Transgenic Technology Based Value Addition in Plant Biotechnology [1 ed.] 0128186321, 9780128186329

Table of contents :
Cover
Transgenic Technology Based Value Addition in Plant Biotechnology
Copyright
Contents
List of Contributors
Preface
one Bioprospecting of biodiversity for improvement of agronomic traits in plants
1.1 Salinity
1.2 Drought
1.3 Low temperature
1.4 Quantitative trait locus–based analysis of traits
1.5 Disease tolerance
Acknowledgment
References
Further reading
two Plant tissue culture: agriculture and industrial applications
2.1 Introduction
2.2 Micropropagation as a multiplication method
2.2.1 Stage 0: Preparation of donor plant
2.2.2 Stage I: Initiation stage
2.2.3 Stage II: Multiplication stage
2.2.4 Stage III: Rooting stage
2.2.5 Stage IV: Acclimatization stage
2.3 Organ cultures
2.4 Somatic embryogenesis and synthetic seeds
2.5 Haploid development via tissue culture
2.6 Pathogen-free plant propagation
2.7 Tissue culture and plant breeding
2.8 Plant tissue culture and development of transgenic plants
2.9 Somaclonal variation and its importance in plant improvement
2.10 Protoplast culture and somatic hybridization
2.11 Elicitation for enrichment of phytocompounds
2.12 Precursor addition
2.13 Hairy root culture and genetic manipulation
2.14 Endophytes and secondary metabolites
2.15 Bioreactor scaling
2.16 Immobilization scaling
2.17 In vitro germplasm storage
2.18 Conclusion and future perspective
References
Three Genome editing technologies for value-added traits in plants
3.1 Introduction
3.2 Techniques of genome editing
3.2.1 Zinc-finger nucleases
3.2.2 Transcription activator-like effector nucleases
3.2.3 Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR associated)
3.3 Application of genome editing systems
3.3.1 Multiplexing and trait stacking
3.3.2 High-throughput mutant libraries
3.3.3 Gene regulation
3.3.4 Targeted structural changes in crop species
3.4 Conclusion and future perspectives
References
four Bioinformatic tools to understand structure and function of plant proteins
4.1 Introduction
4.2 In silico structural and functional characterization proteins
4.3 Sequence-based approach
4.3.1 Biophysical characterization of proteins
4.3.2 Structure prediction
4.3.2.1 Secondary structure prediction
4.3.2.2 Tertiary structure prediction
4.3.2.3 Model validation and evaluation
4.4 Function prediction
4.4.1 Fold recognition/assignment
4.4.2 Structure-based function prediction
4.4.3 Active site prediction
4.5 Summary
References
five Transgenic technology for efficient abiotic stress tolerance in plants
5.1 Introduction
5.2 Transgenic approaches for engineering heat and cold tolerance in plants
5.3 Transgenic approaches for engineering salinity stress tolerance in plants
5.4 Transgenic approaches for engineering drought stress tolerance in plants
5.5 Transgenic approaches for increased flooding stress tolerance in plants
5.6 Improving plant tolerance to nutrient deficiency through genetic engineering
5.7 Improving plant tolerance to heavy metal stress tolerance through transgenic approaches
5.8 Conclusion
5.9 Future prospects
Acknowledgments
References
six Transgenic technologies for efficient insect pest management in crop plants
6.1 Introduction
6.2 Bt genes
6.2.1 Bt strains and toxins
6.2.2 Applications
6.3 First-generation genome editing technology
6.3.1 RNA interference
6.3.2 RNAi pathways and mechanism
6.3.3 Oral delivery method of dsRNA
6.3.3.1 Sprayable RNA interference approach
6.3.3.2 Nanoparticles-coated RNAi
6.3.4 Plant-mediated RNAi
6.4 Second-generation genome editing technology
6.5 CRISPR against insects
6.6 Nematode resistance in crop plants
6.7 Conclusions
Acknowledgment
References
seven Transgenic plants with improved nutrient use efficiency
7.1 Nitrogen
7.1.1 Nitrogen use efficiency
7.1.2 Transgenic crops with elevated nitrogen use efficiency
7.2 Phosphorus
7.2.1 Phosphorus utilization efficiency
7.2.2 Transgenic with elevated phosphorus utilization efficiency
7.3 Sulfur
7.3.1 Transgenic with elevated SUE
References
eight Genome editing of staple crop plants to combat iron deficiency
8.1 Introduction
8.2 Iron uptake and transport
8.2.1 Root uptake: iron uptake Strategy I and Strategy II
8.2.2 Chelators and long-distance transport of iron
8.2.3 Iron storage and vacuole sequestration
8.3 Genetic engineering to improve iron content in crops
8.3.1 Enhancing iron storage
8.3.2 Increasing iron translocation
8.3.3 Improving iron uptake
8.3.4 Multigene expression
8.4 Conclusion
References
nine Transgenic technology to improve therapeutic efficacy of medicinal plants
9.1 History of medicinal plants and natural products
9.2 Natural products: biosynthesis and classification
9.2.1 Terpenes
9.2.2 Alkaloids
9.2.3 Phenolics
9.3 Use of medicinal plants and secondary metabolites in traditional and modern medicine
9.4 Technologies for enhancement of secondary metabolites
9.4.1 Elicitors
9.4.1.1 Abiotic elicitors
9.4.1.2 Biotic elicitors
9.4.2 Homologous overexpression of therapeutic molecule/secondary metabolite biosynthesis key genes
9.4.3 Ectopic expression of genes to produce therapeutic molecule/secondary metabolite
9.4.4 Role of miRNAs in increasing the production of secondary metabolites
9.4.5 Artificial miRNAs for secondary metabolites enhancement
9.4.6 Regulating the expression of transcription factors
9.4.7 Regulating the endogenous levels of phytohormones involved in terpenoid biosynthesis
9.4.8 Regulating interrelated primary metabolic pathways
9.5 New approaches of engineering plant metabolic pathways to enhance secondary metabolites
References
ten Application of transgenic technologies in biofuel production through photosynthetic chassis—new paradigms from gene min...
10.1 Introduction
10.2 Metabolic engineering and synthetic biology
10.3 Improving photosynthesis
10.4 Formation of essential products via photosynthetic chassis
10.4.1 Sugars
10.4.2 Lipids
10.5 Terpenes
10.6 Muconic acid
10.7 Gene mining to genome editing
10.8 Challenges and future opportunities
Acknowledgments
References
eleven Genetic engineering of horticultural crops contributes to the improvement of crop nutritional quality and shelf life
11.1 Introduction
11.2 Conventional strategies to prolong the shelf life
11.3 The metabolic basis underlying fruit ripening and shelf life
11.4 Metabolic alterations incorporating the increased shelf life
11.5 Transgenic technology as a promising tool for crop nutritional quality and shelf life improvements
11.6 Resistance to biotic stress factors
11.7 Resistance to abiotic stress factors
11.8 Biofortification of fruits and vegetables
11.9 Genome editing as an efficient approach to develop crops with better nutritional qualities
11.10 Commercialization of GM fruits and vegetables
11.11 Conclusion
References
twelve Transgenic food crops: public acceptance and IPR
12.1 Transgenic technology for genetic modification of plants
12.2 Adoption and commercial benefits of biotech crops
12.3 Transgenic hybrids in India
12.4 Perceived risks of genetically modified crops
12.4.1 Consumption of foreign DNA
12.4.2 Allergenicity
12.4.3 Horizontal transfer of genetic material
12.4.4 Super plants an environmental risk
12.4.5 Effect on nontarget organism
12.4.6 Contamination of environment with genetically modified proteins
12.5 Safety assessment of genetically modified technology
12.5.1 Codex Alimentarius and Codex Alimentarius Commission
12.5.2 Framework for safety assessment
12.6 Assessment of possible allergenicity
12.6.1 Source of the gene
12.6.2 Sequence homology studies
12.6.3 Physiochemical stability
12.6.4 Serum screening
12.6.5 Testing models
12.7 Potential accumulation of substances significant to human health
12.8 Intellectual property rights in transgenic agriculture biotechnology
12.8.1 Trade secrets
12.8.2 Geographical indications
12.8.3 Trademarks
12.8.4 Copyright and related rights
12.8.5 International organization and agreements for intellectual property rights protection
12.8.6 Patents
12.8.7 Indian legislation on Protection of Plant Varieties and Farmers’ Rights
12.9 Conclusion and future prospects
References
Index
Back Cover

Citation preview

TRANSGENIC TECHNOLOGY BASED VALUE ADDITION IN PLANT BIOTECHNOLOGY

TRANSGENIC TECHNOLOGY BASED VALUE ADDITION IN PLANT BIOTECHNOLOGY Edited by

USHA KIRAN Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India Bioinformatics Institute of India, Noida, India

MALIK ZAINUL ABDIN Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India

KAMALUDDIN Department of Genetics and Plant Breeding, Banda University of Agriculture and Technology, Banda, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-818632-9 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Editorial Project Manager: Rachel Pomery Production Project Manager: Selvaraj Raviraj Cover Designer: Victoria Pearson Typeset by MPS Limited, Chennai, India

Contents List of contributors Preface

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1 Bioprospecting of biodiversity for improvement of agronomic traits in plants

1

K.R Ranjisha, Karuna Surendran, R. Aswati Nair and Padmesh P. Pillai 1.1 Salinity 1.2 Drought 1.3 Low temperature 1.4 Quantitative trait locus based analysis of traits 1.5 Disease tolerance Acknowledgment References Further reading

2 10 13 16 17 20 20 23

2 Plant tissue culture: agriculture and industrial applications

25

Basit Gulzar, A. Mujib, Moien Qadir Malik, Jyoti Mamgain, Rukaya Syeed and Nadia Zafar 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16

Introduction Micropropagation as a multiplication method Organ cultures Somatic embryogenesis and synthetic seeds Haploid development via tissue culture Pathogen-free plant propagation Tissue culture and plant breeding Plant tissue culture and development of transgenic plants Somaclonal variation and its importance in plant improvement Protoplast culture and somatic hybridization Elicitation for enrichment of phytocompounds Precursor addition Hairy root culture and genetic manipulation Endophytes and secondary metabolites Bioreactor scaling Immobilization scaling

Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00015-0

© 2020 Elsevier Inc. All rights reserved.

