Tandem mass tag-based proteomics analysis Thioredoxin M4 Thioredoxin-peroxiredoxin Thioredoxin X Ultraviolet-B

Z. Cankui Department of Agronomy and Purdue Center for Plant Biology, Purdue University, West Lafayette, IN 47907, USA e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_7

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7.1

L. Yan et al.

Introduction

Mulberry tree (Morus alba, Moraceae) is an economically important perennial woody species that is cultivated all across the world. The leaves of mulberry have been used as solo feed for the monophagous silkworm for more than 5000 years (He et al. 2013), which plays a vital role in sustaining the development of the sericultural industry. Because of its great healthbeneficial nutrients and secondary bioactive metabolites, various parts of mulberry, such as leaves, fruits, and seeds, are either directly consumed as a functional food ingredient or as raw material for pharmaceutical chemicals (Ramappa et al. 2020; Chen et al. 2021a). Additionally, the mulberry plant is also adopted in landscaping and natural environment due to its considerable ecological importance (Qin et al. 2010; Liu and Willison 2013; Fan et al. 2014). With its characteristics of rapid growth and biomass production and plant–insect interaction, mulberry is also treated as a potential model species for trees (Dhanyalakshmi and Nataraja 2018). The proteomic technique offers one of the best options to investigate the physiological mechanisms of superior cultivars under different regimes (Komatsu 2019). It provides opportunities for systematically functional analysis of the protein expression pattern changes in an organism (Khurana and Checker 2011). Since the mulberry genomes have been successfully sequenced, an interesting exploration is to identify the proteome of all predicted genes. Various proteomics methods, such as SDS-PAGE, twodimensional polyacrylamide gel electrophoresis (2D-PAGE), isobaric tags for relative and absolute quantification (iTRAQ), and tandem mass tag-based proteomics analysis (TMT), have been used to profile proteins (Tang et al. 2016; Liu et al. 2019b). These works have facilitated our understanding of the physiological mechanisms at the translational level. In this review, we focus on the current mulberry proteomics with functions in growth, development, biotic and abiotic stresses, and interactions between mulberrysilkworm. We also discussed the challenges and

perspectives for future improvement of mulberry productivity and adaptation. An overview of current proteomics in mulberry was shown in Table 7.1.

7.2

Proteomics of Different Mulberry Tissues

Different tissues of mulberry plants vary with the constitutions of secondary metabolites, such as flavonoids, alkaloids, terpenoids, polysaccharides, phenolic acids, coumarins, and stilbenoids (Chan et al. 2016). Proteomics approach becomes increasingly important in uncovering the molecular mechanisms in relation to the accumulation of different compounds in the leaf, branch, and root (Zhu et al. 2019).

7.2.1 Leaf Mulberry leaves are a type of traditional Chinese medicine and are the sole food source for the domesticated silkworm for thousands of years. The nutrition of the mulberry leaf plays important role in the growth of silkworm. Using 2Dgel electrophoresis, Hirano (1982) found that the protein profiles in leaves of 17 varieties of mulberry differed. Little differences were found between the protein profiles of 10, 20 and 40day-old leaves, while remarkable differences existed between 2-day and 10*40-day-old leaves (Hirano 1982). To further understand the protein expression profiles in different mulberry varieties, Li (2013) reported that mature leaf protein content of Da-Ye variety was the highest, while Yu 711 was the lowest among the measured 51 varieties. An RNA polymerase II carboxyl-terminal domain phosphorylase protein was distinctly identified only in the JIALING30 variety (Li 2013). Using a Gel-Free/Label-Free proteomic technique, Zhu et al. (2019) reported 492 proteins related to photosynthesis (13%), protein metabolism (17%), and redox ascorbate/ glutathione metabolism (6%) (Zhu et al. 2019). This study also reported that the leaf protein was

7

Proteomics in Mulberry

169

Table 7.1 Overview of the mulberry proteomic Analysis material

Separation methods

Different expression proteins

Typical proteins or enrichment pathway

References

1. Normal growth condition 1.1 Leaf Leaves from 17 varieties

2-DE

23

Similarity of the profile among the varieties

Hirano (1982)

Leaves from 51 varieties

2-DE, MALDITOF-TOF

4

RNA polymerase II carboxylterminal domain (CTD) phosphorylase protein

Li (2013)

Leaves

GelFree/LabelFree

492

Protein metabolism (17%), photosynthesis (13%), and redox ascorbate/ glutathione metabolism (6%)

Zhu et al. (2019)

Leaves at different stem positions

Label-free LC2MS

852

Photosynthesis, cellular amino acid metabolic catabolic, carbohydrate metabolic glycosyl compound metabolic processes, nitrogen compound metabolic and translation processes

Hou et al. (2021)

Leaves of mutant variety Cty-Ym

IEF /SDSPAGE

36

Large subunit of a RuBisCo

Tan et al. (2005)

Leaves of mutant variety Cty-Sm

1DE-LC–MS

24

Rubisco large and small subunits, light-harvesting complex II, plastocyanin and ATP synthase reduced; phosphoglycerate kinase, rubisco activase, nucleoside diphosphate kinase, CuZnSOD, 17.3 kD class I heat shock protein, 18 kD protein A, and mannose-binding lectin upregulated

Fang et al. (2009)

Mulberry leaves and silkworm foliar

Shotgun, LC– MS

2076 (leaves) and 210 (silkworm) foliar proteins

Primary metabolite, proteinase inhibiting, cell wall remodeling, redox regulating, and pathogenresistant processes

Wang et al. (2017)

GelFree/LabelFree

414

Protein metabolism (16%), stress (8%), and photosynthesis (7%)

Zhu et al. (2019)

Roots

GelFree/LabelFree

355

Protein metabolism (16%), stress (9%), cell wall (6%)

Zhu et al. (2019)

Base cortex during rooting of mulberry hardwood cuttings stage

iTRAQ, LCMS/MS

595 (planting and expanding stage); 660 (planting and rooting stage), and 231 (expanding and rooting stage)

Biosynthesis of secondary metabolites, starch, and sucrose metabolism, protein processing in the endoplasmic reticulum, phenylpropanoid biosynthesis, glycolysis, and gluconeogenesis

Du et al. (2015)

1.2 Branch Branch

1.3 Roots

(continued)

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Table 7.1 (continued) Analysis material

Separation methods

Different expression proteins

Typical proteins or enrichment pathway

References

1.4 Flower and fruit Flowers of variety

2D-PAGE

26

Ethylene metabolism, plant defense, photosynthetic reaction, and oxidation– reduction reaction that related to pollen germination, pollen tube growth, or defense against xenobiotic intrusion

Chen et al. (2013)

Different ripening fruits

2-DE, MALDITOF/TOF

31

Photosynthesis, stress, and glucose metabolism related

Liu et al. (2011)

Da10 and SG01 after pollination

2D-PAGE

471 (Da10) and 545 (SG01)

Cell structure, plant response, photosynthesis, protein and nucleic acid metabolism

Niu et al. (2013)

Pollen

1D-gel, MALDITOF/TOF

18

Allergenic proteins

Cetereisi et al. (2019)

Mulberry sap

LC–MS/MS

712

Stress and defense, signal transduction, secondary substances metabolism

Qin (2020)

Mulberry sap

2D-PAGE

27

Nitrogen nutrition, but also the photosynthesis, protein phosphorylation, and defense metabolism process

Tan and Lou (1999)

Mulberry sap

SDS-PAGE

/

MLX56

Wasano et al. (2009)

1.5 Others

2. Biotic stress condition 2.1 Mulberry yellow type dwarf disease Infected and healthy leaves

2D-PAGE

500

Photosynthetic protein in phytoplasma infected leaves

Ji et al. (2009)

Phytoplasma

Shotgun, LCMS/MS

209

Amino acid biosynthesis, cell envelope, cellular processes, energy metabolism, nucleosides and nucleotide metabolism, replication, transcription, translation, transport and binding as well as the proteins

Ji et al. (2010)

Mulberry sap

iTRAQ

136

Signal, hormone metabolism, and stress responses

Gai et al. (2018)

145

Plant hormone and signal transduction, phenylpropanoid pathway, transcription factors, and suppressing photosynthesis, cellular growth-related metabolism

Dai et al. (2019)

2.2 Mulberry fruit sclerotiniosis Fruit at the early and middle stage

iTRAQ

(continued)

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171

Table 7.1 (continued) Analysis material

Separation methods

Different expression proteins

Typical proteins or enrichment pathway

References

3.Abiotic stress condition 3.1 NaCl ATP (susceptible cultivar), S1 (tolerant cultivar)

2-DE

54 (S1), 51 (ATP)

/

Kumari et al. (2007)

Seedlings

TMT

/

PSII oxygen-evolving complex, LHCII antenna and PSI proteins, Pro, Put, GABA and GSH, SOD, CAT, and Trx-Prx related enzymes and pathway, CEF, xanthophyll cycle and Fddependent ROS metabolism, and nitrogen metabolism

Zhang et al. (2019, 2020a, b)

Guisagnyou12 (salt tolerant), Jiang3 (salt sensitive)

TMT

787 DEPs (Guisangyou12), 700 DEPs (Jisang3)

Amino acid transport and metabolism and posttranslational modification, protein turnover, phenylpropanoid biosynthesis

Gan et al. (2021)

TMT

/

PSII and PSI activity and carbon assimilation, nitrogen assimilation, Glu-Gln cycle, AsA-GSH, Trx-Prx pathway, CEF, xanthophyll cycle, and FTR, Fd-NiR, and Fd-GOGAT proteins

Zhang et al. (2019, 2020a, b)

iTRAQ

1893 DEPs

Flavonoid biosynthesis pathways, hormone signaling pathways, lignin metabolism

Li et al. (2022)

TMT

577 (leaves) and 270 (roots)

Carbon metabolism, photosynthesis, redox, secondary metabolism, and hormone metabolism

Liu et al. (2019b)

TMT

297 ER proteins

Cytochrome P450, cell redox homeostasis, N-glycan biosynthesis

Liu et al. (2022)

3.2 NaHCO3 Seedlings

3.3 Drought Leaves

3.4 Salt and drought Seedlings

3.5 UV-B and dark Endoplasmic reticulum from leaves

richer than that from branch or root. However, in our previous proteome analysis, 3615 proteins were identified from leaf tissue using TMT

technique, which is less than those identified from root tissue (Zhu et al. 2019; Liu et al. 2019b). A possible explanation was that both

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experimental method and plant growth conditions were responsible for the aforementioned difference. The protein abundance varied among mulberry leaves from a different position, which indicated their differential photosynthetic capacities, nutrient substance, and feeding impact to silkworms. Using a label-free LC-2MS approach, protein dynamic profiles of mulberry leaves at different stem positions were investigated. Among the commonly detected proteins, 417 proteins exhibited significantly differential expression in the first leaf (L1) contrasting with the sixth leaf (L6), while 305 proteins were identified with a significant difference in that of the twentieth leaf (L20) and sixth leaf (L6). Besides photosynthesis, proteins were involved in glycosyl compound metabolic and carbohydrate metabolic processes, nitrogen compound metabolic and translation, and cellular amino acid metabolic catabolic processes. The study showed that the first leaf contained fewer proteins related to photosynthetic apparatus; however, the protein biosynthesis activities in the first leaf is very active. The sixth leaf displayed a fully developed photosynthetic apparatus with maximum protein abundance and highest function related to photosynthesis, e.g., LHCb1, HCF101, and FNR. The twentieth leaf contained more channel proteins and oxidoreductases that were involved in secondary metabolisms. Three proteins (RBD1, TerC, and LPA3) involved in the D1 repair cycle were only detected in the twentieth leaf. The relationship of these differential expression proteins with photosynthesis was further verified in etiolated mulberry seedlings. The results indicated that different photosynthesis control mechanisms exist in leaves at different stem positions (Hou et al. 2021). Using isoelectric focusing-sodium dodecyl sulfate–polyacrylamide gel electrophoresis (IEF/SDS-PAGE), Tan et al. (2005) separated 184 protein spots from the mutant and wild type of mulberry to dissect the leaf etiolation mechanism. 36 spots differ remarkably in wild type and mutant, including the spot SSP 2801, a large subunit of Rubisco, which may play important roles in the leaf etiolation in mulberry mutant

L. Yan et al.

Cty-Ym (Tan et al. 2005). In another leaf etiolation of mulberry mutant Cty-Sm, the expression of rubisco large and small subunits, lightharvesting complex II, and plastocyanin and ATP synthase was significantly reduced, while the phosphoglycerate kinase, rubisco activase, nucleoside diphosphate kinase, Cu-ZnSOD, 17.3 kD class I heat shock protein, 18 kD protein A, and mannose-binding lectin was up-regulated detected by the 1DE-LC-MS technology (Fang et al. 2009). Based on the physiological roles of the aforementioned proteins, they might function in maintaining photosynthesis, sugar metabolism, and energy balance to ensure the growth and development of mulberry mutant Cty-Sm.

7.2.2 Branch A total of 414 proteins were identified via the Gel-Free/Label-Free proteomic technique from Morus branch. The top three functional categories were related to protein metabolism (16%), stress (8%), and photosynthesis (7%). The fructose-bisphosphate aldolase, phosphoglycerate kinase, and enolase proteins playing important roles in glycolysis were higher in abundance in the branch than those in the root and leaf. The isoflavone reductase was most abundant in branches, which was approximately three times higher than that in the root and leaf. This indicated that more intense isoflavonoid biosynthesis occurs in the mulberry branch. Additionally, other secondary metabolism associated proteins in the branches included chalcone flavanone isomerase, a key branch-point enzyme in the phenylpropanoid, and flavonoid pathways (Zhu et al. 2019). This enzyme catalyzes the synthesis of flavonoids and isoflavonoids (Dastmalchi and Dhaubhadel 2015).

7.2.3 Root The total flavonoid contents in roots were high in mulberry. For example, the contents of kuwanone H, mulberroside A, morusin, and chalcomoracin are over 10 times higher in the root than

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Proteomics in Mulberry

those in the leaf or branch. To analyze the underlined mechanisms, a total of 355 proteins were discovered in the Morus root using gelfree/label-free proteomic technique, including proteins related to metabolism (16%), stress (9%), and cell wall (6%). It was found that the root-specific proteins related to the secondary metabolism were mainly associated with the flavonoid pathway. For example, flavonoid 3,5hydroxylase and chalcone flavanone isomerase with functions in the biosynthesis of chalcones and dihydroflavonols were highly accumulated in roots (Zhu et al. 2019). Du et al. (2015) reported that 4427 proteins were identified in the cortex when mulberry cuttings were rooted by iTRAQ-based proteomic approach. These proteins were mostly involved in hormone regulation, glucose metabolism, cell wall modification, and flavonoid biosynthesis. Among which, proteins of GDP-D-mannose-3′, 5′-epimerase, lectin KM+, auxin IAA hydrolase, heat shock protein hsp70, allene oxide cyclase, cytochrome b6, and peroxidase were deduced to be closely related to rooting (Du et al. 2015).

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and SG01 after pollination using 2D-PAGE technology, respectively. These components are mainly involved in cell structure, plant response, photosynthesis, protein and nucleic acid metabolism (Niu et al. 2013). There were 441, 222, and 328 protein spots obtained from three different ripening stages. Liu et al. (2011) identified eight differential proteins related to photosynthesis, stress, and glucose metabolism. These proteins are suggested to play a specific physiological role during fruit ripening in mulberry (Liu et al. 2011). Mulberry pollen has been considered an agent related to food allergy or respiratory allergy. Two distinct Ig-E binding proteins around 10 and 18 kDa protein was previously proposed to be similar to Bet V1 allergen and its homologs (Navarro et al. 1997). Methionine synthase which has an apparent molecular weight of 80– 85 kDa was also suggested as one of the allergens in Morus alba pollen by gel electrophoresis and immunoblotted with sera from allergenic patients (Cetereisi et al. 2019).

7.2.5 Other Tissues 7.2.4 Flower and Fruit Mulberry fruit is nourishing with a higher protein content (10.15–13.33%) (Sánchez-Salcedo et al. 2015), which has been recommended as a food ingredient because of its capability in contributing to protein’s dietary allowance (Chen et al. 2021a). With fruit mulberry variety Da10 as material, 2D-PAGE patterns were performed from mulberry flowers at full-bloom stage. A total of 382 and 348 protein spots were obtained from the style (with stigma) and ovary tissues, respectively. Twenty proteins expressed in style were mostly involved in biosynthetic metabolism of ethylene, plant defense, photosynthetic reaction, and oxidation–reduction reaction and were related to pollen germination, pollen tube growth, or defense against xenobiotic intrusion (Chen et al. 2013). To compare the fruit maturation mechanism between different mulberry varieties, Niu et al. (2013) identified 471 and 545 spots from Da10

Vascular tissues (xylem and phloem) are known to play important roles in transporting nutrients, growth and defense signals, mRNA, and proteins between underground and aerial tissues in higher plants (Liu et al. 2021). In mulberry, LC-MS was conducted to analyze the mulberry phloem sap proteome. A total of 712 proteins had been identified to be involved in stress and defense, signal transduction, and secondary substance metabolism. A phloem specific Mul-BRD1 was further found interacting with six proteins by yeast two-hybrid assay, including the glyceraldehyde-3-phosphate dehydrogenase B, COP9 signalosome complex subunit 5A isoform X1(CSN5A), aldehyde oxidase GLOX, GDSL esterase/lipase APG, histone acetyltransferase of the MYST family 1, and an uncharacterized protein. These proteins participate in the pathways such as carbohydrate, photomorphogenesis, oxidation, lipid, and mediated acetylation (Qin 2020). Phloem sap protein expression was also

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analyzed to reveal the infection mechanism of phytoplasma in mulberry (Gai et al. 2018). Mulberry can ooze milky latex from laticifer cells after injury. Latex is supposed to exert rapid roles in wound closure and pathogens defense. Tan and Lou (1999) analyzed the proteins of sap flow in mulberry and reported that there was a significant difference between cultivated and wild-type mulberry varieties using the 2D-PAGE technique. These sap proteins play roles not only related to nitrogen nutrition but also the photosynthesis, protein phosphorylation, and defense metabolism process (Tan and Lou 1999). A latex protein MLX56 with a molecular mass of 56 kDa was purified from mulberry latex using the SDSPAGE method. This protein is highly glycosylated and has a unique structure with an extensin domain that was surrounded by two chitinbinding domains in its N′ and C′ regions. The extensin domain is proline-rich and highly arabinosylated (Wasano et al. 2009). MLX56 and LA-b are chitin-binding defense proteins that can strongly suppress the growth of the larvae of a variety of lepidopteran species (Konno 2011; Konno and Mitsuhashi 2019; Liu et al. 2017b; Gai et al. 2017). Interestingly, MLX56 ectopic expression in tomatoes showed not only strong resistance against the larvae of the common cutworm (Lepidoptera), but also to western flower thrips (Thysanoptera) and hadda beetle (Coleoptera: Henosepilachna) (Murata et al. 2021). Due to the possible function as a barrier in digesting processes of insects, MLX56 is suggested as a promising target in developing pestresistant crops in the future.

7.3

Mulberry Proteomics Under Stress Condition

Plant resistance is the ability to adapt to unfavorable environmental conditions, including biotic (e.g., bacterium, fungal, and insects) and abiotic (e.g., salt, cold, and drought) stressors. Breeding mulberry germplasm with stable resistance is of utmost importance for mulberry industry.

7.3.1 Biotic Stress Biotic stresses, such as bacterial and fungal diseases, are major threats to reducing mulberry leaf productivity (10–20%) and quality (Khurana and Checker 2011). As the natural food for silkworms that is very sensitive to pesticides, it is of great importance to breed resistant varieties to defend mulberry from diseases.

7.3.1.1 Mulberry Yellow Type Dwarf Disease (MD) Mulberry yellow dwarf disease is one of the most serious infectious diseases caused by phytoplasma, which resulted in yellowing phyllody, proliferation, stunting, and witches broom. Ji et al. (2009) identified a total of 500 protein spots from both infected and healthy leaves by twodimensional electrophoresis. Among these, 17 up-regulated and 20 down-regulated responsive spots were with significant changes. The disappearance of sedoheptulose-1,7-bisphosphatase, rubisco activase, and rubisco large subunit was consistent with the loss of the activities of these specific enzymes, indicating the existence of the degradation of photosynthetic protein in phytoplasma infected leaves (Ji et al. 2009). MD phytoplasmas were purified from infected tissues and surveyed by 1D SDS-PAGE and nanocapillary LC-MS. A total of 209 proteins were identified, involving in the function of amino acid biosynthesis, cellular processes, cell envelope, nucleosides and nucleotide metabolism, energy metabolism, replication, translation, transport and binding transcription, as well as the proteins with other functions (Ji et al. 2010). Since phytoplasmas are strictly confined to the phloem compartment and are in close contact with the sieve element (SE), Gai et al. (2018) profiled the transcriptomes and proteins in mulberry in the sap of healthy and infected plants. A total of 136 proteins involved in hormone metabolism, signal, and stress responses were found differentially expressed. A MuMLPL329 protein was deduced to act as receptor to bind plant hormone and activate transduction signals, which then increased the plant resistance to

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Proteomics in Mulberry

pathogen infection (Gai et al. 2018). Another upregulated putative F-box protein was identified to repress the synthesis of auxin and hormone signal transduction in the infected mulberry leaves. Both GA3 and IAA were found significantly lower in infected leaves than that in healthy plants (Ji et al. 2009). These suggested that hormones might be considered responsible for defense reaction in phytoplasma infected mulberry.

7.3.1.2 Mulberry Fruit Sclerotiniosis Mulberry sclerotiniose is a devastating disease of mulberry (Morus alba L.) fruit. This disease is caused by Scleromitrula shiraiana, Ciboria shiraiana, or Ciboria carunculoides. The mycelium of the diseased fruit brought extensive damage and productivity loss (Dai et al. 2019; Yin et al. 2017). The differential gene expression during fungal infection was successively screened and recorded in recent years (Bao et al. 2020). It was found that a total of 145 genes were consistently altered at both the transcriptome and proteome levels after C. carunculoides infection at the early stage (stage 1) and middle stage (stage 2). Differentially accumulated proteins (DAPs) were mainly related to simulating plant hormone and signal transduction, transcription factors, photosynthesis suppression, phenylpropanoid pathway, and cellular growth-related metabolism (Dai et al. 2019). Among these, the up-regulated callose synthase in stage 1 might act as a physical barrier and was deposited at wound sites in plants to resist the pathogen’s attack. In addition, several hormonal signaling pathways, including jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) played pivotal roles in plantinduced defense responses (Glazebrook 2001). In mulberry, both transcription factors NPR1 and TGA are central regulators of SA signaling and were regulated in response to C. carunculoides infection. The EIN3 protein abundance was increased approximately tenfold in infected fruit. Signal transduction pathways for BR and GA may be partially promoted, whereas signaling pathways for ABA and ethylene were partially inhibited by C. carunculoides infection in mulberry (Dai et al. 2019). More molecular

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mechanisms are needed to trigger new targets for regulating defense responses to fungal pathogens in mulberry fruits. Extensive studies have elucidated the molecular pathways in plants attacked by pathogens and herbivores. After infection, a series of signals, including immune and wounding responses, are turned on to prioritize resources and energy for defense. The balance between growth and defense is crucial for a plant’s survival and growth. One example of the tradeoff is Flagellinsignaling mediating with BR and auxin pathways. JA level increased rapidly after wounding. Mulberry is regularly pruned to encourage leaf growth in the industry. JA responsive genes including JA ZIM-domain (JAZ) proteins, coronatine insensitive1 (COI1), and DELLA-JAZ interaction might play roles in ensuring the optimal balance on priority of environmental challenges (Chaiwanon et al. 2016).

7.3.2 Abiotic Stress Salinity and alkalinity, low or high temperature, drought, and heavy metals are the major abiotic stresses which cause reductions in potential foliage yield and quality. Mulberry is increasingly attracting attention because of its stress resistance and environmental adaptability. It is suitable for ecological restoration to utilize marginal, problematic soil, and non-traditional area (Zeng et al. 2019; Shi et al. 2011). Various studies were performed on physiological and molecular mechanisms in response to abiotic stress, which has involved the interaction among several genes through signal transduction pathways and promotes mulberry resistance breeding in the future (Sarkar et al. 2014, 2017; Liu et al. 2015).

7.3.2.1 Salt Soil salinization, primarily due to the excessive Na+ levels, is one of the most serious abiotic stress adversely affecting plant growth and physiological function. Increases in the soil Na+ concentration affect plant growth and development and can lead to plant death (Hanin et al.

176

2016). Kumari et al. (2007) studied the effect of salinity on 2-DE proteomic changes in cultivar ATP (susceptible) and S1 (tolerant) that were subjected to different concentrations of salt for 7 days. The abundance of 33 proteins increased and 21 proteins decreased in cultivar S1, while in ATP cultivar, the abundance of 8 proteins increased and 43 proteins decreased (Kumari et al. 2007). The protein composition of the sensitive cultivar showed more traumatizing than that of the tolerant cultivar under salt stress conditions. Zhang et al. (2019) reported that 100 mmol L−1 NaCl stress had no significant effect on photosystem II (PSII) activity in M. alba seedling leaves. The expressions of the LHCII antenna (CP24 10A, CP26, and CP29), the PSII oxygen-evolving complex (OEE3-1 and PPD4), and PSI proteins (such as PsaF, PsaG, PsaH, PsaL, PsaN, and Ycf4) were enhanced to varying degrees. In addition, the expression of subunits (atpA and atpB) in ATP synthase also increased significantly under NaCl stress. It had little effect on the enzymes and proteins related to ribulose-1,5-bisphosphate (RuBP) regeneration and glucose synthesis during dark reactions (Zhang et al. 2019). These showed a great impact on the assembly of PSI and its stable attachment to the thylakoid membrane (Krech et al. 2012). 100 mmol L−1 NaCl stress seemed to have little effect on the accumulation of proteins for Chl synthesis and had no significant changes in Chl content and Chl a:b ratio in stressed mulberry leaves (Zhang et al. 2020a). Under NaCl stress conditions, superoxide dismutase (SOD) activity, and the expression of related antioxidant proteins in leaves, such as SOD, Fe-SOD, CAT, increased by varying degrees. Excessive production of superoxide anion (O2⋅−) might be effectively scavenged by CAT, AsA-GSH cycle and thioredoxinperoxiredoxin (Trx-Prx) pathway (Ruban et al. 2012; Zhang et al. 2020b). The accumulation of proline, putrescine, and spermidine were also promoted in mulberry leaves. The genes of nitrite reduce, ferredoxin-nitrite reductase, and glutamate decarboxylase were up-regulated, which resulted in promoting the synthesis of caminobutyric acid mechanism for mulberry to

L. Yan et al.

cope with NaCl stress (Zhang et al. 2020c). Simultaneously, the increase of nitrogen metabolism in chloroplast dependent on ferredoxin (Fd) was significantly increased. The expression of ndhH, ndhI, ndhK, ndhM, violaxanthin deepoxidase, ferredoxin-thioredoxin reductase, and ferredoxin-nitrite reductase was up-regulated under NaCl stress, which resulted in the enhancement of xanthophyll cycle, cyclic electron flow (CEF), and Fd-dependent ROS metabolism and nitrogen metabolism. These processes play important roles in alleviating photoinhibition under NaCl stress (Zhang et al. 2020c; Wang et al. 2019). Phenylpropanoid biosynthesis, an important plant secondary metabolic pathway, was reported highly responsive to salt tolerance in mulberry. By comparison of the two varieties after salt stress, the proteins of glycosyltransferase, aldehyde dehydrogenase, cinnamyl alcohol dehydrogenase, and peroxidase were down-regulated, and the proteins of quinate hydroxycinnamoyl transferase and caffeic acid 3O-methyltransferase were induced in salt-tolerant Jisang3. Meanwhile, these proteins showed contrary expression levels in salt-sensitive mulberry varieties Guisangyou12. Phenylpropanoids were indicated to be a candidate to improve salt resistance in mulberry (Gan et al. 2021).

7.3.2.2 Alkaline Stress The damaging effect of alkaline growth conditions may be due to its severe osmotic stress and ion toxicity as well as high pH stress (Song et al. 2017; Yin et al. 2019). Chen et al. (2021b) recently reported that the threat of alkaline stress is mainly dependent on the specificities of the weak acid ions rather than high pH (Chen et al. 2021b). Under 100 mmol L−1 NaHCO3 stress, the photosynthetic apparatus was significantly inhibited in mulberry. The photosynthetic carbon assimilation and electron transfer rate significantly decreased. One of the important reasons was related to the significantly reduced expression of CP47, photosystem II CP43 reaction center protein (CP43), D2, Photosystem II protein D1 (D1), PsbE, and PBH, which could not effectively receive excitation energy. This eventually led to an increase in ROS production and

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Proteomics in Mulberry

oxidative damage (Zhang et al. 2019). The inhibition of PSII electron transfer and carbon assimilation were suggested to be related to the damage of CEF and Fd-dependent ROS metabolism, xanthophyll cycle, and nitrogen metabolism, where the expression of NDH-dependent CEF related proteins subunit (ndhI, ndhH, ndhK, ndhM, and ndhN), VDE, FTR, ZE, Fd-GOGAT, and Fd-NiR was down-regulated in mulberry leaves under NaHCO3 stress (Zhang et al. 2020a). Photosynthesis, especially the photoinhibition of PSII and PSI, is closely related to the production of reactive oxygen species (ROS) (Imtiaz et al. 2018). In response to NaHCO3 stress, the expression of catalase (CAT), the electron donor of ferredoxin-thioredoxin reductase (FTR), and other Trx-related proteins such as thioredoxin M4 (TrxM4), thioredoxin M (TrxM), TrxF, thioredoxin X (TrxX), and Trx CSDP32 were significantly down-regulated. It was suggested that the scavenging of H2O2 by CAT and Trx-Prx pathway in mulberry seedling leaves was inhibited (Zhang et al. 2020b).

7.3.2.3 Low Temperature Stress Low temperature can damage most plant species, particularly young leaves and buds. Sustained low temperature stress brings more severe injuries to the leaf cells, including the rapid rise of the relative electrolyte leakage, declined photosynthesis, swollen chloroplast, and disintegrated membrane system. During early fall to winter period, some of the electrophoresis of plasma membrane protein changes in banding pattern could be related to growth cessation and defoliation in mulberry bark cells (Yoshida 1984). Two purified endoplasmic reticulum (ER)-enriched proteins (WAP20 and WAP27) were indicated to play a significant role in freezing tolerance in cortical parenchyma cells of mulberry trees (Ukaji et al. 1999). As a crucial traditional Chinese medicine in quenching heat, the mulberry leaves after frost showed a better antipyretic effect than those before frost. This may partially be attributed to the significant accumulation of isoquercitrin and astragalin in leaves during frost (Qu et al. 2019). Lipid unsaturation and increase of phospholipids

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are altered to cold acclimation in many plants. Chen et al. (2018b) reported that SOD1, KCS1 (a very-long chain fatty acids biosynthesis enzyme) and Ankyrin-repeat containing protein 2 A (AKR2A) expression in the chilling-tolerant variety was reduced more than that in the chilling-sensitive variety, whereas desaturation related protein (e.g., FADII) expression increased in the chilling-tolerant variety, suggesting that the molecular chaperon AKR2A mediated the chilling-related protein responses in mulberry (Chen et al. 2018b).

7.3.2.4 Drought Droughts stress usually leads to low cell water content, disrupts normal cellular activities, compromises photosynthesis, and ultimately decreases plant yield. Like in other plants, mulberry productivity is also remarkably affected by drought. Protein changes and functions under drought conditions were of great consideration for breeding researchers. Drought tolerant mulberry genotypes exhibited small plasticity in foliar gas exchange and can sustain higher rates of stomatal conductance and photosynthesis (Guha et al. 2010a, b). By complementary transcriptomic and iTRAQ analysis, 1893 DEPs were identified from mulberry leaves under drought stress for 9 days. Of which, pathways related to proline and ABA biosynthesis were active in response to drought resistance of mulberry. The transcription and translation levels of MaArg, MaP5CS1, MaproC, MaZEP, MaNCED, and protein phosphatase 2C were significantly up-regulated. An up-regulated and induced MaWRKYIII8 was suggested to play a key in the mulberry response to drought stress (Li et al. 2022). Drought and salinity are frequently considered to impose similar effects on plants. This is mainly due to the reductions in external water potentials derived from both stresses. To explore the integrated physiological and proteomic responses of mulberry trees subjected to combined salt and drought stress, both leaves and roots were analyzed. A total of 577 and 270 differentially expressed proteins (DEPs) were identified from the stressed leaves and roots, respectively, which

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were both distinctly assigned to photosynthesis, carbon metabolism, redox, hormone metabolism, and secondary metabolism (Liu et al. 2019b). Leaves (source) and roots (sink) are two distinct organs that are connected by vascular system (phloem and xylem) in the plant. The enriched differential proteins involved in starch and sucrose metabolic pathways are of great interest for further exploring the signal transduction and interaction regulation in plants in response to abiotic stress. In addition, several mulberry proteins such as G-proteins (Liu et al. 2017a, 2018), ethyleneinsensitive3 (EIN3)/EIN3-like (Liu et al. 2019a) and NPR4 (Xu et al. 2019) had been heterologous expressed and their functions had been verified in drought stress tolerance in Arabidopsis and tobacco. To combat drought stress, more adaption related traits and mechanisms need to be further performed in mulberry.

7.3.2.5 Other Stresses As one of the most economically important tree species, mulberry also has great potential for the remediation of heavy metal contaminated soils (Huang et al. 2018). To investigate the heavy metal accumulation and their detoxification mechanisms in mulberry, two mulberry phytochelatin synthase were conferred to play important roles in zinc/cadmium tolerance (Fan et al. 2018a). The natural resistance and macrophage proteins, the iron-regulated transporterlike proteins, the heavy metal ATPases, and the metal tolerance or transporter proteins families are involved in cadmium (Cd) uptake, translocation, and sequestration in plants (Fan et al. 2018b). More functional and mechanisms related research still needs to be performed in the future.

7.4

Interaction Between Mulberry and Insect

Mulberry leaves have been selected as the sole diet for silkworms for over 5000 years. The interaction mechanism between mulberry and silkworm is of great interest to be explored. A growing body of evidence at the protein level

had implicated the defensive activities of mulberry leaves against herbivorous insects. Wang et al. (2017) identified 2076 and 210 proteins by analyzing mulberry leaves and silkworm foliage at the fifth larval instar using shotgun liquid chromatography-tandem mass spectrometry. Most of the foliage secretory proteins were found to be involved in primary metabolite, proteinase inhibiting, cell wall remodeling, redox regulating, and pathogen-resistant processes (Wang et al. 2017). Several defensive enzymes were discovered, such as tryptophan aminotransferase, dipeptidyl aminopeptidase, lysine-specific demethylase, insoluble cell-wall-bound invertase, dihydrolipoyllysine-residue acetyltransferase, and b-fructofuranosidase, in the mulberry leaves (Lawrence and Koundal 2002). Interestingly, MLX56, a mulberry latex protein with two heveinlike 4, is non-poisonous to the silkworm and its role in defense avoidance employed by silkworms has been partially elucidated. Silkworm expressed b-fructofuranosidases instead of a-glycosidases in the gut to circumvent the damage of mulberry sugar-mimic glycosidase inhibitors (Hirayama et al. 2007; Daimon et al. 2008; Nakagawa et al. 2010). In addition, 1-Deoxynojirimycin (DNJ) is the most abundant poly-hydroxylated alkaloid in the latex of mulberry leaves and it protects mulberry from insect predation. 30K, GST, and thiol peroxiredoxin genes had a positive correlation with DNJ accumulation, which might play very important roles in the physiological process of DNJ accumulation to adapt to mulberry-defense mechanism (Chen et al. 2018a). More detailed mechanisms are still needed to be explored. Furthermore, to gain insight into the effect of fresh mulberry leaves and artificial diet on silkworm, proteomic studies showed that different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrients, energy metabolism, and silk synthesis (Zhou et al. 2008). Ultraviolet-B (UV-B) treated mulberry leaves improved the BmNPVresistance of silkworm, which was closely related to the improved levels of ribosomal proteins, V-ATPase, ATP synthase, and apoptotic-related proteins, and immunity related proteins (Hu et al. 2017). After successive UV-B and dark

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Proteomics in Mulberry

incubation, secondary metabolites (e.g., chalcomoracin) increased in mulberry leaves. TMTbased proteomics of ER enriched fraction was performed because of its important role in protein biosynthesis and quality control. Studies indicated that ER proteins related to Cytochrome P450, cell redox homeostasis, N-glycan biosynthesis were activated to resist the stress damage. The heat shock-related proteins were increased, while UDP-glucose glycoprotein glucosyltransferase, disulfide isomerase, CNX, and calreticulin were decreased under stress (Liu et al. 2022).

7.5

Future and Prospects

Proteomics knowledge provides important clues for promising candidate genes with putative functions in plant productivity and adaptation. Although there have been significant advances in mulberry proteomics over the years, researches on proteome are still in its infancy for mulberry. Proteomic approaches are still needed and expected to integrate with transcriptomic, genomic, and metabolome studies to identify and characterize novel genes involved in growth or stress signal pathways. Due to the lack of large-scale sequence resources to match the sequences of peptides to their protein origins, a big gap that still exists between the identities of proteins and their corresponding phenotypes. Mulberry has high heterozygosity and ploidy level. The study of mulberry tree functional genomics is still far behind other model plants. Additionally, because of the lack of an efficient transgenic platform in mulberry, the genetic manipulation and functional analysis for important genes and proteins in mulberry is still limited. Therefore, it is urgently important to develop an efficient mulberry transgenic technique. More efforts should be made in the system-wide characterization of the proteomes and function of corresponding genes. Future advances in studies of organelle function, post-translational modifications, protein–protein interactions, and how these proteins may be connected to downstream changes in gene expression, metabolism, and physiology

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will provide deeper insight into the molecular functions that these proteins play.

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180 perennial model system. Plant Signal Behav 13(8): e1491267 Du W, Ban YY, Tang Z, Du XL, Guo XJ et al (2015) Analysis on differential proteome in the base cortex during rooting of mulberry hardwood cuttings. Sci Sericulture 41(4):0593–0602 (in Chinese) Fan Y, Ling H, Piao H (2014) Effects of symbiosis of mulberry (Morus alba) with arbuscular mycorrhizae on absorption of heavy metals (Fe, Mn, Zn, Cu and Cd). Ecol Env Sci 23:477–484 Fan W, Guo Q, Liu C, Liu X, Zhang M et al (2018a) Two mulberry phytochelatin synthase genes confer zinc/cadmium tolerance and accumulation in transgenic Arabidopsis and tobacco. Gene 645:95–104 Fan W, Liu C, Cao B, Qin M, Long D et al (2018b) Genome-wide identification and characterization of four gene families putatively involved in cadmium uptake, translocation and sequestration in Mulberry. Front Plant Sci 9:879 Fang XM, Jia JL, Tan JZ, Kan XQ, Liu YY et al (2009) Analysis of differential proteome of a leaf-variegated mutant Cty-Sm in mulberry( Morus L.). Science of Sericulture 35(1):1–5 Gai YP, Zhao YN, Zhao HN, Yuan CZ, Yuan SS et al (2017) The latex protein MLX56 from mulberry (Morus multicaulis) protects plants against insect pests and pathogens. Front Plant Sci 8:1475 Gai YP, Yuan SS, Liu ZY, Zhao HN, Liu Q et al (2018) Integrated phloem sap mRNA and protein expression analysis reveals phytoplasma-infection responses in mulberry. Mol Cell Proteomics 17(9):1702–1719 Gan T, Lin Z, Bao L, Hui T, Cui X et al (2021) Comparative proteomic analysis of tolerant and sensitive varieties reveals that phenylpropanoid biosynthesis contributes to salt tolerance in mulberry. Int J Mol Sci 22(17):9402 Glazebrook J (2001) Genes controlling expression of defence responses in Arabidopsis–2001 status. Curr Opin Plant Biol 4(4):301–308 Guha A, Rasineni GK, Reddy AR (2010a) Drought tolerance in mulberry (Morus spp.): a physiological approach with insights to growth dynamics and leaf yield production. Exp Agric 46:471–488 Guha A, Sengupta D, Reddy AR (2010b) Physiological optimality, allocation trade-offs and antioxidant protection liked to better leaf yield performance in drought exposed mulberry. J Sci Food Agric 90(15):2649–2659 Hanin M, Ebel C, Ngom M, Laplaze L, Masmoudi K (2016) New insights on plant salt tolerance mechanisms and their potential use for breeding. Front Plant Sci 7:1787 He N, Zhang C, Qi X, Zhao S, Tao Y et al (2013) Draft genome sequence of the mulberry tree Morus notabilis. Nat Commun 4:2445 Hirano H (1982) Varietal differences of leaf protein profiles in mulberry. Phytochem 21(7):1513–1518 Hirayama C, Konno K, Wasano N, Nakamura M (2007) Differential effects of sugar-mimic alkaloids in mulberry latex on sugar metabolism and disaccharidases of Eri and domesticated silkworms: enzymatic

L. Yan et al. adaptation of Bombyx mori to mulberry defence. Insect Biochem Mol Biol 37(12):1348–1358 Hou Z, Xu D, Deng N, Li Y, Yang L et al (2021) Comparative proteomics of mulberry leaves at different developmental stages identify novel proteins function related to photosynthesis. Front Plant Sci 12:797631 Hu J, Zhu W, Li Y, Guan Q, Yan H et al (2017) SWATHbased quantitative proteomics reveals the mechanism of enhanced Bombyx mori nucleopolyhedrovirusresistance in silkworm reared on UV-B treated mulberry leaves. Proteomics 17:13–14 Huang RZ, Jiang YB, Jia CH, Jiang SM, Yan XP (2018) Subcellular distribution and chemical forms of cadmium in Morus alba L. Int J Phytoremediation 20 (5):448–453 Imtiaz M, Ashraf M, Rizwan MS, Nawaz MA, Rizwan M et al (2018) Vanadium toxicity in chickpea (Cicer arietinum L.) grown in red soil: effects on cell death, ROS and antioxidative systems. Ecotoxicol Environ Saf 158:139–144 Ji X, Gai Y, Zheng C, Mu Z (2009) Comparative proteomic analysis provides new insights into mulberry dwarf responses in mulberry (Morus alba L.). Proteomics 9(23):5328–5339 Ji X, Gai Y, Lu B, Zheng C, Mu Z (2010) Shotgun proteomic analysis of mulberry dwarf phytoplasma. Proteome Sci 8:20 Khurana P, Checker VG (2011) The advent of genomics in mulberry and perspectives for productivity enhancement. Plant Cell Rep 30(5):825–838 Komatsu S (2019) Plant proteomic research 2.0: trends and perspectives. Int J Mol Sci 20(10):2495 Konno K (2011) Plant latex and other exudates as plant defence systems: roles of various defence chemicals and proteins contained therein. Phytochemistry 72 (13):1510–1530 Konno K, Mitsuhashi W (2019) The peritrophic membrane as a target of proteins that play important roles in plant defence and microbial attack. J Insect Physiol 117:103912 Krech K, Ruf S, Masduki FF, Thiele W, Bednarczyk D et al (2012) The plastid genome-encoded Ycf4 protein functions as a nonessential assembly factor for photosystem I in higher plants. Plant Physiol 159(2):579–591 Kumari GJ, Kumar SG, Thippeswamy M, Annapurnadevi A, Naik ST et al (2007) Effect of salinity on growth and proteomic changes in two cultivars of mulberry (Morus alba L.) with contrasting salt tolerance. Indian J Biotechnol 6:508–518 Lawrence PK, Koundal KR (2002) Plant protease inhibitors in control of phytophagous insects. EJB Electron J Biotechnol 5(1):573–580 Li S (2013) Proteomics analysis of mulberry leaves. Dissertation for master’s degree of Southwest University (in Chinese) Li R, Su X, Zhou R, Zhang Y, Wang T (2022) Molecular mechanism of mulberry response to drought stress revealed by complementary transcriptomic and iTRAQ analyses. BMC Plant Biol 22(1):36

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Liu Y, Willison JH (2013) Prospects for cultivating white mulberry (Morus alba) in the drawdown zone of the Three Gorges Reservoir, China. Environ Sci Pollut Res Int 20(10):7142–7151 Liu WQ, Zhou LL, Su M, Chi XJ, Tan JZ (2011) Separation and identification of proteins related to fruits ripening in mulberry (Morus alba). Adv Mater Res 175–176:25–29 Liu XQ, Liu CY, Guo Q, Zhang M, Cao BN et al (2015) Mulberry transcription factor MnDREB4A confers tolerance to multiple abiotic stresses in transgenic tobacco. PLoS ONE 10(12):e0145619 Liu C, Xu Y, Long D, Cao B, Hou J et al (2017a) Plant Gprotein b subunits positively regulate drought tolerance by elevating the detoxification of ROS. Biochem Biophys Res Commun 491(4):897–902 Liu Y, Ji D, Chen J, Lin T, Wei J et al (2017b) Overexpression of the mulberry latex gene MAMLXQ1 enhances defence against Plutella Xylostella in Arabidopsis thaliana. Arch Biol Sci 69(2):269–276 Liu C, Xu Y, Feng Y, Long D, Cao B et al (2018) Ectopic expression of mulberry G-proteins alters drought and salt stress tolerance in tobacco. Int J Mol Sci 20(1):89 Liu C, Li J, Zhu P, Yu J, Hou J et al (2019a) Mulberry EIL3 confers salt and drought tolerances and modulates ethylene biosynthetic gene expression. Peer J 7: e6391 Liu Y, Ji D, Turgeon R, Chen J, Lin T et al (2019b) Physiological and proteomic responses of mulberry trees (Morus alba. L.) to combined salt and drought stress. Int J Mol Sci 20(10):2486 Liu Y, Lin T, Valencia MV, Zhang C, Lv Z (2021) Unraveling the roles of vascular proteins using proteomics. Molecules 26(3):667 Liu S, Ou Y, Li Y, Sulaiman K, Tao M (2022) TMTbased proteomic analysis of endoplasmic reticulum proteins in mulberry leaves under UV-B and dark stress. Physiol Plant e13667 Murata M, Konno K, Wasano N, Mochizuki A, Mitsuhara I (2021) Expression of a gene for an MLX56 defence protein derived from mulberry latex confers strong resistance against a broad range of insect pests on transgenic tomato lines. PLoS ONE 16(1):e0239958 Nakagawa K, Ogawa K, Higuchi O, Kimura T, Miyazawa T et al (2010) Determination of imino sugars in mulberry leaves and silkworms using hydrophilic interaction chromatography-tandem mass spectrometry. Anal Biochem 404(2):217–222 Navarro AM, Orta JC, Sánchez MC, Delgado J, Barber D et al (1997) Primary sensitization to Morus alba. Allergy 52(11):1144–1145 Niu RH, Chen YY, Yin PF, Zheng BP, Chen C et al (2013) Comparative analysis of the fruit proteome of mulberry varieties Da10 and SG01. China Sericulture 34(4):18–22 (in Chinese) Qin J, He NJ, Huang XZ, Xiang ZH (2010) Development of mulberry ecological industry and sericulture. Sci Seric 36:984–989 (in Chinese) Qin RL (2020) Identification of phloem sap proteins of mulberry and functional study of Mul-BRD1.

181 Dissertation for master’s degree of Shangdong Agricultural University (in Chinese) Qu Y, Wang L, Guo W (2019) Screening and identification of antipyretic components in the postfrost leaves of Morus alba based on multivariable and continuous-index spectrum-effect correlation. J Anal Methods Chem 16:2019 Ramappa VK, Srivastava D, Singh P, Kumar U, Kumar D et al (2020) Mulberries: a promising fruit for phytochemicals, nutraceuticals, and biological activities. Int J Fruit Sci 20(sup3):S1254–S1279 Ruban AV, Johnson MP, Duffy CD (2012) The photoprotective molecular switch in the photosystem II antenna. Biochim Biophys Acta 1817(1):167–181 Sánchez-Salcedo EM, Mena P, García-Viguera C, Martínez JJ, Hernández F (2015) Phytochemical evaluation of white (Morus alba L.) and black (Morus nigra L.) mulberry fruits, a starting point for the assessment of their beneficial properties. J Funct Foods 12:399–408 Sarkar T, Thankappan R, Kumar A, Mishra GP, Dobaria JR (2014) Heterologous expression of the AtDREB1A gene in transgenic peanut-conferred tolerance to drought and salinity stresses. PLoS ONE 9 (12):e110507 Sarkar T, Mogili T, Sivaprasad V (2017) Improvement of abiotic stress adaptive traits in mulberry (Morus spp.): an update on biotechnological interventions. 3 Biotech 7(3):214 Shi X, Zhang X, Chen G, Chen Y, Wang L et al (2011) Seedling growth and metal accumulation of selected woody species in copper and lead/zinc mine tailings. J Environ Sci (China) 23(2):266–274 Song T, Xu H, Sun N, Jiang L, Tian P et al (2017) Metabolomic analysis of alfalfa (Medicago sativa L.) root-symbiotic rhizobia responses under alkali stress. Front Plant Sci 8:1208 Tan JZ, Lou CF (1999) Two-dimensional electrophoresis analysis of sap flow proteins in mulberry, Morus. Sci Sericulture 25(2):65–69 (in Chinese) Tan JZ, Liu MJ, Zhang GY, Li XM, Wu ZP (2005) Analysis of leaf proteins from mulberry mutant Cty-Ym by using mass spectrometry and two-dimensional electrophoresis. Sci Sericulture 31(1):8–13 (in Chinese) Tang Z, Du W, Du X, Ban Y, Chen J (2016) iTRAQ protein profiling of adventitious root formation in mulberry hardwood cuttings. J Plant Growth Regul 35:618–631 Ukaji N, Kuwabara C, Takezawa D, Arakawa K, Yoshida S et al (1999) Accumulation of small heatshock protein homologs in the endoplasmic reticulum of cortical parenchyma cells in mulberry in association with seasonal cold acclimation. Plant Physiol 120 (2):481–490 Wang D, Dong Z, Zhang Y, Guo K, Guo P et al (2017) Proteomics provides insight into the interaction between mulberry and silkworm. J Proteome Res 16 (7):2472–2480 Wang Y, Jin WW, Che YH, Huang D, Wang JC et al (2019) Atmospheric nitrogen dioxide improves

182 photosynthesis in mulberry leaves via effective utilization of excess absorbed light energy. Forests 10 (4):312 Wasano N, Konno K, Nakamura M, Hirayama C, Hattori M et al (2009) A unique latex protein, MLX56, defends mulberry trees from insects. Phytochemistry 70:880–888 Xu YQ, Wang H, Qin RL, Fang LJ, Liu Z et al (2019) Characterization of NPR1 and NPR4 genes from mulberry (Morus multicaulis) and their roles in development and stress resistance. Physiol Plant 167 (3):302–316 Yin P, Li X, Zeng Q, Shen C, Gao L et al (2017) Effect of popcorn disease infected leaves on silkworm performance and differential proteome analysis of mulberry popcorn disease. Pak J Zool 50 Yin ZP, Zhang H, Zhao Q, Yoo MJ, Zhu N et al (2019) Physiological and comparative proteomic analyses of saline-alkali NaHCO3-responses in leaves of halophyte Puccinellia tenuiflora. Plant Soil 437(1–2):137– 158 Yoshida S (1984) Chemical and biophysical changes in the plasma membrane during cold acclimation of mulberry bark cells (Morus bombycis Koidz. cv Goroji). Plant Physiol 76(1):257–265 Zeng P, Guo Z, Xiao X, Peng C (2019) Effects of treeherb co-planting on the bacterial community composition and the relationship between specific microorganisms and enzymatic activities in metal(loid)contaminated soil. Chemosphere 220:237–248

L. Yan et al. Zhang H, Shi G, Shao J, Li X, Li M et al (2019) Photochemistry and proteomics of mulberry (Morus alba L.) seedlings under NaCl and NaHCO3 stress. Ecotoxicol Environ Saf 184:109624 Zhang H, Wang Y, Li X, He G, Che Y et al (2020a) Chlorophyll synthesis and the photoprotective mechanism in leaves of mulberry (Morus alba L.) seedlings under NaCl and NaHCO3 stress revealed by TMTbased proteomics analyses. Ecotoxicol Environ Saf 190:110164 Zhang H, Li X, Guan Y, Li M, Wang Y et al (2020b) Physiological and proteomic responses of reactive oxygen species metabolism and antioxidant machinery in mulberry (Morus alba L.) seedling leaves to NaCl and NaHCO3 stress. Ecotoxicol Environ Saf 193:110259 Zhang H, Huo Y, Xu Z, Guo K, Wang Y et al (2020c) Physiological and proteomics responses of nitrogen assimilation and glutamine/glutamine family of amino acids metabolism in mulberry (Morus alba L.) leaves to NaCl and NaHCO3 stress. Plant Signal Behav 15 (10):1798108 Zhou ZH, Yang HJ, Chen M, Lou CF, Zhang YZ et al (2008) Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and artificial diet. J Proteome Res 7 (12):5103–5111 Zhu W, Zhong Z, Liu S, Yang B, Komatsu S et al (2019) Organ-specific analysis of Morus alba using a gelfree/label-free proteomic technique. Int J Mol Sci 20 (2):365

8

Importance and Current Status of DUS Testing in Mulberry M. R. Bhavya, P. Sowbhagya, Belaghihalli N. Gnanesh , G. S. Arunakumar, and H. B. Manojkumar

8.1

Introduction

cies that resulted in the formation of innumerable number of recombinants and varieties exhibiting continuous variation in many of the phenotypic characters. Production of good silkworm cocoons for high-quality gradable silk is of prime importance in sericulture industry for its sustainability and profitability, which depends to a great extent on the quality of mulberry leaf the silkworms feed on. Mulberry variety and cultivation practices are the two crucial factors which decides both leaf yield and quality. In India, mulberry is mostly propagated clonally through planting of stem cuttings or very rarely by bud grafting in order to retain true to parental characteristic of a variety. The plants are mostly maintained as bush or sometimes as small trees by regular pruning. In a typical tropical/ subtropical condition of southern India, 5–6 times leaf is harvested per annum to rear the silkworm. Different plant spacing and package of practices have been recommended to suit to the agro-climatic conditions and varieties grown.

Silkworm (Bombyx mori L.) a monophagous insect feeds only on mulberry for the production of highly valued silk. Therefore, mulberry (Morus spp.) has greater importance in sericulture industry. Besides, food of silkworm, mulberry tree is also commercially utilized for timber, fruits, fodder for animals, and medicinal purposes (Vijayan et al. 2011). Silk production is highest in China followed by India, and the share of mulberry silk in India is more than 65%. Major states in India that cultivate mulberry are Karnataka, Andhra Pradesh, and Tamil Nadu in tropical zone and West Bengal, Himachal Pradesh, and the North Eastern states in subtropical zone. Morphological variability in mulberry resources is significant. In India, mulberry is represented by four species, viz., Morus indica, M. alba, M. laevigata, and M. serrata of which first two are cultivated types and the latter two are the wild forms. Mulberry is a perennial tree plant, generally dioecious, highly out breeding, and heterozygous. Mulberry has higher reproductive compatibility across the recognized spe-

8.2

M. R. Bhavya (&)
P. Sowbhagya
B. N. Gnanesh
G. S. Arunakumar
H. B. Manojkumar Central Sericultural Research and Training Institute, Manandavadi Road, Srirampura, Mysuru, Karnataka 570008, India e-mail: [email protected]

According to World Intellectual Property Organization “Intellectual property (IP) refers to creations of the mind, such as inventions; literary and artistic works; designs; and symbols, names and images used in commerce”. There are different types of legal rights called intellectual

Genesis of Plant Variety Protection System

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_8

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property rights to protect the intellectual property viz., patents, trademarks, copyrights, or industrial designs. Intellectual property is broadly divided into two categories: (1) industrial property that includes patents for inventions, trademarks, industrial designs, and geographical indications; and (2) copyright that covers literary works (novels, poems, plays), films, music, artistic works, and architectural design (Hariprasanna 2019). With the rediscovery of Mendelian principles during 1900, systematic plant breeding begins and leads to the development of new crop varieties. Growers of new crop varieties and agricultural industry as a whole started harvesting benefits of improved varieties. During this period, lack of an effective protection or reward system to the plant breeder or developer of new varieties was first felt as there was no plant patent protection in several of the countries (Hariprasanna 2019). Since, plant breeding is a costly affair and involves lot of resource and time to breed desirable varieties for yield, quality, biotic and abiotic stress, etc. The varieties thus developed can easily be reproduced. To secure the investment return and safeguard the interest of the breeders, to support further breeding activities, and to provide the best varieties constantly, protection of plant varieties is necessary. This led to the formation of International Union for the Protection of New Varieties of Plants (UPOV), 1961 which is an intergovernmental international organization, which provides plant variety protection in its 78 member states (as of November 2021; upov_pub_423.pdf, accessed on 30 May 2022).

8.3

option to protect plant varieties “either through patent protection or a system created specifically for the purpose (“sui generis”), or a combination of the two”. India being a member of WTO and International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), chosen sui generis system of plant protection and enacted Protection of Plant Varieties and Farmers Right Act, 2001 to maintain a balance between “Breeders rights” and “Farmers rights”. Further, PPV & FR Authority was established at New Delhi on 11 November 2005 for implementation of the act for registration of plant varieties. Govt. of India has notified 172 crop species on the recommendations of PPV & FR Authority for plant variety registration which includes cereals, pulses and legumes, fibre, sugar, oilseed, spices, vegetables, flowers, medicinal and aromatic crops, tree, forest and plantation crops, fruit, and cash crops (https://plantauthority.gov.in/cropdus-guidelines, accessed on 01 June 2022). Objectives of the PPVFR Act are (1) facilitate an effective system for the protection of plant varieties and the rights of farmers, (2) encourage the development of new varieties of plants, (3) protect the rights of the farmers in respect of their contribution made at any time in conserving, improving, and making available plant genetic resources for the development of the new plant varieties, and (4) facilitate the growth of the seed industry which will ensure the availability of high-quality seeds and planting material to the farmers. For the protection of plant varieties under PPV&FR Act 2001, plant variety must satisfy the criteria of novelty (N) (only for new varieties), distinctness (D), uniformity (U), and stability (S) and should also have distinct denomination.

Protection of Plant Varieties and Farmers Right Act 2001 8.4

World Trade Organization (WTO) established during Uruguay Round of General Agreement on Tariffs and Trade and came into effect from 1st January 1995. For the protection of plant varieties, all the members of WTO need to stick to the norms framed in Trade-Related Aspects of Intellectual Property Rights (TRIPS) agreement. Article 27.3(b) of TRIPS agreement provides an

Novelty, Distinctness, Uniformity, and Stability

According to UPOV, 1978 [Article 6(1)a] distinctness refers “if variety is clearly distinguishable by one or more important characteristics from any other variety whose existence is a matter of common knowledge at the time of application for the protection of the variety”.

8

Importance and Current Status of DUS Testing in Mulberry

According to UPOV, 1978 [Article 6(1)b] novelty refers “if variety not offered for sale or marketed not longer than one year in the territory of the state, not longer than six years in the case of vines, forest trees, fruit trees and ornamental trees or for longer than four years in the case of all other plants in the territory of any other State”. According to UPOV, 1978 [Article 6(1)c] uniformity refers “if variety is sufficiently homogeneous, having regard to the particular features of its sexual reproduction or vegetative propagation”. According to UPOV, 1978 [Article 6(1)d] stability of variety refers “if essential characters remain true to its description after repeated reproduction or propagation at the end of each cycle”.

8.5

DUS Guidelines in Mulberry (Morus Spp.)

Effort by the federal agencies for mulberry crop improvement has culminated in many improved varieties of mulberry contributing to the sericulture development in the country. Protection of these mulberry varieties under IPR by PPV & FR Act 2001 is essential to prevent from infringement, encourages others to develop new varieties, it stimulates the investment from private and public sectors thereby accelerates the sericulture growth of the country by providing the high-quality planting material of improved varieties. In this connection, Central Sericulture Research and Training Institute, Mysuru has been identified as nodal centre with co-nodal centre being proposed at CSRTI, Berhampore. These centres involve in testing distinctness, uniformity, and stability (DUS tests) on mulberry cultivars mainly based on growing tests on behalf of the Protection of Plant Varieties and Farmers’ Rights Authority, Ministry of Agriculture and Farmers Welfare, Government of India for a grant of Plant Breeders’ Rights. DUS test helps in generating varietal descriptions using relevant characters (e.g. plant growth habit, phyllotaxy, leaf base and leaf colour, etc.) by which it can be defined as a variety (UPOV

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2002). Principals involved in DUS testing was given by UPOV for harmonized examination of varieties throughout the members of the union. Harmonization is essential for facilitating cooperation in DUS testing and also helps to provide effective protection through development of harmonized, internationally recognized descriptions of protected varieties (Hariprasanna 2019). As mulberry is propagated by vegetative methods and they exhibit uniformity and stability in expression of morphological traits but, show plasticity in different environments. The phenotypic distinction among different varieties can be clear cut but, quite often obscured due to continuous variation of characters under consideration. In this context, it is very essential to develop basic description of the varieties in terms of distinctness, uniformity, and stability using relevant characteristics to enable comparison with reference varieties and to differentiate the candidate variety from varieties of common knowledge. This warrants an accurate documentation of true genetic variation for their safe guard by registering the variety under Protection of Plant Varieties and Farmers’ Right’s Act 2001. In this connection, identified DUS descriptors and developed the DUS test guidelines for mulberry for DUS testing of new/extant/farmer’s mulberry varieties for their entry into the national list for the protection of plant breeders’ and farmers’ right. Mulberry DUS guidelines have been published in Plant Variety Journal of PPV&FRA in Vol. 10, No. 10 during 2016 (https:// plantauthority.gov.in/sites/default/files/mulberry. pdf). This creates the way for the registration of mulberry varieties and grant of plant variety protection. These test guidelines shall apply to all vegetatively propagated varieties, mutants, polyploids, hybrids, and transgenics of mulberry.

8.6

Characteristics Used in DUS Testing

To use the characteristics for DUS testing or characterization or for production of a variety description, characteristics must fulfil basic

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requirements viz., the expression of characteristics must be “results from a given genotype or combination of genotypes, sufficiently consistent and repeatable in a particular environment, capable of precise definition and recognition, allows uniformity requirements to be fulfilled, allows stability requirements to be fulfilled” (UPOV 2002). There is no necessary of having commercial value for a character, if it satisfies the mentioned criteria for inclusion, it may be considered in the normal way (UPOV 2002). In mulberry, 35 morphological characteristics were included in DUS guidelines (Table 8.1). Generally, among all the characteristics, morphological characteristics have been recognized as more acceptable descriptors for DUS testing (Das 2012; Bhat 2001).

8.7

States of Expression of the Characteristics

To test the variation and to establish variety description, expression range of each character is divided into a different state and numerical “note” is given to the wording of each state for statistical analysis and inference (UPOV 2002). Based on the type of expression, characteristics are classified into three types namely qualitative, quantitative, and pseudo-qualitative. Qualitative characteristics are highly heritable, less influenced by the environment and expressed in discontinuous states. Each state is clearly different and independently meaningful from other states of expression. For example, plant sex is classified as gynoecious (1), androecious (2), bisexual (3), andromonoecious (4), gynomonoecious (5), and androgynomonoecious (6) and stipule nature is classified as bud scale (1), free lateral (2), and foliaceous (3). In case of quantitative characteristics, the expression covers the full range of variation from one extreme to the other, variation is continuous. These characters are less heritable, and expression is highly influenced by the environment. For example, shoot thickness can be classified as thin (3), medium (5), and thick (7) and petiole length can be classified as short (3), medium (5), and long

(7). Pseudo-qualitative characteristics, expression range is partly continuous, but varies in more than one dimension. Plant growth habit is classified as, erect (1), semi-erect (2), spreading (3), and drooping (4) and shape of the mulberry leaf can be classified as cordate (1), wide ovate (3), ovate (5), narrow ovate (7), and lanceolate (9). Mulberry DUS guidelines include six qualitative, 15 quantitative, and 14 pseudo-qualitative characteristics. In DUS guidelines, some characteristics are considered as grouping and asterisked characteristic. Grouping characters are more stable characters known by experience and used to group similar candidate varieties together for DUS testing to assess distinctiveness and also to select varieties of common knowledge, i.e. reference varieties similar to candidate varieties to include in the growing trail, they also help to know dissimilar varieties of common knowledge to exclude from growing trail. In mulberry, following are the grouping characteristics: internodal distance, phyllotaxy, leaf base, sex, and mature inflorescence length. Asterisked characteristics are important for variety descriptions, should be included in every growing season for the description of the variety, except when there is no expression of a character due to unfavourable environmental conditions of the testing regions. Under such situation, adequate explanation shall be provided.

8.8

Example Varieties for States of Expression of the Characteristics

To provide clarification for states/note, example varieties are included in DUS guidelines. For example, in mulberry “French variety” is an example for acute leaf base. Leaf base of “French variety” can be compared with other varieties to know which of the varieties have acute leaf base. They also help to compare DUS test data recorded at different locations and helps new examiners during DUS testing of the species. Example varieties should be easily and freely accessible. If possible, for many characters single example variety can be used. In

8

Importance and Current Status of DUS Testing in Mulberry

187

Table 8.1 List of characteristic for DUS testing https://plantauthority.gov.in/sites/default/files/mulberry.pdf Sl. No

Characteristic

1 QN (*)

Plant: vigor

2 PQ (*) (+)

Plant: growth habit

3 QN

Sprouting (days)

4 QN

Survival % of cuttings (rooting)

5 PQ (+)

Shoot: type

6 QN

Shoot: thickness (cm)

7 PQ

8 QN (*)

Mature shoot: color

Inter-nodal distance (cm)

State

Note

Example varieties

Stage of observation (Day—on or after)

Type of assessment

45

VG

60

VS

8

MG

90

MG

60

VS

60

MS

90

VS

90

MS

Low

3

Kajli, Surat, Harmutty

Medium

5

Kanva-2

High

7

M. laevigata (Hybrid)

Erect

3

Philippines, Kanva-2, Mysore Local

Semi-erect

5

M. multicaulis, Kosen

Spreading

7

Kajli, Bilidevalaya, Doomar Nali, Mizusawa

Drooping

9

Creeping mulberry

Early (15)

7

Urgam-1, French

Low (80)

7

Kajli

Straight

3

Kanva-2

Slightly curved

5

Kosen

Curved

7

Doomar Nali

Thin (1.5)

7

Mizusawa, Lazuraso, Kosen, Gajapathipur-2

Yellow-Green Group 147

1

Kokuso

Greyed-Green Group 195

3

China-34

Grey-Brown Group 199

5

Asiyoake, Lazuraso, Birds Foot

Brown Group N200

7

K2xBC (P11)

Grey Group 201

9

Barbat Farm

Short (6)

7

Phillipines, Doomar Nali, Moreti (Syringe), Birds Foot (continued)

188

M. R. Bhavya et al.

Table 8.1 (continued) Sl. No

Characteristic

State

Note

Example varieties

Stage of observation (Day—on or after)

Type of assessment

9 PQ (*) (+)

Phyllotaxy

Distichous (1/2)

3

Birds Foot, Doomar Nali, M. laevigata (Hybrid)

60

VS

Tristichous (1/3)

5

Bilidevalaya

Pentastichous (2/5)

7

Lazuraso, Kosen, Harmutty, Mizusawa

Mixed type {(1/2 and 1/3), (1/2 and 2/5), (1/3 and 2/5), (1/2, 1/3, and 2/5)}

9

French, Mysore Local, Kanva-2, Asiyoake

Acute

3

Kanva-2, Mysore Local

45

VG

Horizontal

5

Moreti (Seringe)

Obtuse

7

Philippines

Short (5)

7

Railway Quarter, Doomar Nali, Creeping CP x V-1 (P5)

Thin (0.4)

7

M. multicualis, Phillipines, Doomar Nali, China-34 45

VS

60

MS

60

MS

10 QN (+)

Leaf: angle

11 QN (+)

Petiole: length (cm)

12 QN

13 QL

14 QN (*)

15 QN (*)

Petiole: thickness (cm)

Stipule: nature

Leaf lamina: length (cm)

Leaf lamina: width (cm)

Bud scale

1

Surat, Acc.106

Free lateral

2

Mysore Local, Railway Quarter

Foliaceous

3

Barbat Farm

Short (20)

7

M. multicaulis, Doomar Nali, Phillipines, M. laevigata (Hybrid)

Narrow (15)

7

Doomar Nali (continued)

8

Importance and Current Status of DUS Testing in Mulberry

189

Table 8.1 (continued) Sl. No

Characteristic

State

16 QN (*) (+)

Leaf: size (sq. cm)

Small (< 200) Medium (200–400) Large (> 400)

7

Doomar Nali, M. laevigata (Hybrid)

Cordate (L/W ratio = < 1:1)

1

Kanranjotli-1

Wide ovate

3

M. multicaulis

Ovate (L/W ratio = 1.5:1)

5

Doomar Nali, Kanva2, Mysore Local

Narrow ovate (L/W ratio = 2:1)

7

Harmutty

Lanceolate (L/W ratio = 3:1)

9

French

Light Green-141 D

3

Kanva-2

17 PQ (+)

Leaf: shape

Note

Example varieties

Stage of observation (Day—on or after)

Type of assessment

3

Surat

90

MG

5

Kosen, Kanva-2, Punjab Local

60

MS

60

VS

90

VS

90

VS

60

VS

60

VS

(L/W ratio = 1.2:1)

18 PQ (+)

19 PQ

20 PQ

21 PQ (*) (+)

22 PQ (*) (+)

Leaf: color

Leaf: hairiness

Leaf: texture

Leaf: base

Leaf: apex

Green-137C

5

Philippines

Dark Green-137A

7

Railway Quarter, Kajli, M. multicaulis

Glabrous

3

Kosen

Sparsely hairy

5

Birds Foot, Badodhi, Malakai Local, Ranchi-5

Hairy (pubescent)

7

Urgam-1

Membranaceous

3

Philippines

Charataceous

5

Kajli, Surat, Mysore Local

Coriaceous

7

French, Barbat Farm

Acute

3

French

Truncate

5

Kanva-2, M. laevigata (Hybrid)

Cordate

7

Mizusawa, M. multicaulis, Punjab Local

Lobate

9

Kosen, Malakai Local

Acute

3

Philippines, French, China-34

Acuminate

5

Punjab Local, Mysore Local

Caudate

7

Barbat Farm, Harmutty, Badodhi, Ranchi-5

Obtuse

9

– (continued)

190

M. R. Bhavya et al.

Table 8.1 (continued) Sl. No

Characteristic

23 PQ (*) (+)

Leaf: margin

24 PQ (*)

25 QN (+)

Leaf: type

Mature bud size

26 QL (+)

Bud attachment

27 PQ (*) (+)

Mature bud shape

State

Note

Example varieties

Stage of observation (Day—on or after)

Type of assessment

60

VS

60

VG

90

VG

60

VS

90

VG

60

VS

20

VG

Crenate

3

Philippines, Kosen

Dentate

5

Surat, Acc.106, Malakai Local

Serrate

7

M. laevigata (Hybrid)

Repand

9

Lamia Bay

Unlobed (entire)

1

M. multicaulis

Lobed

2

Kajli, Bilidevalaya

Mixed type

3

Mysore Local, Badodhi, Ranchi-5

Small

3

Philippines, Surat, Punjab Local

Medium

5

Kosen, M. multicaulis, Kajli

Large

7

Doomar Nali

Adhering to branch

1

Philippines, Surat, Mysore Local

Slanting out ward

2

M. multicaulis, Kajli, Barbat Farm

Tilting to one side

3

–

Round

3

Acc.106, Gajapathipur-2

Acute triangle

5

Philippines, Mysore Local, Punjab Local

Long triangle

7

Mizusawa, M. multicaulis, Birds Foot, Badodhi

Spindle

9

Doomar Nali

28 QL (+)

Accessory bud

Absent

1

French

Present

9

M. multicaulis, Mysore Local

29 QL (*) (+)

Sex

Gynoecious

1

Kajli,, Doomar Nali, M. multicaulis

Androecious

2

Lamia Bay

Bisexual

3

Gajapathipur-2

Andromonoecious

4

–

Gynomonoecious

5

–

Androgynomonoecious

6

– (continued)

8

Importance and Current Status of DUS Testing in Mulberry

191

Table 8.1 (continued) Sl. No

Characteristic

State

Note

Example varieties

Stage of observation (Day—on or after)

Type of assessment

30 QN (*) (+)

Mature inflorescence: length (cm)

Short (4)

7

Birds Foot, Doomar Nali, M. laevigagta (Hybrid)

Pubescent

3

Mysore Local, Railway Quarter

20

VS

Papillate

7

Philippines, Doomar Nali, Lazuraso

Erect

3

Lasuraso, M. multiculis, Kajli, Birds Foot

20

VS

Spreading

5

Punjab Local, Kanva2, Bilidevalaya

Divaricate

7

Mysore Local, Surat, Harmutty, Karanjotli-1

Twisted

9

Doomar Nali, Ranchi5 40

MS

40

MS

31 QL

32 QL

33 QN (+)

34 QN

Stigma: nature

Stigma: type

Mature fruit: length (cm)

Mature fruit: width (cm)

Short (8)

9

Doomar Nali

Narrow (1.5)

7

Railway Quarter (continued)

192

M. R. Bhavya et al.

Table 8.1 (continued) Sl. No

Characteristic

State

Note

Example varieties

Stage of observation (Day—on or after)

Type of assessment

35 PQ (+)

Mature fruit: color

Black Group 203—Bluish Black C

1

M. multicaulis, Kajli, Bilidevalaya, Surat, Kanva-2, Mysore Local

40

VG

Greyed-Orange Group 172 —Dark Reddish Orange B

2

M. laevigata (Hybrid)

Purple Group 76—Very Pale Purple C

3

Punjab Local, RC-1

Yellow-Green Group 145 —Light Yellow Green D

4

Moreti (Seringe)

White

5

Ranchi-5

Green

6

Saravathi Tea Estate

The type of assessment of characteristics indicated in the table is as follows: MG: Measurement by a single observation of a group of plants or parts of plants MS: Measurement of a number of individual plants or parts of plants VG: Visual assessment by a single observation of a group of plants or parts of plants VS: Visual assessment by observations of individual plants or parts of plants

addition to example varieties, photographs and drawings are used for illustrating the characteristics with their states of expression. Mulberry DUS guidelines consist of 34 example varieties for different states of expression (Table 8.2). Frequency distribution of 34 genotypes into different descriptor states is given in (Table 8.3). The dendrogram generated by SPSS hierarchical cluster analysis divided the genotypes into four main clusters at a rescaled distance of over 20. Cluster 1 consists of 8 genotypes, cluster 2 consists of 9 genotypes, cluster 3 consists of 16 genotypes, and mulberry genotype Philippines in cluster 4 (Fig. 8.1, top to bottom). The distribution of genotypes into different clusters indicates the presence of diversity and distinctiveness among 34 example genotypes.