25 27 29 29 31 31 32 32 36 37 38 40 40 41 41 42

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Contents

2.17 In vitro germplasm storage 2.18 Conclusion and future perspective References

3 Genome editing technologies for value-added traits in plants

42 43 44

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Usha Kiran, Malik Zainul Abdin and Kamaluddin 3.1 Introduction 3.2 Techniques of genome editing 3.3 Application of genome editing systems 3.4 Conclusion and future perspectives References

4 Bioinformatic tools to understand structure and function of plant proteins

51 53 59 64 65

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Ahmad Abu Turab Naqvi, Usha Kiran, Malik Zainul Abdin and Md. Imtaiyaz Hassan 4.1 Introduction 4.2 In silico structural and functional characterization proteins 4.3 Sequence-based approach 4.4 Function prediction 4.5 Summary References

5 Transgenic technology for efficient abiotic stress tolerance in plants

69 71 84 89 90 91

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Arun Gokul, Lee-Ann Niekerk, Mogamat Fahiem Carelse and Marshall Keyster 5.1 5.2 5.3 5.4 5.5 5.6

Introduction Transgenic approaches for engineering heat and cold tolerance in plants Transgenic approaches for engineering salinity stress tolerance in plants Transgenic approaches for engineering drought stress tolerance in plants Transgenic approaches for increased flooding stress tolerance in plants Improving plant tolerance to nutrient deficiency through genetic engineering 5.7 Improving plant tolerance to heavy metal stress tolerance through transgenic approaches 5.8 Conclusion 5.9 Future prospects Acknowledgments References

95 96 101 104 107 109 112 114 115 116 116

Contents

6 Transgenic technologies for efficient insect pest management in crop plants

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Faisal Saeed, Muneeb Hassan Hashmi, Md. Jakir Hossain, Muhammad Amjad Ali and Allah Bakhsh 6.1 Introduction 6.2 Bt genes 6.3 First-generation genome editing technology 6.4 Second-generation genome editing technology 6.5 CRISPR against insects 6.6 Nematode resistance in crop plants 6.7 Conclusions Acknowledgment References

7 Transgenic plants with improved nutrient use efficiency

123 127 130 141 144 145 148 148 148

157

Sadia Iqrar, Kudsiya Ashrafi, Usha Kiran, Saman Fatima, Kamaluddin and Malik Zainul Abdin 7.1 Nitrogen 7.2 Phosphorus 7.3 Sulfur References

8 Genome editing of staple crop plants to combat iron deficiency

157 166 172 178

187

Mather A. Khan and Nga T. Nguyen 8.1 Introduction 8.2 Iron uptake and transport 8.3 Genetic engineering to improve iron content in crops 8.4 Conclusion References

9 Transgenic technology to improve therapeutic efficacy of medicinal plants

187 188 196 201 202

207

Monica Saifi, Shazia Khan, Usha Kiran, Saman Fatima and Malik Zainul Abdin 9.1 History of medicinal plants and natural products 9.2 Natural products: biosynthesis and classification 9.3 Use of medicinal plants and secondary metabolites in traditional and modern medicine 9.4 Technologies for enhancement of secondary metabolites

207 209 212 213

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9.5 New approaches of engineering plant metabolic pathways to enhance secondary metabolites References

10 Application of transgenic technologies in biofuel production through photosynthetic chassis—new paradigms from gene mining to genome editing

220 221

227

Kashif M. Shaikh, Iqra Mariam, Asha A. Nesamma, Malik Zainul Abdin and Pannaga P. Jutur 10.1 Introduction 10.2 Metabolic engineering and synthetic biology 10.3 Improving photosynthesis 10.4 Formation of essential products via photosynthetic chassis 10.5 Terpenes 10.6 Muconic acid 10.7 Gene mining to genome editing 10.8 Challenges and future opportunities Acknowledgments References

11 Genetic engineering of horticultural crops contributes to the improvement of crop nutritional quality and shelf life

227 229 230 231 234 234 235 238 239 240

247

Saber Delpasand Khabbazi, Afsaneh Delpasand Khabbazi, Volkan Cevik and Ali Ergül 11.1 11.2 11.3 11.4 11.5

Introduction 247 Conventional strategies to prolong the shelf life 248 The metabolic basis underlying fruit ripening and shelf life 249 Metabolic alterations incorporating the increased shelf life 250 Transgenic technology as a promising tool for crop nutritional quality and shelf life improvements 252 11.6 Resistance to biotic stress factors 253 11.7 Resistance to abiotic stress factors 254 11.8 Biofortification of fruits and vegetables 257 11.9 Genome editing as an efficient approach to develop crops with better nutritional qualities 259 11.10 Commercialization of GM fruits and vegetables 262 11.11 Conclusion 263 References 264

Contents

12 Transgenic food crops: public acceptance and IPR

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Usha Kiran and Nalini Kant Pandey 12.1 Transgenic technology for genetic modification of plants 12.2 Adoption and commercial benefits of biotech crops 12.3 Transgenic hybrids in India 12.4 Perceived risks of genetically modified crops 12.5 Safety assessment of genetically modified technology 12.6 Assessment of possible allergenicity 12.7 Potential accumulation of substances significant to human health 12.8 Intellectual property rights in transgenic agriculture biotechnology 12.9 Conclusion and future prospects References Index

273 274 278 279 284 289 292 292 303 304 309

List of Contributors Malik Zainul Abdin Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India Muhammad Amjad Ali Department of Plant Pathology, Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan Kudsiya Ashrafi Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, New Delhi, India Allah Bakhsh Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, Nigde, Turkey Mogamat Fahiem Carelse Environmental Biotechnology Laboratory, Department of Biotechnology, University of the Western Cape, Bellville, South Africa Volkan Cevik Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom Ali Ergül Ankara University, Biotechnology Institute, Ankara, Turkey Saman Fatima Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India Arun Gokul Environmental Biotechnology Laboratory, Department of Biotechnology, University of the Western Cape, Bellville, South Africa Basit Gulzar Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi, India Muneeb Hassan Hashmi Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, Nigde, Turkey Md. Imtaiyaz Hassan Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India Md Jakir Hossain Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, Nigde, Turkey Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00016-2

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List of Contributors

Sadia Iqrar Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, New Delhi, India Pannaga P. Jutur Omics of Algae Group, Integrative Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India Kamaluddin Department of Genetics and Plant Breeding, Banda University of Agriculture and Technology, Banda, India Marshall Keyster Environmental Biotechnology Laboratory, Department of Biotechnology, University of the Western Cape, Bellville, South Africa; DST-NRF Centre of Excellence in Food Security, University of the Western Cape, Bellville, South Africa Afsaneh Delpasand Khabbazi Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran Saber Delpasand Khabbazi Ankara University, Biotechnology Institute, Ankara, Turkey Mather A. Khan Division of Plant Sciences, C.S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States Shazia Khan Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India Usha Kiran Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India; Bioinformatics Institute of India, Noida, India Moien Qadir Malik Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi, India Jyoti Mamgain Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi, India Iqra Mariam Omics of Algae Group, Integrative Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India A. Mujib Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi, India

List of Contributors

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R. Aswati Nair Department of Biochemistry and Molecular Biology, Central University of Kerala, Kasaragod, India Ahmad Abu Turab Naqvi Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India Asha A. Nesamma Omics of Algae Group, Integrative Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India Nga T. Nguyen Division of Plant Sciences, C.S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States Lee-Ann Niekerk Environmental Biotechnology Laboratory, Department of Biotechnology, University of the Western Cape, Bellville, South Africa Nalini Kant Pandey Mitakshara IP Services, Ghaziabad, India Padmesh P. Pillai Department of Genomic Science, Central University of Kerala, Kasaragod, India K.R Ranjisha Department of Genomic Science, Central University of Kerala, Kasaragod, India Faisal Saeed Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, Nigde, Turkey Monica Saifi Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India Kashif M. Shaikh Omics of Algae Group, Integrative Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India; Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India Karuna Surendran Department of Genomic Science, Central University of Kerala, Kasaragod, India Rukaya Syeed Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi, India Nadia Zafar Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi, India

Preface Genetic engineering has led to greater understanding of gene structure, composition, and expression opening new doors for manipulation of the genome for the production of desired product. Transgenic plant technology is a growing field worldwide with scientists conducting experiments of different magnitudes, both in terms of direction and scalability. A gap, however, has cropped in between the work being done and the dissemination of generated knowledge about the products and processes used in plant biotechnology. Very limited text is available, which describes these products and processes with their principle, methodology, and applications. With the compilation of this book, we continue our pursuit to bring the latest technologies and their application to fill the knowledge gap for our scientists and research scholars. This book is aimed to disseminate the existing knowledge and help in broadening the horizons of understanding of the subject, leading to the creation of new processes and products. The book consists of 12 chapters. Each chapter is formulated by one or more eminent scientists across the globe, to ensure uniqueness and applicability. The book shall be useful for undergraduates and postgraduate students of biotechnology, agricultural science, environmental sciences, molecular biologist, and working professionals working in academia and industries. Chapter 1, Bioprospecting of Biodiversity: Mining of Novel Gene Sources for Improvement of Agronomic Traits in Crop Plants, describes the genes and metabolites produced by different plants during their sessile life to nullify the vulnerability to hostile conditions including salinity, salinity, drought, low temperature, and pest and pathogen attack, which can be used for conferring tolerance in other plants as well. Apart from emphasizing the importance of bioprospecting plant genomes for potential genes conferring resistance/tolerance in the crop and medicinal plants, the chapter also describes their effective transfer in targeted plants through biotechnological interventions. Chapter 2, Plant Tissue Culture: Agriculture and Industrial Applications, highlights the dependence of agriculture and plant breeding on plant tissue culture (PTC) in realizing the economic potentials of medicinal and crop plants through micropropagation, synthetic seed