8.9

candidate variety from all existing reference varieties. At present, mulberry have 12 reference varieties (Table 8.2) which are collected, established, and maintained in field at nodal mulberry DUS centre, CSR&TI, Mysore. Theoretically reference collection refers to varieties of common knowledge “world-wide basis”. But, practically this not likely to happen due to limitations in collecting all varieties on a national basis https:// www.cicr.org.in/pdf/dus_test_manual.pdf.

8.10

Candidate Varieties

Varieties applied for registration under Plant Variety Protection Act are called candidate varieties. At present, mulberry has six candidate varieties (Table 8.2).

Reference varieties

All released and notified varieties are called reference varieties or varieties of common knowledge (varieties in the public domain and known to people). For grant of plant variety protection, candidate variety must be compared with reference varieties to establish distinctiveness of the

8.11

Plant Material Required

In mulberry, applicant should supply normally 50 stem cuttings of 12–15 cm length and 1.0– 1.5 cm diameter from 6 to 8 months mature shoots with 2–3 healthy buds. Stem cuttings must

8

Importance and Current Status of DUS Testing in Mulberry

193

Table 8.2 Example, reference, and candidate varieties of mulberry Sl. No

Variety name

Accession number

Species

Country/State

Pedigree/Source

Example varieties 1

Acc.106

MI-0082

Morus spp.

Karnataka, India

Collection

2

Asiyoake

ME-0120

Morus spp.

Japan

CPH collection

3

Badodhi

MI-0340

M. laevigata

Madhya Pradesh, India

Collection

4

Barbat Farm

MI-0104

Morus spp.

Uttar Pradesh, India

Clonal collection

5

Bilidevalaya

MI-0042

Morus indica

Karnataka, India

Collection

6

Birds Foot

MI-0079

M. laevigata

Karnataka, India

Collection

7

China-34

ME-0214

Morus spp.

China

Introduction

8

Creeping CPXV1 (P5)

MI-0812

Morus spp.

Tamilnadu, India

Hybrid

9

Creeping Mulberry

ME-0075

Morus spp.

Japan

Introduction

10

Doomar Nali

MI-0365

M. laevigata

Andaman &Nicobar, India

Collection

11

French

ME-0090

Morus spp.

France

Introduction

12

Gajapathipur-2

MI-0135

Morus spp.

Uttar Pradesh, India

Collection

13

Harmutty

MI-0360

M. indica

Arunachal Pradesh, India

Collection

14

K2 X BC (P11)

MI-0470

Morus spp.

Karnataka, India

Hybrid

15

Kajali

MI-0068

Morus spp.

West Bengal, India

Local land race

16

Kanva-2

MI-0014

M. indica

Karnataka, India

OPH of Mysore Local

17

Karanjotli-1

MI-0633

M. laevigata

Jharkhand, India

Collection

18

Kokuso

ME-0113

Morus alba

Japan

Introduction

19

Kosen

ME-0066

M. latifolia

Japan

Introduction

20

Lamia Bay

MI-0364

M. laevigata

Andaman &Nicobar, India

Collection

21

Lazuraso

ME-0016

Morus spp.

Japan

Introduction

22

M. laevigata (Hybrid)/Hosur C3

MI-0673

Morus spp.

Tamilnadu, India

Hybrid

23

M. multicaulis

ME-0168

Indonesia

Introduction (continued)

194

M. R. Bhavya et al.

Table 8.2 (continued) Sl. No

Variety name

Accession number

Species

Country/State

Pedigree/Source

M. latifolia 24

Malkai Local

MI-0454

M. indica

Meghalaya, India

Collection

25

Mizusawa

ME-0034

M. bombycis

Japan

Introduction

26

Moreti (Seringe)

ME-0096

Morus latifolia

Japan

Introduction

27

Mysore Local

MI-0052

Morus indica

Karnataka, India

OPH selection

28

Philippines

ME-0011

Morus latifolia

Philippines

Introduction/Clonal selection

29

Punjab Local

MI-0026

Morus alba

Punjab, India

OPH selection

30

Railway Quarter

MI-0777

M. indica

Maharastra, India

Collection

31

Ranchi-5

MI-0629

M. laevigata

Bihar, India

Collection

32

Saravathi Tea Estate

MI-0380

M. laevigata

West Bengal, India

-

33

Surat

MI-0003

Morus spp.

Gujarat, India

Collection

34

Urgam-1

MI-0408

M. serrata

Uttar Pradesh, India

Collection

MI-0014

M. indica

Karnataka, India

Selection

Reference varieties 1

K-2

2

S-34

MI-0160

M. indica

Karnataka, India

OPH

3

AR-11

˗

M. indica

Karnataka, India

OPH

4

S-146

MI-0045

Morus spp.

West Bengal, India

Selection from OPH

5

RFS-135

MI-0048

M. indica

Karnataka, India

OPH

6

S-36

MI-0013

M. indica

Karnataka, India

MB (EMS treatment) from Berhampore local

7

MR-2

MI-0025

M. sinensis

Tamilnadu, India

Selection from OPH

8

S-13

MI-0012

M. indica

Karnataka, India

OPH

9

RFS-175

MI-0066

M. indica

Karnataka, India

OPH

10

S-1635

MI-0173

M. indica

West Bengal, India

OPH

11

Vishala

˗

M. species

Karnataka, India

Clonal selection

12

V-1

MI-0308

M. indica

Karnataka, India

Hybrid from S30  Ber. C776 (continued)

8

Importance and Current Status of DUS Testing in Mulberry

195

Table 8.2 (continued) Sl. No

Variety name

Accession number

Species

Country/State

Pedigree/Source

Candidate varieties 1

G-4

˗

M. species

Karnataka, India

Hybrid from M. multicaulis  S-13

2

RC-1

˗

M. species

Karnataka, India

Hybrid from Punjab local  Kosen

3

G-2

˗

M. species

Karnataka, India

Hybrid from M. multicaulis  S-34

4

Sahana

MI-0524

M. indica

Karnataka, India

Hybrid from Kanva2  Kosen

5

AR-12

MI-0799

M. indica

Karnataka, India

PB, S-41(4X)  Ber. C776

6

MSG-2

˗

˗

Karnataka, India

Hybrid from BR-4  S-13

*K-2 is both example and reference variety Cross pollinated hybrid (CPH), open pollinated hybrid (OPH), polyploidy breeding (PB), and mutation breeding (MB)

be healthy, vigorous and free from pest, disease and mechanical damage. They should not have undergone any treatment that affects the expression of the character of the variety.

8.12

Conduct of Tests

In mulberry, DUS test is for two years with two independent growing cycle (duration of 90 days from pruning) per year (June–August and September–November). Test will be conducted at two locations or in on-site testing (DUS test of candidate variety may be conducted at places specified by the applicant).

8.13

Reasons for Two Location

Two make available DUS test results in shortest time. Varieties developed and recommended for different geographical regions may require different agro-climatic growing conditions. To make available suitable growing conditions, test will be conducted at different locations. Due to some unfavourable conditions, if DUS test at primary location fails, data of second location will be available to take the decision. The conclusions made on results of more than one location are strong.

8.14

Methods and Observation

DUS test can be conducted using characters mentioned in table of characters (Table 8.1), and explanation for characters is also given in the chapter for easy understanding. For the assessment of distinctiveness and stability, observations shall be made on nine plants (e.g. plant vigour, plant growth habit, etc.) or parts of nine plants (e.g. leaf size, mature fruit length, etc.), which shall be equally divided among three replications. For the assessment of uniformity of vegetatively propagated varieties, a population standard of 1% and an acceptance of probability of at least 95% shall be applied. Grouping characteristics can be used to group the candidate varieties to facilitate the assessment of distinctiveness.

8.15

Recommendation and Grant

DUS data for all the DUS characters has been recorded for two years, and at the end of the second year, the data will be analysed by the examiner and gives recommendation on whether or not each candidate variety meets the DUS criteria.

Petiole: thickness (cm)

Petiole: length (cm)

Leaf: angle

Phyllotaxy *

Inter-nodal distance (cm) *

Mature shoot: color

Shoot: thickness (cm)

Shoot: type

Survival % of cuttings (rooting)

Sprouting (days)

Plant: growth habit *

Plant: vigour *

Descriptors

Medium (25)

Thin (2)

Medium (18)

Short

(8)

(25) (7)

Horizontal

Acute

(3)

(8)

(20) Tristichous

Distichous

(4)

(13) Medium

(5) Short

(6)

Grey-Brown Group 199

Medium (9)

Thin (24)

Slightly curved (21)

Straight

(14)

(17) (10)

Medium

Low

Medium (12)

Early (12)

Semi-erect (16)

Erect

(13)

(7)

Medium

5+

(15)

4+

Low

3+

Greyed-Green Group 195

2+

Yellow-Green Group 147

1+

Descriptor state and number of varieties in parenthesis

Table 8.3 Frequency distribution of thirty-four example genotypes into different descriptor states 6+

(7)

Thick

(9)

Long

(1)

Obtuse

(5)

Pentastichous

(10)

Long

(9)

Brown Group N200

(1)

Thick

(3)

Curved

(3)

High

(10)

Late

(9)

Spreading

(6)

High

7+

(continued)

(18)

Mixed type

(1)

Grey Group 201

(2)

Drooping

9+

196 M. R. Bhavya et al.

Leaf: type *

Leaf: margin *

Leaf: apex *

Leaf: base *

Leaf: texture

Leaf: hairiness

Leaf: color

Leaf: shape

Leaf: size (sq. cm) *

Leaf lamina: width (cm) *

Leaf lamina: length (cm) *

Stipule: nature

Descriptors

Table 8.3 (continued)

Lobed (7)

Unlobed

(17)

Ovate

(4)

(2) (10)

Mixed type

Dentate

(22)

(6) Crenate

Acuminate

(11)

Acute

(1)

(16) Truncate

Acute

(9)

(13) Charataceous

Membranaceous

(17)

(16) Sparsely Hairy

Glabrous

(10)

(25) Green-137C

(3) Light Green141D

Wide ovate

(2)

Medium (8)

Small

(15)

(22)

Medium

(9)

(22)

Narrow

Medium

5+

(1)

4+

Short

(2)

Foliaceous

3+

Cordate

Free lateral (29)

Bud scale

2+

(33)

1+

Descriptor state and number of varieties in parenthesis 6+

(27)

Serrate

(6)

Caudate

(13)

Cordate

(9)

Coriaceous

(4)

Hairy

(8)

Dark Green137A

(3)

Narrow ovate

(4)

Large

(10)

Broad

(11)

Long

7+

(continued)

(1)

Repand

(0)

Obtuse

(9)

Lobate

(1)

Lanceolate

9+

8 Importance and Current Status of DUS Testing in Mulberry 197

*

Essential descriptors,

Mature inflorescence: length (cm) *

Sex *

Accessory bud

Mature bud shape *

Bud attachment

Mature bud size

Descriptors

Table 8.3 (continued)

+

Andro monoecious Medium (16)

(0)

(10)

(1)

Short

(0)

Notes (1–9) for the different state of different characteristics

(10)

(20)

Gyno monoecious

Androgyno monoecious (3) (8)

Long

(16)

(1)

Spindle

9+

(16) Androecious

Gynoecious

Bisexual

(15)

(2)

Long triangle

(4)

Large

7+

(18)

Acute triangle

Round

(0)

6+

Present

(10)

(24)

Tilting to one side

Medium (13)

5+

Small

4+

(17)

3+

Absent

Slanting outward

2+

Adhering to branch

1+

Descriptor state and number of varieties in parenthesis

198 M. R. Bhavya et al.

8

Importance and Current Status of DUS Testing in Mulberry

199

Fig. 8.1 Dendrogram of thirty-four mulberry genotypes using nearest neighbour cluster method, generated by SPSS hierarchical cluster analysis

200

8.16

M. R. Bhavya et al.

Explanation on the Table of Characteristics

The characters and its states of expression mentioned in table of characters (Table 8.1) are described in detail with explanations, drawing, and photographs for thorough understanding (Figs. 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 8.10, 8.11, 8.12 and 8.13). http://csgrc.res.in/ downloads/muberry_manual.pdf Plant vigour—to record plant vigour a group of plants are observed visually for above ground biomass of the plant after 45 days of pruning. Plant growth habit—each plant is observed visually for the angle between the foliage branches to the main trunk after 60 days of pruning (Fig. 8.2). Sprouting (days)—group of plants in each accession are observed starting from eighth day after pruning and the number of days taken for uniform bud sprouting > 75% may be recorded. Survival % of cuttings (rooting)—sixty cuttings per accessions will be planted in the nursery, and survival % of cuttings will be recorded after 90 days of growth (Thangavelu et al. 1997, 2000). Shoot type—the longest shoot was visually observed for straightness or curved nature after 60 days of pruning. Shoot thickness (cm)—recorded after 60 days of pruning by measuring the diameter of the longest shoot in lower 1/3rd portion at about 10 cm above the bottom of the longest shoot using Vernier calliper. Mature shoot colour—visually assessed after 90 days of pruning in the lower 1/3rd portion of the longest shoot using standard Royal Horticultural Society (RHS) colour chart. Inter-nodal distance (cm)—recorded on 90th day after pruning by measuring the total length of the longest shoot divided by the total number of nodes on the longest shoot (Fig. 8.3) (Tikader et al. 2006; Borpuzari et al. 2013). Phyllotaxy—observed visually after 60 days of pruning in the middle 1/3rd portion of the longest shoot for arrangement of leaves on a shoot. Consider lowest leaf in the middle 1/3rd

portion of the longest shoot as first leaf and move upward by counting the leaves, if third leaf is above first leaf, fourth is on second leaf, and so on then that arrangement is called distichous (1/2). If fourth leaf is above first leaf, fifth is on second leaf, sixth is on third leaf, and so on then that arrangement is called tristichous (1/3). If sixth leaf is above first leaf, seven is on second leaf, eighth is on third leaf, and so on then that arrangement is called pentastichous (2/5) and if observed combination of any two of above arrangement or all the three then called mixed type (Fig. 8.4) (Thangavelu et al. 2000; Tikader et al. 2013). Leaf angle—observed for the angle between the leaf petiole at leaf base and the main stem after 45 days of pruning in the middle 1/3rd portion of the longest shoot (Fig. 8.5). Petiole length (cm)—recorded after 60 days of pruning in the leaves collected from middle portion of the longest shoot. Measure the length of the petiole after separating petiole portion from the base of the leaf blade (Fig. 8.6). Petiole thickness (cm)—Petiole thickness was measured using Vernier callipers in the middle portion of the petiole from the same petioles used for petiole length. Stipule nature—observed the stipules present both sides of the bud at base of the petiole after 45 days of pruning in upper 1/3rd portion of the longest shoot. If stipules are short, thin and pointed, then called bud scale, if they are freely hanging from both sides of the nodal portion, then called free lateral. If broad and like miniature leaves, then called foliaceous. Leaf lamina length (cm)—recorded after 60 days of pruning in the leaves collected from middle portion of the longest shoot. Measure the leaf lamina length from leaf tip to the leaf base at the juncture of the petiole attachment. Leaf lamina width (cm)—recorded after 60 days of pruning in the leaves collected from middle portion of the longest shoot. Measure the width of the leaf from the widest point on both sides of the leaf margins. Leaf size (sq. cm)—measured after 90 days of pruning from fully expanded matured leaves

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Fig. 8.2 Characteristic of plant growth habit Fig. 8.3 Characteristic of inter-nodal distance

Distinctiveness in Inter-nodal Distance

Short

Medium

Long

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Fig. 8.4 Characteristic of phyllotaxy

Distinctiveness in Leaf: Angle

Acute Fig. 8.5 Characteristic of leaf angle

Horizontal

Obtuse

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Distinctiveness in Petiole: length (cm)

Short

Medium

Long

Fig. 8.6 Characteristic of petiole length

in the middle portion of the longest shoot using leaf area metre. Leaf shape—it is a leaf lamina length to leaf lamina width ratio calculated by using measurements of leaf lamina length and leaf lamina width. Leaf colour—visually assessed after 60 days of pruning in the leaves collected from middle portion of the longest shoot using standard Royal Horticultural Society (RHS) colour chart. Leaf hairiness—observed after 90 days of pruning from fully expanded matured leaves in the middle portion of the longest shoot based on feeling by touch. If upper leaf surface is smooth without hairs, then called glabrous, surface with few hairs called sparsely hairy and surface with more hairs and feels rough called pubescent. Leaf texture—recorded after 90 days of pruning from fully expanded matured leaves in

the middle portion of the longest shoot. If leaves are thin and semi-transparent like a fine membrane, then called membranaceous. If leaves are opaque and like a writing paper, then called charataceous, if leaves are like leathery, then called coriaceous. https://www.fao.org/3/ AD107E/ad107e0v.htm#TopOfPage Leaf base—recorded after 60 days of pruning by collecting leaves from the middle 1/3rd portion of the longest shoot and observing the shape of the leaf base. Leaf margin at the base form acute angle to the mid vein then called acute leaf base. In truncate leaf base, leaf margin is perpendicular to the mid vein or nearly so. Cordate leaf base looks like a heart shape embed in a sinus whose sides are straight or convex. Leaf base with small to large rounded projections (lobes) whose inner margins towards the petiole and are in partly concave shape (Fig. 8.7) (Alok et al. 2016).

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Distinctiveness in Leaf: Base

Acute

Truncate

Cordate

Lobate

Fig. 8.7 Characteristic of leaf base

Distinctiveness in Leaf: Apex

Caudate Acute

Fig. 8.8 Characteristic of leaf apex

Acuminate

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Importance and Current Status of DUS Testing in Mulberry

Fig. 8.9 Characteristic of leaf margin

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Distinctiveness in Leaf: Margin

Serrate Dentate Crenate

Fig. 8.10 Characteristic of stigma nature

Stigma nature

Leaf apex—recorded after 60 days of pruning by collecting leaves from the middle 1/3rd portion of the longest shoot and visually observing the shape of the leaf apex. The tip portion is straight to convex margins forming an

angle of 45° to 90°, then called acute. Leaf tip is slightly extended, margins are straight to concave and angle less than 45°, then called acuminate. The leaf tip extended greatly forming a tail like structure with concave margins, then called

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Stigma type

Fig. 8.11 Characteristic of stigma type

Distinctiveness in Fruit Size

Short

Medium Long

Fig. 8.12 Characteristic of distinctiveness fruit size

caudate. Margins form terminal angle more than 90°, then called obtuse (Fig. 8.8) (Alok et al. 2016). Leaf margin—recorded after 60 days of pruning by selecting leaves from the middle

1/3rd portion of the longest shoot and visually observing the margin of the leaf for its smoothness and serrations. If crenations are smoothly rounded without a pointed apex, then called crenate. A serrate margin is when a leaf has

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Distinctiveness in Fruit colour

Black Group 203 – Bluish Black C

Yellow – Green Group 145 – Light Yellow Green D

Purple Group 76 –Very Pale Purple C

Fig. 8.13 Characteristic of mature fruit colour

sharp, “saw-like” teeth. Serrations are pointed with their axis approximately perpendicular to the trend of the margin. Dentate margin is when a leaf has triangular, tooth-like edges. Dentations are pointed with their axis approximately perpendicular to the trend of the margin. Margins form a smooth line or arc without noticeable projections called repand (Fig. 8.9) (Alok et al. 2016). Leaf type—recorded after 60 days of pruning by general observation on whole plant leaves of group of plants for presence or absence of lobations. If all the leaves are unlobed, then called unlobed leaf type, if all the leaves are lobed, then called lobed leaf type, and if both type of leaves are present in single branch or different branch, then called mixed type. Mature bud size—recorded after 90 days of pruning in middle 1/3rd portion of the longest shoot and the buds were visually observed for their size. Bud attachment—recorded after 60 days of pruning in middle 1/3rd portion of the longest shoot and buds are observed for its attachment to the main stem. Adhering to branch: bud attached to stem, slanting outward: bud projected away

from stem, and tilting to one side: tip of the tilted to one side. Mature bud shape—recorded after 90 days of pruning in middle 1/3rd portion of the longest shoot and a group of buds are observed visually for their shape. Round: buds are circular, acute triangle: bud is like triangle and tip of the bud acute, long triangle: bud is like triangle and tip of the bud is slightly elongated, and spindle: basal portion of bud is tapered to look like spindle. Accessory bud—recorded after 60 days of pruning in middle 1/3rd portion of the longest shoot for the presence or absence of accessory bud (s) to the main axillary bud. Sex—recorded by visual observation of matured or fully developed inflorescence in the natural flowering season (January-March) or about 2–3 weeks after pruning. If plant bears only the female inflorescence in all the branches, then called gynoecious. If plant bears only the male inflorescence in all the branches, then called androecious. If plant bears only the bisexual inflorescence in all the branches, then called bisexual. If plant bears predominantly male inflorescence with few bisexual inflorescence, then called andromonoecious, if plant bears

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predominantly female inflorescence with few bisexual inflorescence, then called gynomonoecious, and if plant bears predominantly male inflorescence with few female and bisexual inflorescence, then called androgynomonoecious. Mature inflorescence length (cm)—observations to be made on fully matured inflorescence, i.e. before anthesis in male inflorescence and at receptive stage in female inflorescence. The length of the inflorescence should measure including pedicel of the inflorescence. Stigma nature—recorded in fully matured inflorescence by visually observing the stigma surface under the microscope. If stigma surface has long hair, then called pubescent, and if surface is plain without any hairs/minute hairs, then called papillate (Fig. 8.10). Stigma type—recorded in fully matured inflorescence by visually observing the bifid stigma under the microscope. Erect: both the stigmas are straight and close to each other, spreading: both the stigmas are spreading forming wide angle, divaricate: both stigmas are away and bent towards style, and form curve like structure and twisted: both stigmas are twisted each other (Fig. 8.11). Mature fruit length (cm)—recorded after 40 days of pruning in fully matured/ripened fruit in the longest shoot by measuring the length including the peduncle of the fruit using scale or Vernier calliper. Mature fruit width (cm)—same fruits used for measuring fruit length to be used to measure fruit width Vernier calliper. Distinctiveness in fruit size is presented in Fig. 8.12. Mature fruit colour—visually assessed after 40 days of pruning in fully ripened fruits using standard Royal Horticultural Society (RHS) colour chart (Fig. 8.13).

8.17

DUS Test Plot

Established and maintaining DUS test plot with 34 example varieties and at present DUS test plot also consists 12 reference varieties and 6 candidate varieties, the number of reference and candidate varieties may change time to time. DUS

test plot is used for testing of candidate varieties for distinctiveness, uniformity, and stability by comparing against varieties of common knowledge (reference varieties).

8.18

Application for Registration of Mulberry Varieties Under PPVFRA 2001

Application of a plant variety for registration can be filed by any person claiming to be a breeder, successor of breeder, any institute, farmer or group of farmers, any person being assignee of any of above, convention country, and any other. Application forms are different for filing application under PPV&FRA 2001 for registration of new and extant varieties, farmer varieties, and essentially derived varieties and links to access applications are given below: https://plantauthority.gov.in/sites/default/files/ newextantvariety2013.pdf https://plantauthority.gov.in/hi/registrationessentially-derived-variety-edv-variety https://plantauthority.gov.in/sites/default/files/ farmervariety2013.pdf

For submission of applications to PPV &FR Authority, following points are to be kept in mind: 1. Correctly filled copy of Form 1 (Form II is applicable only to essentially derived varieties including the Transgenic varieties). 2. Correctly filled copy of Technical Questionnaire (TQ). 3. Each page of the application form and technical questionnaire to be signed by the authorized signatory which could be Project Director/Project Coordinator/Director of the crop-based institute of the concerned crop in case of ICAR or SAUs. 4. Copies of PV 2 (authorization by breeders to the applicant, i.e. ICAR or SAUs) and or PV 1 (authorization by applicant (ICAR or SAUs) to the authorized signatory for filing application on behalf of the breeders). ICAR has issued a letter authorizing PDIPCs/Directors to file applications of plant variety protection. This letter can be used as PVI (Annexure 1).

8

5.

6.

7. 8.

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reproduced, e.g. Pus a Bold, Pusa-Bold, Signature and official seal of the authorized PusaBold, Pusa/Bold are considered different signatory as mentioned in 3 above, on last denominations and the application shall be page of Form 1, Form 2, and TQ. liable for revision if such difference in A witness has to sign with full name, denominations are observed by the PPV address/seal on last page of TQ. Witness can &FR Authority. be any scientist breeder from the institute/Project Directorate/Project Coordi- 10. Details of payment made towards registration fee as to be mentioned in the application. nator Unit or the University. All applications should be in triplicate (one In mulberry, varieties which were authorized original with 2 photocopies). The photocopies of all enclosures including demand for release by mulberry variety authorization draft, affidavits, etc., to be included on sec- committee (MVAC) Central Silk Board, Bengaluru and suo-moto authorized varieties by ond and third copy. Print on single side (one side) of the paper. MVAC which were developed at CSR&TI, Mysuru from 1997 to 2018 were considered for Photocopy also on single side. Ensure availability of pure seed as per filing application for protection of varieties under guidelines (follow breeding standards extant varieties category. During the year 2019, required for maintenance of pure seeds, and CSR&TI, Mysore, filed application for the first also submit the quantity required for each time in mulberry in India for the registration of crop as and when requested by the PPV &FR V-1 and G-4 mulberry varieties which were Authority as given in the crop specific released for high leaf yield potential. From the guidelines published by the authority and same institute, mulberry varieties viz., G-2, appearing on the PPV &FR Authority web- Sahana, MSG-2, RC-1, and AR-12 which were released for specific conditions like chawki site from time to time). Denomination: The denomination (name) silkworm rearing, intercrop under trees, varieties given to the variety being filed for protection for sub-optimal conditions, resource constraint shall be such that it is capable of identifying areas, and alkaline soils, respectively, were filed the variety. It should not solely numbers or during the year 2020. Submitted applications were examined by figures and should not mislead regarding characteristics, value of the identity of the PPV&FR authority, V-1 mulberry variety which breeder. It should be different from every was released during 1997 was not accepted for other denomination which designates a registration because one of the conditions for variety of the same botanical species or of a acceptance is that the date of release of variety closely related species registered under the should not be more than 18 years at the time of Act. The name should not hurt religious filing application. All other varieties filed for sentiments of any class or section of citizen registration were found satisfactory and accepted of India and should not use any emblem for DUS testing, then prescribed DUS testing fee (mentioned in the emblems and names) has been deposited. DUS testing of candidate mulberry varieties (prevention of improper use) Act 1950. It (G-4, G-2, AR-12, Sahana, RC-1, and MSG-2) is should not solely or partly use geographical names. It is to be noted that the denomination under taking at CSR&TI, Mysore, its being a filled in the application form should be same nodal DUS centre for mulberry. After two years in all parts of the application form including of DUS testing, DUS data and final recommenTQ and affidavits. Use of white space dation on candidate varieties whether or not met between two parts of the name, use of DUS criteria will be submitting to PPV&FRA hyphen (“-”), or a comma (“,”) should be and final decision will be taken by the registrar, uniform wherever the denomination is to be PPV&FRA.

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Passport data of the candidate variety/varieties will be published in Plant Variety Journal of India (PVJI) for calling objections, if any, within a specified time frame. If there are no objections, registration certificate will be issued to the applicant. This certificate is valid for nine years in the case of tree and vines and six years for other crops and may be renewed for remaining period on payment of prescribed fee. The total period of validity of certificate of registration shall not exceed 18 years in case of trees and vines and 15 years in case of other crops, from the date of registration of the variety (www. plantauthority.gov.in).

8.19

Use of Molecular Markers in DUS Testing

DUS test guidelines developed in mulberry is based on morphological descriptors, these descriptors have disadvantages such as expression of trait is influenced by environment and stage specific and limited in number, and therefore, DNA markers can be used as supporting tool for DUS testing and rapid identification of varieties (Gray and Call 1994; Pinto et al. 2018). Even though several molecular markers have been applied to understand the genetic difference in mulberry, but application of these markers in DUS testing is very limited. Simple sequence repeats (SSRs) are the most preferred markers as mulberry is a highly cross-pollinated and heterozygous (Mathithumilan et al. 2016; Pinto et al. 2018; Arunakumar et al. 2021; Shinde et al. 2021; Gnanesh et al. 2023; Vijayan et al. 2022a, b). Pinto et al. (2018) developed 24 specific SSR markers for application in cultivar identification and DUS testing. In our study, we selected 18 representative varieties, example (06), reference (07), and candidate (5) and genotyped using 20 polymorphic SSRs. The dendro analysis arranged all accessions into two major clusters at the distance coefficient of 0 to 0.1 as shown in

Fig. 8.14. Cluster-I and Cluster-II were subdivided into two groups Cluster IA and Cluster IB, Cluster IIA and IIB. Cluster IA and IIA had three mulberry varieties; similarly cluster IB and IIB had six mulberry varieties (Table 8.4). G-2 is a mulberry variety developed for chawki rearing obtained from the crosses made between M. multicaulis  S-34 (M. indica), but based on the genomic regions distribution G-2 and their parents are grouped in the different sub-cluster of major cluster I. Correspondingly, G-4 mulberry variety developed from Morus multicaulis  S-13, G-4 and their parents are widely dispersed (S-34 in Cluster II), this genetic differentiation might be due to different allele combination or migration of alleles. Interestingly, mulberry varieties AR-12 and Vishala which are good agronomic superior triploids have grouped in the Cluster II with V-1 mulberry variety having good yield potential (promising and ruling variety), its foliage is of excellent quality and ideal for silkworm rearing in all seasons also ruling high yielding variety of India. Except Kosen, all other example varieties are grouped at cluster I (Green highlighted) likewise reference varieties are grouped in the cluster II except S-34 (Pink highlighted) but candidate varieties are present in both the clusters (blue highlighted). Kosen germplasm utilized generally used as a parent in mulberry breeding programme, MR-2 (OPH variety) evolved at Coonoor Sericulture Farm, Tamil Nadu, and it mildew resistant variety was closely grouped with S-36 mulberry variety which was EMS mutant diploid developed of Berhampore local germplasm, however, this variety is nutritionally superior mainly used for silkworm rearing. Even though the SSRs used were able to categorize the example, reference, and candidate varieties but still they were intermixed with the other clusters. This indicates use of single nucleotide polymorphism (SNPs) will be more beneficial for DUS testing in mulberry.

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Fig. 8.14 Neighbour-joining tree of the 18 representative (example, reference, and candidate) mulberry cultivars based on 20 SSRs

Table 8.4 Clustering of example, reference, and candidate mulberry varieties based on molecular marker analysis Sl no

Clusters

Sub-clusters

Number of germplasm

Germplasm

1

Cluster I

IA

03

Punjab local, RC-1 and S-34

IB

06

G-2, G-4, M. multicaulis, Birds foot, Kokuso and Ranchi-5

2

Cluster II

IIA

03

S-13, V-1 and Vishala

IIB

06

S-36, Kosen, MR-2, Sahana, AR-12 and K-2

8.20

Conclusion

Efforts made towards the development of mulberry DUS guidelines under PPV&FRA is the first step towards registration of mulberry varieties. The DUS guidelines include 35 characters, 34 example genotypes, and different states of expression for each character to describe the

variety. Explanation on table of characteristics can be used for recording data of the variety for filing application for registration and protection of the variety under PPV&FR Act, 2001. Furthermore, some modifications are required in the present mulberry DUS guidelines, i.e. instead of mature bud size which is recorded by visual assessment which is differ on people perception can be removed and mature bud length can be

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included which is through measurable assessment, plant vigour which is through visual assessment can also be assessed through measuring length and width of plant portion that grown after pruning. Moreover, need to add two more states of expression to sex character for genotypes that bear both male and female flowers on same inflorescence or on same plant and for non-flowering genotypes. Some more traits like stomatal frequency and lenticel density can be added to the guidelines after validating stability of these traits in commonly known varieties. Protection of mulberry varieties and channelizing the availability of true to type planting material through Kissan nurseries will accelerates the sericulture growth of the country by providing the high-quality planting material of improved varieties. Use of protected varieties for development of improved mulberry varieties through gene editing and transgenic technologies may fetch the royalty to the protected variety. At present, examination and grant of protection is entirely based on morphological characters although the possible use of molecular markers in the examination of DUS is under active consideration. With the plenty of availability of genomic resources in mulberry, SNPs are the choice of markers for exploiting genetic variation and in DUS testing.