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formation, somaclonal variation, hybridization, genetic transformation, haploid culture, and pathogen eradication. PTC also releases the pressure of overexploitation of medicinally important plants growing naturally, by providing the alternative way of production of economical plants or plant parts at large scale in the laboratory, under controlled conditions. Chapter 3, Genome Editing Technologies for Value-Added Traits in Plants, describes the importance of restructuring plant genome to meet the nutritional and medicinal needs of ever-growing world population. The chapter highlights the importance of targeted delivery of desired traits within the species or across genetic barriers, using zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9). Chapter 4, Bioinformatic Tools to Understand Plant Proteins, highlights the use of computer-based protein research by the scientific community. The development of computation tools has enabled mining of enormous data generated from omics projects to obtain the solution for problems like hunger and diseases. The chapter provides a comprehensive overview of recent advances in computational approaches developed to study the structural and functional characterization of proteins and their use in crop improvement. Chapter 5, Transgenic Technology for Efficient Abiotic Stress Tolerance in Plants, highlights the effective use of transgenic approach to minimize the losses incurred in crop and medicinal plant productivity, due to abiotic stress. The chapter describes the selection and incorporation of the appropriate gene using transgenic technology to engineer plants to tolerate heat, cold, salinity, drought, flooding, nutrient deficiency, and heavy metal stresses efficiently. Chapter 6, Transgenic Technologies for Efficient Insect Pest Management in Plants, highlights the importance of transgenic technologies in minimizing the yield losses in crops. Different transgenic approaches have been employed by the scientific community to develop pest-resistant crops that have better crop productivity and are highly advantageous to the farming community worldwide. The chapter provides comprehensive insights and discussion on the usage of modern transgenic technologies in today’s agriculture and integration to efficient integrated pest management. Chapter 7, Transgenic Plants With Improved Nutrient Utilization Efficiency, highlights the importance of transgenic technology to develop

Preface

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varieties with improved nutrient utilization efficiency, even in the limited supply of nutrients. The chapter suggests incorporation of some important genes related to the use-efficiency, coordinate uptake, redistribution, assimilation, and storage of nitrogen, phosphorus, and potassium to reduce the dependence on fertilizers. Chapter 8, Genome Editing of Staple Crop Plants to Combat Iron Deficiency, highlights the importance of iron (Fe) as an essential micronutrient for plants and plants as a major source of Fe for humans and livestock. The chapter describes the molecular mechanism adopted by Arabidopsis, rice, and maize for iron uptake and assimilation as well as the different strategies on genome editing to enhance the iron content of crop plants. Chapter 9, Transgenic Technology to Improve Therapeutic Efficacy of Medicinal Plants, highlights the use of transgenic technology to modulate the metabolic pathways in plants to produce medicinally important compounds, which otherwise is produced by plants in very low amounts. Chapter 10, Application of Transgenic Technologies in Biofuel Production Through Photosynthetic Chassis—New Paradigms From Gene Mining to Genome Editing, highlights the importance of gene mining and its incorporation in plants as an important tool for engineering plant genome and directing the metabolic pathways toward the production of value-added biomass and enable environmental adaptability for the next-generation biofuels. This chapter mainly focuses on recent advancements in the applications of transgenic technologies in plants and algae through photosynthetic chassis for biofuel production. Chapter 11, Genetic Engineering of Horticultural Crops Contributes to the Improvement of Crop Nutritional Quality and Shelf Life, describes the importance of genetic modifications for sustainable delivery of sufficient nutrients to the populations suffering from limited food resources as well as reducing the pre- and postharvest losses. This chapter gives the current state of tools of transgenic plant development for fruits and vegetables with better nutritional quality and shelf life. Chapter 12, Transgenic Food Crops: Public Acceptance and IPR, describes conflicts and controversies surrounding the usage of transgenic technology in agriculture to meet the increasing demands of energy on the shrinking of cultivable land. The chapter describes the state-of-art regarding the intellectual property rights issues related to the control over the economics of production and selling, infringement of rights of

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developer, producer, and consumer and overall technological dependence on the process. The compilation of this book is an effort of editors to present the science behind the technology in simplest way. The book is mix of both basic science and biotechnological applications. The topics covered in the book are based on consultation with scientists and researchers, about the relevance of the subject to the current context. I extend my sincere thanks to every author for their tremendous work to present the most valuable achievements of their specific activity fields in very unpretentious manner. I really hope the scientific content of each chapter would be appreciated equally by postgraduates and research students, plant biotechnologist, molecular biologist, geneticists, and plant breeders in academia and industry. Usha Kiran, Malik Zainul Abdin and Kamaluddin New Delhi, India

CHAPTER ONE

Bioprospecting of biodiversity for improvement of agronomic traits in plants K.R Ranjisha1, Karuna Surendran1, R. Aswati Nair2 and Padmesh P. Pillai1 1

Department of Genomic Science, Central University of Kerala, Kasaragod, India Department of Biochemistry and Molecular Biology, Central University of Kerala, Kasaragod, India

2

Improvement in agriculture for quality traits shares the long history of selection and domestication of wild plants, by the human race. Improvisation for better yield coupled with resistance to biotic and abiotic stresses was done either knowingly or unknowingly by the civilizations, as a continuous process resulting in fine selection of cultivars and landraces. Nevertheless, this resulted in the process of elimination, retention, and acquisition of certain traits, which proved to be beneficial. Changes in biotic and abiotic factors, whether propelled by climatic changes, have severely affected agricultural productivity at the global level. For instance, soil fertility, emergence, and re-emergence of soil/waterborne pathogens have not only caused hardships to the farmers but also decreased the productivity at an alarming pace. Therefore it has necessitated improvement at every step including infrastructure developments, production and judicious usage of fertilizers and pesticides, disease diagnostics and building resistance, molecular screening, and marker-assisted selection, to name a few, to boost agricultural productivity. The specific characters that farmers consider for continuous and sustained improvement in the crop plants are known as agronomic traits of the taxa. These agronomic traits include high yield, disease resistance, tolerance to both biotic and abiotic stresses, better physiological, and morphological features. The measures of improvement as discussed are targeted toward better yield meant for sustained agriculture practice, resulting in the generation of improved or high-yielding crop varieties.

Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00001-0

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Therefore some of the desired agronomic traits that entail improvement through biotechnological interventions are briefly discussed in this chapter.

1.1 Salinity Natural processes like weathering of rocks and sea salt carried by rainwater through the estuaries are some of the common reasons for primary salinity. Secondary salinization or irrigation-induced salinization is caused due to human activities. It has been noticed that within a short period of time, hectares of land were lost in Punjab, India due to water logging and subsequent salinity, caused as a result of irrigation (Reynolds et al., 2005). On the contrary, recent studies have also claimed that in the last 50 years, 40% of increase in food production came from irrigated land (Prakash and Stigler, 2012). Nevertheless, according to the UNEP report, about 20% of irrigated agricultural lands were affected with severe salinity problems (Nellemann et al., 2009). Experiments in salinity tolerance have shown that low or moderate salinity is preferred and adapted by most plant species and their growth shows severe retardation above the critical range of 200 mM NaCl (Sengupta and Majumder, 2009). Another major predicament experienced by the plant is salt-induced physiological drought due to which the roots are unable to absorb water from the soil, owing to high concentration of salt in the outside environment (Munns and Tester, 2008). In most salt-sensitive field crops, the effect of severity of salt stress depends upon the developmental stages and the concentration of salt. The variation in salt concentration can lead to the induction of ionic stresses, physiological drought, secondary stresses like cell damage, and nutritional disorder. By maintaining the cell wall turgor pressure, plants, however, create protective barriers against salinity by increasing the accumulation of osmolytes like proline, sorbitol, mannitol, pinitol, glycine-betaine, myo-inositol, and trehalose (Joshi et al., 2013). According to the study conducted by Chen et al. (2016), the overexpression of Apocynum venetum DEAD-box helicase 1 (AvDH1) gene in a transgenic cotton plant caused increased accumulation of proline, compared to the wild-type plants. Apart from the normal functions such as cellular signaling and biogenesis, myo-inositol also plays crucial role in hypertonic conditions caused by salt stress.

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The expression of myo-inositol-1-phosphate synthase (MIPS) encoding gene (INO1) has been extensively studied in salt-tolerant Mesembryanthemum crystallinum. The expression studies showed upregulation of INO1 gene in M. crystallinum wherein the level of expression was low in Arabidopsis, a salt-sensitive plant. Myo-inositol acts as a facilitator for the uptake and transport of sodium ions in halophyte Spartina alterniflora (smooth cordgrass) (Joshi et al., 2013). Interestingly, the overexpression of SaINO1 (gene encoding myo-inositol from Spartina alterniflora) in Arabidopsis thaliana resulted in increased salt tolerance and less sensitivity to photoinhibition at different developmental stages in transgenic plants. It was further shown that the competition to achieve better photosynthesis efficiency could result in improved seedling and vegetative growth. Thus expression of SaINO1 gene can increase the myo-inositol production that could finally enhance the sodium sequestration and protection of photosystems, especially PSII (Joshi et al., 2013). The imbalance of ion distribution and water potential caused due to high salt concentration can disrupt homeostasis, at both cellular and whole-plant levels, causing hazardous impact on plant functions including growth arrest and cellular as well as molecular damages, eventually leading to death of the plant. It is identified that various plant functions such as nutrient acquisition, activity of photosynthesis apparatus, and activity of various enzymes are severely affected by salt stress. An important cause of damage identified under salt stress condition is the increased production of ROS. The oxidative protection under stress condition was identified with the expression of specific genes. A regulatory protein called NPK1, a member of MAP kinase family, is reported to have functional role in the oxidative stress response. The role of pst1, a negative regulator of oxidative stress responses, was identified in pst1 mutant Arabidopsis. The studies suggest that pst1 mutant plants have more resistance against salt stress (Zhu, 2001). It is interesting to note that glycophytic plants like Arabidopsis and rice generally do not have much critical defense mechanism against salinity, but halophytic plants have adaptive mechanism and, hence, serve as good model for the study of salt tolerance. They have been useful for mining stress-tolerant genes, which can be extrapolated to heterologous systems for understanding of salt tolerance. Dicot and monocot plants have different anatomical structures and express different regulatory adaptations. Generally, halophytic plants among grass species with superior allele or specialized regulatory mechanisms can be used for genetic

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engineering especially in food crops (Baisakh et al., 2012). Wild plant varieties among halophytic plants often have strong resistant mechanisms to all types of stresses and provide source of gene pool that can be used in the field of stress research. A salt-tolerant plant known as wild Asian rice, Porteresia coarctata, has a surviving capacity up to 30 40 dSm and provides an excellent source for the isolation of specific salt-tolerant genes or proteins. The genes from functionally enriched genotype from P. coarctata can be engineered into cultivated varieties of rice to achieve salt tolerance. For example, engineering of inositol methyl transferase gene from P. coarctata has a role in creating a pinitol pool during salt stress (Sengupta and Majumder, 2009). Detailed understanding by physiological and proteomic analysis of salt tolerance mechanism of P. coarctata in contrast to saltsensitive Oryza sativa varieties showed noticeable changes both internally and externally during salt stress conditions. During elevated salt concentration, P. coarctata adapted various regulations that finally caused changes in plant cell characters at qualitative and quantitative levels. This included changes in growth character development, leaf biomass enhancement and cellular water status in terms of leaf RWC (relative water content). P. coarctata survives better in higher salt concentrations with 90% RWC. Even at higher salt concentration, the plant survives through reduction in the internal salt concentration (specifically through extrusion of salt by two types of hairs present on the plant body), increased osmolyte production, increased protein degradation, as well as increased expression of many functional proteins. This study provides the information that the wild varieties have more stress tolerance regulations as compared to domesticated variety and they will be useful for further experiments using genetic engineering tools to make resistant varieties among salt-sensitive crops. Also, P. coarctata (wild halophytic rice) is a good model wild plant variety to study stress adaptations in various cultivated crop plants (Sengupta and Majumder, 2009). It is well known that salt-tolerant grasses (STGs) including halophytes, salt-tolerant glycophytes, and facultative halophytes are potent source of salt-resistant genes (Roy and Chakraborty, 2014). Therefore they are also excellent models to study the mechanism of salt tolerance and to explore ways and means to introduce the novel traits into cereal crops. The list of genes imparting salinity tolerance isolated from various plant species is shown in Table 1.1. Basically, two methods are adopted by plant species to lower the in vivo salt concentration under high salinity conditions. This is done either by the exclusion of excess salts through salt glands or

Table 1.1 List of stress-related genes imparting tolerance to salinity isolated from various plant species. Category

Transporters

Enzymes

Gene

Character

Source: plant species 1

1

a

Target organism

AeNHX1

Vacuolar Na /H antiporter

Agropyron elongatum

Arabidopsis sp.

AlNHX1

Vacuolar Na1/K1 antiporter

Aeluropus littoralisa

Nicotiana tabacum

HvNHX2

Vacuolar Na1/H1 antiporter

Hordeum vulgare

Arabidopsis thaliana

OsNHX1

Na1/H1 antiporter

Oryza sativa

Populus sp.

PgNHX1

Vacuolar Na1/H1 antiporter

Pennisetum glaucuma

Oryza sativa

SaVHAC1

Spartina alternifloraa

Oryza sativa

TaNHX2

Vacuolar H1 ATPase subunit C1 Vacuolar Na1/H1 antiporter

Triticum aestivum

Medicago sativa

PutHKT2;1

K1 transporter

Puccinellia tenuiflora

Arabidopsis

PvUGE1

Putative UDP-galactose epimerase Synthesis of L-myo-inositol-1 phosphate synthase

Paspalum vaginatuma

Oryza sativa

Porteresia coarctataa

Nicotiana tabacum

PcINO1

Observed features

Reference 1

Compartmentation of Na Roy and Chakraborty ions specifically in roots (2014) High Na1 compartmentation in roots Comparatively high K1/ Na1 ratio in leaves Showed salt tolerance at 200 mM NaCl Capable to thrive at 200 mM/L NaCl Successful completion of life cycle of transgenic plants at 150 mM NaCl Induction of extensive root system Increased salinity tolerance in transgenic rice Enhanced tolerance toward salinity Enhanced salt tolerance by Subudhi and controlling Na1 Baisakh (2011) homeostasis Increased salt tolerance in Roy and transgenic plants Chakraborty (2014) Showed growth at high salt concentrations (200 300 mM NaCl)

(Continued )

Table 1.1 (Continued) Category

Gene

PutAPx SaSce9

AvDH1

Transcription factors

Character

Source: plant species

Ascorbate peroxidase coding Puccinellia tenuiflora gene Small ubiquitin-related Spartina alternifloraa modifier (SUMO) conjugating enzyme Apocynum venetum DEAD-box helicase1

SaINO1

Myo-inositol 1-phosphate synthase gene

Spartina alternifloraa

McIMT1

Myo-inositol O-methyl transferase gene

M. crystallinum

LcDREB3a

Dehydration-responsive element-binding transcription factor

Leymus chinensisa

LcMYB-1

MYB-related transcription factor

Leymus chinensisa

OrbHLH001

Basic helix-loop-helix (bHLH) Oryza rufipogon protein gene

a

Target organism

Saccharomyces

Observed features

Reference

Able to resist oxidative stress induced by salinity Arabidopsis thaliana Stress-responsive genes mediated tolerance to salt stress Gossypium hirsutum L. Decreased membrane ion Chen et al. (2016) leakage Increased yield as a result of increased salt tolerance Arabidopsis thaliana Improved salt tolerance Joshi et al. (2013) during germination, seedling growth, and development Tobacco Salinity tolerance in Subudhi and transgenic plants due to Baisakh (2011) the presence of high amount of myo-inositol, increased accumulation of D-ononitol, etc. Arabidopsis thaliana Enhanced salinity tolerance Roy and Chakraborty Proper development of (2014) transgenic plants without any retardation in growth Arabidopsis thaliana Increased accumulation of proline and soluble sugars showed tolerance toward salinity Arabidopsis thaliana Enhanced salinity tolerance

Other functional proteins

ChlMT1

Metallothionein

Chloris virgataa

Saccharomyces

HVA1

Encodes a group of three late embryogenesis-abundant (LEA) proteins

Hordeum vulgare

Morus alba

PcSrp

Root-specific cDNA-encoding Porteresia coarctataa serine-rich protein Two conserved zinc-finger Aeluropus littoralisa domains A20 and AN1 Nodulin 26-like intrinsic Triticum aestivum protein (novel aquaporin gene)

AlSAP TaNIP

a

Eleusine coracana Nicotiana tabacum Arabidopsis thaliana

TaST

Unknown salt-induced gene

Triticum aestivum

Arabidopsis thaliana

ZmPMP3-1

Plasma membrane protein

Zea mays

Arabidopsis thaliana

Grasses considered as STG model plants.

Enhanced tolerance toward the harmful effects of ROS Increased accumulation of proline and thereby enhanced tolerance to salt stress Capable to grow and set seed at 250 mM NaCl Able to set seed at up to 350 mM NaCl Salt tolerance in transgenic plants due to increased amount of K1 and proline Accumulation of high amount of Ca21, soluble sugar, proline, and less Na1 increased salinity tolerance Tolerance to salt stress by regulation of ion homeostasis and ROS scavenging

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by reducing the uptake of salts. There are various mechanisms developed by plants with the aim to reduce the total salt concentration inside the plant body like the selectivity of K1 over Na1, where toxic Na1 is replaced with K1 and it is calculated as net selectivity (SK:Na). As expected, this ratio varies among different plant species and was found to be highest in the order poales with 60 as compared with other flowering plants. Similarly, the chloridoid grasses have adopted a mechanism to reduce the salt level by the exclusion of salt using salt glands in their leaf epidermis followed by the accumulation of solutes like glycine-betaine, an important osmolyte. The knowledge about the expression of specific genes during salt stress via Na1 accumulation in the vacuole and Na1 exclusion from the root or leaves was used for various transgenic approaches against salt tolerance (Roy and Chakraborty, 2014). The differential expression of genes remains a key mechanism in regulating the plant response to the salt stress. Under salt-stressed condition, a clear upregulation in the expression of genes like plasma membrane protein 3 (SaPMP3), vacuolar ATPase (SaV-ATPase), transport protein (SaCTP), and myo-inositol-1-phosphate synthase (SaMIPS) from leaf and root tissues of Spartina alterniflora was analyzed through reverse transcription PCR (RT-PCR) (Baisakh et al., 2008). Furthermore, a number of candidate genes encoding osmoprotectants, transcription factors, antioxidants, and ion transporters from salt-tolerant Spartina alterniflora were found to be upregulated during salt stress (Subudhi and Baisakh, 2011) and thereby contribute potential source of salt-tolerant gene for crop improvement. Therefore an integrated approach outlining genomic, transcriptomic, proteomic, and metabolomics (Fig. 1.1) is recommended for the isolation and expression of salt-tolerant genes in crop plants. The many proteins that are involved in the normal functioning of a cell also have significant role in the regulatory mechanisms under stress conditions. For example, a class of motor proteins called helicases, considered as important group of proteins that takes part in the central dogma of life, have highly conserved sequence motifs and classified under the DEAD-box protein superfamily. They are already characterized in yeast, animals, and plants. An association of helicases during abiotic stresses such as salt stress, temperature, and light has been studied in various plants (Chen et al., 2016). The involvement of two DEAD-box helicases, stress response suppressor 1 and stress response suppressor 2, in ABA-dependent as well as ABA-independent abiotic signaling pathway was identified in Arabidopsis (Kant et al., 2007). The transgenic rice and sugarcane with the

Figure 1.1 Strategy based on omic’s technology showing isolation and introgression of salt-tolerant genes in target crop plants (ref: http://www.as-botanicalstudies.com/content/55/1/31).