References Alok S, Jhansi Lakshmi K, Saraswathi P, Sekar S (2016) Manual of mulberry gene bank operations and procedures. http://csgrc.res.in/downloads/muberry_manual. pdf Arunakumar GS, Gnanesh BN, Manojkumar HB, Doss SG, Mogili T, Sivaprasad V, Tewary P (2021) Genetic diversity, identification, and utilization of novel genetic resources for resistance to Meloidogyne incognita in mulberry (Morus spp.). Plant Dis 105 (10):2919–2928 Bhat KV (2001) DNA fingerprinting and cultivar identification. National Research Centre on DNA finger printing, NBPGR, New Delhi, pp 101–109 Borpuzari MM, Rao AA, Ramesh SR, Jhansilakshmi K, Saraswathi P, Mohan Ram Rao D, Radhakrishnan R, Sekar S, Shankar PBV (2013) Catalogue on Mulberry

M. R. Bhavya et al. (Morus spp) germplasm, vol (5) Published by Dr. A. Manjula, CSGRC, Central Silkboard, Hosur, India Das KD (2012) Genetic evaluation and characterization of Jute (Corchorus spp. L) genotypes using DUS parameters. SAARC J Agri 10(2):147–153 Gnanesh BN, Mondal R, Arunakumar GS, Manojkumar HB, Singh P, Bhavya MR, Sowbhagya P, Burji SM, Mogili T, Sivaprasad V (2023) Genome size, genetic diversity, and phenotypic variability imply the effect of genetic variation instead of ploidy on trait plasticity in the cross-pollinated tree species of mulberry. bioRxiv 2023.04.02.535280; https://doi.org/ 10.1101/2023.04.02.535280 Gray E, Call NM (1994) Effects of induced plant injury on leaf lobation in red mulberry (Morus rubra L.). Castanea 59:167–175 Hariprasanna K (2019) Aruna C, Visarada KBRS, Venkatesh Bhat B, Tonapi VA (eds) Distinctness, uniformity and stability (DUS) testing in sorghum breeding sorghum for diverse end uses. Woodhead publishing, pp 341–365 Mathithumilan B, Sajeevan RS, Biradar J, Madhuri T, Nataraja K, Sreeman SM (2016) Development and characterization of genic SSR markers from Indian mulberry transcriptome and their transferability to related species of Moraceae. PloS one 11(9):e0162909 Pinto MV, Poornima HS, Sivaprasad V, Naik VG (2018) A new set of mulberry-specific SSR markers for application in cultivar identification and DUS testing. J Genet 97(1):31–37 PPV & FRA (2005) Training manual on DUS test in cotton with reference to PPV & FR legislation 2001. https://www.cicr.org.in/pdf/dus_test_manual.pdf PPV & FRA (2016) DUS test guidelines for Mulberry (Morus spp.). Plant Variety J India 10(10). https:// plantauthority.gov.in/sites/default/files/mulberry.pdf Shinde BB, Manojkumar HB, Arunakumar GS, Bhavya MR, Gnanesh BN (2021) Assessment of statistical software to analyze genetic diversity in mulberry germplasm. Sericologia 61(3&4):19–22 Thangavelu K, Mukherjee P, Tikader A, Ravindran S, Goel AK, Rao AA, Naik VG, Sekar S (1997) Catalogue on Mulberry (Morus spp) germplasm, vol 1, Published by Dr P Mukherjee, Silkworm and Mulberry Germplasm Station, Central Silkboard, Hosur, India Thangavelu K, Tikader A, Ramesh SR, Rao AA, Naik VG, and Sekar S and Deole AL (2000) Catalogue on Mulberry (Morus spp) germplasm, vol 2, Published by Dr K Thangavelu CSGRC, Central Silkboard, Hosur, India Tikader A, Chandrasekhar M, Borpuzari MM, Saraswat RP, Rao AA, Sekar S, Deole AL(2006) Catalogue on Mulberry (Morus spp) germplasm, vol 3 & 4, Published by Dr. C. K. Kamble, CSGRC, Central Silkboard, Hosur, India Tikader A, Saratchandra B, Vijaya K, Singh R (2013) Mulberry Germplasm management and utilization. A. P. H. Publishing corporation, New Delhi

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UPOV (2002) General introduction to the examination of distinctness, uniformity and stability and the development of harmonized descriptions of new varieties of plants, TG/1/3*. International Union for the Protection of New Varieties of Plants, Geneva. http://www.upov. int/en/publications/tg-rom/tg001/tg_1_3.pdf Vijayan K, Tikader A, Teixeira da Silva JA (2011) Application of tissue culture techniques for propagation and crop improvement in mulberry (Morus spp.). Tree For Sci Biotechnol 5(1):1–13

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Vijayan K, Gnanesh BN, Shabnam AA, Sangannavar PA, Sarkar T, Zhao W (2022a) Genomic designing for abiotic stress resistance in Mulberry (Morus spp.). In: Genomic designing for abiotic stress resistant technical crops 2022b. Springer, Cham Vijayan K, Arunakumar GS, Gnanesh BN, Sangannavar PA, Ramesha A, Zhao W (2022b) Genomic designing for biotic stress resistance in Mulberry (Morus spp.). In: Genomic Designing for Biotic Stress Resistant Technical Crops 2022a. Springer, Cham

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Molecular Diagnostics of Soil-Borne and Foliar Diseases of Mulberry: Present Trends and Future Perspective Belaghihalli N. Gnanesh , G. S. Arunakumar, A. Tejaswi, M. Supriya, Anil Pappachan, and M. M. Harshitha

9.1

Introduction

The cultivation of mulberry leaves for the rearing of silkworm (Bombyx mori L.) is called Moriculture. The mulberry is derived from Latin word Morus and originated in the sub-Himalayan tract. It is the fast-growing woody perennial plant that grows faster. In tropical countries, mulberry is grown as a shrub and as a tree in temperate countries like Japan. There are 68 species of Morus, and over 1000 varieties are under cultivation. The most common species are M. alba (white mulberry), M. nigra (black mulberry), and M. rubra (red mulberry) which are grown under a wide range of agro-climatic conditions (Vijayan et al. 2022a). The cultivation of mulberry is mainly distributed in China, India, Japan, Russia, Korea (DPR and South), Brazil, Thailand, Vietnam, and distributed in Europe, North and South America and Africa (Awasthi et al. 2004). The perfect temperature range varies from 24 to 28 °C,

B. N. Gnanesh (&)
G. S. Arunakumar
A. Tejaswi
M. Supriya
M. M. Harshitha Molecular Biology Laboratory-1, Central Sericultural Research and Training Institute, Mysuru, Karnataka 570008, India e-mail: [email protected] A. Pappachan P2 Basic Seed Farm, National Silkworm Seed Organization (NSSO), Yelagiri Hills, Tamil Nadu 635853, India

humidity within the range of 65–80%, and the optimum pH is 6.5–6.8 (Dandin et al. 2003). The silkworm B. mori feeds only on mulberry to produce silk. Mulberry leaves protein is used by the silkworm to biosynthesize the silk, which is composed of two proteins, i.e., fibroin and sericin, and its leaf and cell wall together contain structural carbohydrates that are highly digestible. India is the second largest leading producer of mulberry raw silk, next to China, and it produces all four kinds of natural silk (Mulberry, Eri, Muga, and Tassar). Cultivation of these varieties differs greatly in their adaptability to various soil types and climatic conditions, leaf quality, and leaf yield as well as resistance to diseases. Mulberry being highly nutritious, and perennial is affected by various diseases, under congenial climatic conditions (Arunakumar et al. 2018; Govindaiah et al. 1993). Several fungi, bacteria, virus, nematode, and mycoplasma like organisms cause different types of diseases frequently and affects their yield (Sukumar and Padma 1999). These diseases are usually airborne and soil-borne and cause up to 5–10% loss in leaf yield by defoliation and an additional loss of 20–25% by deterioration in leaf quality. Molecular characterization using universal primers has been widely recommended for classification and identification of soil-borne and foliar disease causing pathogens (Bautista-Cruz et al. 2019; Crous and Groenewald 2005). Precise identification of causal agents at species level can be achieved by using the combination

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_9

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of two or more genes, like internal transcribed spacer—ITS, b-tubulin -TUB, and translation elongation factor 1 − a (TEF1) genes (Chen et al. 2013, 2021; Marques et al. 2013; Rosado et al. 2016; Vijayan et al. 2022b). This strategy can avoid misidentifications (Cruywagen et al. 2017; de Silva et al. 2019). Hence, accurate detection of pathogens is very crucial and essential for the development of effective management approaches (Choudhary et al. 2021; Hariharan and Prasannath 2021; Srivastava 2016). This chapter summarizes the available and emerging molecular techniques used in diagnosing soil-borne and foliar diseases of mulberry.

9.2

Soil-Borne Diseases

Soil-borne diseases are the most serious threat that affects the root which leads to high economic losses and plants become vulnerable to other diseases. Mulberry being perennial crop supports multiplication, inoculum build-up, and survival of certain soil-borne pathogens that cause cutting rot, stem-canker, collar rot, and dieback in the nursery and root rot and root-knot in established gardens. These diseases affect the leaf quality and reduce leaf productivity by 20% (Arunakumar et al. 2021; Gnanesh et al. 2021). The survey has revealed that the mortality of plants with a leaf yield loss of about 14–30% is due to various types of root rot in countries like Japan, China, India, Philippines, Iran, and Thailand (Aoki 1971; Gangwar and Thangavelu 1991; Minamizawa 1997; Telan and Gonzales 1998; Sharma et al. 2003; Ertian 2003; Chowdary 2006). Considering the economic losses caused by mulberry root rot, various chemical and biological methods were recommended for disease management (Sharma and Gupta 2005). The non-judicious use of fungicides has allowed pathogens to gain resistance (Leroch et al. 2011), impacted environment and human health (Komárek et al. 2010), and exhibited a toxic effect on silkworms (Sharma and Gupta 2005) making chemical control measures undesirable. Biocontrol measures were not very effective as various factors can influence their efficacy (Nelson 2004), whereas wild species

have unique alleles for tolerance to biotic and abiotic stresses (Vijayan and Gnanesh 2022). Genetic improvement by breeding resistant varieties ensures a cost-effective, sustainable, and environment-friendly feasible way of disease management (Arunakumar et al. 2021; Pandey and Bansadrai 2021; Basandrai et al. 2021; Vijayan et al. 2022a, b).

9.2.1 Root Rot Disease A complex of pathogens is associated with root rot of mulberry; Fusarium solani, F. oxysporum, Lasiodiplodia theobromae, Rhizopus oryzae, Macrophomina phaseolina, Sclerotium rolfsii and Phytophthora megasperma and Athelia rolfsii (Sutthisa et al. 2010; Pane et al. 2017; Pappachan et al. 2020; Gnanesh et al. 2021; 2022, Saratha et al. 2022a; b). The disease was first reported in 1954, and several types of root rot have been reported in mulberry, such as dry root rot caused by Fusarium solani (Mart.) Sacc., F. oxysporum Schlecht. black root rot—Botryodiplodia theobromae Pat. [syn. Lasiodiplodia theobromae (Pat.) Griffon & Maubl.] and charcoal root rot—Macrophomina phaseolina (Tassi.) Goid. [syn. Rhizoctonia bataticola (Taub.) Butler] (Sharma et al. 2003). In India, root rot is a major problem in mulberry cultivation states of Karnataka, Tamil Nadu, Andhra Pradesh, and Telangana (Philip et al. 1995; Mallikarjuna et al. 2010; Rajeswari and Angappan 2018; Sowmya et al. 2018; Gnanesh et al. 2021, 2022) and Kashmir (Gani et al. 2017). The impact of root rot disease has reached epidemic proportions due to the involvement of more than one pathogen (microbial complexity) with the potential to kill the plants and even wipe out entire plantations (Sharma et al. 2003). The disease can spread rapidly with devastating effects and results in losses of life yield of up to 31.5% (Chowdary and Govindaiah 2009) and even rendering the land unsuitable for further cultivation. The initial symptoms of root rot disease are yellowing of leaves, withering of leaves followed by sudden wilting, and drooping of branches. The affected plants can be pulled out

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easily, and the affected roots are brown and later turn to a black color resembling charcoal, and tissues become weak breaking off easily. In severe conditions, the sclerotial bodies are seen scattered on the affected root tissues. The infected plants fail to sprout after pruning and dry up completely. The pathogens that cause root rot remain dormant, and when soil microclimates are altered by a variety of factors, they become contagious, causing severe damage to plantations in a short period. In addition to the dominant root rot pathogens, there are many saprophytic or weak pathogens associated with root rot in mulberry (Gnanesh et al. 2021; Sharma et al. 2011). Information on the effects of saprophytes under field conditions and their interactions with pathogens associated with root rot in mulberry is partial. Presently, very limited varieties are resistant to root rot disease, and the majority of the commercially grown varieties are found to be susceptible, while some of the local mulberry varieties have shown moderate resistance to the disease having low leaf productivity (Sharma et al. 2003; Sharma and Gupta 2005; Gnanesh et al. 2022).

9.2.1.1 Black Root Rot (BRR) The causative agent of BRR in mulberry is L. theobromae Pat. (Syn: Botryodiplodia theobromae (Pat.) Griff. and Maubl.). This fungus causes black salsify rot and has been reported to occur in China and different states of India (Sukumar and Padma 1999; Xie et al. 2014; Sowmya et al. 2018; Pappachan et al. 2020; Gnanesh et al. 2022). When mulberry plants become susceptible to infection, the fungus becomes dominant in the roots and rapidly multiplies hyphae in the cortical tissue, extending to the pith. It penetrates xylem vessels, and browning is observed as the disease progresses, followed by plant death (Sukumar and Padma 1999; Sharma and Gupta 2005). Sowmya et al. (2018) used RAPD and SSRs for studying the variability among the ten isolates of L. theobromae causing mulberry BRR; similarly, Pappachan et al. (2020) characterized one isolate of L. theobromae. However, Gnanesh et al. (2022) studied the molecular

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phylogeny of isolates and identified resistant sources to L. theobromae.

9.2.1.2 Dry Root Rot In India, earlier mulberry dry root rot was described as white/violet root rot (Rangaswami et al. 1976), and the surveys by different researchers indicated that dry root rot is caused by F. solani and F. oxysporum (Philip et al. 1995, 1997; Amreen et al. 2017). Worldwide, it is considered one of the most dreaded fungal pathogens causing root rot disease on > 500 plant species. This fungus is difficult to control due to the thick-walled, resistant sclerotia that persist in soil and plant debris. Symptoms include deteriorating root bark and darkening of the skin due to mold spores and mycelium under the bark. Many affected plants lose their footing on the ground and are easily uprooted (Arunakumar and Gnanesh 2023). In severe cases, the entire root system rots, and the plant dies. Affected plants either do not grow after pruning or have small, pale-yellow leaves with a rough surface on the plant. Favored by warm climatic conditions and water stress, dry root rot has become a major disease across different parts of the world (Sutthisa et al. 2010; Pappachan et al. 2020; Saratha et al. 2022a, b). 9.2.1.3 Rhizopus Rot: Caused by R. oryzae was found in grafted saplings stocks of mulberry in Tsukuba in 1999 (Yoshida et al. 2001) and later reported in India Gnanesh et al. (2021). Roots are initially brown, later blackened, weakly structured, and easily broken (Yoshida et al. 2001; Fang et al. 2011; Gnanesh et al. 2021). 9.2.1.4 Charcoal Root Rot M. phaseolina is a common pathogen found in the sericulture belt of South India (Yadav et al. 2011; Sowmya et al. 2018). The majority of the mulberry cultivars are susceptible to charcoal rot which reduces the leaf yield by up to 35% due to poor leaf quality and mortality of the plant affecting the profit of sericulture farmers (Chowdary 2006; Pinto et al. 2018).

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9.2.2 Root-Knot Nematodes (RKN) RKN is extremely adaptable and causes significant damage to crop plants. The RKN was first reported from USA by Bessey in 1911, and later, it was reported from various countries. The species like Meloidogyne incognita, M. arenaria, M. javanica, and M. hapla are found mainly worldwide (Saucet et al. 2016). In India, RKN for the first time was reported by Swamy and Govindu (1966) and later by Narayanan et al. (1966) at Mysore on mulberry. Several RKN species were reported on mulberry M. incognita, M. javanica, M. hapla, M. arenaria, M. arenarithamsi, M. mali, and M. enterolobii (Toida and Yaegashi 1984; Ertian 2003; Paes-Takahashi et al. 2015). Originally, M. incognita was found to be associated with mulberry, and, later M. javanica was also reported (Sujathamma et al. 2014; Gnanaprakash et al. 2016). Out of these two species, M. incognita is very serious and chronic to the mulberry (Govindaiah and Kumar 1991; Arunakumar et al. 2021). The severity and damage depend on the soil and climatic conditions of different localities (Ramakrishnan and Senthilkumar 2003). The disease is widespread and commonly occurs in sandy soils under irrigated conditions. M. incognita causes severe damage to the crop and also it predisposes the plant to other soilborne pathogens. Young galls are small and yellowish, while older galls are large and dark brown (Arunakumar et al. 2018). Above-ground symptoms include stunted plant growth and slight chlorosis and necrosis of leaves. Leaves often lose turgidity during hot sunny days. While below ground, dry root tips and swelling of the roots are the early symptoms. Affected plants become weaker and less productive due to reduced uptake of water and nutrients (Caillaud et al. 2008). Disease severity increases with plantation age, with estimated losses in leaf yield of up to 20%; the leaf quality is also affected significantly which affects silkworm rearing (Devi and Kumari 2014). Thus, the nematode has the potential to render the entire garden unproductive. Previously, identification of root-knot nematode at the species level was largely

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dependent on a perineal pattern which is not accurate because of its large variance. At present, many molecular diagnostic tools have been developed to facilitate easy and rapid identification of different species of root-knot nematodes. For instance, M. enterollobii from mulberry in Hainan provinces of China was successfully identified using ribosomal DNA (rDNA) IGS2 (Sun et al. 2019); similarly, a modified DNA extraction method was developed for identification of RKN collected from rootknot galls at species level using species-specific diagnostic markers and sequenced 28S rDNA for further confirmation with phylogenetic analysis (Manojkumar et al. 2022). Hence, molecular diagnosis by using specific markers for different species provides a quick and easy diagnostic technique for RKN of mulberry.

9.3

Foliar Diseases

The quality of mulberry leaves plays a major role in the production of silk, which is affected by various pathogens. The major foliar diseases in mulberry are leaf spot, leaf rust, leaf blight, powdery mildew, and bacterial blight which cause economical loss by affecting leaf yield and quality (Reddy et al. 2009; Shree and Nataraj 1993).

9.3.1 Leaf Spot The leaf spot of mulberry is one of the major diseases occurring prominently during the winter and rainy seasons. It causes significant losses in leaf yield in addition to reducing the quality of leaf (Philip et al. 1991; Peris et al. 2012). The disease causes a direct leaf yield loss of about 5– 10% due to defoliation and added loss of 20– 25% by the damage to leaf area and quality (Srikantaswami et al. 1996). Hence, feeding silkworms with low quality diseased affected leaves will greatly influence the cocoons quality and yield (Noamani et al. 1970; Sullia and Padma 1987). Different types of leaf spots are detailed below.

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9.3.1.1 Cercospora moricola In the initial stages, the symptoms of C. moricola are represented by the presence of small brownish, uneven spots on the leaves which slowly increase in size and turn dark brown. Under severe conditions, a shot hole lining a yellow circle was formed by fall off of the dead tissues from the spot. Later, leaves turn yellowish and fall off before maturity. Leaf spot disease due to C. moricola Cooke is the most serious disease of mulberry which causes leaf yield loss. It was reported that Cercospora infection induces changes in the biochemical constituents which may affect the quality of leaves (Siddaramaiah and Hegde 1990). The temperature range of 20–28 °C and continuous wetness of 36–72 h plays a significant role in the highest infections. The disease intensity will increase with the increase in the density of the plantation and also reported that altered agronomical practices: spacing, crown height, and method of harvesting will influence the disease incidence (Ghosh et al. 2012). 9.3.1.2 Setosphaeria rostrata It was reported for the first time from, Karnataka, India. Initially, symptoms appear as specks later the spot enlarges into a brownish irregular shape encircled by a yellow halo. These spots start from the leaf margins and are inter-veinal in nature. Further, spots enlarge and coalesce, leading to a blighted appearance. With the increase in the severity of the disease, leaves turn yellowish and fall off before maturity. Based on the molecular (ITS) and morpho-cultural characteristics, S. rostrata was characterized in India (Arunakumar et al. 2019a). 9.3.1.3 Nigrospora sphaerica The association of N. sphaerica with shot hole symptoms was identified in India based on cultural, morphological, and ITS-rDNA sequencing. Initially, symptoms appear as specks, circular, or irregular shapes with a brownish center surrounded by a yellow halo. Later, the larger lesion dries and fell out and appears as a shot hole (Arunakumar et al. 2019b).

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9.3.1.4 Curvularia lunata The symptoms were brownish to black lesions starting from the tips or edges of the lamina. These symptoms are mainly seen in younger leaves. Severe symptoms lead to the falling of leaves. To confirm the identification of the causal organism, the internal transcribed spacer (ITS) region of ribosomal DNA was amplified with universal primers ITS4/ITS5 and sequenced directly (Bussaban et al. 2017). 9.3.1.5 Pseudocercospora mori It causes black leaf spot in mulberry and affected leaves develop small to medium size velvety black spots that appear primarily on the ventral surface of leaves. Subsequently, the spots coalesce with each other forming larger spots. Severely infected leaves turn yellowish and defoliate prematurely. It usually occurs from May to February which peaks during November and causes a 5–10% reduction in foliage yield. The fungus can cause significant damage to leaves of Morus spp. causing necrotic spots, which become brittle, resulting in a shot hole, leaf yellowing, and defoliation (Babu et al. 2002). 9.3.1.6 Paramyrothecium roridum It causes brown leaf spot, which is a major disease of mulberry, especially in Eastern and North-Eastern India (Pappachan et al. 2019). Typical symptoms of brown leaf spot include brown necrotic spots, which turn to dark brown or black color surrounded by yellow hallow which varies in shape from round to irregular. As the disease progress, smaller spots coalesce to form blighted areas. In advanced stages, highly infected leaves turned yellowish and defoliate prematurely (Belisario et al. 1999; Kim et al. 2003). Irregularly shaped, raised, black sporodochia are also observed with a white fringe of mycelia. The spore structures appear in concentric rings within the necrotic areas and on the lower side of diseased leaves (Chase 1992; Byrne and Raymond 2007). A temperature of 30–32˚C, 80–90% relative humidity, and > 10 rainy days/month are highly favorable to

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increase the disease and results in 10–12% yield loss (Qadri et al. 1999). Sprinkler irrigation, dense planting, and susceptible variety also enhance disease incidence. The disease occurs in the Gangetic plains of West Bengal, Assam, and valley districts of Manipur. It is prevalent from June to November in the plains of West Bengal.

9.3.1.7 Cladosporium pseudocladosporioides It causes leaf spot with the symptoms of round, tan or necrotic lesions bordered by a dark margin, in other cases, the lesions coalesced to form relatively large necrotic regions. The pathogen was identified as Cladosporium pseudocladosporioides based on the morphological characters of the pathogen and phylogenetic analysis of ITS and TEF gene sequences (Heo et al. 2021). 9.3.1.8 Xanthomonas campestris Pv. Mori It causes bacterial leaf spot (BLS) a devastating foliar disease predominant during monsoon (June–September) months in all the mulberry growing areas (Maji et al. 1998; Sharma et al. 2000). The foliage loss during the peak season is from 10 to 15% (Maji et al. 2000). The disease manifests as water-soaked small spots on the lower surface of a younger leaf. With time, isolated, irregularly shaped desecrate blackishbrown patches surrounded by a yellow halo appear in the leaf lamina. Eventually, necrotic spots shed off, and shot holes become visible on the leaves. In highly susceptible varieties, leaf blight symptom is also observed. The disease is responsible for 12–14% of foliage loss. Besides it also reduces the nutritional value of the leaves. The BLS disease appears during May and continues up to November in West Bengal. Bacterial leaf spot is reported from Manipur and Assam during late spring and early autumn crops. The spread of disease occurs through rain droplets and mechanical injury. Dense plantation and 70–80% RH are conducive to disease development.

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9.3.2 Powdery Mildew Caused by the fungus Phyllactinia corylea, the disease appears as white powdery patches on the lower surface of the moderately mature to mature mulberry leaves. The disease is largely severe in the autumn and spring in most areas and also expands to monsoon in the hilly areas of India (Govindaiah and Gupta 2005). White powdery patches appear on the lower surface of the leaves, and the concomitant upper surface develops chlorotic lesions. In the later stage, white powdery patches turn to brownish-gray black; leaves turn yellowish, leathery, and defoliate prematurely. The disease reduces leaf yield by 10–15% and adversely affects the feeding quality of the leaf due to luxuriant mycelial growth on a lower surface (Noamani et al. 1970). Feeding of powdery mildew infected leaves prolongs larval duration and decrease larval weight which ultimately results in smaller-sized cocoons as well as poor-quality silk (Dandin et al. 2000; Manimegalai and Chandramohan 2007; Monir et al. 2017).

9.3.3 Leaf Rust 9.3.3.1 Black Leaf Rust It is caused by Cerotelium fici (Cast). Arthur and also called Peridiospora mori Barclay (Prasad et al. 1993). Leaf rust belonging to the Family— Uredinaceae, Order-Uredinales, Class-Imperfect fungi. The pathogen produces pin-head-sized circular to oval, brownish to black eruptive lesions on the surface of the leaves. The affected leaves become yellowish and as the disease becomes severe, the leaves wither off prematurely. The disease appears on mature leaves and can cause losses of 5–10%; it also affects the quality of the leaf by reducing moisture, crude protein, sugars, and total sugars (Philip et al. 1994). 9.3.3.2 Brown Leaf Rust The brown leaf rust disease is caused by the fungus Peridiospora mori and manifests as numerous small pin head-shaped brown to black

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rusty pustules on the lower surface of the mature leaves. Affected leaves gradually become yellowish, margins dry up, and the leaves wither off prematurely. This disease causes up to 15% leaf loss besides a reduction in leaf quality (Sengupta et al. 1990; Philip et al. 1994, Teotia and Sen 1994; Pratheesh Kumar et al. 2000). It is prevalent from October–February crops in the plains of Eastern states. In the ‘Tarai’ and foothills, the disease appears from September and lasts up to Falguni seed crop (December). Infection reduces moisture, crude protein, reducing sugars, and total sugar content in the leaves. A temperature of 22–26 °C and > 70% relative humidity favors the disease development.

9.3.3.3 Red Rust Red rust is caused by Aecidium mori Barclay and causes leaf loss of up to 15% (Teotia and Sen 1994; Pratheesh Kumar et al. 2000). The symptoms are numerous round shiny spots appear on both surfaces of the leaf which later protrude gradually into yellow. Infected young shoots swell and curl up abnormally with densely and slightly protruded yellow spots on the malformed buds. Red rust is more prevalent in China, Japan, Korea, and other sub-tropical countries cultivating mulberry. However, in India, it is observed occasionally in northern and north-eastern regions. Red rust affects young buds, leaves, petioles, and shoots. Upon infection, the buds swell and curl up in an abnormal shape, giving rise to several slightly protruded yellow spots. The symptoms on leaves appear as numerous small, round, and shiny-yellow colored protruded spots on both the surfaces and on stem and petioles as reddish-brown lesions (Wang 1980; Teotia and Sen 1994). 9.3.3.4 Yellow Rust Fungus Aecidium mori incites yellow rust and affects young buds, leaves, petioles, and shoots. On leaves, numerous small, round, shiny-yellow spots appear on both surfaces. Affected buds become swollen with shiny-yellow protruded pustules. Subsequently, infected parts become

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thick, deformed, and tender twig portion curl up abnormally. As the disease progresses, defoliation of infected leaves is observed. Foliage loss goes up to 8–10%. The disease is prevalent during rainy (June to October) and spring (January to February) seasons in the hilly areas of West Bengal, Sikkim, Manipur, Mizoram, Nagaland, and Meghalaya states. 10–22 °C temperature and relative humidity of 77–85% favor the development of disease. The cloudy and overcast day also enhances the infection.

9.3.4 Anthracnose Disease The mulberry anthracnose is caused by Colletotrichum acutatum and has been observed on the leaves of a particular mulberry tree in Tsukuba. The fungus was isolated from leaves, bark, xylem, and winter buds. The fungus was detected from the bark and bud scales in winter, but not from the xylem or the inside of winter buds. The fungus overwinters on the tree can repeat the infection cycle year after year, suggesting a strong association of the fungus with the host (Yoshida and Shirata 1999). Sreenivasaprasad et al. (1994) identified the isolates of Colletotrichum as C. acutatum by molecular characterization. Colletotrichum aenigma also reported being involved in the anthracnose disease based on the molecular characterization with ITS, GAPDH, CAL, ACT, CHS-1, GS, and TUB2 genes (Zhu et al. 2022). Early symptoms are light brown, with small lesions later coalescing into larger patches. Irregular dark brown or black spots surrounded by a tan border with slightly perforated necrotic lesions. The leaves of mulberry in Dujiangyan, Sichuan Province, China, also found with brown spot lesions caused by Colletotrichum species. These include Colletotrichum fioriniae, C. fructicola, C. cliviae, C. karstii, C. kahawae subsp. ciggaro, and C. brevisporum identified based on morphological and molecular characterization of the ITS, GAPDH, ACT, CHS-1, TUB2, and GS sequences (Xue et al. 2019).

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9.3.5 Blight

9.3.6 Sooty Mold

9.3.5.1 Twig Blight Twig Blight is induced by Fusarium lateritium (order-Moniliales, class-Deuteromycetes). Diseased plants show a bushy appearance, lateral shoots grow profusely, and leaves show slight browning/blackening at first, and become completely charred in later stages, causing severe defoliation. Affected branches show black longitudinal lesions that later lead to branch splitting and desiccation.

Capnodium sp., Metacapnodium sp., Euantennaria sp., Chaetothyrium sp., and Curvularia affinis, etc. have been described to be related to sooty mold. Symptoms appear in the form of a black powdery mass covering the entire upper surface. Highly infected leaves fall off from the shoot. The presence of adult white flies or nymphs in the garden sucks the sap from tender leaves resulting in chlorosis. The fungi involved are saprophytic and do not invade inside the plant tissues but remain on the surface. They grow on honeydew excreted by sucking insects like whitefly. Symptoms appear during August and continue up to December. Foliage losses may reach up to 20–30%.

9.3.5.2 Bacterial Blight Pseudomonas syringae causes by pv. mori causes a 5–10% reduction in leaf yield during the wet season. Numerous water-soaked spots which are irregular appear on the underside of leaves. Under severity, the leaves curl and rot, turning brownish black. Bacterial leaf blight of mulberry was first reported in Uttar Pradesh, India (Sinha and Saxena 1966). Subsequently, the disease was reported in other Indian states like Karnataka (Siddaramaiah et al. 1978), Tamilnadu (Gangwar and Thangavelu 1991), West Bengal (Teotia and Mandal 1993), Andhra Pradesh, Karnataka, and Kerala (Gunasekhar et al. 1994). Following the onset of monsoon, small, water-soaked lesions appear initially on the underside which later develops on the upper surface too. These spots turn brown with a yellow border. The younger leaves get crinkled, distorted, and curved outward and drop prematurely. Vein and veinlet infections are also common. On the stem, the symptoms consist of dark irregular sunken lesions which also coalesce in severe stages. Another type of leaf blight is characterized by the presence of a yellow margin around the blighted portion called halo blight and upon felling off necrotic tissues the spots form a shot hole. It is caused by a new strain of Pseudomonas syringae pv. Mori reported from the Shimane Prefecture in Japan in 1977.

9.3.7 Canker and Twig Dieback Symptoms of the disease begin as small depressions in the shoots, and then branches expand to cover larger areas of infected tissue, leading to dieback and branch death. Brown discoloration can be found in cross sections of the infected stems and trunks. The causal agent was identified as Diplodia seriata using the molecular tools [(ITS)-rDNA and ef-1a gene] and morphocultural characteristics (Arzanlou and Dokhanchi 2013).

9.4

Molecular Diagnostic Methods for the Detection of Plant Pathogens

Pathogens infect plants under natural environmental conditions at all stages from the seedling stage to seed maturation, singly or together with other types of plant pathogens (Narayanasamy 2011). Ensuring food safety through organized plant disease management and control is a

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necessity for a growing world population (Sarrocco and Vannacci 2018). Identifying pathogens can help improve plant vitality and health. Accurate detection of phytopathogens leads to the control of important plant pathogens in the field of crop protection. Additionally, for precise diagnosis of diseases, a list of all the known plant diseases along with their typical signs and symptoms and their known potential pathogens for the exact host is essential (Basandrai et al. 2021; Hariharan and Prasannath 2021; Srivastava 2016; Thind 2015). The analysis of plant pathogens has advanced from using conventional morphological traits to increasing use of molecular strategies with the fast improvement of molecular biology. Identification of fungal species has historically relied on morphological, phenotypic and reproductive characterizations that fulfill the concept of biological species. Although classification based on morphological data is an accepted method in fungal taxonomy, it may not reflect phylogenetic relationships. However, DNAbased methods have become a popular method for accurately diagnosing plant diseases (Choudhary et al. 2021; Hariharan and Prasannath 2021). The advent of DNA sequencing and the accompanying advances in phylogenetic analysis have provided a strong foundation for studying fungal species differentiation at a more fundamental level. Additionally, a combination of new emerging tools such as spectroscopy, imaging, and nanomaterial-based sensors combined with molecular diagnostics offers exceptional sensitivity and spatiotemporal resolution (Kumar et al. 2021). Among the several types of nanomaterials available, carbon nanotubes, graphene, and gold nanoparticles (AuNPs) are amenable to various detection platforms. Nanobased biosensors have already been used in medical diagnostics, food quality aspects, environmental monitoring; however, there are only a few reports on plant biosensors (Kulabhusan et al. 2022).

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9.4.1 Progress in the Application of Diagnostics Tools in Mulberry Recent years have seen remarkable progress in the development of molecular biology tools and techniques. Molecular taxonomy requires a decisive molecular phylogeny of genera. Abe et al. (2010) performed preliminary studies of the phylogeny of Rhizopus grounded on rDNA nucleotide sequences. From that point on, molecular data such as rDNA sequences and DNA-DNA complementarity became accepted by researchers. There are several methods for characterization of fungal flora including ITS, ribosomal gene sequencing and single sequence repeats (SSR) genotyping (Zhang et al. 2010). PCR-based molecular techniques, such as Restriction Fragment Length Polymorphism (RFLP) of rDNA, Amplified Fragment Length Polymorphism (AFLP; Vandemark et al. 2000), and Random Amplification of Polymorphic DNA (RAPD) were used to differentiate fungal strains and assess host specificity (Fuhlbohm et al. 2013; Powell et al. 1996; Sowmya et al. 2018). Advances in standard and variant polymerase chain reaction (PCR) assays including nested PCR, multiplex PCR, quantitative PCR, magnetic and bio-capture hybridization PCR techniques, isothermal and post-amplification methods, development of DNA and RNA-based probe, and next-generation sequencing (NGS) provide unique molecular diagnostics tools in fungal detection and differentiation. These methods are efficient in discriminating both symptomatic and asymptomatic diseases of culturable and non-culturable fungal pathogens in single infections and co-infections. Two common approaches that are used to select target DNA sequences for diagnostic purposes are: developing a method that uses a known conserved gene, common to all fungi, but with useful and readily available sequence variants. The other is to screen random portions of the fungal

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genome for regions of desired specificity. A major DNA region of interest for the development of diagnostic procedures is ribosomal DNA (White et al. 1990). Ribosomal DNA (rDNA) has several useful properties that make it a suitable target for diagnostic procedures. They are available in high copy number in all organisms, allowing very sensitive detection. Comparison of sequence data with similar data available in public databases provides geographically relevant information on fungal species. Detection of pathogen using only ITS region sequence data is incomprehensible (Babu et al. 2010; Gnanesh et al. 2022). Therefore, genome regions like the sequencing of conserved and housekeeping genes are investigated (Gautam et al. 2014). The nucleotide sequence of a particular gene like ITS, 18S rDNA, Cox 1, Cox 2, B-tubulin, Ef-1a is known to be unique and conserved to the particular microbial species and it can be easily identified by analysis of this region. Multigenic phylogenies are often used to identify the genus Rhizopus. (Abe et al. 2006, 2010; Dolatabadi et al. 2014a, b; Gnanesh et al. 2021). Fingerprinting methods allow identification of random genomic regions of pathogen to detect species-specific sequences when conserved genes do not have sufficient variation to successfully identify a species or strain (Patil 2018). This analysis is useful to study the phylogenetic proximity of fungal populations. Moreover, these methods are also insightful for detecting specific sequences that are reliable to identify pathogens at bottom taxonomic levels and virulent isolates in similar species with different host ranges and fitness groups. Enormous sequencing technologies provide intense improvement in affordable sequence output, with an incredible impact on genomic research. Sanger sequencing has been partially superseded by some ‘next-generation’ sequencing technologies that can generate numerous copies of short sequences from many organisms in less time. Some of the bioinformatics databases like the National Center for Biotechnology Information (NCBI)’s GenBank, the European Bioinformatics Institute's (EBI)'s Nucleotide Sequence Database Collaboration, and MycoBank provides

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platforms for documenting novelty of mycological nomenclature, preserving and recovering facilities of plant infecting fungal nucleotide sequences, further accelerating molecular tools to probably diagnose and perform species discrimination between subsisting and emerging mycotic species (Hariharan and Prasannath 2021). Molecular diagnostic tools have been successfully employed in the identification of mycotic pathogens in mulberry (Fig. 9.1) PCR-based molecular diagnosis of soil-borne and foliar fungal pathogens of mulberry identified using gene-specific markers are listed in Table 9.1.