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overexpression of a Pisum sativum helicase gene PDH45 and rice mitochondrial helicase OsSUV3 (suppressor of Var3), respectively, have been found to show salt tolerance. An enhanced detoxification of reactive oxygen species (ROS) and increased expression of stress-responsive genes during salt stress were also studied by the overexpression of rice antigen-Bassociated transcript (OsBAT1) helicase. All these transgenic plants were analyzed for salt stress tolerance under greenhouse-based environment. With the knowledge of helicase activity, another study has been conducted in the cotton plant to identify the role of helicases under growth chamber as well as the saline field. The AvDH1, the gene is expressed in response to NaCl but not to ethylene glycol or ABA, was overexpressed in cotton. The study showed positive influence of the transgene to salt stress and had increased plant height. It also provided the information that transgenic and nontransgenic cotton plants under nonsaline condition have no growth difference, reinstating the results of previous studies. It is also shown in greenhouse and saline field experiments, both resulted in positive regulation of the helicase gene against salt stress, that saline field transplants are characterized with increase boll size, boll weight and seed yield than the wild-type plants grown in the same field. It is already known that the rate of accumulation of ROS during salinity is high and, hence, can cause damage to the cellular macromolecules, imbalance to membrane stability, etc. The overexpression of AvDH1 in cotton plant has led to elevation of SOD (superoxide dismutase) activity, the important ROS scavenging enzyme, resulting in improved plant survival rate through reduced membrane damage (Chen et al., 2016).

1.2 Drought Drought is yet another threat causing decreased agricultural productivity. Global studies reveal that out of 51% of crop loss contributed by abiotic stresses (Boyer, 1982; Bray et al., 2000), 15% is contributed by drought alone (Dey and Upadhaya, 1996). As a sessile organism, plants have developed with more complex mechanisms in order to resist these stresses. Plants manifest a plethora of physiological situations due to drought including cell injury via ROS, increased cellular temperature, which further leads to protein denaturation and aggregation, increased viscosity of cellular components, and alteration of proteins interaction

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(Farooq et al., 2008). Plants have developed various signaling mechanisms mediated through induction and repression of genes that cause decreased transpiration, accumulation of osmolytes, active antioxidant system, etc. (Joshi et al., 2016). In search of better tolerant genes, researchers have developed wide approaches to study about drought stress. An experimental setup created for the cultivation of plants under controlled environmental conditions showed that withdrawal of water from these plants altered the expression of genes and proteins, thereby confirming that differential expression of genes caused the underlying mechanism of tolerance or resistance to abiotic stresses. The expression-based profiling of Arabidopsis genome under drought tolerance has revealed that out of 7000 genes, 277 were upregulated and 79 were downregulated. Furthermore, increased level of expression of genes under water stress was further studied (Seki et al., 2002). The perennial grass plant, Cleistogenes songorica, a dominant species of desert grassland in China, is ideal taxa for various investigations analysis in the area of stress resistance. Its characteristic adaptations to low nutrient and sandy soil conditions along with excellent drought tolerance allows selecting the plant as a good candidate genetic resource for learning about gene-based tolerance. To gain an accurate knowledge about drought tolerance in C. songorica, expressed sequence tags (ESTs) have proven to be an unavoidable tool in the field of functional genomic research and are obtained from four cDNA libraries generated from the roots and leaves of the plant. From a group of 5664 random selected clones, 3579 highquality trimmed sequences were obtained and the trimmed ESTs showed average read length of 613 bp; 805 singleton unigenes and 694 multimember unigenes have been identified from a nonredundant set of 1499 contigs. Study about stress tolerance with these unigenes showed that 63 unigenes were involved in the regulation mechanism and 22 unigenes showed similarity to known genes involved in normal drought tolerance. A detailed study on these 22 genes showed that about 13 transcripts displayed threefold increase in expression. These studies helped to generate an idea about EST and cDNA collections from C. songorica that were expressed during drought conditions (Zhang et al., 2011). In brief, the analysis indicates that physiological, molecular, and biological levels of changes are adopted by plants to achieve tolerance to drought stress (Morari et al., 2015). Second messengers like calcium and calcium sensors (Klimecka and Muszynska, 2007; Kudla et al., 2010; Reddy et al., 2011; Vivek et al., 2013) and abscisic acid (Zeng et al., 2015) are closely

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associated with drought stress. The intracellular protein kinase called calcium-dependent protein kinases (CPKs) has the ability to convert calcium signals into a phosphorylation event and modulated drought stress tolerance. CPKs are group of highly conserved proteins having various functions in stress signaling, defense responses, plant growth, and proteasome regulations. Evidence indicates that CPKs acts as both positive and negative regulators of drought stress tolerance. The accumulation of the compatible osmolyte proline in A. thaliana is positively regulated by AtCPK6. Similarly, the effect of other CPKs like AtCPK10 and OsCPK13 in A. thaliana and rice, respectively, was identified with positive regulatory mechanism in response to drought. The CPK (AtCPK23), which controls two plasma membrane-bound slow voltage-gated ion channels, was reported to be negative regulator of drought and salt tolerance (Ma and Wu, 2007). Based on previous reports about the role of CPK, Cie´sla et al. (2016) evaluated the role of barley (Hordeum vulgare L.) CPK, HvCPK2a, against drought stress in Arabidopsis. The transgenic Arabidopsis showed increased transcripts of HvCPK2a under drought condition, and this overexpression of the gene increased the drought sensitivity. Transgenic Arabidopsis plant displayed decreased relative water content (RWC), reduced nitrogen balance index (NBI), drought sensitivity, and increased amount of total chlorophyll content, thereby suggesting an important role for HvCPK2a in the regulation of leaf growth and biomass under drought condition. The results also suggested that HvCPK2a contributes to regulation of the drought stress response by interacting enzymes involved in formate, malate, and pyruvate metabolism. Further, in vitro kinase assay combined with mass spectroscopy suggests that HvCPK2a is a dual-specificity CPK that auto-phosphorylates on T, S, and Y residues localized within the catalytic and calmodulin-like domain. Thus CPKs may serve as targets for producing transgenic varieties resistance to drought. The stress-responsive genes mainly code for proteins having either metabolic or regulatory activities. The regulatory group primarily comprises of transcription factors (TFs), protein phosphatases, and signaling protein kinases. It is known that regulatory genes including TFs regulate downstream stress-responsive genes at multiple abiotic stresses. The cisregulatory elements present in the promoter region of stress-responsive genes serve as the binding site for the TFs and thereby regulate the gene expression. TFs are regulated by themselves at the level of transcription with the help of other upstream elements. The posttranscriptional

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modifications make them into a complex regulatory network that modulates the expression of stress-responsive genes. The field of genetic engineering mainly deals with the overexpression of these specific TFs, enzymes, and other metabolites to generate drought-tolerant varieties. Based on the nature of DNA-binding domain, the TFs are classified into different gene families such as AREB, DREB, MYB, WRKY, NAC, and bZIP. The TFs that are studied under drought condition show both ABA-dependent and -independent pathways for their functioning (Joshi et al., 2016). Among different traits analyzed, cellular tolerance (CT) was identified as one of the important ways to achieve drought tolerance. It has been reported that the manipulation of CT can be achieved through the overexpression of genes involved in scavenging of ROS, biosynthesis of osmolytes, etc. Overexpression of the TF AtDREB2A in Arabidopsis and independent expression of AtABF3 and AtHB7 in rice and Arabidopsis, respectively, were identified to be involved in stress tolerance. A transgenic approach was conducted in peanut by constitutive coexpression of different validated drought-responsive TFs such as AtDREB2A, AtHB7, and AtABF3. The TF AtDREB2A regulates ABA-independent pathway, whereas AtHB7 and AtABF3 regulate ABA-dependent pathways. The transgenic plants that are reported with the increased activity of SOD enzyme reveal the role of AtABF3 in ROS scavenging machinery. It was also noticed that transgenic plants had better osmotic adjustment, which could be due to increased production of proline and delayed senescence under ethylene-induced stress. The reason for delayed senescence could be the result of the expression of AtHB7 since its role in delayed senescence has been studied previously. The promoter regions of some selected downstream stress-responsive genes were analyzed by the use of STIF (Stress Gene Transcription Factor) database, which reveals that promoter regions contain specific cis-elements (DRE, ABRE, and HDE). The idea of coexpression of TFS that can activate the expression of multiple target genes can be used to produce transgenic lines showing increased stress tolerance (Pruthvi et al., 2014).