9.4.1.1 Polymerase Chain Reaction (PCR) Recent developments use high-yield molecular detection strategies for fungi infecting plants. Which include standard polymerase chain reaction (PCR), real-time PCR, nested PCR, loopmediated isothermal amplification (LAMP), rolling circle amplification (RCA), and nucleic acid sequence-based amplification (NASBA) (Aslam et al. 2017; Cheng et al. 2020; Hariharan and Prasannath 2021; Heo et al. 2019; Wang et al. 2020). PCR-Restriction Fragment Length Polymorphism (PCR–RFLP) and PCRDenaturing Gradient Gel Electrophoresis (PCRDGGE) are preferred techniques for genotyping rather than species detection (Johnston-Monje and Mejia 2020). Additionally, molecular methods include Magnetic Capture Hybridization PCR (MCH-PCR), In situ, collaborative, multiplex PCR, DNA macro- and microarrays, next-generation sequencing (NGS)—RNA-Seqbased (Kumar et al. 2016). DNA-based molecular methods are popular due to the increased reliability, high specificity, sensitivity, and greater accuracy (Capote et al. 2012; Midorikawa et al. 2018), despite the presence of low concentrations of DNA (Luchi et al. 2013; Rollins et al. 2016) molecular methods allows the detection of phytopathogens at early stages of infection. The advent of PCR has restored accurate identification of fungi as well as various plant pathogens in disease control (Ma and Michailides 2007). This in vitro technique uses specific

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Fig. 9.1 Summary of the diagnostic procedures used for identification of fungal pathogens in mulberry

primers, deoxyribonucleotide triphosphates (dNTPs), and thermostable Taq DNA polymerase (Griffiths 2014) in buffer to perform denaturation, annealing, extension, terminal extension, and retention. It amplifies the DNA template exponentially by repeating these cycles at different temperatures (Caetano-Anolles 2013). The first step in PCR is to heat the DNA to 95 °C to denature the two complementary strands. Two short single-stranded DNA primers are designed to match the ends of the amplifying region. They bind or anneal only to complementary regions of DNA typically at 40– 65 °C. Another DNA strand is created by primer extension typically performed at 72 °C with deoxyribonucleoside triphosphates (dNTPs) as

building blocks using a thermostable DNA polymerase. By the end of this first cycle of the PCR reaction, two target DNA copies are present instead of one, which enters consecutive cycles of PCR reaction. After 25 such cycles, millions of copies of the sequence are generated within hours and are checked for the presence of amplified DNA by electrophoresis on an ethidium bromide-stained agarose gel. Other detection methods are also used to confirm the presence of amplified DNA. Molecular diagnosis of Nigrospora sphaerica causing a shot hole leaf spot in the mulberry leaf was identified by Arunakumar et al. (2019a, b) using the rDNA-ITS region a specific primer (NigroF and NigroR) was designed from rDNA-

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Table 9.1 Identification of fungal pathogens in mulberry using gene-specific markers Diseases

Pathogen

Diagnostic marker

Geographic region

References

Leaf spot

Setosphaeria rostrata

ITS

Karnataka, India

Arunakumar et al. (2019a)

Korea

Hong et al. (2011)

Phloeospora maculans

Shot hole leaf spot

Anthracnose on leaf

Cladosporium pseudocladosporioides

ITS, TEF, ACT

South Korea

Heo et al. (2021)

Nigrospora sphaerica

ITS

Santai County, Sichuan Province, China

Chen et al. (2018)

Karnataka, India

Arunakumar et al. (2019b)

Sichuan Province, China

Xue et al. (2019)

Hubei province, China

Zhu et al. (2022)

Colletotrichum fioriniae C. fructicola, C. cliviae C. karstii, C. kahawae subsp. ciggaro and C. brevisporum

ITS, GAPDH, CAL, ACT, CHS-1, GS and TUB2

Colletotrichum aenigma Leaf Blight

Curvularia lunata

ITS

Thailand

Bussaban et al. (2017)

Leaf rust

Cerotelium fici

LSU & ITS

Karnataka, India

Leaf spot/blight Leaf blight

Lasiodiplodia theobromae

ITS

Arunakumar et al. (Unpublished)

Fusarium equiseti

ITS, TEF, b-tubulin

Paramyrothecium roridum

ITS

Berhampore, West Bengal, India

Pappachan et al. (2019)

Powdery mildew

Phyllactinia corylea

ITS

Karnataka, India

Arunakumar et al. (Unpublished)

Sclerotial disease

Ciboria carunculoides C. shiraiana, and Scleromitrula shiraiana

ITS

–

Lv et al. (2022)

Rhizopus root rot

Rhizopus oryzae

ITS, ACT, TEF

South India

Gnanesh et al. (2021)

Root rot

Fusarium solani

ITS

Kolasib, Mizoram, India

Pappachan et al. (2020)

Tamil Nadu, India

Saratha et al. (2022a)

Fusarium species

Macrophomina phaseolina

ITS

Northeastern Thailand

Sutthisa et al. (2010)

ITS, TEF, b-tubulin

South India

Arunakumar and Gnanesh (2023) Gnanesh et al. (Unpublished)

ITS

Tamil Nadu, India

Saratha et al. (2022a)

Rhizoctonia solani Sclerotium rolfsii Athelia rolfsii Phytophthora megasperma P. multivora

Saratha et al. (2022b) ITS, COI

Italy

Pane et al. (2017)

(continued)

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Table 9.1 (continued) Diseases

Pathogen

Diagnostic marker

Geographic region

References

Black root Rot

Lasiodiplodia theobromae

ITS

Kolasib, Mizoram, India

Pappachan et al. (2020)

ITS, b-tubulin

South India

Gnanesh et al. (2022) Arunakumar and Gnanesh (2023)

RAPD

South India

Sowmya et al. (2018)

ITS, EF1-a

Guangxi Province, China

Xie et al. (2014)

Botryosphaeria dothidea

ITS, b-tubulin

Xiajin County Shandong Province, China

Huang et al. (2019)

Diplodia seriata

ITS, TEF

Iran

Arzanlou and Dokhanchi (2013)

Meloidogyne incognita

SCAR

South India

Manojkumar et al. (2022)

Meloidogyne enterolobii

rDNA-IGS2 rDNA-ITS; D2-D3 region of the 28S rRNA

Hainan, Guangdong, Guangxi, and Hunan provinces of China

Sun et al. (2019) Zhang et al. (2020)

Shoot canker and dieback

Root-knot Nematode

Ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), glutamine synthetase (GS), b-tubulin 2 (TUB2) translation elongation factor (TEF), cleaved amplified polymorphic sequences (CAPS), cytochrome c oxidase subunit 1 (COI)

ITS region of the microfungi facilitating its rapid detection using the qPCR platform. If multiple different pathogens need to be detected simultaneously, multiplex PCR using multiple primer pairs in the same PCR reaction can be used. This saves time and money, but care must be taken to optimize conditions to efficiently generate all the different amplicons (Henegariu et al. 1997).

9.4.1.2 End-Point PCR Endpoint PCR enables precise diagnosis of fungal phytopathogens by designing either specific sequences targeting specific fungal species or universal primers that amplify multiple pathogens followed by sequencing. Each set of nucleotide sequences of fungal isolates is analyzed using Basic Local Alignment Search Tool (BLAST) to determine the identity of each isolate by comparison to ex-type cultures available in the NCBI GenBank database. The target sequence present is detected by agarose gel electrophoresis which ensures the presence of the target plant pathogen. Endpoint PCR systems are

counted as an affordable option with reference to other existing molecular diagnostic options for detection of mycotic phytopathogens. Although, endpoint PCR assays are tedious and are difficult to design sets of primers to describe mycotic pathogens that are closely related. Compared to the endpoint PCR approach, a real-time quantitative PCR approach was more sensitive for the rapid diagnosis of fungal phytopathogens (Sikdar et al. 2014).

9.4.1.3 ITS-Conventional PCR Several diagnostic methods for pathogen detection and identification have been developed based on sequence mutation of specific fragments of rDNA clusters. The rDNA gene cluster in fungi consists of three highly conserved ribosomal RNA subunits that encode: 5.8S rRNA, 18S rRNA (known as small subunit or SSU RNA), and 28S rRNA (large subunit or LSU RNA) genes. The segments between 18S to 5.8S and 5.8S to 28S are called ITS regions, including ITS1 and ITS2 (Villa-Carvajal et al.

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2006). rDNA has an extensive conserved and variable region and evolves relatively slowly, which simplifies molecular detection by the design of a wide range of species-specific primers. But effective detection is possible by the detection of specific pathogens in a single step process, without the need for DNA sequencing or sequence comparison. The capability to design specific primers is critical for the purpose particularly when same disease is caused by several closely related pathogens in a culture. Several molecular detection studies used universal ITS regions and primers (such as ITS1 and ITS4) to detect fungal pathogens that infect filamentous plants. ITS sequence data can be considered the primary barcode for identifying fungal species. Its sequencing data can reliably identify 73% of the fungal taxa studied and has high sequencing and PCR success rates (Bridge et al. 2005 and Schoch et al. 2012). Identification of the Curvularia lunata as a causative agent of leaf spot disease in tomatoes is achieved by amplification of ITS region of the rDNA with universal primers ITS 4/ITS5 (Bussaban et al. 2017; Abdelfattah et al. 2021). The fungal pathogen causing brown leaf spotting in mulberry was detected to be Paramyrothecium roridum (Tode) L. Lombard & Crous (syn. Myrothecium roridum Tode ex. Fr.) based on ITS 5.8S rDNA sequence analysis (Pappachan et al. 2019). Sequencing of D2–D3 regions of the 28S rRNA genes and rDNA ITS, combined with morphological features of (Root-Knot Nematode) RKN, revealed Meloidogyne enterolobii as the causative species for RKN in mulberry (Sun et al. 2019; Zhang et al. 2020).

9.4.1.4 Non-Internal Transcribed Spacers Nuclear Genes: PCR-Based Traditional Ways Sequencing of internal transcribed spacers region of ribosomal DNA is routinely utilized to identify fungal pathogens, but in certain cases, the method may be not agreeable for developing molecular markers quite specific at species level. This can be attributed to low sequence divergence among closely interrelated species as well as

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considerable variations in intraspecific sequences. Under these limitations, approaches like the nuclear-encoded housekeeping genes could be explored. For example, genes such as internal transcribed spacers (ITS), Glyceraldehyde-3phosphate dehydrogenase (GAPDH), Calmodulin (CAL), Actin (ACT), Chitin synthase (CHS1), Glutamine synthetase (GS), and b-tubulin 2 (TUB2) have been used to identify anthracnose pathogen (Colletotrichum spp.) in mulberry (Zhu et al. 2022; Xue et al. 2019). Similarly, Heo et al. (2021) identified Cladosporium pseudocladosporioides causing leaf spot disease on mulberry using ITS, ACT and TEF1-a (translation elongation factor 1-alpha) gene sequences. Accurate determination of Lasiodiplodia theobromae could be accomplished by combining two or more genes such as ITS, TUB, and TEF1) genes as evidenced by the works of Chen et al. (2013, 2021), Gnanesh et al. (2022), Marques et al. (2013) and Rosado et al. (2016). These genes are also increasingly being exploited in diagnosis as well as characterizing pathogens. The b-tubulin sequences are one of the most commonly targeted genes for the diagnosis of fungal pathogens. Likewise, elongation factor-1 (EF-1) gene is a single-copy, well conserved nuclear protein encoding sequence with relatively low intraspecific variations that have been used as a subsidiary DNA barcode for various fungi and fungus-like organisms, frequently utilized in studying phylogeny of fungi. Nonetheless, information on these gene sequences is not elaborate in comparison to ITS regions. They often contain more variable nucleotide sites than that of ITS and could be handy when variations in ITS sequences or conservation is unfitting for developing taxon-specific diagnosis for delineating closely related fungi. Though PCR-based methods for detection of pathogens are extremely specific, highly responsive, and swift; advancements in the application of molecular tools for the detection of different diseases in mulberry have been moderate when compared to other crops. There is an immediate need to further develop and apply extremely responsive, distinct, as well as cheaper molecular methods to identify key pathogens.

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9.4.2 Future Molecular Diagnostic Methods for Detection of Mulberry Pathogens 9.4.2.1 DNA or RNA Probe-Based Assays With the emergence of techniques for the isolation, purification, cloning, and hybridization of DNA from various microorganisms, DNA probes are earliest molecular markers used for detecting, identifying and working out phylogeny of soilborne pathogens. Some pathogens remain dormant in plant tissue, which remains symptomless and complicates timely disease management decisions. In such situations, a visual inspection cannot detect the pathogen present in a plant like a fungus, bacteria, or virus (Singh et al. 2013; Tarafdar et al. 2012, 2013). In such cases, species-specific DNA probes have high specificity. Pure culture of the target organism is not necessary for the detection of pathogen. These probes are generated from cloned random DNA fragments generated from DNA sequences that have been cleaved with different restriction endonuclease enzymes and have several advantages over classical approaches. White leaf infection in sugarcane was studied with the help of DNA-based voltammetric electrochemical corroboration of widespread field infections using an immobilized single-stranded DNA (ssDNA) probes as an explicit sensor (Wongkaew and Poosittisak 2015). Several Phytophthora spp. in soil and host tissue, particularly P. parasitica, were detected by applications of cloned chromosomal DNA probes (Goodwin et al. 1989). Mitochondrial DNA (mtDNA) probes were reported to be used for discrimination of Phytophthora species that show intersecting variability of morphological characters (Mills et al. 1991) due to the presence of high copy numbers and the ability to produce more simple restriction fragment patterns. This technology has also been applied to discriminate among special forms of Fusarium oxysporum, the causal agent of vascular wilt of numerous plants (Bridge et al. 1997). Further, Babu et al. (2007) used a similar method in case of M. phaseolina, causing wirestem, seedling blight,

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charcoal root rot, collar rot, and stem rot in different crops. Recently, Li et al. (2019) developed a handheld device smartphone-based volatile organic compound (VOC) sensing platform to detect late blight (Phytophthora infestans).

9.4.2.2 In Situ Hybridization In situ hybridization (ISH) was first applied in locating specific DNA sequences on chromosomes using probes labeled with radioisotopes. It also helps in identifying mRNAs existing in the fixed samples. This method involves annealing single-stranded labeled DNA or RNA probes to single-stranded nucleic acids in cells (sequence of interest). Although, cDNA probes and synthetic oligonucleotide probes can also be used (Jensen 2014). DNA-DNA-ISH enables locating DNA sequences in chromosomes and interphase nuclei. RNA ISH displays gene expression patterns in tissues or cells. Radioactive isotopes like 35S, 125I, and 32P are used to label probes because they are very keen and evaluated easily for detection. But they are very costly and hazardous, which becomes a constraint in their use. Non-isotopic probes such as bromodeoxyuridine, alkaline phosphatase, tyramide, digoxigenin, and biotin can also be used for probe labeling. Photographic signal detection, autoradiography using liquid emulsion, X-ray film, and microscopy procedures are also utilized for detecting the signals (Corthell, 2014). Ellison et al. (2016) used the ISH technique for distinguishing rust pathogens from various isolates using plant tissues fixed in paraffin. These methods help pathologists to understand fungal morphology, life cycle of pathogens and help to determine growth patterns in host tissue. 9.4.2.3 FISH (Fluorescent in Situ Hybridization) Fluorescent in situ hybridization is a relatively new and advanced method to identify and enumerate specific microbial groups in plant disease diagnostics. This technique can be used to determine whether a particular genetic element is present in a sample depending on occurrence or absence of a fluorescent signal. This helps

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determine whether a microorganism has a particular gene and whether that gene is expressed under specific conditions. This blends the distinctness in DNA sequences with simplicity of microscopic observation as well as hybridization based on fluorochromes’ sensitivity for detecting pathogens (Hijri 2009; Cui et al. 2016). Fluorescent probes (DNA/RNA) are developed to attach to distinct genetic sequences of a microbe which uniquely discriminates them from others (Shakoori 2017). This results from extreme sensitivity due to high affinity and selectivity of the probe, as FISH happens under highly specific hybridization conditions sufficient to classify binding of 15–20 oligonucleotide probes with one nucleotide difference in it. FCM or flow cytometry analysis could be done when fluorescent tags are applied to microbial communities. A fluorescence signal is utilized to tally or classify distinct genotypes from a population of cells. rRNA comprises functional gene sequences shared across different species and also sequences highly specific to individual species. The sensitivity of these sequences makes them ideal targets for FISH. Microbial rRNA is hybridized with fluorescent mono-labeled oligonucleotide probes and the cells which are stained are observed by confocal laser scanning or wide-field epifluorescence microscopy (Lukumbuzya et al. 2019). Li et al. (1996) designed the first FISH probe targeting a living microorganism, Aureobasidium pullulans infecting the apple plantlets. FISH retains the intactness of the pathogen by detecting distinct rRNA sequences that are specific to pathogens present in hosts upon infection (Fang and Ramasamy 2015). FISH technology is the same as an amplification tool and can theoretically identify single cells. Milner et al. 2019 effectively detected Sclerotium rolfsii, a southern blight causing pathogen from soil in tomato plantations by using (Cy3 and Cy5) cyanine dye labeled oligonucleotide probe in FISH technique. According to Frickmann et al. 2017, FISH may provide information on resolution, morphological features, as well as help in detecting major pathogens in pooled samples. Although, false-positives due to autofluorescence

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of some materials are a drawback that minimizes specificity in assays (Moter and Göbel 2000; Tsui et al. 2011).

9.4.2.4 High Throughput Sequencing: High throughput sequencing (HTS) also called next-generation sequencing (NGS) is one the most advanced novel diagnostic methods that have opened new ways in the detection and identification of phytopathogens (Chalupowicz et al. 2019). The drastic reduction of its costs and continuous progress of NGS has boosted new and unpredictable developments in plant pathology field (Aragona et al. 2022). The primary phases in DNA-based NGS include isolating and fragmenting DNA, preparing DNA library, sequencing, bioinformatics analysis, annotation as well as interpreting results (Qin 2019). Oligonucleotide ligation detection (SOLID), colony sequencing, pyrosequencing and massive parallel sequencing, etc. are some commonly used innovative sequencing technologies in high throughput sequencing (Rajesh and Jaya 2017). Further methods like RNA-Seq or RNA sequencing enhance coverage and resolution of the transcriptome. Illumina’s HiSeq platform is one of the commonly used HTS platform used in RNA sequencing (Kukurba and Montgomery 2015). RNA sequencing-based HTS has enormous potential for quickly identifying new fungal pathogens in plant hosts. The high-quality draft genome will help in understanding the genome biology of the phytopathogenic fungi of economic importance and provides valuable information for further evolutionary studies within the genus and family (Angelini et al. 2019). The emergence of new pathogens is an example of a situation in which targets cannot be precisely defined. Since NGS does not require any foresight of the pathogen sequence, it can sequence the entire pathogen genome without the need for distinct pairs of primer or amplification using PCR (Hadidi et al. 2016; Malapi-Wight et al. 2016). Recognized and culturable diseases caused by oomycete pathogens are often detected using ITS primers. When diseases are relatively unknown or pathogens are obligate, HTS could come in

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handy (Ramadan ad Kenta 2018). All diseases, particularly key eukaryotic plant pathogens, have a lot of potential for diagnosis with NGS. The method can be used to sequence several hundred or even more DNA sequences at a time, to discover culturable pathogens as well as nonculturable viruses. However, compiling and analyzing NGS data can be time-consuming, difficult, and require expertise (Kumar et al. 2016). Structural variants, INDELS (Insertions and deletions), and SNPs (Single nucleotide polymorphisms) can all be recovered using datasets from NGS-based population genomics (Potgieter et al. 2020). NGS has tremendous potential when it comes to identifying plant diseases. However, the disadvantage of this strategy is the time and effort required to assemble and analyze large volumes of sequences. This difficulty in heterogeneous samples could be solved by enriching specific nucleic acids using TGC oligonucleotide probes, increasing the fraction of NGS reads for low abundance targets. Using metagenomes, the Electronic Probe Diagnostic Nucleic Acid Analysis (EDNA) can be used to simply detect oomycete fungal plant infections. Because electronics probes rely entirely on matches between queries and metagenome readings, EDNA has a higher accuracy for detecting fungal and oomycete plant diseases. The time spent assembling and analyzing vast amounts of sequence data is the most significant restriction in NGS (Espindola et al. 2015). Advances in third-generation sequencing (singlemolecule sequencing) technology have fore headed the second-generation sequencing methods among NGS methodologies (Schadt et al. 2010). Inadequate RNA production and/or integrity, stability of RNA, and contaminations involving salts, DNA, or other chemical compounds limit next-generation applications (Cortés-Maldonado et al. 2020). Though obtaining data is easy and rapid, NGS analysis requires expertise in bioinformatics and mycology. Therefore, an understanding of fungal and bioinformatics analysis is necessary to avoid misunderstandings.

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In addition, disease surveillance and pathogen detection, along with effective disease outbreak prediction, are important goals. A lot of information can be provided using NGS technology in this area. For example, samples that do not require culture, such as those that cannot be cultured and detected by conventional methods, can be directly identified using NGS technology (Grunwald et al. 2016). Indeed, genome-based species identification and recognition using NGS may transform oomycosis diagnosis as the cost and accessibility of analytical platforms decrease (Xu 2020). The majority of the time, early-stage infections in plants caused by various fungal/oomycete diseases are not visible until symptoms appear in the host plant. Many of the above-mentioned serological and molecular approaches are commonly utilized to detect these infections. However, due to its ability to target unique and many pathogen loci in an infected plant metagenome, next-generation sequencing (NGS) can be an efficient diagnostic tool (Sharma et al. 2016).

9.4.2.5 Isothermal Amplification-Based Methods RCA or Rolling Circle Amplification RCA or rolling circle amplification is a kind of isothermal enzymatic reaction, using DNA polymerase or RNA polymerase to generate single-stranded DNA or RNA which was first described by Fire and Xu (1995). RCA experiments require a polymerase, a homology buffer, short DNA or RNA primers, a circular template and dNTPs (Gu et al. 2018). This method uses phi29 DNA polymerase, which has strand displacement activities, to extend single or multiple primers that anneal to the circular DNA template (van Emmerik et al. 2020). The unique strand displacement activity results in DNA templates releasing ssDNA by displacing previously synthesized DNA molecules (Bhat and Rao 2020). After a series of strand displacement events, thousands of long ssDNA copies of original target sequence are formed (Kieser and Budowle 2020).

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Bull's eye rot of apples caused by Neofabraea sp. was identified using padlock probes (PLPNm, PLP-Np, PLP-Nk, and PLP-Nv) that target the translation elongation factor 1-a gene (TEF1a) of the pathogen. Infection was diagnosed using the RCA assay, providing effective and sensitive information for pathogen surveillance in quarantine areas (Lin et al. 2018). Likewise, a padlock probe based on the TEF-1a elongation factor polymorphism was used in isolating Fusarium head blight causing pathogens using RCA (Davari et al. 2012). The RCA test has the advantages of simplicity, efficacy, and lack of temperature cycling equipment (Dong et al. 2013; Goo and Kim 2016). It also enables gene expression studies, SNPs, mRNA splicing, besides post-translational modifications of protein (Gao et al. 2019). Loop-Mediated Amplification (LAMP) In recent times, PCR-based techniques have been the most frequently used methods for diagnosing plant diseases. Nevertheless, they demand considerable time as well as require technical advancements to develop. LAMP is one such method developed by Tsugunori et al. (2000) for amplification of nucleic acid under isothermal conditions. This is a molecular detection technology and uses 4–6 specific primers to amplify the targeted DNA under constant temperature for a short duration of time by using Bst DNA polymerase (Becherer et al. 2020; Francois et al. 2011; Notomi et al. 2000; Wang et al. 2021). LAMP is an outstanding tool for diagnosis and has tremendous potential in the management of plant diseases (Le and Vu 2017; Sun et al. 2022). This is a widely used technique superior diagnostic method due to its great specificity, simplicity, accessibility, efficiency, and speed (Yang et al. 2022; Zhang et al. 2022). It contains two each shorter inner and long outer primers that identify specific 6 sequences in the target. It yields 109–1010 copies of target DNA in 45– 60 min at isothermal conditions of 60–65 °C, an ideal temperature for Bst DNA polymerase (Notomi et al. 2000; Chander et al. 2014). This reaction involves two main steps, a primary

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circular amplification and subsequent extension (Panno et al. 2020). Initiation of DNA synthesis and target sequence hybridization occurs using the first inner primer having sense and antisense sequences. Following complementary strand synthesis by the inner primer, the outer primer undergoes strand displacement DNA synthesis. An ssDNA is generated acting as a template for the second inner and outer primers, thereby forming a DNA with a looped structure. For rapid and sensitive detection, the LAMP method was developed for diagnosis and sustainable management of sunflower black stem (Sun et al. 2022), clubroot (Plasmodiophora brassicae) of cruciferous crops (Yang et al. 2022), sheath rot of rice caused by the seedborne pathogen Sarocladium oryzae (Choudhary et al. 2022), brown root rot caused Phellinus noxius (Zhang et al. 2022) and similarly, Kaczmarek et al. 2019 sugar beet rust-causing fungi, Uromyces betae. Conventional loop-mediated amplification (cLAMP) and a quantitative LAMP (qLAMP) assay have been used in diagnosing Fusarium circinatum a pathogen causing pitch canker disease of conifers and pines. qLAMP tests are revealed to have higher specificity than cLAMP, using LAMP probes targeting TEF-1a for Fusarium circinatum detection (Stehliková et al. 2020). Analysis of raw samples can be performed with LAMP unaffected by the presence of inhibitors (Panno et al. 2019). LAMP technology requires isothermal and energyefficient enrichment, making it ideal for quick and inexpensive alternative assays (Waliullah et al. 2020). LAMP finds applications in the fields of medical, agricultural, and food industries (Mori and Notomi 2009; Guan et al. 2010; Panno et al. 2020). However, isolating closely related species by designing primers is often difficult and requires a lot of resources. Therefore, not all pathogens can be detected using LAMP. The small size fragment of the target, the requirement of complex primers, and other reaction substances inhibiting polymerase and carryover contamination are some of the major disadvantages (Tanner et al. 2015).

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9.5

Conclusions and Future Prospects

Recent breakthroughs in the field of molecular biology have been quite useful in the identification and diagnosis of newly evolving as well as reappearing diseases of a plant. Conventional and variant-based PCR methods, isothermal and postamplification tools, hybridization techniques, and high throughput sequencing (HGS) have been proving their merit in the diagnosis of plant diseases caused by fungi. Quantitative PCR is often used to quantify and isolate pathogens when the sample load is too low to be detected by various PCR-based methods. Furthermore, LAMP is a promising tool for detecting infections caused by fungi in plants, allowing discrimination of multiple species. HGS utilizes several platforms for sequencing genome of fungi without any previous information on the sequence of the pathogens and is thus useful in the identification of new and evolving diseases. In contrast, expertise in mycology and bioinformatics is required to avert misrepresentation of molecular biological analysis results. By combining other new technological developments and molecular techniques for diagnosing fungal diseases, molecular approaches should become point-of-care testing (POCT). More research is intensifying toward the development of in-field sensors to detect plant pathogens on crops. In mulberry, early detection and diagnosis are challenging tasks because soil-borne fungal pathogens occur as species complexes, possessing diverse profiles and varied virulence levels, and also require highly sensitive and specific molecular tests. Therefore, the newly developed novel techniques should be adopted for accurate identification and management of mulberry foliar and soil-borne diseases. Acknowledgements Dr. Gnanesh B.N. acknowledges the Science and Engineering Research Board (SERB), New Delhi, for financial support (SERBSB/S2/RJN049/2015).

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Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges

10

Tanmoy Sarkar, M. K. Raghunath, Vankadara Sivaprasad, and Babulal

10.1

Introduction

Mulberry (Morus spp.) is a cross-pollinated, fastgrowing, perennial, hardy tree, which belongs to the Moraceae family and exhibits extensive variations in ploidy level (2n = 28–308) (AliZade and Achundowa 1970; Sarkar et al. 2017). It is commercially cultivated for its leaves to feed the monophagous silkworm (Bombyx mori L.). Mulberry possesses inherent traits such as high biomass development, rapid growth and medicinal, nutritional and pharmacological properties, biotic and abiotic stress-adaptive traits due to which it could be used as a perennial model system (Dhanyalakshmi and Nataraja 2018). Further, mulberry has the potential for revenue generation, phytoremediation and environmental protection which make it suitable for sustainable development (Rohela et al. 2020). Globally, mulberry is grown under varied climatic, topographical and soil conditions spanning from tropical and subtropical to temperate regions (Jan

T. Sarkar (&)
M. K. Raghunath
Babulal Central Sericultural Research and Training Institute (CSR&TI), Manandawadi Road, Srirampura, Mysuru, Karnataka 570008, India e-mail: [email protected] V. Sivaprasad Seri-Biotech Research Laboratory, Kodathi, Carmelram Post, Bangalore, Karnataka 560035, India

et al. 2021). It is cultivated in China, India, Thailand and many more countries for silkworm rearing and also for multiple purposes (Sarkar et al. 2018). Mulberry is a highly heterozygous tree; hence, its F1 progenies, with high foliage yield and/improved agronomic traits, are selected for commercial cultivation via raising of saplings from stem cuttings (Sarkar et al. 2022). Commercial cultivation of high-yielding cultivars, supplementation of appropriate dosage of chemical fertilizers in the gardens lead to enhancement of foliage productivity of mulberry, and in turn silk production (Sori and Bhaskar 2012). China stands first in mulberry raw silk production; while, India is the second largest producer of mulberry raw silk and ranks first for its raw silk consumption (Sarkar et al. 2017). Silk is mainly made of two proteins, viz. fibroin and sericin, and around 70% of silk protein synthesized by a silkworm is resulted from the protein of mulberry foliage (Fukuda et al. 1959). Quality mulberry leaf alone contributes to 38.2% of quality cocoon production (Matsumara et al. 1958). It was estimated that the cost of mulberry leaf production accounts for more than 60% of the costs of silkworm cocoon production (Vijayan et al. 2009). Hence, the sustainability of silk industry is profoundly reliant on the continuous supply of high-quality mulberry leaves in adequate quantities for silkworm rearing. In India, most of the mulberry-growing areas fall under the arid and semi-arid regions, where abiotic stresses such as terminal and intermittent drought (soil moisture

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_10

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deficit), alkalinity and salinity lead to 50–60% foliage yield loss (Rao 2002; Lal et al. 2008). Burgeoning demand for quality silk, climatic changes, scarcity of water and agricultural land necessitate the development of high-yielding mulberry varieties suitable for various agroclimatic conditions especially marginal land, challenging soils and non-traditional areas affected by various abiotic stresses such as salinity, alkalinity, temperature extremes and moisture deficiency for sustainable development of global sericulture industry (Khurana and Checker 2011; Sarkar et al. 2017; Vijayan et al. 2022a).

10.2

Rationale

Mulberry germplasm including wild spp. exhibits wide genotypic and phenotypic variations for important agronomic, anatomical, morphological and physiological traits, biochemical indices, disease resistance and abiotic stress tolerance (Tikader and Kamble 2008; Chaitanya et al. 2009; Guha et al. 2010a; Chattopadhyay et al. 2011a; Guruprasad et al. 2014; Naik et al. 2015; Rukmangada et al. 2018, 2020). The leaf is the most important parts of mulberry due to its numerous economic applications. Mulberry leaf quality has a significant impact on silkworm growth and quality silk production which is further reliant on the moisture content and nutritional composition of leaf (Rahmathulla 2012; Dhanyalakshmi et al. 2021). Hence, the improvement of foliage productivity is the main criterion for mulberry breeding programmes. Further, the foliage productivity of mulberry is a multi-factorial trait which is influenced by various associated quantitative traits such as length of the longest shoot, total shoot length, number of branches, nodal length, leaf retention capacity, laminar index (weight), dry weight of 100 leaves, total above-ground biomass, etc. (Vijayan et al. 1997a; Tikader and Kamble 2009; Doss et al. 2012; Suresh et al. 2021). Indirect selection method has been adapted for the enhancement of leaf productivity through the improvement of these associated traits via conventional breeding (Doss et al. 2012). Mulberry breeding is

constrained due to long juvenile period, outbreeding and predominant dioecious nature, heterozygosity, asynchronous flowering, linkage drag, quantitative nature of some desirable traits, non-availability of inbred lines, and limited information on the inheritance of the trait(s), heterosis, genotype  environment interaction (G  E), genetic markers, etc. (Vijayan 2010, 2014; Vijayan et al. 1997b, 1998; Arora et al. 2017; Pinto et al. 2018). Further, one of the most important constraints in mulberry genetic improvement programmes through traditional breeding is the limited availability of adequate genetic variations in the cultivated Morus spp. (Khurana and Checker 2011; Dhanyalakshmi et al. 2021). Traditional breeding method is a timeconsuming process as it takes around 15– 20 years to develop a new variety, and it is also laborious. However, it has contributed significantly to the development of superior mulberry varieties with enhanced leaf yield and quality, biotic and abiotic stress tolerance (Mogili et al. 2008; Vijayan et al. 2009; 2022a, b; Banerjee et al. 2011; Khurana and Checker 2011; Doss et al. 2012). Drought, high temperature, low temperature, salinity and alkalinity are experienced in most of the mulberry cultivation areas, which are the key abiotic stresses affecting its growth and metabolic activities resulting in the reduction of foliage yield and quality (Chaitanya et al. 2001; Vijayan et al. 2009; Guha et al. 2010a, b; Ahmad et al. 2014; Sarkar et al. 2017; Chen et al. 2018; Liu et al. 2019). In the plant system, abiotic stress tolerance is a polygenic/ quantitative trait, which involves cross-talk among several genes via signal transduction mechanisms (Khurana and Checker 2011; Sarkar et al. 2014, 2017; Liu et al. 2015). Many foliar diseases such as leaf spot, rust, powdery mildew and root diseases, viz. root rot and root knot, and pests commonly affecting mulberry garden lead to a considerable reduction in leaf productivity and quality (Khurana and Checker 2011; Pinto et al. 2018; Arunakumar et al. 2021; Dhanyalakshmi et al. 2021; Gnanesh et al. 2021, 2022; Vijayan et al. 2022b). Inappropriate use of agrochemicals for the protection of mulberry

10

Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges

gardens from pests and diseases might have deleterious effects on silkworm growth and silk quality (Ji et al. 2008; Jyothi et al. 2019). Unbalanced application of chemical fertilizers to the mulberry fields could lead to deterioration of soil productivity and acceleration of various nutrient deficiencies (Sori and Bhaskar 2012). Under these circumstances, genetic engineering provides an alternate strategy to transfer trait (s)/gene(s) across the taxonomic boundaries, when traditional or molecular breeding approaches may not be enough due to unavailability of variations in the natural populations and/lack of information on a specific gene(s)/QTLs (quantitative trait loci) governing a particular trait(s) of interest (Sarkar et al. 2017; Vijayan et al. 2022a). Genetic engineering techniques minimize the difficulties of traditional breeding and speed up the process of genetic improvement in plants. Adoption of genetic engineering approaches in mulberry improvement programmes may fructify to develop transgenic mulberry varieties with improved desirable traits in a targeted manner.

10.3

Regeneration

Plant tissue culture has a great impact on genetic improvement programmes and in vitro regeneration system for the development of complete plants is the prime prerequisite for genetic manipulation in mulberry. However, the perennial nature and prolonged juvenile period delayed the regeneration process in mulberry (Dhanyalakshmi et al. 2021). Mulberry is a recalcitrant plant as its in vitro regeneration potential is genotype-dependent and explantspecific (Tewary et al. 2000; Sarkar et al. 2022). The success of in vitro regeneration in mulberry is influenced by various aspects such as explant type, age, genetic makeup and origin, media composition, type of growth regulators and dosage, carbon source, gelling agent, pathological and physiological conditions of the mother plant (Vijayan et al. 2011; Sarkar et al. 2018). Ohyama (1970) first reported the in vitro regeneration of mulberry plants from axillary buds inoculated on Murashige and Skoog

245

(MS) medium amended with growth regulators. Complete mulberry plants were regenerated through both direct and indirect (through the callus phase) organogenesis routes (Sarkar et al. 2017). Through somatic embryogenesis, matured cotyledonary embryos were regenerated from zygotic embryos (Agarwal et al. 2004a). However, adventitious shoot induction from a somatic embryo and subsequent development of the complete mulberry plant has not been demonstrated so far. Complete mulberry plants were developed through direct and indirect (via callus phase) organogenesis from various explants (shoot tips, axillary bud, apical bud, petiole, leaf, hypocotyl, cotyledon) of various genotypes/cultivars (AR12, S13, S36, V1, S1, K2, DD, Chinese white, Kokuso27, G4, etc.) belonging to diverse species (Vijayan et al. 2000; Bhatnagar et al. 2001; Bhau and Wakhlu 2003; Chitra and Padmaja 2005; Kavyashree 2007; Raghunath et al. 2008, 2013; Rao et al. 2010; Attia et al. 2014; Aroonpong and Chang 2015; Choudhary et al. 2015; Rohela et al. 2018a, b; Sarkar et al. 2022). MS medium augmented with many combinations and concentrations of plant growth regulators (PGRs) was mostly used for in vitro regeneration of mulberry plantlets (Murashige and Skoog 1962). Two(Chattopadhyay et al. 2011b; Sajeevan et al. 2011; Rohela et al. 2018a) or three-phase (Akram and Aftab 2012; Raghunath et al. 2013; Choudhary et al. 2015; Rohela et al. 2018b; Sarkar et al. 2022) regeneration strategies were used for the development of mulberry plants. Cytokinins such as 6-benzylaminopurine (BAP), thidiazuron (TDZ), kinetin, adenine sulphate or isopentenyl adenosine alone or in combination with silver nitrate (AgNO3) and/auxins such as NAA (anaphthalene acetic acid) or indole-3-acetic acid (IAA) have been used for adventitious shoot induction (Sajeevan et al. 2011; Raghunath et al. 2013; Choudhary et al. 2015; Rohela et al. 2018b; Sarkar et al. 2022). Whereas, elongation and/proliferation of adventitious shoots were observed on culture media supplemented with cytokinin (BAP, TDZ or kinetin) alone or in combination with AgNO3 and/gibberellic acid (Bhatnagar et al. 2001; Rohela et al. 2018b). The

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addition of activated charcoal (AC), additional dosage of calcium chloride (CaCl2.2H2O), and inclusion of putrescine dihydrochloride and AgNO3 in the elongation and/proliferation media minimized the incidence of hyperhydricity/ necrosis/browning of regenerated shoots/leaves (Sarkar et al. 2022). A low concentration (about 0.2–3.0 mg/L) of auxin (IBA: indole-3-butyric acid, NAA, IAA or 2, 4-dichlorophenoxyacetic acid: 2, 4-D) with/without activated charcoal is suitable for root induction irrespective of the explants and genotypes (Bhau and Wakhlu 2001; Chattopadhyay et al. 2011b; Sajeevan et al. 2011; Raghunath et al. 2013; Aroonpong and Chang 2015; Rohela et al. 2018a; Sarkar et al. 2022).