1.3 Low temperature The rate of surviving capacity of a living organism, considering other essential factors too, depends on the temperature fluctuations in

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their habitat. The enormous diversity exhibited by the living organisms depends more or less on the different range of temperatures they used to survive. Low temperature (LT), one of the most prominent abiotic factors, has an important role in growth, distribution, and yield of plants. Considering a good model plant, extensive research has been done on Arabidopsis to understand the changes associated with LT. It, however, has limitations due to lack of known ecotype of the plant with extreme tolerance to LT stress and reduced capacity to survive at this freezing conditions (Bressan et al., 2001). Response against various stresses inside the plant body primarily starts from a signal transduction pathway, which is a ligand receptor-associated mechanism. Predictably, all other metabolic pathways follow the message that is received from initial signal transduction pathway. Studies have revealed that LT tolerance responses are carried out with respect to the LT signal received coordinated by a group of proteins called mitogenactivated protein kinases (MAPKs). MAPKs are present in diverse organisms including plants. Plant responses against various stresses that are processing inside the plant body primarily starts from a pathway called signal transduction pathway which is a ligand-receptor associated mechanism. Predictably, all other metabolic pathways will follow the message that is received from this signal transduction pathway. Many of the biotic and abiotic stresses such as LT, salt, osmotic shock, heat shock and wounding and the signaling molecules that are involved in defense-related mechanisms such as jasmonic acid (JA), salicylic acid (SA) and hydrogen peroxide, are found to be involved in the process of activation of MAPKs. Interestingly, MAPK pathway is a cascade of three kinase proteins and their activities are regulated by phosphorylation &dephosphorylation of specific amino acid in their sequence (Ghawana et al., (2009). Ghawana et al. (2009) identified two genes from Rheum australe, RaMPK1 and RaMPK2, similar to MAPKs using differential display (DD). Both genes were found to be differentially maintained with under stress conditions. The detailed structural analysis reveals that these genes have some additional domains when compared with other MAPKs, which helps them to interact with other domains like SH3, and thereby, facilitating their involvement in various regulatory processes including signal transduction pathways. The RaMPK1 transcript showed upregulation to LT and JA exposure but with ABA and PEG treatment, it showed a downregulation. These results lead to a logical reference to

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that LT and JA might control similar reactions. From this data, it is likely that ABA-independent pathway is selected by RaMPK1 in LT-mediated responses. RaMPK1 homology similarity with SIPK has made a suggestion that it has a role in SA signaling pathway, and the detailed study shows that it is further downregulated during the pathway. From the above characteristics, it is concluded that RaMPK1 and RaMPK2 were downregulated by SA at the level of transcription. This research also provides a space for further insight into the regulatory pathway at the level of protein expression and the resultant data suggest that MAPKs can be utilized to clone promoters to produce transgenic plants having tolerance to LT. Bhardwaj et al. (2010) used Caragana jubata, a perennial shrub of the temperate zone to identify novel genes involved in LT induced stress. These plants grow under the snow where they have extreme temperature variations ranging between 5 C and 37 C during daytime and 2 C and 10 C during the night. Differential display of mRNA (DD) studies identified a potential ribosomal protein gene called QM that was induced by LT in Caragana. QM expression study in response to LT in rice showed no change in expression 1 in root tissue but in the leaf tissue, its expression was upregulated after 6 h of low-temperature treatment followed by the regaining of its normal state at 24 h. Recent studies showed downregulation of QM under LT and in response to abscisic acid. Studies by Bhardwaj et al. (2010) suggested upregulation of Caragana CjQM under LT in response to abscisic acid. These results suggest that the difference in QM expression might be due to the difference in the nature of plant; Caragana flourishes under snow and tea exhibit winter dormancy. It is known that salicylic acid (SA) and methyl jasmonate (MJ) are involved in gene regulation under LT conditions, but CjQM expression study toward SA and MJ shows nonresponsiveness of the gene. This indicates that CjQM has specific regulatory mechanism toward LT apart from SA and MJ pathways. CjQM expression studies were mainly carried out by either early or continuous exposure to LT, both leading to an upregulation of CjQM expression suggesting that continuous requirement is needed under LT condition. It is also known that QM helps to maintain the ribosomal structure by aggregating 40S and 60S ribosomal subunits together and also participates in signal transduction pathways. The increased level of CjQM in response to LT and ABA might be required to produce proteins for various mechanisms that are developed during LT condition (Bhardwaj et al., 2010).

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1.4 Quantitative trait locus based analysis of traits Various studies have been conducted in the area of production of high-yielding crop plants. Quantitative trait loci (QTLs) studies have improved our knowledge in understanding the natural allelic diversity, which can be further used for crop improvement studies. Agronomic traits that are controlled by QTL were identified using a specific molecular marker and also by map-based cloning (Tagle et al., 2016). Genetic regions that are linked to quantitative traits can now be easily identified by using methods such as QTLs mapping, marker-assisted selection (MAS), and QTL pyramiding (Nogué et al., 2016). In QTL pyramiding, combination of two or more QTLs responsible for different traits in different varieties or compatible species was studied by the introgression of those QTLs into same elite line (Ashikari and Matsuoka, 2006). Such introgression studies showed successful results when introgression of five or more QTLs for certain traits into a given elite line is done (Semagn et al., 2006). QTL experiments cannot be conducted directly in rice-breeding programs because of their changing expressions according to the different genetic background and with the environment (Li et al., 2003). To overcome these problems, recently, researchers have conducted an experiment with the information about the elite rice genetic background and succeeded in the genetic dissection of yield-related traits using introgression line (IL)-based studies. ILs carrying small introgressed segments of donor parents were used for the analysis of agronomic traits, specifically for the selected introgressions (Zhang et al., 2013). A study was performed in high-yielding and high-quality rice variety called indica, in which a variety known as IR64 was chosen for the detailed understanding of genetic basis of yield-related traits. For that, a total of 334 ILs were taken from the cross between IR64 and 10 donor varieties, mainly selected from NPT (new plant type) lines that were previously used in the study conducted by IRRI-Japan collaborative research project. The result showed several selected lines, for example, YTH288 having unique agronomic character inherited from the selected NPT lines. Along with these studies, chromosomal analysis associated with agronomic traits using the ILs indicates that ILs are useful in the area that deals with the genetic analysis of unique agronomic features in high-yielding varieties (Tagle et al., 2016).

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Yonemaru et al. (2010) conducted an experiment about the colocalization and distribution of QTL and QTL clusters with different morphological characteristics present on chromosomes 1, 3, 4, 6, and 9 and the long arm of chromosome 4, respectively. Recently, Tagle et al. (2016) reported that four QTLs for CL (culm length), FLL (flag leaf length), FLW (flag leaf width), and FSN (filled spikelet number per panicle) from a rice variety have been colocalized in the long arm of the fourth chromosome. Attributably this colocalization of QTL can suggest that it may represent a gene cluster or impart a pleiotropic effect of gene cluster for different traits underlying the QTL. So, colocalization study of QTL in plants disturbed with various stresses would provide a better way to understand the genes that take part in the process of tolerant mechanisms under stress conditions.

1.5 Disease tolerance The spectacular crop failures in the field of agriculture are mainly noticed with the challenging diseases caused by plant pathogens including fungi, bacteria, and viruses. Pathogens are identified in all plant communities, but their growth and harmful effects are restricted depending on the environment and season where they have to complete their life cycle. The infection of a pathogen can lead to drastic changes at various levels of plant activity and this restricts its growth from obtaining maximum yield and profit. The main outcomes of infection consist of damages to the seeds, stunted plant growth, reduced yield, changes in internal functioning, etc. There are many studies conducted to understand and develop resistant varieties against these diversifying diseases. These studies have suggested that the gene-level approaches can be used to develop disease resistance better than by reducing the disease effects through cultural practices. Crop improvement by generating disease-resistant cultivars is a challenging area of research since the modes of invasion and multiplication of these pathogenic organisms resulting in collapse of plant immunity are yet to be revealed. Plant resistance (R) genes are the class of genes that have been used by breeders in disease-resistant variety production. Plants also have an inducible defense mechanism by the expression of another set of genes called pathogenesis-related (PR) genes (Akhond and Machray, 2009).