10.4

Genetic Transformation in Mulberry

Direct (Agrobacterium-mediated) and indirect (particle bombardment and electroporation) transformation methods have been explored in mulberry for the development of transformed tissues/ transgenic plants (Fig. 10.1 and Table 10.1). However, Agrobacterium-mediated genetic transformation could be preferred over the indirect methods, as the former reduces the chances of multiple transgene integration in the mulberry genome. Machii (1990) for the first time demonstrated genetic transformation in mulberry using leaf disc explants of Morus alba var. Ohyutaka. However, the transformed tissues did not produce complete plantlets. Subsequently, particle bombardment (Machii et al. 1996; Bhatnagar et al. 2002), electroporation (Sugimura et al. 1999) and A. tumefaciens-mediated (Nozue et al. 2000) genetic transformation have been explored in mulberry for the development of transformation protocols. These experiments demonstrated the expression of the heterologous gus gene in the transformed tissues/cells as detected by histochemical assay of the reporter gene. Oka and Tiwary (2000) reported A. rhizogenes-mediated genetic transformation in mulberry, which leads to induction and culture of hairy roots on MS medium.

A. tumefaciens-mediated in planta transformation was found suitable in developing transgenic mulberry plants (Ping et al. 2003). A. tumefaciens-mediated transformation were used for the development of transgenic mulberry containing heterologous glycinin (AlaBlb) and Oryza cystatin (OC) genes (Jianzhong et al. 2001; Wang et al. 2003). Subsequently, attempts have been made to optimize protocols for the development of complete transgenic mulberry through Agrobacterium-mediated genetic transformation (Bhatnagar and Khurana 2003; Bhatnagar et al. 2004). Earlier studies showed that various explants such as cotyledon, hypocotyl, leaf and leafderived callus were used for transformation in mulberry (Fig. 10.1 and Table 10.1). Among these explants, the leaf-derived callus of mulberry cv. K2 was found to be the choice of explant for transformation as it showed the highest transformation efficiency than others (Das et al. 2011). However, in most of the transformation studies, cotyledon and hypocotyl explants of mulberry cv. K2/M5 have been used for the successful development of stable transgenic mulberry plants with improved stress tolerance traits (Lal et al. 2008; Checker et al. 2012; Saeed et al. 2015; Sajeevan et al. 2017). This might be due to the juvenile nature of these explants, their possession of sufficient number of competent cells and high regeneration potential than the others (Bhatnagar et al. 2002; Bhatnagar and Khurana 2003; Saeed et al. 2015; Sarkar et al. 2022). The Agrobacterium-mediated transformation frequency in mulberry is dependent on explant type, Agrobacterium strain, the density of Agrobacterium cells, duration of incubation of the explants with Agrobacterium suspension, duration of co-cultivation in dark condition and plasmid used in the experiments (Bhatnagar and Khurana 2003; Bhatnagar et al. 2004). For example, A. tumefaciens strains, viz. GV2260 and A281, were more effective than LBA4404 for gene transfer. The expression of a heterologous gene in transformed cells/tissues after more than 15 days of transformation could be an indication of stable integration of the gene of interest into their genome (Bhatnagar and

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Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges

247

Fig. 10.1 Integration of in vitro regeneration and genetic transformation in mulberry for development and characterization of transgenic plants with improved stress-adaptive traits

Leaf disc

Immature leaves, hypocotylcallus, suspension culture

Protoplasts (root meristem)

Cotyledoncallus; hypocotyl, root

HypocotylOP seedling, hypocotyl

Hypocotyl, cotyledon, Leaf-callus

Hypocotyl, cotyledon, Leaf-callus

Cotyledon

–

–

–

–

–

–

–

–

M. alba (cv. Ohyutaka)

M. alba (cv. Ichinose)

M. alba (cv. Ficus)

M. indica

M. indica (cv. K2)

M. qlba (cv.M5)

Explant

Transgene/ miRNA

Mulberry genotype

nptII gus

nptII gus

nptII gus

– –

Hpt gus

pBI121

pBI101, pBI121, pCAMBIA1301, pCAMBIA2301

pBI1101, pBI121, pCAMBIA130, pCAMBIA2301

Ri plasmid

pBI121

pBI221

pBI121, pBI131, pBC1, pNG1

– gus

– gus

pBI121

Plasmid

nptII, gus

Plant selectable marker gene and reporter gene

Direct organogenesis

Direct and indirect organogenesis

–

1–3

5

5

–

–

–

–

–

–

10

Preculture (Days)

–

–

Direct organogenesis

Regeneration mode

1–3

3

–

2

2

–

–

2

Cocultivation (Days)

Table 10.1 Genetic transformation protocols for development of transgenic mulberry plants

LBA4404

LBA4404 GV2260 A281

–

MAFF 210,268 MAFF 720,001 MAFF 210,265

EHA101 LBA4404

–

–

LBA4404

Agrobacterium strain

–

Yes (200 µM)

–

–

Yes (275 µM)

–

–

–

Application and dosage of acetosyringone

A. tumefaciens

A. tumefaciens

Particle bombardment

A. rhizogenes

A. tumefaciens

Electroporation

Particle bombardment

A. tumefaciens

Genetic transformation method

Development of transgenic plants

Expression of gus in tissues and explants

Expression of gus in explants

Induction and culture of hairy roots

Expression of gus in callus

Transient expression of gus in protoplasts

Transient expression of gus in explants

Expression of gus in shoots

Objective and/targeted trait

(continued)

Agarwal et al. (2004a, b)

Bhatnagar and Khurana (2003)

Bhatnagar et al. (2002)

Oka and Tiwary (2000)

Nozue et al. (2000)

Sugimura et al. (1999)

Machii et al. (1996)

Machii (1990)

References

248 T. Sarkar et al.

Hypocotyl, cotyledon

Shoot meristems

Hva1 (Barley)

–

bch1 (M. indica)

M. indica (S-36)

M. indica (cv. K2)

Leaf-callus, hypocotyl, cotyledon

Leaf-callus, hypocotyl, cotyledon

–

pCAMBIA2301

nptII gus

pBI121

pBI121

nptII gus

nptII –

nptII –

pCAMBIA2301

Direct organogenesis

Direct organogenesis

Direct organogenesis

Direct and indirect organogenesis

Direct organogenesis

5

–

5

5

5

3

2

3

3

3

1–3

–

Yes (200 µM)

–

Yes (200 µM)

Yes (200 µM)

Yes (200 µM)

–

GV2260

Agl1

Agl1

LBA4404

LBA4404

A. tumefaciens

A. tumefaciens

Tolerance to UV rays, high temperature and irradiance

Development of transgenic plants

Tolerance to drought, salinity and lowtemperature stress

Resistance/ tolerance to fungi, drought and salinity stress

A. tumefaciens

A. tumefaciens

Tolerance to drought and salinity stress

A. tumefaciens

A. tumefaciens

(continued)

Saeed et al. (2015)

Chitra et al. (2014)

Checker et al. (2012)

Das et al. (2011)

Lal et al. (2008)

Agarwal and Kanwar (2007)

Bhatnagar et al. (2004)

Osmotin (Tobacco)

nptII gus

1–3

A. tumefaciens

–

Hypocotyl, cotyledon

Somatic embryogenesis; direct and indirect organogenesis

LBA4404 GV2260 A281

Hva1 (Barley)

pBI121

3

M. indica (cv. K2)

nptII gus

5

References

Cotyledon, Cotyledoncallus, mbryogenic clumps

Direct and indirect organogenesis

Objective and/targeted trait

–

pB1121, p35SGUSINT

Genetic transformation method

Application and dosage of acetosyringone

M.alba (cv.M5)

nptII gus

Agrobacterium strain

Hypocotyl, cotyledon; leaf, leafcallus

Cocultivation (Days)

–

Preculture (Days)

M. indica (cv. K2)

Regeneration mode

Explant

Transgene/ miRNA

Mulberry genotype Plasmid

Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges

Table 10.1 (continued)

Plant selectable marker gene and reporter gene

10 249

Transgene/ miRNA

SHN1 (A. thaliana)

premiR166f (M. multicaulis)

MmSK interference sequence (M.alba)

Mulberry genotype

M. indica (cv. M5)

M. multicaulis

M. alba

Table 10.1 (continued)

pBI121

pCAMBIA2301

pTRV2

– –

– –

Leaf of mulberry seedlings

Cotyledon of mulberry seedlings

Plasmid

nptII –

Plant selectable marker gene and reporter gene

Hypocotyl, cotyledon

Explant

–

–

Direct organogenesis

Regeneration mode

–

–

5

Preculture (Days)

–

–

3

Cocultivation (Days)

GV3101

LBA4404

EHA105

Agrobacterium strain

Yes (150 mM)

Yes (150 mM)

Yes (250 mM)

Application and dosage of acetosyringone

A. tumefaciens

A. tumefaciens

A. tumefaciens

Genetic transformation method

Transient transformation, VIGS and sensitivity to drought stress

Transient transformation and tolerance to drought stress

Reduced postharvest water loss and increased leaf wax content

Objective and/targeted trait

Li et al. (2018b)

Li et al. (2018a)

Sajeevan et al. (2017)

References

250 T. Sarkar et al.

10

Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges

Khurana 2003). Further, A. tumefaciens strain LBA4404 harbouring pBI121 plasmid showed a higher frequency of transient expression of gus gene (20–25%) in all the four types of transformed explants (cotyledon, hypocotyl, leaf and leaf-derived callus) than the other plasmids (pBI101, p35SGUSINT), after 15 days of cocultivation (Bhatnagar and Khurana 2003). Various types of explants of mulberry showed varying sensitivity to a selective agent such as kanamycin. For example, adventitious bud formation from the explants, viz. cotyledon, hypocotyl and leaf inoculated on MS medium plus kanamycin (50 mg/L), was inhibited, whereas leaf-derived callus produced adventitious buds even on kanamycin (75 mg/L) after 30 days of culture (Bhatnagar et al. 2004). In most of the genetic transformation studies, neomycin phosphotransferase II (nptII) gene was used as a selectable marker gene, and kanamycin (50 mg/L) had been used as an optimum selection pressure for identifying transformed tissues (Bhatnagar and Khurana 2003; Bhatnagar et al. 2004; Checker et al. 2012; Sajeevan et al. 2017). Nozue et al. (2000) used hygromycin phosphotransferase (hpt) gene as a selectable marker in the transformation study, and hygromycin (about 12.5 mg/L) was added in callus induction medium for selection of transformed calli. Agrobacterium cells of early log phase are suitable for the co-cultivation step, and the optimum cell density required is explant-specific in mulberry. Bhatnagar et al. (2004) reported that bacterial density of 8  l08 cells/ml was optimum for cotyledon and hypocotyl, while a relatively higher density of 10  l08 cells/ml was optimum for leaf and leaf-derived callus. The optimum time of incubation for the explants in bacterial suspension was 30 min (Bhatnagar and Khurana 2003; Lal et al. 2008; Saeed et al. 2015). Most of the studies demonstrated that the duration of cocultivation required for successful transformation in mulberry was 3 days (Bhatnagar et al. 2004; Das et al. 2011; Checker et al. 2012; Saeed et al. 2015; Sajeevan et al. 2017). Incubation of explants with bacterial suspension beyond

251

90 min and co-cultivation of explants with Agrobacterium cells beyond 5 days lead to overgrowth of bacteria on explants, thus making it difficult to recover the explants from bacteria after washing with cefotaxime (Bhatnagar et al. 2004). Varying frequency of transformation was recorded in various stages of the transformation process in mulberry. For example, just after 3 days of co-cultivation, the frequency of transient gus expression was 90–100% in the tested explants, viz. leaf, cotyledon, hypocotyl and leafderived callus (Bhatnagar and Khurana 2003). On selection medium, the transformation efficiency for leaf-derived callus was 90%, while for cotyledon and hypocotyl it was 18.60–60% and 20%, respectively (Agarwal et al. 2004b; Das et al. 2011). Chitra et al. (2014) reported transformation efficiency (62.2%) from shoot meristem (cv. S-36) cultured on a selection medium. Transformation frequency (6%) was reported in mulberry based on the number of transgenic plants containing heterologous gene in their genome, which were developed from the total number of explants co-cultivated (Bhatnagar et al. 2004). The addition of phenolic substance, viz. acetosyringone (200 µM–250 mM) in bacterial suspension and co-cultivation medium might have enhanced transformation frequency in mulberry (Bhatnagar et al. 2004; Raghunath et al. 2010; Saeed et al. 2015; Sajeevan et al. 2017; Li et al. 2018a, b). Pre-culturing/pre-conditioning of explants on TDZ or BAP plus 2, 4-D amended medium for 2 or 5 days just before transformation, enhanced the susceptibility of cells to Agrobacterium infection and subsequent T-DNA transfer in the genome (Agarwal et al. 2004b; Bhatnagar et al. 2004; Das et al. 2011; Checker et al. 2012; Sajeevan et al. 2017). The age and physiological state of explants at the time of co-cultivation with Agrobacterium is crucial for the success of the transformation. The explants should contain adequate cells competent for regeneration and DNA uptake during genetic transformation (Bhatnagar et al. 2004).

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10.5

T. Sarkar et al.

Development of Stable Transgenic Mulberry Plants

Complete transgenic mulberry plants were regenerated via direct and indirect organogenesis routes. Stable transgenic mulberry plants were developed through direct organogenesis from cotyledon, hypocotyl and leaf explants, whereas, through indirect organogenesis, transgenic plants were developed from leaf-derived callus of K2/M5 cultivar (Table 10.1 and Fig. 10.1). The integration of heterologous gene in the genome of stable transgenic plants was ascertained by polymerase chain reaction (PCR) and/Southern blot analysis (Bhatnagar and Khurana 2003; Bhatnagar et al. 2004; Agarwal and Kanwar 2007; Das et al. 2012; Sajeevan et al. 2017). Agarwal and Kanwar (2007) demonstrated the development, maturation and germination of transformed somatic embryo from embryogenic clumps via somatic embryogenesis, but regeneration of whole transgenic plants from this regeneration system is yet to be achieved.

10.6

Characterization of the Transgenic Mulberry Plants

Most of the studies generally focused on the development of genetic transformation protocols in mulberry, followed by histochemical assay of gus gene expression, and the transgene (selectable marker and/reporter gene) integration was confirmed by PCR from transformed explants or regenerated tissues obtained from transformed explants (Machii 1990; Machii et al. 1996; Sugimura et al. 1999; Nozue et al. 2000; Oka and Tiwary 2000). As mulberry is highly heterozygous due to out-breeding reproductive system, the transgenic mulberry plants so far developed by various research groups have been assessed for tolerance/targeted traits in T0 generation, followed by clonal propagation for development of subsequent vegetative generation (Lal et al. 2008) as reported in transgenic pineapple (Yabor et al. 2016). Genetic manipulation has been attempted in mulberry via gain-of-function approach, where

the heterologous gene/MicroRNA was introduced in its genome (Bhatnagar and Khurana 2003; Lal et al. 2008; Das et al. 2011; Saeed et al. 2015; Sajeevan et al. 2017; Li et al. 2018a); and loss-of-function approach, where the function of endogenous gene was silenced by expressing a small interfering sequence of the target gene (Li et al. 2018b). Stable integration of the heterologous gene of interest in transgenic mulberry was ascertained by PCR and Southern blot analysis (Bhatnagar and Khurana 2003; Lal et al. 2008; Checker et al. 2012; Sajeevan et al. 2017), its expression at transcription level was assessed by Northern blot analysis (Lal et al. 2008), reverse transcription (RT)-PCR (Sajeevan et al. 2017) and quantitative RT-PCR (qRT-PCR) (Das et al. 2011), and the protein product of the transgene was analysed by Western blot assay (Checker et al. 2012).

10.7

Abiotic Stress Tolerance in Transgenic Mulberry

The performance of transgenic mulberry plants was compared with non-transformed (NT)/wildtype (WT) counterparts at different growth stages, viz. tissues (detached leaf), whole plant (saplings) and in vitro condition (via axillary bud culture) under various stresses under the contained facility as per biosafety guidelines (Lal et al. 2008; Saeed et al. 2015; Negi and Khurana 2021). In vitro regeneration potential of transgenic lines was compared with NT plants by culturing axillary buds on MS medium amended with plant growth regulators and polyethylene glycol (Lal et al. 2008). The axillary buds of transgenic lines showed a higher rate of sprouting (55–80%) than NT plants when cultured on MS medium amended with 1% polyethylene glycol (Lal et al. 2008). As the development of a transgenic mulberry plant, its transfer and establishment in the soil is a prolonged process (3–4 years) (Lal et al. 2008), and due to its inherent heterozygous nature (Khurana and Checker 2011), further development of its successive filial generations or backcrossing for many generations has not been employed so far.

10

Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges

For testing whether the targeted trait/ heterologous gene was introduced in primary (T0 generation) transgenic plant in its hemizygous/ heterozygous condition (James et al. 2002; Passricha et al. 2016; Sattar et al. 2021), detached leaf of transgenic mulberry and NT plants were exposed to simulated abiotic stresses, viz. dehydration, drought and salinity, methyl viologen, high light, high temperature and ultraviolet rays (Lal et al. 2008; Saeed et al. 2015). Subsequently, the physio-biochemical parameters such as proline, relative water content (RWC), cellular membrane stability (CMS) and photosynthetic yield of transgenic and NT plants were assessed to analyse levels of tolerance to stress. Further, saplings of transgenic mulberry and NT plants maintained in pots were exposed to drought by withholding irrigation for 20 and 15 days, respectively for evaluation of their physiobiochemical traits (Lal et al. 2008). Constitutive (Actin1, CaMV35S) and stress-inducible (rd29A) promoters have been used for the regulation of heterologous gene expression in transgenic mulberry plants (Lal et al. 2008; Das et al. 2011; Checker et al. 2012; Sajeevan et al. 2017). Lal et al. (2008) developed transgenic mulberry plants overexpressing Hordeum vulgare-abundant protein 1 (HVA1), which is a group-3 late embryogenesis-abundant (LEA) protein functions as a molecular chaperone. These transgenic plants (Actin1::HVA1) showed tolerance to drought and salinity stresses as indicated by improved membrane stability, photosynthetic yield and water use efficiency (decreased carbon isotope ratio: d13C), and less photo-oxidative damage (Lal et al. 2008). Further, transgenic mulberry plants (rd29a::HVA1) showed tolerance to drought, salinity and low-temperature stresses which might have resulted from better membrane stability, higher photosynthetic yield and proline content (Checker et al. 2012). Xanthophylls function as accessory pigments which play an important role in the protection of photosynthetic machinery of the plants under high light irradiance, and xanthophylls are synthesized from the carotenoid precursor (b-carotene) through the

253

enzymatic activity of b-carotene hydroxylase (Saeed et al. 2015). Overexpression of heterologous b-carotene hydroxylase 1 (bch1) gene from M. indica in mulberry cv. K2 showed a higher level production of carotenoids and enhanced tolerance to various oxidative stresses (high light, heat and UV irradiation) as compared to NT plants, which successfully validates the cisgenic approach for augmenting climate resilience in mulberry (Saeed et al. 2015). Plant MicroRNAs (miRNAs) and up-stream transcription factors (TFs) regulate the expression of downstream stress-inducible genes in response to abiotic stresses (Sarkar et al. 2014; Li et al. 2018a). The downstream regulatory functions of heterologous gene/miRNA in transgenic mulberry plants were analysed via qRT-PCR (Checker et al. 2012; Li et al. 2018a). Expression of Hva1 gene, under the regulatory control of constitutive and stress-inducible promoters in transgenic mulberry plants, showed activation of downstream stressresponsive genes (viz. MidnaJ, MiERD10, MiERD15, MiMYB60, MiWCOR413, MiSRC1 and Mi2-cysperoxidin) under abiotic stress (Checker et al. 2012). The expression levels of downstream stress-responsive genes were higher in transgenic lines (rd29A::Hva1 and Actin1:: Hva1) than in the NT mulberry plants. The expression level of these stress-responsive heterologous genes was higher in transgenic plants with rd29A promoter than transgenic lines with Actin1 promoter. However, the transgenic mulberry lines with Actin1 promoter performed better than those with rd29A promoter under severe stress conditions (Checker et al. 2012). The surface wax load of mulberry leaf is responsible for moisture retention capacity (Mamrutha et al. 2010). SHINE1/Wax Inducer1 (SHN1/WIN1) gene coding for a transcription factor that coordinates the expression of downstream target genes associated with wax biosynthesis was introduced in mulberry. Transgenic mulberry plants (CaMV35S::AtSHN1) showed dark green and shiny appearance; and enhanced leaf surface wax content and leaf moisture retention capacity (Sajeevan et al. 2017).

254

10.8

T. Sarkar et al.

Biotic Stress Tolerance in Transgenic Mulberry

Genetic transformation had been attempted in mulberry mostly to develop transgenic plants with abiotic stress tolerance, except for one (Table 10.1). Heterologous expression of osmotin gene (which codes for a protein belonging to PR5 group of plant system) from tobacco under the control of constitutive promoter (CaMV35S) and stress-inducible promoter (rd29A) conferred tolerance to abiotic and biotic stresses in transgenic mulberry (Das et al. 2011). Transgenic mulberry plants with stress-inducible promoter showed better tolerance to salinity and drought stresses than those plants with the constitutive promoter. However, transgenic lines with constitutive promoter showed more resistance to fungal pathogens, viz. Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium. Altogether, transgenic plants (CaMV35S::osmotin) are better suited for both abiotic and biotic stresses, while the transgenic plants (rd29a::osmotin) are more tolerant to abiotic stresses (Das et al. 2011).

10.9

Transient Expression System in Mulberry

A transient transformation system has been developed for mulberry for functional analysis of novel genes/miRNAs (Table 10.1 and Fig. 10.1). Transient overexpression of pre-miR166f from mulberry in the leaves of M. multicaulis showed improved physio-biochemical indices (relative water, proline and soluble protein contents, and superoxide dismutase and peroxidase activities), lower level of malondialdehyde content and tolerance to drought (Li et al. 2018a). In these transient transgenic lines, target genes such as HD-Zips and histone arginine demethylase showed a lower level of expression, indicating miR166f could negatively regulate the expression of target genes under drought stress. This study indicated that miR166f functioned as a positive regulator for drought stress tolerance in mulberry.

Virus-induced gene silencing (VIGS) approach has been used in mulberry for functional analysis of novel gene(s) from mulberry itself, thus paving the way for reverse genetics approach in this tree species. In plants, shaggylike protein kinase plays crucial roles in the growth and development, regulation of substrate metabolism, signal transduction and responses to abiotic and biotic stresses (Li et al. 2018b). Transient expression of interference sequence of shaggy-like protein kinase of Morus alba (MmSK) gene, via VIGS in mulberry, showed down-regulation of endogenous MmSK gene, sensitivity to drought stress and deterioration of physio-biochemical indices such as soluble protein and proline contents, and superoxide dismutase and peroxidase activities (Li et al. 2018b).

10.10

Nutritional Attributes of Transgenic Mulberry

Biochemical constituents and moisture retention capacity of mulberry leaves are important for silkworm growth and quality silk production. Nutritional status, viz. protein and carbohydrate content (mg/g fresh weight of leaf: FWL), and moisture retention capacity in leaves of NT mulberry cv. V1 and mulberry transgenic lines (rd29a::HVA1, CaMV35S::HVA1, CaMV35S:: bch1, CaMV35S::osmotin, rd29a::osmotin) were analysed (Manjunatha et al. 2020a). Maximum protein content (14.25 mg/g FWL) was recorded in the transgenic line (CaMV35S::osmotin) followed by the transgenic line with rd29A::osmotin (14.00 mg/g FWL), whereas V1 showed protein content (11.50 mg/g FWL). Similarly, the highest carbohydrate content (0.28 mg/g FWL) was recorded in the transgenic line (CaMV35S:: HVA1) followed by NT mulberry cv. V1 (0.27 mg/g FWL), and the transgenic line (rd29a::osmotin) recorded 0.24 mg/g (FWL) of carbohydrate (Manjunatha et al. 2020a). Further, the transgenic lines (CaMV35S::AtSHN1) showed significantly higher leaf moisture retention capacity and slower degradation of proteins in detached leaves as compared to NT plants

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Transgenic Mulberry (Morus Spp.) for Stress Tolerance: Current Status and Challenges

(Sajeevan et al. 2017). These studies indicate that the nutritional status of most of the transgenic lines is on par with or even slightly better than the NT mulberry plants.

10.11

Bioassay of Silkworm and Mulberry Pests

Due to biosafety regulations, physiological perspectives of silkworm and ecological concerns, the leaves of transgenic mulberry were used to feed silkworm to study the effects of transgene protein on larval growth and development, and cocoon quality and quantity. Feeding bioassay revealed that expression of the heterologous gene in mulberry leaves has no undesirable effects on the growth of silkworm larvae, cocoon yield and silk quality (Lal et al. 2008; Das et al. 2011; Saeed et al. 2015; Sajeevan et al. 2017; Manjunatha et al. 2020b). Similarly, the growth and development of two common mulberry pests, viz. leaf roller (Diaphania pulverulentalis) and mealybug (Macconellicoccus hirsutus), were assessed by rearing on shoot tips and tender leaves of five different transgenic mulberry lines (CaMV35S:: HVA1, rd29a::HVA1, CaMV35S::BCH1, rd29a:: osmotin and CaMV35S::osmotin) along with NT mulberry cv. V1 (Manjunatha et al. 2021). Both the pests (leaf roller and mealybug) were able to complete their life cycle on transgenic mulberry lines as did on NT mulberry plants. The transgenic mulberry lines, expressing the transgenes under the regulatory control of stress-inducible or constitutive promoter, did not upset the growth and development of mulberry pests.

10.12

Conclusions

Considerable achievements have been made towards the enhancement of mulberry foliage yield and quality through traditional breeding approaches. Till date, genetic improvement of mulberry through molecular breeding approaches

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showed partial success, which might be due to limited desired genetic variability, the complex nature of agronomic and yield traits associated with leaf productivity and stress tolerance, long juvenile period, and non-availability/limited availability of molecular markers tightly linked to QTLs. Under climate change scenario, the foliage productivity and quality are challenged by various biotic and abiotic stresses, which may directly affect silk productivity. Transgenic technology paved the way for the manipulation of targeted traits by single-gene introduction/ multi-gene stacking approaches through the introduction of a single-action gene or gene codes for a desirable transcription factor. Transgenic mulberry lines with improved nutritional status and biotic/abiotic stress-adaptive traits so far developed need to be assessed under confined field trials for trait evaluation, foliage yield potential and nutritional status as per biosafety guidelines. Further, the introduction of stressresponsive heterologous genes from wellcharacterized transgenic mulberry lines could be introgressed into different genetic backgrounds of high-yielding cultivars through traditional and marker-assisted breeding strategies, as reported in bread wheat (Shavrukov et al. 2016). However, genotype-dependent and explant-specific regeneration protocols in mulberry show slight hindrances and necessitate constant efforts to develop genotypeindependent, robust, and reproducible in vitro regeneration and genetic transformation protocols. The development of cisgenic and/transgenic lines from suitable somatic tissues (explants) retaining the genetic makeup of the mother plant except for introduced/heterologous gene(s) is the need of the hour. Genome editing tools such as clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) systems (CRISPER/Cas9) can be used in mulberry to understand the molecular basis of biotic/abiotic stress tolerance and other agronomic traits of interest and to widen genetic variability in the existing germplasm for further trait improvement.

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Tikader A, Kamble CK (2009) Development of core collection for perennial mulberry (Morus spp.) Germplasm. Pertanika J Sci Technol 17:43–51 Vijayan K (2010) The emerging role of genomic tools in mulberry (Morus) genetic improvement. Tree Genet Genomes 6:613–625 Vijayan K, Chakraborti SP, Roy BN (2000) Plant regeneration from leaf explants of mulberry: Influence of sugar, genotype and 6-benzyladenine. Indian J Exp Biol 38(5):504–508 Vijayan K, Chauhan S, Das NK, Chakraborti SP, Roy BN (1997a) Leaf yield component combining abilities in mulberry (Morus spp.). Euphytica 98:47–52 Vijayan K, Tikader A, Das KK, Chakraborti SP, Roy BN (1997b) Correlation studies in mulberry (Morus spp.). Indian J Genet 57:455–460 Vijayan K, Das KK, Chakraborti SP, Roy BN (1998) Heterosis for yield and related characters in mulberry. Indian J Genet 58(3):369–374 Vijayan K, Doss SG, Chakraborti SP, Ghosh PD (2009) Breeding for salinity resistance in (Morus spp.). Euphytica 169(3):403–411 Vijayan K, Tikader A, da Silva JAT (2011) Application of tissue culture techniques propagation and crop improvement in mulberry (Morus spp). Tree Forestry Sci Biotechnol 5(1):1–13 Vijayan K, Raju PJ, Tikader A, Saratchnadra B (2014) Biotechnology of mulberry (Morus L.)—a review. Emir J Food Agr 26(6):472–496 Vijayan K, Gnanesh BN, Shabnam AA, Sangannavar PA, Sarkar T, Zhao W (2022a) Genomic designing for abiotic stress resistance in mulberry (morus spp.). In Kole C (ed) Genomic designing for abiotic stress resistant technical crops. Springer Nature, Switzerland, pp 157–244. https://doi.org/10.1007/978-3-03105706-9_7 Vijayan K, Arunakumar GS, Gnanesh BN, Sangannavar PA, Ramesha A, Zhao W (2022b) Genomic designing for biotic stress resistance in mulberry (Morus spp.). In Kole C (ed) Genomic designing for biotic stress resistant technical crops. Springer Nature, Switzerland, pp 285–336. https://doi.org/10.1007/9783-031-09293-0_8 Wang H, Lou C, Zhang Y, Tan J, Jiao F (2003) Preliminary report on Oryza cystatin gene transferring into mulberry and production of transgenic plants. Can Ye Ke Xue 29:291–294 Yabor L, Valle B, Rodríguez RC, Aragón C, Papenbrock J, Tebbe CC, Lorenzo JC (2016) The third vegetative generation of a field-grown transgenic pineapple clone shows minor side effects of transformation on plant physiological parameters. Plant Cell Tiss Organ Cult 125:303–308

Application of Mulberry and Mulberry Silkworm By-Products for Medical Uses

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Ravindra M. Aurade, Y. Thirupathaiah, V. Sobhana, Dhaneshwar Padhan, B. Kishore Kumar, and Babulal

11.1

Introduction

According to the World Health Organization poll, about 80% of the public in developing nations believes in natural medicines, which ensure patient safety and improve the knowledge and abilities of medicine providers for health safety. Plants are involved in about 25% of medicines, both directly and indirectly (Cragg et al. 1997) and are employed as medications (Rawat and Uniyal 2003). To address the WHO primary health needs worldwide, almost 80% of the population in rural regions relies on wild plants (Alves Rômulo and Ierecê 2005). Contrary to popular belief, many different species of plants are employed as medication in traditional healthcare systems (Prusti 2008). Medicinal plants, according to pharmacological evaluations, are a possible source of important antioxidants and bioactive chemicals. Nutraceuticals contain phytochemicals isolated from medicinal plants (Ncube et al. 2008). Mulberry is a member of the Moraceae family and can be found in a wide range of environmental conditions, from temperate to tropical.

R. M. Aurade (&)
Y. Thirupathaiah
V. Sobhana
D. Padhan
B. K. Kumar
Babulal Central Sericultural Research and Training Institute, Central Silk Board, Srirampura, Mysuru, Karnataka 570008, India e-mail: [email protected]

Moraceae, generally known as the mulberry, is a flowering plant family that includes more than twenty-four species, each with one subspecies and at least a hundred variants (Ercisli and Orhan 2007). The name Morus comes from the Latin word “mora,” which means “delay,” most likely due to the sluggish growth of its buds. It is a common woody plant with tremendous economic significance even outside of sericulture, giving it various distinct characteristics. Morus nigra (black mulberry), Morus alba (white mulberry), and Morus rubra (red mulberry) are the most widely used species in the genus Morus because they have the most therapeutic characteristics. M. alba is the most common of all the species (Ercisli and Orhan 2007). The culinary, medical, and cosmetic industries can employ the bioactive components found in mulberry roots, leaves, bark, stem twigs, and fruits. Mulberry fruits, particularly the black and red types, are traditionally thought to be beneficial to the human body (Ercisli and Orhan 2007). Nearly, all mulberry plant varieties have been recognized as having a variety of pharmacological characteristics in Ayurveda, Unani, and Chinese medicine (Jan et al. 2021). Mulberry (Morus spp.) is a vitally important plant that contains a variety of bioactive components, including cancer-prevention substances such as flavonoids, phenolics, and dietary fibres. It has a strong effect on diseases such as diabetes, cardiovascular infection, and viral infections due to the presence of bioactive components. It can

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_11

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be deduced from the proximity of bioactive sections and has a significant impact on diseases including diabetes, cardiovascular disease, and viral activity. Mulberry investigates a wide range of pharmacological activities, including antidiuretic, antimicrobial, antimutagenic, developmental neutralizing activity, anxiolytic, anthelmintic, antistress, immunomodulatory, hypocholesterolemic, anticholesterolemic, and anticholesterolemic, as well as specific effects like adaptons, hyperlipidemia, and melanin biosynthesis containment (Stand and Oliver 2012). This plant also had antiatherogenic growth and HIV activity that was unfavourable. Also, sericulture silk by-products such as sericin and fibroin proteins are having various applications in cosmetic, nutraceutical, and clinical. About 20–30% of the weight of a silk strand is made up of the globular protein sericin. Its function in worm cocoons is to cover and connect the fibroin filaments (Aghaz et al. 2015). Silk fibre contains 70–80% fibroin protein. To maintain its compact structure, it has crystalline and amorphous domains with short amino acids chains (Koh et al. 2015). Additionally, sericin also shields the cocoon from wind, UV radiation, rain, and cold (Cao and Zhang 2016).

11.1.1 Anti-Diabetic Properties of Mulberry Leaf Mulberry leaf is found to greatly reduce peak-totrough oscillations in blood glucose (Mudra et al. 2007). Within 120 min, mulberry leaf extracts combined with sugar lowered elevated glucose levels in blood. The capacity of fahomine in mulberry leaves to suppress insulin is also a strategic reason for ingestion. Lipid peroxidation may be reduced as a result of this. The difficulty with diabetic individuals is a lack of response or very low insulin production, which leads to “hyperglycemia,” or high blood sugar. According to Naowaboot et al. (2009), mulberry leaves extract has antihyperglycaemic, antioxidant, and antiglycation properties, making it a good beverage for treating hyperglycemia. He also examined the antihyperglycemic,

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antioxidant, and antiglycation properties of mulberry leaf extract in animal models and discovered a significant reduction in the risk of hyperglycemia. Red mulberry leaf extract has been shown to improve rat health (Sharma et al. 2010). The 1-Deoxynojirimycin (DNJ) was crucial in lowering blood sugar levels. It works by inhibiting intestinal-glucosidases, lowering blood sugar levels (Hansawasdi and Kawabata 2006). Furthermore, Lown et al. (2017) investigated the effects of mulberry leaf extract on glucose tolerance. The mulberry leaf extract significantly reduced total blood glucose rise after 120 min of maltodextrin (Maize starch) consumption. The mulberry leaf’s hybrid 1-Deoxynojirimycin (DNJ) and polysaccharide help modulate the expression of enzymes such as phosphoenolpyruvate carboxykinase, glucokinase, and glucose 6-phosphatase (Li et al. 2011). The enzyme alpha-amylase catalyses the first steps in the hydrolysis of starch into smaller oligosaccharides. According to Sudha et al. (2011), the mulberry leaf extract has considerable alpha-amylase inhibitory action. Furthermore, Doi et al. (2000) found that leaf extracts were effective at scavenging the radical and inhibiting oxidative alteration of rabbit and human. Bajpai and Rao (2014) recently reported that 1-Deoxynojirimycin (DNJ) found in mulberry leaves has active pharmacokinetic principles and the potential to control hyperglycemia.