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While considering the scenario of wheat production, one of the economically important cereal crop, the major diseases that severely affects the plant growth and yield are leaf, stem and stripe rust caused by fungal pathogens Puccinia triticina, Puccinia graminis, and Puccinia striiformis respectively. Among these three rust diseases, leaf rust is the common disease that can cause yield loss of about 30% 70%, depending upon the stage at which the disease emerges (Huerta-Espino et al., 2011). When compared with the nature of race-specific protection with the help of effectortriggered immunity of plant resistance (R) gene along with quantitative resistance (QR) gene, the QR genes are found to be more desirable for plant breeding programs as QR genes are broad-spectrum, race-nonspecific and confers durable protection. Studies have identified three durable multi-pathogen rust resistance genes from wheat viz., Lr34, Lr46 & Lr67, which not only confer resistance against the three wheat rusts but also gives resistance to powdery mildew disease. Further, Lr34 also confers resistance to spot blotch. Two functional homologous copies of Lr34 were identified from hexaploid wheat chromosomes of 7D and 4A. The 7D copy was found to have two predominant alleles named as resistant allele Lr34res and susceptible allele Lr34sus. Recently, transformation of Lr34 into barley provided resistance against barley rust and barley powdery mildew. The expression of Lr34res showed that it is expressed in both seedling and adult stages in barley, whereas its action is expressed only in adult plant stage in wheat. No resistance was observed with the transformation of Lr34sus into barley. The expression of Lr34res transgene in barley resulted in strong leaf tip necrosis (LTN)-type senescence, retardation of growth, reduced grain yield, etc. In order to find out the reason for this negative result, transcriptomics and targeted metabolite analysis of Lr34res lines of barley were carried out to identify the changes in the molecular components of barley in the presence of Lr34res. It was found that even in the absence of infection, Lr34res are involved to induce various defense mechanisms (Chauhan et al., 2015). The gene expression studies conducted in uninfected Lr34 transgenic barley lines indicated that genes from multiple defense pathways have role in disease resistance and were active in seedling and mature leaves, along with an increased hormonal levels of jasmonic acid and salicylic acid, increased concentration of lignin and hordatines, etc. These observations suggest important role of Lr34 in strong, constitutive reprogramming of metabolism. Analysis of protein product of Lr34res and Lr34sus indicates that Lr34res allele differs by two amino acid polymorphisms from Lr34sus allele and deletion of a

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single phenyl alanine residue from Lr34sus resulted in the induction of Lr34-based responses (Chauhan et al., 2015). It has been identified that more than 400 plant species were infected with a necrotrophic fungal pathogen known as Sclerotinia sclerotiorum (Lib.) de Bary, which has the ability to produce a long-lived melanized resting structure sclerotia. These nonhost-specific pathogen activities were noticed in many oil crops such as oilseed rape, sunflower, and soybean, accompanied with the disease conditions like stem rot, severe yield damage, decrease in total oil content, and changes in fatty acid profile. The devastating disease sclerotinia stem rot caused by the resting sclerotia under favorable condition in Brassica napus (oilseed rape) has made it difficult to understand the proper time for applying fungicide. This led the researchers to concentrate on other areas of defense such as breeding and resistant variety production. The known reasons that make difficulties in breeding process in B. napus are the lack of highly resistant germplasm and unknown interactive mechanism of plant and pathogen during infection. Within these limitations, many studies were conducted with this partial resistant germplasm and succeeded to produce improved resistant cultivars. Microarray studies for the gene expression analysis showed that defense-related and phytohormone-responsive genes, transcription factors, genes involved in cell wall structure, and secondary metabolism are differentially regulated after infection (Wu et al., 2013). With the sense of developing disease-resistant breeding products, a recent study succeeded in identifying the major QTLs responsible for the sclerotinia stem rot in B. napus. A complex quantitative trait controlled by minor polygenes was analyzed in the resistant mechanism against S. sclerotiorum in B. napus, and this characteristic quantitative defense mechanism has already been studied in other plants such as soybean, common bean, and sunflower. By mapping the QTLs for stem resistance (SR) at the mature plant stage and leaf resistance (LR) at the seedling stage under different environmental condition led to the identification of two important QTLs such as SRC6 on LG C6 and LRA9 on LG A9. The plants with these two QTLs have reduced disease characteristic features after pathogen infection. The effects of SRC on SR and LRA9 on LR were analyzed for two different genotypes Hua5 (susceptible parent) and J7005 (resistant parent) in the experimental farm of Huazhong Agricultural University, China in the seasons of 2009 10 and 2010 11 for SRC and 2010 11 and 2011 12 for LRA9. Furthermore, the effect of SRC on SR for the same genotypes was also analyzed in the experimental farm of Huanggang Academy of Agricultural

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Sciences, China in the season of 2010 11. The overall result showed that the size of disease lesion developed for SRC is higher for J7005 than that of Hua5. But the lesion size developed for LRA9 is smaller for J7005 than that of Hua5. To gain more precise knowledge about these QTLs, further analyses were conducted. A candidate gene BnaC.IGMT5.a for QTL SRC6 was also identified through homologous cloning, comparative mapping with Arabidopsis, and extensive data mining from previous profiling experiments. The differential expression pattern of BnaC.IGMT5.a gene that was exhibited by two parental lines in response to pathogen infection suggests that in future, BnaC.IGMT5.a can be used as a candidate gene for the resistant QTL of SRC6 in B. napus for studying and developing resistance against S. sclerotiorum (Wu et al., 2013). Rice is one of the major staple food crops in the world. As known, Indica variety constitutes 80% of the total cultivated rice. However, one of the indica varieties, HR-12, has good agronomic traits but has the drawback of high susceptibility to blast disease (Gowda et al., 2015) caused by Magnaporthe oryzae. To understand the mechanism of resistance against the rice blast, an experiment was conducted wherein highly susceptible varieties of indica such as C0-39 and HR-12 and highly resistant varieties such as Tetep and Tadukan were used for genome sequencing. From the sequenced genome, allele mining for resistant (R) genes showed that R genes are conserved with point mutation and InDel mutation in resistant varieties, while in susceptible ones, R genes were noticed to experience with the loss of these mutations. This study has extended a good genomic resource for future studies by providing information about global rice diversity and genetics for molecular breeding (Mahesh et al., 2016).

Acknowledgment The authors acknowledge with thanks the Vice Chancellor of Central University of Kerala for providing all facilities required for the preparation of this manuscript.

References Akhond, M.A.Y., Machray, G.C., 2009. Biotech crops: technologies, achievements and prospects. Euphytica 166, 47 59. Ashikari, M., Matsuoka, M., 2006. Identification, isolation and pyramiding of quantitative trait loci for rice breeding. Trends Plant Sci. 11, 344 350. Baisakh, N., Subudhi, P.K., Bhardwaj, P., 2008. Primary responses to salt stress in a halophyte, smooth cordgrass (Spartina alterniflora Loisel.). Funct. Integr. Genomic 8, 287 300.

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Baisakh, N., Mangu, V.R., Rajasekaran, K., Subudhi, P., Janda, J., Galbraith, D., et al., 2012. Enhanced salt stress tolerance of rice plants expressing a vacuolar H1-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Loisel. Plant Biotechnol. J. 10, 453 464. Bhardwaj, P.K., Ahuja, P.S., Kumar, S., 2010. Characterization of gene expression of QM from Caragana jubata, a plant species that grows under extreme cold. Mol. Biol. Rep. 37, 1003 1010. Boyer, J.S., 1982. Plant productivity and environment. Science 218, 443 448. Bray, E., Bailey-Serres, J., Weretilnyk, K., 2000. Responses to abiotic stresses. In: Buchannan, B., Gruissem, W., Rockville, J. (Eds.), Biochemistry and Molecular Biology of Plants. American Society of Plant Biologists, pp. 1158 1203. Bressan, R.A., Zhang, C., Zhang, H., Hasegawa, P., Bohnert, H., Zhu, J.K., 2001. Learning from the Arabidopsis experience. The next gene search paradigm. Plant Physiol. 127, 1354 1360. Chauhan, H., Boni, R., Bucher, R., Kuhn, B., Buchmann, G., Sucher, J., et al., 2015. The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley. Plant J. 84, 202 215. Chen, J., Wan, S., Liu, H., Fan, S., Zhang, Y., Wang, W., et al., 2016. Overexpression of an Apocynum venetum DEAD-Box Helicase Gene (AvDH1) in Cotton confers salinity tolerance and increases yield in a saline field. Front. Plant Sci. 6, 1227. ´ ´ Cie´sla, A., Mituła, F., Misztal, L., Fedorowicz-Stronska, O., Janicka, S., Tajdel-Zielinska, M., et al., 2016. A role for barley calcium-dependent protein kinase CPK2a in the response to drought. Frontiers in Plant Science, 7. Dey, M.M., Upadhaya, H.K., 1996. Yield loss due to drought, cold, and submergence in Asia. In: Evenson, R.E., Herdt, R.W., Hossain, M. (Eds.), Rice Research in Asia, Progress and Priorities. Oxford University Press, Cary, NC, pp. 231 242. Farooq, M., Basra, S.M.A., Wahid, A., Cheema, Z.A., Cheema, M.A., Khaliq, A., 2008. Physiological role of exogenously applied glycinebetaine to improve drought tolerance in fine grain aromatic rice (Oryza sativa L.). J. Agron. Crop. Sci. 194, 325 333. Ghawana, S., Kumar, S., Ahuja, P.S., 2009. Early low-temperature responsive mitogen activated protein kinases RaMPK1 and RaMPK2 from Rheum australe D. Don respond differentially to diverse stresses. Mol. Biol. Rep. 37, 933 938. Gowda, M., Shirke, M.D., Mahesh, H.B., Chandarana, P., Rajamani, A., Chattoo, B.B., 2015. Genome analysis of rice-blast fungus Magnaporthe oryzae field isolates from southern India. Genom. Data 5, 284 291. Huerta-Espino, J., Singh, R.P., German, S., McCallum, B.D., Park, R.F., Chen, W.Q., et al., 2011. Global status of wheat leaf rust caused by Puccinia triticina. Euphytica 179, 143 160. Joshi, R., Ramanarao, M.V., Baisakh, N., 2013. Arabidopsis plants constitutively overexpressing a myo-inositol 1-phosphate synthase gene (SaINO1) from the halophyte smooth cordgrass exhibits enhanced level of tolerance to salt stress. Plant Phys. Biochem. 65, 61 66. Joshi, R., Wani, S.H., Singh, B., Bohra, A., Dar, Z.A., Lone, A.A., et al., 2016. Transcription factors and plants response to drought stress: current understanding and future directions. Front. Plant Sci. 7, 1029. Kant, P., Kant, S., Gordon, M., Shaked, R., Barak, S., 2007. STRESS RESPONSE SUPPRESSOR1 and STRESS RESPONSE SUPPRESSOR2, two DEAD-box RNA helicases that attenuate Arabidopsis responses to multiple abiotic stresses. Plant Physiol. 145, 814 830. Klimecka, M., Muszynska, G., 2007. Structure and functions of plant calcium-dependent protein kinases. Acta Biochem. Pol. 54, 219 233.