11.1.2 Antioxidative Properties of Mulberry Leaf The term “antioxidant” refers to a substance that reduces the effects of oxidation in the human body. Mulberry leaves have a good antioxidative property due to the presence of several types and a wide spectrum of flavonoids. Mulberry leaves are high in antioxidants, such as phenolics and flavonoids, which have been found to be effective antioxidants. The fractions of mulberry leaf extract with higher levels of phenolic and flavonoid components have stronger antioxidant properties (Iqbal et al. 2012; Flaczyk et al. 2013).

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Application of Mulberry and Mulberry Silkworm By-Products for Medical Uses

When compared to the petiole, mature fruit, unripe fruit, and stem, the mulberry leaf extract has the highest antioxidant (DPPH and hydrogen peroxide radical scavenging) characteristics (Lim and Choi 2019; Lim and Teo 2019). Enkhmaa et al. (2005), Chen and Li (2007), Katsube et al. (2010) and Iqbal et al. (2012) revealed that increased levels of quercetin in mulberry leaves were responsible for reducing the oxidation process. Oxyresveratrol and 5, 7-dihydroxycoumarin 7-methyl ether is found in the ethanolicas and aqueous extract of mulberry leaves. It essentially aids in the scavenging of superoxide and has antioxidant properties (Oh et al. 2002). Samuel et al. (2016) investigated the antioxidant activity of mulberry leaves aqueous extract from four varieties (S-146, AR-14, BR-2, and S-1) in the brains of experimented animals, finding that it reduced malondialdehyde (50.49%, 36.14%, 41.36%, and 37.13%) and superoxide dismutase (54.01%, 40.18%, 34.82%, and 29%). According to Iqbal et al. (2012), M. nigra has a distinct advantage over other mulberry species in terms of disease prevention by scavenging phenolics, DPPH radicals, and ABTS radical cations. The scavenging activity of DPPH and ABTS radicals are the two most often used methods for determining antioxidant capabilities in mulberry leaves. Yigit et al. (2008) investigated the potential of mulberry leaves to scavenge DPPH radicals and reduce lipid peroxidation, finding a statistically significant link between DPPH radical scavenging and total phenolic compounds.

11.1.3 Role of Mulberry Leaf in Prevention of Cardiovascular Diseases Cardiovascular disease (CVD) is an illness that affects the heart or blood vessels (Mendis et al. 2011). According to Kadam et al. (2019), mulberry leaves contain a large amount of iron (Fe), which aids in the better distribution of oxygen by increasing the synthesis of red blood cells. Another key flavonoid found in mulberry leaves is resveratrol, which works directly to relieve blood vessel constriction, lowering the risk of

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heart failure, and increasing the generation of nitric oxide, a vasodilator. This means it relaxes blood arteries, lowering the risk of blood clots and consequent heart problems like strokes or heart attacks. The leaf extracts of the mulberry (M. alba and M. bombycis) have been proven to reduce hypertension in rodents, lower serum cholesterol, and prevent atherosclerosis (Doi et al. 2000; Oh et al. 2007). Ma et al. (2019) reported that 1-Deoxynojirimycin (DNJ) in mulberry leaves extract improved antioxidant and anti-inflammatory functions, reducing coronary heart disease (CHD) and blood stasis syndrome (BSS) in patient.

11.1.4 Anticancer Effects of Mulberry Leaf Cancer, or abnormal cell proliferation in the human body, is a calamity. Abnormal cell proliferation has the ability to spread throughout our body. Despite the fact that there is no cure for cancer, mulberry leaves can be an excellent alternative for mitigating or reducing the risk of cancer due to their high antioxidant and other therapeutic characteristics. The ethanol extract of the mulberry leaf can eradicate the neuroblastoma stem cell-like population, which is one of the main causes of this fatal disease (Park et al. 2012). Mulberry leaf extract at 10–40 µg/L effectively promoted differentiation by elongating neurites, lowering clonogenicity, and preventing sphere formation. This was demonstrated by a drop in stem cell markers and an increase in differentiation markers. Polyphenols found in mulberry leaves have been shown to decrease tumour cell proliferation, invasion, and metastasis (Yang et al. 2012). Mulberry leaf polyphenols extract can also cause cell death in hepatocellular cancer cells. Mulberry leaf extract also inhibits the proliferative signal pathway of adipocytes’ inflammatory response in hepatocelluar carcinoma (HCC) and can prevent obesity-related liver cancer (Chang et al. 2017). Similarly, Fallah et al. (2018) found that the deoxyribonucleic acid (DNA) from Iranian mulberry leaves can be employed as an anti-cancer medicine.

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Experiments revealed that oxidative flavonoids derived from mulberries were particularly helpful in extending the life span of a rat with a malignant tumour.

11.1.5 Anti-Inflammatory Effects of Mulberry Leaf Inflammation is a biological response that occurs when the body attempts to cure itself by removing harmful or irritating effects in various sections of the body. The anti-inflammatory qualities of the mulberry tree are largely found in the bark and roots, while only a small quantity is found in the leaves. Mulberry leaf extract has diaphoretic and emollient properties, making it useful for preparing a decoction that may be used as a gargle and greatly reducing throat discomfort (Lim et al. 2013). The mulberry leaf extract acts as anti-inflammatory function in obese mice fed with diet contains high fat. Nitric oxide synthase and manganese superoxide dismutase, proteins that are indicators of inflammation and oxidative stress, were reduced by this medicine in the liver and adipose tissue after 12 weeks without causing liver damage. In addition, the mulberry leaves have inhibitory capabilities for inducible nitric oxide synthase (iNOS) (70% with 10 µg/L), which are particularly beneficial for inflammation (Hong et al. 2002). According to Park et al. (2013), mulberry leaves extract may act anti-inflammatory agent to block nuclear factor kappa-light-chain enhancer of activated B cells or NF-kB-mediated inflammatory response and also activities of pro-inflammatory mediators and cytokines.

11.1.6 Neurological Disorders, Skin Diseases, Gastrointestinal Disorders Alzheimer’s disease is a neurological illness caused by amyloid beta peptides in the brain. Mulberry leaves contain Kaempferol-3-Oglucose and Kaempferol-3-O-(6-malonyl0 glucoside, (Niidome et al. 2007; Khaengkhan et al.

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2009), and their ingestion helps to prevent peptide synthesis inside the brain. Yadav and Nade (2008) found that the methanol extract of the leaf has an anti-dopaminergic action, blocking the dopamine (D2) receptors. Kang et al. (2006) reported that the gamma-aminobutyric acid (GABA) found in the extract has the potential to protect the PC12 (model for neural development) cells against various free hydroxyl radicals. Shih et al. (2010) also discussed the effect of antioxidants in defence and enhancing the human body’s defence system. Mulberry leaf extract contains oxyresveratrol (a tyrosinase inhibitor), which acts as a neuroprotective component (Zhang and Shi 2012) and is a promising treatment for acute ischaemic stroke (Horn et al. 2003). Breuer et al. (2006) showed that oxyresveratrol can pass the blood–brain barrier, providing direct brain protection. Mulberry leaf extract can be used as a natural skin care treatment in addition to being consumed as a tea. The leaves have anti-ageing properties and are excellent for acne-prone or pimple-prone skin (Cheng 2016). It lowers irritation and sebum production on the skin. Mulberry leaves include anti-tyrosinase action, making them useful for skin whitening and bright complexion (Suisse 2017). Tyrosinase, an enzyme, has the ability to regulate the formation of melanin. Singh et al. (2013) investigated the effects of mulberry, kiwi, and Sophora angustifolia extracts on melanogenesis and melanin transfer in human melanocytes. Mulberry leaf extract lightens the complexion by inhibiting tyrosinase activity. Gastrointestinal disorders are diseases that affect the gastrointestinal tract. Mulberry leaves have a lot of potentials to help with digestion. Furthermore, it contains an excessive number of dietary fibres, which aid in the thickening of stool and improved digestion (Kadam et al. 2019). By inoculating mulberry leaves extract with carbon tetrachloride, Hogade et al. (2010) described the defensive action on liver (rats) (induced). Mulberry leaves are also abundant in minerals and vitamin C, as well as antioxidants, and contain no anti-nutritive or harmful substances. As a result, mulberry leaves’ bioactive

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Application of Mulberry and Mulberry Silkworm By-Products for Medical Uses

components are extremely beneficial increasing metabolism (Li et al. 2019).

for

11.1.7 Antimicrobial Effects of Mulberry Leaf The term “antimicrobial” refers to a substance that protects against microorganisms. Antibiotic is essential in the preservation of the human body against dangerous microorganisms. Mulberry leaf extract contains saponins, tannins, alkaloids, and flavonoids, all of which have antibacterial properties. Microbes such as E. coli, Salmonella Typhimurium, Staphylococcus epidermis, Staphylococcus aureus, Candida albicans, and Saccharomyces cerevisiae are all susceptible to the flavonoid extract from mulberry leaves (Paiva et al. 2010). Chalcomoracin, a mulberry leaf phytoalexine, has been shown to have potent antibacterial properties against Staphylococcus aureus (Fukai et al. 2005). Similarly, Oliveira et al. (2015) found that an ethanolic extract of mulberry leaves protects mice from oral toxicity caused by Artemiasalina (L.). Ayoola et al. (2011) investigated the antibacterial activity of ethanol extracts of M. alba leaves and discovered that Gram-negative bacteria like E. coli, Pseudomonas aeruginosa, and Neisseria gonorrheae, as well as Gram-positive bacteria like Proteus vulgaris, Staphylococcus aureus, and Streptococcus faecium, and fungi.

11.2

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11.2.1 Immunological Response of Sericin Silk fibres have been employed as sutures in the biomedical area because of their biocompatibility (Moy et al. 1991). A few types of research have shown that silk proteins activate the immune system (Soong and Kenyon 1984; Celedon et al. 2001; Zaoming et al. 1996). In the past, sericin was held accountable for hypersensitivity reactions (Altman et al. 2003). While having no impact on the release of interleukin 10 (IL-10) or TNF, sericin inhibits the proliferation of peripheral blood mononuclear cells that have been activated in vitro and decreases the release of interferon gamma (IFN-) (Chlapanidas et al. 2013; Fabiani et al. 1996). The sericin protein effectively added to the cell culture media without having any cytotoxicity effect (Terada et al. 2003, 2005).

11.2.2 Antioxidant Role of Sericin The sericin protein’s surrounding layers contain phenolic and flavonoid compounds, which support the antioxidant activity of the sericin protein as well as the colouring of the cocoon (Aramwit et al. 2010; Prasong 2011; Zhao and Zhang 2016; Napavichayanun et al. 2017). Sericin has a large number of amino acids with hydroxyl groups, primarily serine, which act as chelators and boost its antioxidant power (Micheal and Subramanyam 2014).

Sericin Properties and Biomedical Applications 11.2.3 Sericin in Cosmetology

The physicochemical properties and molecular heterogeneity of sericin regulate its functionality, and these characteristics are directly impacted by extraction techniques. Sericin is immunologically inert, proving its safety and opening up a wide range of applications in biomedicine, including the food and cosmetic industries, supplementation in culture media, cryopreservation, wound healing, antitumour effect, various metabolic effects in organic systems, and tissue engineering.

The availability of amino acids with hydrophilic side groups (*80%), out of which serine accounts nearly 30%, which has a high water absorption capacity. The use of sericin protein in cosmetic formulations, such as creams and shampoos, leads to an increase in elasticity, hydration, cleaning with less irritation, antiageing, and antiwrinkle effects, and sericin may build a silky and smooth coating on the skin’s surface, reducing water loss (Voegeli et al. 1993;

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Patel and Modasiya 2011; Singh et al. 2011; Singh 2014).

11.2.4 Supplement of Sericin in Culture Media and Cryopreservation Cell culture studies are frequently used to evaluate new findings or technologies, especially in cell therapy and regenerative medicine research (Terada et al. 2005). The most commonly used media is bovine serum albumin, which can be infected by viruses such bovine spongiform encephalopathy (Terada et al. 2003). Furthermore, cryopreservation of cell lines is a major focus of tissue engineering research, with BSAsupplemented medium containing 10% DMSO (dimethyl sulfoxide) being widely employed (Sasaki et al. 2005). Terada et al. (2005) reported that serum is the most expensive component of cell culture, developing innovative methods of supplementing and cryopreservation, particularly sericin protein in place of serum.

11.2.5 Sericin in Wound Healing The various studies have revealed that sericin protein has therapeutic properties because it enhances the proliferation, migration, and synthesis of collagen. This function is influenced by molecular weight and amino acid composition of sericin protein (Aramwit and Sangcakul 2007; Aramwit et al. 2009, 2010; Matsuura et al. 1968).

11.2.6 Antitumour Effect of Sericin Chemotherapy for cancer treatment has significant cytotoxicity, affecting both normal and cancerous cells, limiting its clinical utility (Tsubouchi et al. 2005). Another issue is chemotherapeutic resistance (Cheok 2012), necessitating the quest for antitumour medicines with lower toxicity and biocompatibility, such as sericin.

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11.3

Wound Healing Applications of Silk Fibroin Protein

There is a lot of interest in fibroin protein as a wound dressing material, because of its biocompatibility and wound healing functions. The skin fibroblasts, an important part and involved in the process of wound healing function, may adhere on fibroin fibres comparable to greater than collagen. Minoura et al. (1995) and Yamada et al. (2004) utilized a proteolytic enzyme to degrade fibroin and looked at how the peptide fragments affected fibroblast adhesion and proliferation. Fibroin has also been demonstrated to help human keratinocytes, which are responsible for wound reepithelization, adhere, and proliferation (Min et al. 2004a, b). When used as a wound dressing, the adhesion and development of both fibroblasts and keratinocytes on fibroin should aid in wound healing and healing time. The healing capacities of silk fibroin, alginate, and silk fibroin/alginate (SF/AA) mix sponges were investigated by Roh et al. (2006). The biocompatibility and biodegradability of fibroin have sparked renewed interest in the substance as a biomedical material. Fibroin is gaining popularity in two areas: (1) as a contact lens material and (2) as a wound dressing. Biocompatibility, as well as oxygen permeability, is required for both applications (Sashina et al. 2009; Min et al. 2012). The degree of regenerated epithelium, collagen deposition, and cell proliferation was investigated after these sponges, as well as a gauze control, were subjected to circular wounds surgically created in rats. When compared to the gauze control, these sponges considerably reduced wound healing time (by about 50%), boosted collagen deposition, and increased cell proliferation. Individual silk fibroin or alginate sponges have marginally greater healing capacities than the SF/AA sponge. Min et al. (2012) demonstrated that a silk fibroin sponge dressing coated with nano-Ag has excellent wound healing and antibacterial activity, allowing for the growth of a normal epidermal layer over the wound and reducing wound healing time.

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Application of Mulberry and Mulberry Silkworm By-Products for Medical Uses

Inflammation, the production of new tissue, and tissue remodelling are just a few of the many overlapping phases that occur during the multistep process of wound healing (Singer and Clark 1999). Depending on how severe the injury was, it may take a year or longer for the extracellular matrix and epidermis to regenerate during the remodelling or maturation stage of the wound healing process (Midwood et al. 2004; Gurtner et al. 2008). The initial phase of wound healing is inflammation, which happens shortly after an injury and can remain for up to two days. To stop infection, stop further fluid and blood losses, eliminate dead and dying tissues, and prevent future fluid and blood losses, the immune system, inflammatory pathways, and coagulation cascade must all be activated. Neutrophils and macrophages, which are inflammatory cells, have a number of functions in the healing process, including phagocytosis and the release of many cytokines and growth factors, and the second phase of wound healing is new tissue development, which is linked to collagen/matrix deposition, reepithelization, angiogenesis, and wound contraction (Gonzalez et al. 2016; Midwood et al. 2004). The maturation phase of the wound healing process is connected with the rebuilding of the epidermis and extracellular matrix, which can take a year or longer depending on the severity of the injury. The second phase of wound healing is new tissue development, which is linked to collagen/matrix deposition, reepithelization, angiogenesis, and wound contraction (Gonzalez et al. 2016; Midwood et al. 2004; Gurtner et al. 2008). Scar tissue is inferior to normal tissue in both function and appearance. When an animal has a wound, a regenerative process replaces the normal tissue structure, and the goal of skin restoration is still skin regeneration (Reinke and Sorg 2012; Wood 2014). Extracellular matrix is produced in excess as a result of damage; collagen I, a crucial protein that affects extracellular matrix architecture during wound healing, is principally produced by fibroblasts and is mostly regulated by the cytokine transforming growth

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factor 1 (TGF)1 (Ferguson 1998; Border and Ruoslahti 1992; Agarwal et al. 2016). However, many biopolymers’ material properties cannot be changed enough for most controlled release applications. A material must possess a number of critical material features in order to be successful in implanted controlled release applications. Not only must the material be biocompatible, but it must also be biodegradable and free of toxic breakdown products. Controllable biodegradability is essential. Furthermore, the material must be compatible with the medications or biomolecules being supplied, and its release kinetics must be controlled (Pritchard and Kaplan 2011). All of these characteristics appear to be present in silk fibroin, making it an attractive biopolymer for controlled release applications. Silk fibroin fibres and films offer exceptional biocompatibility and mechanical qualities that are unique and noteworthy (Altman et al. 2003). The crystallinity of regenerated silk fibroin films can be changed to alter their mechanical properties (Motta et al. 2002). The rate of biodegradation can be controlled from days to years by modifying the sheet crystallinity, processing solvent, silk concentration, and porosity. Silk fibroin is biodegradable in vivo, has harmless degradation products of amino acids and small peptides, and the rate of biodegradation can be controlled from days to years by modifying the sheet crystallinity, processing solvent, silk concentration, and porosity (Lucas et al. 1957; Horan et al. 2005; Wang et al. 2008; Pritchard et al. 2011). Through intermolecular forces between the silk and biomolecules, fibroin films and matrices can encapsulate and stabilize proteins and enzymes, then release the proteins undenatured and the enzymes with full activity (Lu et al. 2010). Unprocessed fibres, films, nanolayers, hydrogels, sponges, and microspheres are just some of the ways silk fibroin can be processed and used in drug delivery (Pritchard and Kaplan 2011). Changes in the sheet crystalline content can affect mass transfer and release kinetics in regenerated fibroin materials (Karve et al. 2011). The polymer shape and other factors such as molecular mass can also regulate

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drug diffusion within the fibroin (Pritchard and Kaplan 2011). Medication release kinetics can also be altered by altering degradation behaviour as described above, or by co-releasing proteinase inhibitors such as ethylenediaminetetraacetic acid with the target drug to disrupt proteolytic action (Pritchard et al. 2011). Medical sutures were one of the first applications for silk fibres. Choi et al. (2004) demonstrated that controlled release can be used to improve fibroin sutures and generate an infection-resistant suture. Wang et al. (2007) used a silk nanolayer to coat numerous medicines and proteins and studied their release kinetics in vitro. The coatings had excellent mechanical properties for the application, and the release kinetics could be altered by altering the coating’s sheet crystallinity as well as its thickness. Fibroin hydrogels have also been developed for applications requiring controlled release. Silk fibroin has outstanding mechanical, degrading, and biocompatibility qualities, making it ideal for producing highly loaded grafts, particularly in the musculoskeletal sector. In this context, investigations on a silk fibroin-based anterior cruciate ligament (ACL) graft (Hohlrieder et al. 2013). A procedure for fully removing contaminated sericin from the silk fibroin-based textile designed scaffold had been created prior to this investigation. Simple boiling stages of the raw textile-engineered scaffold in an alkaline borate buffered solution (pH 9.0) dissolve the sericin while preserving the underlying silk fibroin with its favourable mechanical properties (Teuschl et al. 2014). A bioreactor system has been designed to test cell compatibility under mechanical stress (Hohlrieder et al. 2013). Up to ten samples can be cultured at the same time with this system, and they can be cyclically tensioned with a set longitudinal force for certain time patterns. Adipose tissue-derived stem cells were cultivated on silk scaffolds and mechanically strained for 3 weeks in pre-vivo experiments. Cells from the neighbouring tissues had totally penetrated the silk-based graft, partially degrading silk strands and replacing them with ligament-like tissue. We built a textile-engineered so-called

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nerve guiding conduit based on silk threads in another study (Teuschl et al. 2015). The purpose of these scaffolds is to serve as guide structures for the regeneration of peripheral nerves. Silk fibroin was produced after the silkworm cocoon was degummed with 0.02 M Na2CO3 for 40 min at 95 °C. The sericin proteins were then removed by washing three to four times with distilled water (Lee et al. 2014; Chung et al. 2015; Martínez-Mora et al. 2012). The degummed silk fibroin was then solubilized for 50 min at 98 °C using calcium chloride, ethanol, and water (at a molar ratio of 1: 2: 8) before being subjected to dialysis to remove the calcium and ethanol. Both in in vitro and in vivo, the silk fibroin solution significantly accelerated the wound healing process (Park et al. 2018; AbdelNaby et al. 2017).

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Application of Green Synthesized Nanoparticles in Sustainable Mulberry Production: Current Trends and Opportunities

12

G. S. Arunakumar, Akhil Suresh, P. M. N. R. Nisarga, M. R. Bhavya, P. Sowbhagya, and Belaghihalli N. Gnanesh Abbreviations

AFM Ag AgNPs ATCC B. mori BmCPV Cl− CNTs CS-PMAA Cu DAP DNA EDTA Fe FTIR HSP K

Atomic force microscopy Silver Silver nanoparticles American type culture collection Bombyx mori Bombyx mori cytoplasmic polyhedrosis virus Chloride ion Carbon nanotubes Chitosan polymethacrylic acid Copper Diammonium phosphate Deoxyribonucleic acid Ethylenediaminetetraacetic acid Iron Fourier-transform infrared spectroscopy Heat shock proteins Potassium

G. S. Arunakumar (&)
A. Suresh
P. M. N. R.Nisarga
M. R. Bhavya
P. Sowbhagya
B. N. Gnanesh Central Sericultural Research and Training Institute, Manandavadi Road 570 008, Srirampura, Mysuru, Karnataka, India e-mail: [email protected]

KCl kDa Kg L MALDI-TOF

mg MIC mL mM MTCC N N 2O NAA nm NPs P PGPR pH PMAA ppm ROS SEM SOD SSP

Potassium chloride Kilodalton Kilogram Liter Matrix-assisted laser desorption/ionization-time of flight Milligram Minimum inhibitory concentrations Millilitre Millimolar Microbial Type Culture Collection Nitrogen Nitrous oxide Naphthalene acetic acid Nanometres Nanoparticles Phosphorus Plant growth-promoting rhizobacteria Potential of hydrogen Polymethacrylic acid Parts per million Reactive oxygen species Scanning electron microscope Superoxide dismutases Single superphosphate

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_12

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TEM TiO2 UV–VIS XRD Zn ZnO ZnONPs µg

12.1

Transmission electron microscopes Titanium dioxide Ultraviolet–visible spectroscopy X-ray diffraction analysis Zinc Zinc oxides Zinc oxide nanoparticles Microgram

Introduction

Sericulture is a very important occupation in many countries of the world. India and China are world’s leading silk producers. In global silk production, these two countries account for over 60% contribution (Narzary et al. 2022). Mulberry is a fast-growing perennial plant belonged to the family Moraceae and genus Morus; this crop is used as a feed for the silkworm (Bombyx mori L.) for production of silk throughout the world (Kwon et al. 2018). Nanoparticles are defined as the material with size range of 10–100 nm which has their unique physiochemical and biological properties which are uniquely distinct from their macrocounterparts. There are three different factors that can uniquely distinguish any kind of nanoparticles, namely size, properties and interaction in system (Gour and Jain 2019). In the material science, nanotechnology is an important subject and manufacture of nanoparticles (NPs) is growing quickly across the globe. As a consequence of precise merits like shape, size and structure, NPs exhibit totally new or improved properties (Nejatzadeh 2021; Taran et al. 2017). Although nanotechnology has very much been advanced in different fields such as medicine, material science, space science, etc., in agriculture, application of nanotechnology still remains in its infant stage in sericulture. However, lot of research on the agriculture application of nanoparticle has been completed, and there are still many in

progresses (Worrall et al. 2018). The potential of nanotechnology in agriculture is huge and wide. Some of the important applications include detection of plant disease, plant production, delivery of fertilizers, reducing the soil wastage and improvement of soil health. There are various types and methods of synthesis of nanoparticles, but most important method of synthesis of nanoparticle is green synthesis method that has attracted the attention of agriculture researchers considerably. Green synthesis of nanoparticles has been seen as the safer way of preparation of synthesizing nanoparticles with least/nil harm to nature. The green method of synthesis of nanoparticle utilizes biological entities such as plant, microbes and their metabolites to synthesize nanoparticles. This method has been providing economic, viable and easily scalable process when compared to chemical method and physical method. Chemical method and physical method are methods as the later methods are costly and are not environmental friendly (Chhipa 2019). In sericulture, the production of silkworm silk with higher quality is very important to gain more acceptability in the global market. Excessive use of chemical fertilizers and pesticides affect significantly the quality of the silk. The unique properties of nanomaterials have great potential to advance the development and growth of different areas including cosmetics, medicine, agriculture and allied sectors. In sericulture, nanotechnology helps to enhance the silkworm survival rate, improvement of growth and development thereby increasing the quality silk production. However, the optimistic aid of nanomaterials is masked by some apprehension about the safety of environment and human health as some of the nanomaterials showed toxicity not only to silkworms but also to the environment, though a number of other nanomaterials displayed therapeutic and antimicrobial properties (Fometu et al. 2021; Naikoo et al. 2021) and, hence, are being used in the food packaging materials to avoid food spoilage (Chaudhry and Castle 2011) to manage diseases and pests in agriculture (Bhattacharyya et al. 2014). In the present era, green synthesis of nanoparticles has gained a great attention due to

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Application of Green Synthesized Nanoparticles in Sustainable …

low production cost, safe to nature and the simplicity in manufacture. Green synthesis is a consistent method for developing a broad range of nanostructures, and it is alternative to conventional method of synthesis. Antimicrobial properties of nanoparticles have been unravelled by recent studies (Naikoo et al. 2021).

12.2

Green Synthesis of Nanoparticles and Their Use in Mulberry Production

The synthesis of nanoparticles involves several chemical methods, which are toxic and hazardous to nature. Therefore, a greener and ecofriendly approach has been adopted for controlling a variety of microbes like fungal, bacterial, actinomycetes, viruses, plants and plant parts through green synthesis of nanoparticles (Duhan et al. 2017). For precision farming, various bioresources were utilized for green nanoparticles synthesis (Bansal et al. 2014). In the recent past, scientists were started studies on utilization of green synthesized nanoparticles along with mulberry leaves and their effects on silkworm growth and development. These studies showed an encouraging results.

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nanodevices and their use in the field crops can prevent the nutrient loss of the water, soil and air, thereby reducing the environmental pollution (DeRosa et al. 2010). By using nanozeolites, nitrogen source can be supplied uninterruptedly in a regulated manner to the plants (Leggo 2000). The controlled supply of nitrates can be achieved by using surfactant-modified zeolite as fertilizer carrier (Li 2003; Liu et al. 2007). Recently, new strategies and technologies have been developed to provide sustained supply of fertilizers based on environmental changes. Under nutrient constraint situation, plants exudate certain carbon compounds into rhizosphere region for the release of nitrogen and other nutrients from the soil organic matter. This root exudate can be utilized as the signals by the nanobiosensors for sensing the environmental changes and to release the required fertilizers from the nanofertilizerreleasing devices. Other applications of nanofertilizers include titanium dioxide which can be incorporated into fertilizers to act as a bactericidal compound, nanosilica to form a protective layer around the cell walls of root to provide protection against the pathogens and resistance during the stress conditions (DeRosa et al. 2010).

12.3.1 Macronutrients

12.3

Green Synthesized Nanofertilizers

The nanoscale and nanostructured materials can be utilized as fertilizer carriers for the slow and controlled release of fertilizers to improve the absorption efficiency of mineral elements by plants and to decrease the application cost of fertilizers (Chinnamuthu and Boopathi 2009; Cui et al. 2010). Fertilizers can be encapsulated within a nanoparticle which can be done in three different ways: (a) fertilizer can be encapsulated inside the porous materials of nanoscale; (b) fertilizer can be coated with a surrounding thin polymeric film; and (c) fertilizers are released as small particles or emulsions of same size as that of nanoparticles (Rai et al. 2012). The synchronization of release of nanofertilizers with the

Huge amounts of fertilizers’ usage has increased production of food significantly but found numerous detrimental effects to the useful soil microorganisms. Fertilizers’ availability is less to plants due to evapotranspiration, leaching, runoff and ultimately polluting the environment (Wilson et al. 2008). Nanofertilizers can resolve these problems by slow release of fertilizers. In India, after green evolution, use of urea form of nitrogen fertilizer has been utilized in increased manifold (29%). Thus, it has greater contribution to greenhouse gas finally leads to global warming (Park et al. 2012). The scarcity of NPK content in the crop production has been met by application of chemical fertilizers. However, majority of the applied fertilizers are lost due to run-off/volatilization. A huge loss has

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been estimated among the applied fertilizers; it causes loss of resources to the nation and polluting the environment (Trenkel 1997; Ombodi and Saigusa 2000). In order to circumvent this huge loss, a current approach adopted in the modern agriculture is to use nanourea. Slow release of nanocoated urea fertilizers helps to absorb the urea efficiently by the plant roots (Wu and Liu 2008). Sulphur nanocoating (  100 nm layer) fertilizers are useful as slow release mechanism for sulphur-deficient soils (Brady and Weil 1999; Santoso et al. 1995). In India, majority of soils are deficient in macronutrients, and nanofertilizers will be advantageous to achieve soil and crop requirements. The sustained release of fertilizers has been achieved by the usage of numerous synthetic and natural polymers. Chitosan nanoparticles (  78 nm) with biodegradable polymer properties have showed slow release behaviour of fertilizers (Corradini et al. 2010). Unnecessary nutrients interfaced with microbes, air and water can be avoided by the utilization of nanofertilizers which stabilizes the release of N and P fertilizer thereby preventing the nutrient losses (Emadian 2017). By the application of nanofertilizers, nutrient absorption of plants from soil can be increased. Growth of plant under high temperature and humidity may be improved by application of nanofertilizers encapsulated nanosilica. It can also give protection against diseases caused by fungus and bacterial infections (Wang et al. 2002). Application of silicon dioxide nanoparticles increased the plant resistance by increasing root development and seedling growth (Hutasoit et al. 2013). A non-toxic TiO2 or titanium is used as supplement in fertilizers to raise food production, and also it can increase water retention (Emadian 2017). A broad range of slow/controlled release of fertilizers were manufactured by using synthetics/biopolymers to reduce the rapid solubility of many N fertilizers (particularly in the nitrate form) (Shaviv 2001; Tarafdar and Subramanian 2011). Many techniques were developed to produce controlled-release fertilizers using the various adsorbents (Sharmila 2010). In last

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decade, for loading NPK fertilizers, polymethacrylic acid (PMAA) was used along with chitosan nanoparticles. The strength of the chitosan polymethacrylic acid colloidal suspension was reported to be higher along with nitrogen, potassium and phosphorus nutrient fertilizers (Hasaneen et al. 2014). The firmness of the solution was established with the addition of potassium (400 ppm) for dispersion (Wu et al. 2006; de Vasconcelos et al. 2006; de Moura et al. 2008; Emadian 2017).

12.3.2 Micronutrients Green synthesis of ZnO nanoparticles for agriculture application from leaf extract is economical and environmentally safe. Agricultural production in alkaline soils is affected by deficiency of zinc which is a most common problem (Takkar and Walker 1993). Zinc deficiency in soils can be corrected by application of ZnO and zinc sulphates (Mortvedt 1992). Nevertheless, zinc application to soil is limited and leads to unavailability of Zn to plants for proper growth and development. This problem may be solved by introduction of green synthesized ZnO nanoparticles. The bioavailability of zinc in soils with calcium carbonate can be increased by application of zinc oxide nanofertilizers. Micronutrients (Zn, Mg, Cu, B, Fe, Mb) are very essential for plant growth and a considerable boost in crop production from green revolution. Novel agricultural activities were gradually decreased the availability of micronutrients in soil (Alloway 2009). Providing micronutrients through foliar spray can improve uptake by the leaves (Martens and Westermann 1991). Nanotechnology is the best option for making plants to get required micronutrients. Nanomicronutrients can be applied on plants through foliar spray or direct application to soil to improve soil health and plant vigour (Peteu et al. 2010). The releasing behaviour of NAA hormone of chitosan nanoparticles was examined at various temperature and pH, and it was found that these hormones help slow release of fertilizers (Tao et al.

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Application of Green Synthesized Nanoparticles in Sustainable …

2012). To apply adequate quantity of micronutrients to plants, various nanoparticles have been tested. Under higher pH and calcareous soils, iron deficiency is a major problem for plants. It can be easily addressed by foliar spray of iron with the advantage of nanotechnology. It was assessed to check wheat growth/yield and grain quality by spraying of FeO nanoparticles. The five levels of FeO nanoparticles solution were used (0, 0.01%, 0.02%, 0.03% and 0.04%) to test the result on spike/1000 grain weight, biologic/grain yield and protein content of grains. It was found that there was an increase in these traits (Bakhtiari et al. 2015). From the study of Ghafariyan et al. (2013) found that the use of FeO nanoparticles as a source of Fe to solve iron deficiency in soybean. In black-eyed peas, iron nanoparticles resulted considerable increase in the number of pods per plant (by 47%), weight of 1000-seeds (by 7%), the iron content in leaves (by 34%) and chlorophyll content (by 10%) over controls by foliar application of 500 mg/litre (Delfani et al. 2014). Majority of the plants demands iron in soil for an optimal growth and development (Hoagland and Arnon 1950). Growth of mung bean was increased by application of manganese nanoparticles (Vigna radiata) and photosynthesis (Pradhan et al. 2013). For the agricultural sustainability, it is very much essential to adapt application of nanofertilizers in agriculture with a purpose to reduce nutrient losses and increase the yield by nutrient management. FeO nanoparticles sprayed on squash fruits have shown the highest content of protein, lipids and energy (Shebl et al. 2019). Micronutrients like Fe, Mg and Zn are the important for many plant growth including squash (Rui et al. 2016; Novillo et al. 2001; Bhattacharya et al. 2007; Bandyopadhyay et al. 2014). In mulberry (Morus alba L.), it was estimated that the requirement of Fe for growth and development can be met by applying two concentrations of Fe nanoparticles in a pot experiment (Soil and foliar application). These green synthesized nanoparticles showed significant effects on morpho-physiochemical parameters (Haydar et al. 2021).

12.4

277

Green Synthesized Nanopesticides

The positive effects application of Ag nanoparticles as pesticides to manage pests of crop plants has been well established. Biological methods are widely used for synthesis of Ag nanoparticles than physical/chemical methods. Since, silver nanoparticles synthesized through physical/ chemical methods are toxic and require the extreme conditions for synthesis. Silver nanoparticles synthesized from plants and plant products, fungi, bacteria on the other hand are much safer and economically viable and have been used to control disease causing microorganisms (Duhan et al. 2017). The antimicrobial activity of Ag nanoparticles is greatly be used in plant diseases management (Mishra et al. 2014). Photocatalytic activity of TiO2 has been greatly used for degradation of pesticide residues (Pelaez et al. 2012). Pathogen disinfection efficiency of TiO2 has a wide scope for applications in plant protection in sericulture industry where broadspectrum disinfectants have generally been used to protect both silkworm and host plants. It is high time to explore the use of nanomaterials in production of food and crop protection. Insect pests are causing huge loss during crop production and storage of its produce; therefore, nanoparticles have major roles in the management of crops pests and pathogens (Khot et al. 2012). The nanoencapsulated pesticide has slowreleasing properties with improved stability, solubility, specificity and permeability (Bhattacharyya et al. 2016). The dosage of pesticides and human beings exposure to nanoencapsulated pesticides formulation is greatly reduced thereby becomes ecofriendly crop protection (Nuruzzaman et al. 2016). In recent days, required chemicals can be developed into quality products by nanoencapsulation. Of late, few chemical companies promote nanopesticides for sale. Products from Syngenta and pesticides from BASF may fighting fit for nanoscale (Gouin 2004). In Raphanus sativus var. aegyptiacus, green synthesized silver nanoparticles of size range

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6–38 nm has been noticed. Reduced activity and death of snails were observed on exposure to 20% silver nanoparticles (Ali et al. 2015). Insect pest resistance is a new challenge arising from excessive use of synthetic agrochemicals. However, use of nanoparticles has the advantage of controlling the pest problem with least environmental hazards. It has been found that nanoparticles enhance the efficacy of pesticides insecticidal activity of garlic essential oil against red flour beetle (Tribolium castaneum) which was enhanced by polyethylene glycolcoated nanoparticles (Yang et al. 2009). The 80% efficacy of nanoformulation against adult T. castaneum was recorded due to the sustained and slow release of the active ingredient from the nanoparticles. The applications of various types of nanoparticles in the control of rice weevil and silkworm grasserie disease were studied (Goswami et al. 2010). Viral load was significantly decreased after feeding mulberry leaves treated with ethanolic suspension of hydrophobic alumino-silicate nanoparticle to silkworm (Rai and Ingle 2012). Significant death of Sitophilus oryzae L. and Rhyzopertha dominica was observed after 3 days of continuous exposure to wheat treated with alumina nanoparticles which showed insecticidal property of nanostructured alumina. Thus, insecticides available at market, inorganic alumina nanostructured products may give an economical and reliable option to control insect pests. The entomotoxicity of silica nanoparticles were tested against rice weevil Sitophilus oryzae and compared the efficacy with bulk-sized silica (Debnath et al. 2011) and found 90% mortality of rice weevil in unstructured silica nanoparticles, revealed silica nanoparticles effectiveness against insect pest. Slow and sustained release of nanopesticides permits proper assimilation of the chemical by the plants and is longlasting as compared to other agrochemicals (Shyla et al. 2014). In pest management, by considering the detrimental effects on environment and human health by usage of synthetic pesticides, it can be replaced with green synthesized nanoparticles.

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12.5

Nanofungicides and Bactericides

The Ag+ nanoparticles’ treatment in a lab study decreased the occurrence of fungal load in the adjacent soil (Ali et al. 2015). The experiment on management of wheat spot blotch disease causing pathogen (Bipolaris sorokiniana) has been carried out using spherical shaped silver nanoparticles (  10–20 nm in size range) and exhibited strong antifungal action against pathogen (Mishra et al. 2014). Better antimicrobial activity was exhibited by zinc oxide nanoparticles (Xie et al. 2011). The antimicrobial activity of zinc oxide nanoparticles from Moringa oleifera leaf extract was tested against bacteria and fungus. ZnO nanoparticles with spherical and hexagonal shape from Parthenium hysterophorus L. were synthesized. The size-dependent antifungal activity against plant fungal pathogens (Aspergillus flavus and Aspergillus niger) was explored by using zinc oxide nanoparticles. A highest zone of inhibition against Aspergillus niger and Aspergillus flavus was found in 27 ± 5 nm size zinc oxide nanoparticles. ZnO nanoparticles synthesized from parthenium leaf extract demonstrated to be ecofriendly and ideal antifungal agents (Rajiv et al. 2013). Zinc oxide nanoparticles were synthesized using leaves of Catharanthus roseus (L.) of spherical shape an average size of 23– 57 nm. Antibacterial activity of these nanoparticles against Pseudomonas aeruginosa (ATCC 15,442), Escherichia coli (ATCC 25,922), Bacillus thuringiensis (ATCC 10,792) and Staphylococcus aureus (ATCC 6538) was tested. B. thuringiensis showed the resistance to ZnO nanoparticles followed by E. coli while P. aeruginosa was found more susceptible. It was opined that antibacterial formulations of ZnO nanoparticles may be used against P. aeruginosa (Bhumi and Savithramma 2014). Plants and plant materials are the primary choice for researchers for green synthesis of TiO2 nanoparticles. TiO2 nanoparticles of spherical shape and clustered by using aqueous leaf extract

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Application of Green Synthesized Nanoparticles in Sustainable …

of Psidium guajava have been synthesized (Santhoshkumar et al. 2014). TiO2 nanoparticles were synthesized successfully with spherical clusters and quite polydispersed shape of size range from 36 to 68 nm by Eclipta prostrata leaf extract (Rajakumar et al. 2012). Green synthesis of nanoparticles offered potential and sustainable option compared to conventional synthesis of nanoparticles. From the past research, it was revealed that nanoparticles have great potential for antimicrobial and antiviral activities. In this chapter, the improvement in green synthesis of nanoparticles using natural sources and other relevant sources was focused. These nanoparticles exhibit varied chance to counteract life-threatening viral and other antimicrobial infections (Naikoo et al. 2021). An illustrative diagram of biological synthesis of nanoparticles is given in Fig. 12.1. Many studies showed antiviral properties of silver nanoparticles against the potato virus Y (Noha et al. 2018), yellow mosaic virus (Elbeshehy et al. 2015), sunhemp rosette virus (Jain and Kothari 2014) and tomato mosaic virus. These studies

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showed that silver nanoparticles stop replication of viral nucleic acid in the plant cell and also found stimulation of plant resistance and improved the release of reactive oxygen species (Jiang et al. 2014; Tripathi et al. 2017). In other similar kind of study, the synthesis of ZnO nanoparticles and their antiviral action contrary to potato virus Y and tobacco mosaic virus was reported (Cai et al. 2019). ZnO nanoparticles exhibited defensive and curative activity against tobacco mosaic virus (Abdelkhalek and Al-Askar 2020). In present days, green synthesis of nanoparticles has turned into a popular procedure because of their many antimicrobial properties (Zhu et al. 2019). The green synthesis of nanoparticles methodology can be determined. It has sole kind for the improvement of nanoparticles (Zhu et al. 2019; Anastas and Warner 1998; El-Batal et al. 2018). Typically, smaller size of nanoparticles develops improved antimicrobial activities (Mosallam et al. 2018). Additionally, the dimension of the nanoparticles should be under 50 nm to become an efficient antimicrobial factor (Wong et al. 2020; Ashour et al. 2018).

Fig. 12.1 An illustrative diagram of green synthesis of nanoparticles using beneficial fungus/bacteria, plant and plant materials. Created using Biorender

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Broad antibacterial features of silver nanoparticles have attracted the attention of many scientists in different fields. The green synthesized TiO2 nanoparticles exhibited significant antifungal activity against Aspergillus niger (Wang et al. 2000). In eggplants, growth of Verticillium dahliae was significantly suppressed by silver nanoparticles (Jebril et al. 2020). Synthesized spiral-shaped CuO and FeO nanoparticles using Euphobia heliscopia leaves extract through microwave-assisted method. These oxide nanoparticles were used to study the fungal inhibition. It was revealed that FeO was superior in antifungal properties against Cladosporium herbarum as compared to CuO nanoparticles (Henam et al. 2019). Ag+ nanoparticles were synthesized using peanut shell extract and studied the antifungal activities. These nanoparticles supressed the growth of Phytophthora capsici and Phytophthora infestans (Velmurugan et al. 2015). Green synthesis of Ag+ nanoparticles was performed by using xylan from corn cobs which contains nanoxylan. These nanoparticles were tested against C. parapsilosis (MIC = 7.5 mg/mL), Cryptococcus neoformans (MIC = 7.5 mg/mL) and Candida albicans (MIC = 7.5 mg/mL) for antifungal activities and found improved antifungal activities of nanoparticles with nanoxylan compared to silver nitrate (Silva Viana et al. 2020). Crop production is severely affected by many fungal diseases which leads to greater loss. Application of commercially available fungicides causes detrimental effects on plants and hazardous to humans and environment. These problems may be addressed by the advent of nanotechnology which plays a very important role in agriculture at present. Antifungal activity of nanoparticles of Ag+ (20–80 nm), TiO2 (85– 100 nm) and ZnO (35–45 nm) was tested against Macrophomina phaseolina. The maximum antifungal activity was noticed in Ag+ nanoparticles at low concentrations as compared to ZnO and TiO2 nanoparticles (Shyla et al. 2014). Silicananoparticles may serve as an alternative potent antifungal agent against phytopathogens (Suriyaprabha et al. 2014).

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Silver nanoparticles have much greater antifungal properties as compared to other metals particles because silver ions cause the inactivation of cell-wall thiol groups of fungus leading to disruption of transmembrane, energy metabolism and electron transport chain. And also other mechanisms like fungal DNA mutations, dissociation of the enzyme complexes that are essential for the respiratory chain, reduced membrane permeability and cell lysis (Velmurugan et al. 2009). However, the particle size plays an important role in the efficacy of silver nanoparticles; it decreases with increasing particle size and also the shape of the particles observed key role; it was found that truncated triangular particle shape resulted greater “cidal” effect than spherical and rod-shaped nanoparticles (Pal et al. 2007; Panáček et al. 2006). Antifungal activities of Ag nanoparticles for disease management in plant were used (Karimi et al. 2012). Silver nanoparticle solution will perform as an outstanding fungicide due to good sticking on fungal and bacterial cell surface (Kim et al. 2009). Nanoparticles of other metals have also been used for controlling diseases as the disease symptoms in cotton seedling under greenhouse conditions were considerably reduced by using ZnONPs. Significant increase in antifungal effect by the new formulation of a trichogenic ZnONPs was also observed. Utilization of Trichoderma harzianum—a biocontrol agent for the synthesis of a medium-scale zinc nanoparticles—could be a safe strategy and further it can be utilized in cotton for fungal disease control (Zaki et al. 2021). Magnesium dioxide nanoparticles were reported to advance the height and weight of tobacco plants. In tobacco plants, bacterial wilt disease severity caused by Ralstonia solanacearum was also greatly reduced (Cai et al. 2018).

12.6

Nanoherbicides

Weeds are a major problem in crop production, as it competes with crop plants for the nutrients and largely reduces the crop yield. Weed

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Application of Green Synthesized Nanoparticles in Sustainable …

management by following old methods is time consuming. At present, great numbers of weedicides are available in the market, but they damage crop plants also to a certain extent while killing the weeds. Herbicide also causes soil pollution and reduces soil fertility. These problems of commercially available herbicides can be mitigated by the usage of nanoherbicides which can control weeds in an environment friendly approach, without forming any harmful residues in soil and environment (Pérez-de-Luque an Rubiales 2009). Herbicides can encapsulate with polymeric nanoparticles by which environmental safety can be achieved (Kumar et al. 2015). Weeds also establish resistance against herbicides under repeated use of similar herbicide for longer duration. Efficiency of nanozero-valentiron wasted to detoxify atrazine from atrazinepolluted soil and water (Satapanajaru et al. 2008). A nanoparticle with unique properties for target specific delivery of herbicide to roots of weeds was developed. These nanoparticles enter into the roots of weeds leads to death of plants by translocating to weed plant cells and inhibit glycolysis metabolic pathways (Nair et al. 2010; Ali et al. 2014). Toxicity of polynanocapsules containing ametryn and atrazine was tested and found lower toxicity to the algae and higher toxicity to the microcrustacean as compared to the herbicides alone (Clemente et al. 2014). Zinc oxide nanoparticles induce reactive oxygen species production, leading to cell death when the antioxidative capacity of the cell is exceeded (Xia et al. 2006; Ryter et al. 2007; Long et al. 2006; Lovrić et al. 2005; Lewinski et al. 2008). In wheat, induction of free radical was observed upon application of zinc nanoparticles, resulting in enhanced malondialdehyde and lower levels of reduced glutathione (Panda et al. 2003) and reduced chlorophyll contents (Aarti et al. 2006).

12.7

281

Role of Green Synthesized Nanoparticles in Abiotic Stress Tolerance

In the worldwide environment, majority of the cultivable crops including mulberry undergo abiotic stresses like drought, heat, salinity, alkalinity and low temperature, and these stresses have risen across the globe (Vijayan et al. 2022). However, nanoparticles have greater solubility, reactivity and surface tension than bulk materials. As a result, the damage caused by abiotic stress can be addressed to a great extent to achieve sustainable agriculture globally. The abiotic stress defence by the different forms of silver nanoparticles in agriculture was investigated, and these characters of nanoparticles are gaining wide popularity. The important features of these AgNPs include enhancing crop stress tolerance by overcoming nutrient deficiencies, increasing enzymatic activity, and helping in the adhesion of plant growth-promoting rhizobacteria (PGPR) to roots of plants under abiotic stresses. Under unfavourable environmental conditions, these preliminary results on nanoparticles were encouraging to enhance crop production. Soil salinity is the very important abiotic stress in plants, which causes a severe impact on crop productivity (Alharbi et al. 2021). Due to salinization and sodification of soil, arable land nearly of 1.5 million hectares becomes uncultivable in each year (Abou-Zeid and Ismail 2018). In this condition, a new approach to decrease the harmful effects of the stresses on plants is urgently needed. By the application AgNPs, it can substantially enhance chloride, sodium and potassium of plants. It was noticed that silver nanoparticles have more stable in water with low salinity (Banan et al. 2020). Plant growth was affected due to higher salinity in soil (Sagghatol-Islami 2010).

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Another major abiotic stress is low water availability which had a higher negative impact on crop production. Lack of sufficient water in the soil hampers transport, osmo-regulation, and single-cell expansion through cellular membranes to achieve higher crop production (Iwuala et al. 2022). This kind of stress can be solved by partially regulating the water permeability. Development of drought-tolerant varieties to tackle drought stress is an easy way to ensure food security. Though many studies have demonstrated that silver nanoparticles have successfully decreased salinity stress (Abou-Zeid and Ismail 2018; Almutairi 2016), few studies could be conducted on use of silver nanoparticles to address drought stress in plants. In lentil crop, the presence of nanoparticles facilitated to keep water balance under drought condition by enhancing the growth and yield attributes (Hojjat and Hojjat 2016). Low temperature tolerance of plants was observed in many crops through application of silver nanoparticles (Prazak et al. 2020). Application of low levels of silver nanoparticles on green beans improved the plant height, fresh and dry weight and photosynthesis. And also rapid and identical germination of green beans seeds under lab and field conditions observed (Prazak et al. 2020). The green synthesized silver nanoparticles reduced the damaging effects of heat stress in wheat (Iqbal et al. 2019). Silver nanoparticles decreased the malondialdehyde concentration, hydrogen peroxide and improved antioxidant defence in the wheat plants during high-temperature conditions. Silver nanoparticles reduced oxidative stress in plants by their abnormal plasmon-resonance optical scattering character against high temperature. Thus, it could be concluded that the application of green synthesized silver nanoparticles is a useful method to increase the productivity in agriculture and also to exploit the large areas that are affected by high temperature and salinity across the globe.

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12.8

Impact Assessment on Silkworm

Sericulture is the art and science of silkworms rearing through feeding mulberry leaves to get the cocoons from which silk is produced. It is a sector of agriculture, and cottage industry deals with the mass rearing of silkworms (Ganga and Chetty 2019). The silk industry was affected by a serious disease caused by Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), and till date, no control measures could be developed once the disease affects the silkworm. A recent study revealed that silkworm immunity could be enhanced against the damage of cytoplasmic polyhedrosis virus through pretreatment of silkworm larvae using titanium dioxide nanoparticles. The resistance of silkworm to BmCPV was enhanced by nanotitanium dioxide pretreatment which could inhibit the proliferation of BmCPV in the midgut of silkworm, trigger JAK/STAT and PI3K-AKT immune signalling pathways and upregulate the expression of important immune genes, thereby as immunity of silkworm is improved. Similarly, the growth and development of Drosophila (egg to adult stage) were affected by dietary uptake of carbon black and single-walled nanotubes as it affected the locomotor function of these flies (Liu et al. 2009). The genotoxicity in the wing spot assay of fruit flies was stimulated by low concentrations (0.1, 1, 5 and 10 mM) of silver nanoparticles through somatic recombination (Demir et al. 2011). An exposure of the fruit fly larvae to titanium nanoparticles affects the cytotoxicity in the imaginal and midgut disc tissues (Carmona et al. 2015). It was also found that titanium nanoparticles cause more DNA damage than the DNA damage caused by bulk TiO2. Fruit flies exposed to 20 mg/L of silver nanoparticles were unable to complete their life cycle (Panacek et al. 2011). Increased production levels of ROS among the silkworms fed with 100 ppm of silver nanoparticles were noticed, which caused DNA

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damage, cell apoptosis and necrosis (Foldbjerget al. 2011). The concentrations of silver nanoparticles greater than 800 mg/L improve the growth rate of silkworms, in addition to silkworm death (Meng et al. 2017). Higher concentrations of silver nanoparticles positively reflected on the bodyweight of the silkworm and also adverse effects on tissues on primary organs (Nouara et al. 2018). The harmful effects of three different concentrations (100, 200 and 400 mg/L) of silver nanoparticles on the silkworm midgut tissues were studied by Chen et al. (2019). The increase in concentrations of silver nanoparticles (400 mg/L) affects the development and causes damage to the tissues of the silkworms. It is reported that feeding of titanium nanoparticles could ameliorate the ill effects of phoxim residue. It was also found that phoxim residue hampers functioning of fibroin and sericin genes (ser2 and ser3), due to vacuolation in the silk gland leading to the decreased cocooning rate (Li et al.2014). The application of silver nanoparticles synthesized with Spirulina platensis was proficient in controlling a Bombyx mori nuclear polyhedrosis virus (BmNPV) infection (Govindaraju et al. 2011). Introducing silver nanoparticles in the haemolymph of the silkworm reports that 3.9 lg was lethal to the haemocytes than groups exposed to 0.39 and 0.039 lg of silver nanoparticles (Xing et al. 2016). The survival rate of the silkworm increased from 22 to 67% when compared to untreated control group due to the efficacy in using AgNPC to reduce BmNPV infection to silkworms. Silkworms feeding with nanoparticles like titanium dioxide, copper, carbon nanotubes (CNTs) and grapheme were documented to develop the mechanical characters and secondary structures of silk (Shearer et al. 2014; Wang et al. 2015). The production of fundamentally modified silk fibres was carried out by feeding silkworms with a titanium oxide in artificial diet with four different concentrations (1, 2, 3, and 4%) (Cai et al. 2015). The ultraviolet resistance of the silk fibre and mechanical characters were significantly enhanced by titanium oxide than control. Higher concentrations of titanium oxide decrease

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the breaking strength and ultraviolet resistance. The application of one percent titanium oxide nanoparticles conforms the transition of silk fibroin from a random a-helix to b-sheet, which interprets to limited crystallization effect. Gold nanoparticles were synthesized by using onions (Allium cepa L.), confirmed the identity by UV–Vis spectrophotometer, XRD, FTIR, SEM, TEM and AFM and also tested on silkworm physiology to study the function of green gold nanomaterials. From the study, it was clearly indicated that they have a tremendous effect on enhancement of silk proteins and thus the enhancement of the cocoon weight in silkworms (Patil et al. 2017). To evaluate the toxic effects of silver nanoparticles on silkworm (B. mori), leaf extract of Morus alba (mulberry) was used to prepare bionanoparticles. Hence, B. mori was a monophagous insect, and the exact lethal level can be identified using the sole food. Observations revealed that maximum larval weight was in 1 ppm treatment and pupal weight was highest at 100 ppm concentration. And also cocoon and shell weight were quite high in all the treated levels than control lots. The protein profile of treated haemolymph envisaged the expression of a 20 kDa protein identified as glutathione-Stransferase, when subjected to MALDI-TOF analysis (Pandiarajan et al. 2016). A list of nanoparticles and their specific usage in mulberry and silkworm was given in Table 12.1.

12.9

Future Perspectives and Conclusion

As detailed in the chapter, many nanoparticles may be created using different biological systems as green synthesis. Out of several nanoparticles listed and documented in the chapter, the researchers gave utmost importance to the novel bioinspired noble metals. It is because of various advantages with many biological properties like antimicrobial and antiviral properties of green synthesized nanoparticles. More studies need to be taken up in mulberry production as very limited number of studies were noticed.

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Table 12.1 Details of nanoparticles and their applications in sericulture Crop/Animal

Source/Types of nanoparticles

Mulberry

Nanofungicides or nanobactericides

AgNPs

Advantages

References

Rates of bacterial pathogens growth decreased with the increase in AgNP concentrations

Some et al. (2019)

The antimicrobial activity studies

Roy et al. (2019)

Different parts of mulberry species such as fruits and leaves have been shown to be rich in phytochemicals, particularly the phenolic compounds and flavonoids

Prabhu and Poulose (2012)

Enhance the larval and pupal growth and quantity of silk production than control

Prabu et al. (2011)

Abiotic factors

AgNPs

Various mulberry species have been shown to be rich in these bioactive compounds and have high antioxidant capacity

Bae and Suh (2007)

Nanoparticles extraction using mulberry leaf

AgNPs

Used for synthesis of different nanomaterials such as gold, silver and iron nanoparticle

Liem et al. (2019)

Act as reducing and stabilizing agent and effective against Vibrio cholera (gramnegative) and Staphylococcus aureus (grampositive) pathogens

Adavallan and Krishnakumar (2014)

Antibacterial effects against the human pathogens using mulberry leaf extract gold nanoparticles Silkworm

Abiotic and biotic factors

Green NPs

Spirulina

AgNP

TiO2

Control of entomopathogenic fungi and their effectiveness on the larval growth, survivability and mass production of silk

Pandey and Kachhwaha (2021)

Provides nutrition supplement

Amala and Ranjith (2011)

Beneficially influence the energy and economic parameters of B. mori, which can be exploited in commercial cocoon production

Dharanipriya (2019)

Protection against heat stressinduced apoptosis

Sakano et al. (2006)

Low concentrations of TiO2 NPs were effective for

Li et al. (2016) (continued)

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Application of Green Synthesized Nanoparticles in Sustainable …

285

Table 12.1 (continued) Crop/Animal

Source/Types of nanoparticles

Advantages

References

feed efficiency, weight gains and cocoon mass, whereas higher concentrations had an inhibitory effect on the growth rate AgNP

(a) Promoted the growth and cocoon weights of B. mori (b) Changes of B. mori body weights, survival rates and proteomic differences (c) Beneficial effects on the survivability, body weights of the Bombyx mori L. larvae, pupae, cocoons and shells weights via enhancing the feed efficacy

Some et al. (2019)

TiO2 NP

Promote silkworm growth and improve its resistance to organophosphate pesticides

Xu et al. (2015)

AgNP

(a) Active against several types of viruses, even against pebrine as well (b) More effective against CPV than that of NPV

Li et al. (2013)

Therefore, it is required to explore the possible advantages of the nanotechnology in mulberry production for augmenting silk production in India. Additionally, the research in this field should be continuous while addressing the problems of diseases and pests. It is well documented that there may be a chances of getting microbial resistance over time, and also these nanoparticles may become ineffective against certain strains of microbes. Therefore, further research requires in this field for targeting resistant strains of the pathogens using nanoparticles. Furthermore, the outcome of the research on nanoparticles varies due to the chemical components present in plant extracts may vary across the world. Thus, it is a disadvantage which will hamper in reaching homogeny for generating green synthesized nanoparticles. Nevertheless, a specific component in the plant could be studied,

and similar results can be obtained. The term “bioinspiration” is allocated to the practice that uses biological plan for advantageous technologies, viz. antifungal, antibacterial and antiviral activities. There was an incredible dedication towards generation of green synthesis materials from the past few decades. Current chapter revealed the new advancements in the green synthesis of nanoparticles using various plants and microbes as source material for application as nanofertilizers, nanopesticides, nanofungicides, nanoherbicides and nanomaterials for abiotic stress management in the crop production. It also detailed the impact on silkworm and silk production. Hence, the green synthesized nanoparticles play a vital role in future to fulfil the requirements of plant protection and production.

286

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Future Perspectives of Mulberry Genomic Research

13

Belaghihalli N. Gnanesh , Raju Mondal, and Kunjupillai Vijayan

13.1

Introduction

Post-green revolutionary challenges, like global warming, erratic climate changes, and the evolution of new pests and insects, have immensely affected mulberry productivity and the sustainability of sericulture across the world. It is presumed that one of the major challenges for the sustainability of sericulture would be to sustain an economically viable leaf production using the limited resources (Jain et al. 2022). Hence, it is necessary that a complex genetic adaptation of mulberry under drought conditions is considered to be a future goal in sericulture research. Very little effort has been made to understand the genetics of complex traits and their interactions with environment (Checker et al. 2012). For instance, to develop a mulberry variety with high

B. N. Gnanesh (&) Molecular Biology Laboratory-1, Central Sericultural Research and Training Institute, Mysuru, Karnataka 570008, India e-mail: [email protected] R. Mondal Mulberry Tissue Culture Lab, Central Sericultural Germplasm Resources Centre (CSGRC), Ministry of Textile, Government of India, Hosur, Tamil Nadu 635109, India K. Vijayan International Sericultural Commission, Central Silk Board Complex, BTM Layout, Madiwala, Bangalore, Karnataka 560068, India

water use efficiency (WUE), it is essential to find accessions with traits that are associated with better water use as parents to be used in strategic trait-based breeding. The process of identification of genotypes with high WUE is constrained by the complexity of polygenic inheritance, as well as their interactions with environments (Mondal et al. 2023a). Further, it is evident that variety development through conventional breeding is slow in mulberry due to (i) the perennial growth habits coupled with a long juvenile period and (ii) insufficient molecular markers to use for marker-assisted selection breeding (Mathithumilan et al. 2013; Pinto et al. 2018). Additionally, lack of trait-specific mutants, homozygous, and overexpressed lines as well as low-cost mining of publicly available resources (to elucidate comprehensive interactions, co-expression, and metabolic/biosynthetic grid with the integration of advanced artificial intelligence) limit understanding of complex phenomena at a multi-dimensional level. Hence, genomic research could be an excellent platform and most efficient system for a better understanding of the complex genetic mechanisms in mulberry. Highly precise and targeted breeding methodologies like genome editing toolkits can be adopted for quick and effective variety development (Savadi et al. 2021). Toward this end, Jain et al. (2022) reported several gene ontology (GO) terms such as cellular functions related to abiotic stress, transport, developmental, and reproduction process by genome sequencing of M. indica (Kanva-2). Recent studies also

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 B. N. Gnanesh and K. Vijayan (eds.), The Mulberry Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-031-28478-6_13

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unraveled several transcription factors (TFs) including basic helix–loop–helix (bHLH), myeloblastosis (MYB), APETALA2-ethyleneresponsive element binding proteins (AP2EREBP), Lateral Organ Boundaries (LOB), and NAC (NAM, no apical meristem, Petunia— ATAF1–2, Arabidopsis thaliana activating factor —CUC2, cup-shaped cotyledon, Arabidopsis) have significant contribution in a tissue-specific manner. In this chapter, efforts were made to summarize the recent developments in genomic research of mulberry along with the future perspectives of genome research to aid breeding for developing tailor-made varieties in mulberry.

13.2

Improvement of Mulberry Through Genomic Research

Plant genome sequencing has several applications as—firstly, whole-genome sequencing (WGS) serves as an anchor genome for understanding phylogenetic relationships, evolutionary consequences, adaptability, etc. Secondly, a complete genome sequence can serve as the foundation for a comprehensive study of complex traits. Thirdly, developing near-one-plant sequence information can also be useful for relative crop/species identifications. Fourthly, good quality annotation genome sequence provides functional characterization (EST development, FISH/GISH study, etc.). Fifthly, genome editing methods like transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats associated (Cas) can produce desirable changes in the genome. Sixthly, genome sequence can be used in DNA bar coding, which played a crucial role in molecular taxon. Lastly, WGS exposed the mutational landscape (which generates a huge number of SNP markers) for genome-wide association studies (GWAS).

13.2.1 Next-Generation Sequencing and Genotyping In 2013, the first nuclear genome sequence with a tissue-specific transcriptional profile of natural haploid Morus notabilis was reported by He et al. (2013), and in 2020, chromosomal-level genome information was reported in diploid Morus alba (Jiao et al. 2020). Recently, Jain et al. (2022) reported a high-quality genome sequence complemented with RNA-seq data of Indian cultivar Kanava-2 belonging to Morus indica, which offers a source for functional and translational genomics. In this connection, two important repository databases MorusDB (Li et al. 2014a) and MindGP (Jain et al. 2022) will act as the prime source of molecular biology-related work in mulberry. Additionally, 21 accessions of mulberry were re-sequenced which in turn revealed *2.5 million SNPs and *0.2 million InDels (insertions/deletions). Feasible and less expensive allele-specific markers development is a critical step for the effective use of highthroughput MAS in any breeding program (Gnanesh et al. 2013). The huge availability of SNPs can be exploited in mulberry breeding programs by using uniplex or multiplex genotyping platforms that combine different chemistries, detection techniques, and reaction compositions (Semagn et al. 2014). In addition to fixed SNP genotyping arrays, there are many high-throughput technologies that are accessible for quick screening of SNP markers. Competitive allele-specific PCR (KASP) is the most used uniplex SNP genotyping assay globally (Thomson 2014). For conducting SNP assays in the laboratory, the best method is to use PCR-based fluorescently labeled SNP markers like KASP. It could be run as single marker at a time using real-time PCR or examined using fluorescent plate readers.

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Future Perspectives of Mulberry Genomic Research

13.2.2 Linkage Mapping and Genome-Wide Association Studies (GWAS) Despite the accessibility of the three Morus genome whole sequences, there has not been any significant progress in genome-assisted breeding of mulberry. A genetic linkage map was first generated with 50 F1 full-sib progeny by Venkateswarlu et al. (2006) in mulberry. However, due to the low density of markers, a comprehensive linkage map with high-density markers is needed for the precise identification of QTLs to use for marker-assisted breeding (MAB). Fasttracking genetic enhancement is possible only when robust DNA-based molecular markers governing important traits associated with specific traits such as leaf yield, biotic and abiotic stress tolerance, and nutrient use efficiency are to be discovered (Vijayan et al. 2022a, b). The recent availability of a large number of highquality SNP can be made used to construct such dense linkage map in the recent future for faster development of varieties with specific traits. Genome-wide association studies (GWAS) with the aid of next-generation sequencing (NGS) is a powerful tool for understanding the genetic basis of yield and drought at the allelic level (Esvelt Klos et al. 2016). GWAS makes use of historic recombination and is as powerful as we can identify10–100 loci linked with a particulate trait and the loci comprised of hundreds of single-nucleotide polymorphisms (SNPs) (McMullen et al. 2009). Interestingly, once the association between SNPs and superior traits was identified, desired alleles can be used in a broader application to anticipate agronomic traits. Unfortunately, in mulberry very limited studies have been carried out using GWAS. The largescale availability of SNP markers can be employed in association studies with complex polygenic traits for increasing allelic diversity and mapping resolution, thus providing an enhanced resolution compared to biparental mapping with a limited number of markers (Vijayan and Gnanesh 2022). Whatever, we will never get benefited from genomic research, until

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we will capitalize on the benefits of gene regulation and expression. Hence, in addition to structural genomics, information on gene expression atlas, epigenome maps, proteome maps, and metabolome maps has been developed for a few crop species.

13.2.3 Genomic Research for Understanding Complex Traits in Mulberry Breeders mostly target complex traits like nitrogen use efficiency (NUE) for improvement of quality and quantity of yield, because it is the most essential nutrient for plant growth and development. Excess fertilizer use is becoming more common in order to increase productivity, leading to decreased nitrogen acquisition, affecting soil health as well as imposing drastic negative impacts on the environment (Sun et al. 2020). Interestingly, estimation suggests that a *1% augment in crop NUE can annually save $1.1 billion (Kant et al. 2011; Mondal et al. 2021). Hence, for achieving greater sericulture sustainability, the development of mulberry with more efficient nitrogen use efficiency is an important research challenge. A recent efficient nutrient management strategy suggested that increased NUE can maximize mulberry production (Rajendran et al. 2021; Mahesh et al. 2021). However, the genetic/molecular mechanism including co-expression network/interaction cis/ trans-elements mediated regulation is still to be understood.

13.2.4 Pangenome A “pangenome” is the collective whole-genome sequence of many individuals representing the genetic diversity of the species (Wang et al. 2022). The first crop pangenome was reported by Li et al. (2014b) in soybean (Glycine soja). Low and cost-effective next-generation sequencing technologies have resulted in multiple developments of pangenomes in Brassica napus L.,

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Glycine max (L.) Merr., Oryza sativa L., Triticum aestivum L., Cajanus cajan, Helianthus annuus, Sorghum bicolor, Zea mays, and many more crops (Zanini et al. 2022). Pangenome allows detection of precise genetic variation existing in a particular species by uniting genomic data with many accessions as compared to a single reference genome. Pangenome also provides beneficial resources for studying evolution and functional genomics. Besides SNPs and InDels, other structural variations (SVs) including presence or absence variations (PAVs), copy number variations (CNVs), inversions, transversions, and translocations contribute to the genetic diversity in plants (Jayakodi et al. 2021; Savadi et al. 2021). Hence, having an extensive pangenome is very important in mulberry. Currently, in mulberry, we do not have any pangenomes, but with the availability of three genomes of Morus species (M. indica, M. alba, and M. notabilis) and a lot of resequencing data, we can plan for a pangenome. As and when the genomic data in mulberry continue to surge, we can develop high-density SNP maps and identification of genetic variations in mulberry genomes and their associations with major traits facilitating genome-assisted breeding (GAB).

13.3

Functional Genomics

Mulberry is a tree plant, and because of its long life span, it is mostly cultivated through vegetative propagation for commercial purposes (Mondal et al. 2023b). Therefore, in comparison with other model systems like arabidopsis, rice, etc., functional study and characterization of genes in and phenotypic associations are quite difficult. Nonetheless, for a basic understanding of the genetic architecture of complex traits, characterization of gene family could be understood through in silico gene expression, cis-element mediated gene regulation, miRNA mediated posttranscriptional regulation, co-expression network analysis, and prediction of protein–protein interaction, etc.; in this concern, expression data stress of different species like M. indica, M. laevigata, M. notabilis, and M. serrata

was available in different databases like MindGP (http://tgsbl.jnu.ac.in/MindGP; Jain et al. 2022) and MorusDB (https://morus.swu.edu.cn; Li et al. 2014a). Additionally, MindGPMorusDB is also useful for comprehensive analysis of tissuespecific expression including leaf, root, stem, winter bud (DB), male inflorescence (MIN), female inflorescence (FINF), and fruit. Simultaneously understanding transcriptional, posttranscriptional, translational regulation mediated through CREs, miRNA, interaction with other proteins (PPI) can also be performed using a different database. Database like PlantRegMap (http://plantregmap.gao-lab.org; Tian et al. 2020), PlantCARE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html; Lescot et al. 2002), and STRING (https://string-db.org) is important to predict functional aspect of genes. Although, the major limitation to understanding the role of genes is due to the unavailability of a transcription library in different stress conditions. Hope in the future using the present repository of information mulberry crop improvement will be initiated.

13.4

Gene-Editing Technologies (CRISPR/Cas9)

Regulation in the guidelines made by the Government of India will now permit the use of genome-edited organisms/plants devoid of “foreign” genes. As a result, the gene-editing technology in India will speed up and lead to the development of breeding new crop varieties resistant to disease and drought tolerance. Among the three classes of gene editing, Sitedirected Nuclease (SDN1, SDN2, and SDN3), only two SDN1 and SDN2 are exempted from biosafety assessment which is free from exogenous introduced DNA. The SDN1 and SDN2 involve “knocking off” or “overexpressing” certain traits without any insertion of a gene, which will be protected by the new changes, whereas the SDN2 involving foreign genes will be considered a GMO. The most common technology adopted widely in genome editing is CRISPRCas9 (clustered regularly interspaced short

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Future Perspectives of Mulberry Genomic Research

palindromic repeats)-Cas9 (CRISPR-associated protein), and it has been used in more than 20 crops to modify several traits such as yield and to develop resistance against abiotic/biotic stresses (Fiaz et al. 2009; Ahmad et al. 2020; Tyagi et al. 2021). Besides genome editing tool can revolutionize the development of perennial tree crops like mulberry (Chen et al. 2020). Recently, base editing is an emerging efficient tool for highly precise gene-editing systems (Bharat et al. 2020), and in mulberry, with all the available genomic resources these tools can be exploited for breeding resistant/tolerant varieties against biotic and abiotic stresses (Vijayan et al. 2022a). Thus, the recent developments in mulberry genome research show that in the coming days it would be possible to develop well-adapted, high yielding, and robust varieties to sustain a vibrant silk industry to meet the burgeoning demands of the world.

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