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Kudla, J., Batistic, O., Hashimoto, K., 2010. Calcium signals: the lead currency of plant information processing. Plant Cell 22, 541 563. Li, Z.K., Yu, S.B., Lafitte, R., Huang, N., Courtois, B., Hittalmani, S., et al., 2003. QTL 3 environment interactions in rice. I. Heading date and plant height. Theor. Appl. Genet. 108, 141 153. Ma, S.Y., Wu, W.H., 2007. AtCPK23 functions in Arabidopsis responses to drought and salt stresses. Plant Mol. Biol. 65, 511 518. Mahesh, H.B., Shirke, M.D., Singh, S., Rajamani, A., Hittalmani, S., Wang, G.L., et al., 2016. Indica rice genome assembly, annotation and mining of blast disease resistance genes. BMC Genomics 17, 242. Morari, F., Meggio, F., Lunardon, A., Scudiero, E., Forestan, C., Farinati, S., et al., 2015. Time course of biochemical, physiological, and molecular responses to field mimicked conditions of drought, salinity, and recovery in two maize lines. Front. Plant Sci. 6, 314. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651 681. Nellemann, C., Devette, M.M., Manders, T., 2009. The Environmental Food Crisis: The Environment’s Role in Averting Future Food Crisis: A UNEP Rapid Response Assessment. United Nations Environment Programme of the United Nations, Nairobi. Nogué, F., Mara, K., Collonnier, C., Casacuberta, J., 2016. Genome engineering and plant breeding: impact on trait discovery and development. Plant Cell Rep. 35(7), 1475–1486. Prakash, A., Stigler, M., 2012. FAO Statistical Yearbook. Food and Agriculture Organization of the United Nations, Rome. Pruthvi, V., Narasimhan, R., Nataraja, K.N., 2014. Simultaneous expression of abiotic stress responsive transcription factors, AtDREB2A, AtHB7 and AtABF3improves salinity and drought tolerance in peanut (Arachis hypogaea L.). PLoS One 12, e111152. Reddy, A.S., Ali, G.S., Celesnik, H., Day, I.S., 2011. Coping with stresses: roles of calciumand calcium/calmodulin-regulated gene expression. Plant Cell 23, 2010 2032. Reynolds, M.P., Mujeeb-Kazi, A., Sawkins, M., 2005. Prospects for utilizing plantadaptive mechanisms to improve wheat and other crops in drought- and salinityprone environments. Ann. Appl. Biol. 146, 239 259. Roy, S., Chakraborty, U., 2014. Salt tolerance mechanisms in salt tolerant grasses (STGs) and their prospects in cereal crop improvement. Bot. Stud. 55, 31. Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., et al., 2002. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J. 31, 279 292. Semagn, K., Bjørnstad, A., Ndjiondjop, M.N., 2006. Progress and prospects of marker assisted backcrossing as a tool in crop breeding programs. J. Biotechnol. 5, 2588 2603. Sengupta, S., Majumder, A.L., 2009. Insight into the salt tolerance factors of a wild halophytic rice, Porteresia coarctata: a physiological and proteomic approach. Planta 229, 911 929. Subudhi, P.K., Baisakh, N., 2011. Spartina alterniflora Loisel, a halophyte grass model to dissect salt stress tolerance. In Vitro Cell. Dev. Biol. Plant 47, 441 457. Tagle, A., Fujita, D., Ebron, L., Telebanco-Yanoria, M., Sasaki, K., Ishimaru, T., et al., 2016. Characterization of QTL for unique agronomic traits of new-plant-type rice varieties using introgression lines of IR64. The Crop J. 4(1), 12–20. Vivek, P.J., Tuteja, N., Soniya, E.V., 2013. CDPK1 from ginger promotes salinity and drought stress tolerance without yield penalty by improving growth and photosynthesis in Nicotiana tabacum. PLoS One 8, e76392.

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Wu, J., Cai, G., Tu, J., Li, L., Liu, S., Lou, X., et al., 2013. Identification of QTLs for resistance to sclerotinia stem rot and BnaC.IGMT5.a as a candidate gene of the major resistant QTL SRC6 in Brassica napus. PLoS One 8 (7), e67740. Yonemaru, J., Yamamoto, T., Fukuoka, S., Uga, Y., Hori, K., Yano, M., 2010. QTARO: QTL annotation rice online database. Rice 3, 194 203. Zeng, H., Xu, L., Singh, A., Wang, H., Du, L., Poovaiah, B.W., 2015. Involvement of calmodulin and calmodulin-like proteins in plant responses to abiotic stresses. Front. Plant Sci. 6, 600. Zhang, J., Ulrik, P.J., Wang, Y., Li, X., Gunawardana, D., Polotnianka, R.M., et al., 2011. Targeted mining of drought stress-responsive genes from EST resources in Cleistogenes songorica. J. Plant Physiol. 168, 1844 1851. Zhang, H.H., Wang, Y.Q., Xia, J., Li, Z., Shi, Y., Zhu, L., et al., 2013. Simultaneous improvement and genetic dissection of grain yield and its related traits in a backbone parent of hybrid rice (Oryza sativa L.) using selective introgression. Mol. Breed. 31, 181 194. Zhu, J.K., 2001. Plant salt tolerance. Trends Plant Sci. 6 (2), 66 71.

Further reading Analiza, G., Tagle, D.F., Leodegario, A., Ebron, M.J.T., Kazuhiro, S., Tsutomu, I., et al., 2016. Characterization of QTL for unique agronomic traits of new-plant-type rice varieties using introgression lines of IR64. Crop. J. 4, 12 20. Ardie, S.W., Liu, S., Takano, T., 2010. Expression of the AKT1-type K(1) channel gene from Puccinellia tenuiflora, PutAKT1, enhances salt tolerance in Arabidopsis. Plant Cell Rep. 29 (8), 865 874. Bayat, F., Shiran, B., Belyaev, D.V., 2011. Overexpression of HvNHX2, a vacuolar Na1/ H1 antiporter gene from barley, improves salt tolerance in Arabidopsis thaliana. Aust. J. Crop. Sci. 5 (4), 428 432. Cheng, L., Li, X., Huang, X., Ma, T., Liang, Y., Ma, X., et al., 2013. Overexpression of sheep grass R1-MYB transcription factor LcMYB1 confers salt tolerance in transgenic Arabidopsis. Plant Physiol. Biochem. 70, 252 260. Endo, N., Yoshida, K., Akiyoshi, M., Yoshida, Y., Hayashi, N., 2005. Putative UDPgalactose epimerase and metallothioneine of Paspalum vaginatum enhanced the salt tolerance of rice, Oryza sativa L. from transplanting to harvest stages. Breed. Sci. 55, 163 173. Fabien, N., Kostlend, M., Cécile, C., Josep, M.C., 2016. Genome engineering and plant breeding: impact on trait discovery and development. Plant Cell Rep. 35, 1475 1486. Fu, J., Zhang, D.F., Liu, Y.H., Ying, S., Shi, Y.S., 2012. Isolation and characterization of maize PMP3 genes involved in salt stress tolerance. PLoS One 7 (2), e31101. Gao, Z., He, X., Zhao, B., Zhou, C., Liang, Y., Ge, R., et al., 2010. Overexpressing a putative aquaporin gene from wheat, TaNIP, enhances salt tolerance in transgenic Arabidopsis. Plant Cell Physiol. 51 (5), 767 775. Guan, Q., Li, L., Tetsuo, T., Liu, S., 2011. Cloning of an ascorbate peroxidase gene from Puccinellia tenuiflora and its expression analysis. Genom. Appl. Biol. 28 (4), 631 639. Huang, X., Wang, G., Shen, Y., Huang, Z., 2012. The wheat gene TaST can increase the salt tolerance of transgenic Arabidopsis. Plant Cell Rep. 31 (2), 339 347. Karan, R., Subudhi, P.K., 2012. A stress inducible SUMO conjugating enzyme gene (SaSce9) from a grass halophyte Spartina alterniflora enhances salinity and drought stress tolerance in Arabidopsis. BMC Plant Biol. 12, 187.

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Lal, S., Gulyani, V., Khurana, P., 2008. Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica). Transgenic Res. 17 (4), 651 663. Li, F., Guo, S., Zhao, Y., Chen, D., Chong, K., Xu, Y., 2010. Overexpression of a homopeptide repeat-containing bHLH protein gene (OrbHLH001) from Dongxiang Wild Rice confers freezing and salt tolerance in transgenic Arabidopsis. Plant Cell Rep. 29, 977 986. Mahalakshmi, S., Christopher, G.S.B., Reddy, T.P., Rao, K.V., Reddy, V.D., 2006. Isolation of a cDNA clone (PcSrp) encoding serine-rich-protein from Porteresia coarctata T. and its expression in yeast and finger millet (Eleusine coracana L.) affording salt tolerance. Planta 224 (2), 347 359. Majee, M., Maitra, S., Dastidar, K.G., Pattanaik, S., Chatterjee, A., Hait, N.C., et al., 2004. A novel salt tolerant L-myo-Inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt tolerance phenotype. J. Biol. Chem. 279, 28539 28552. Nishiuchi, S., Liu, S., Takano, T., 2007. Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerance to oxidative, salinity and carbonate stress in Saccharomyces cerevisiae. Biotechnol. Lett. 29 (8), 1301 1305. Qiao, W.H., Zhao, X.Y., Li, W., Luo, Y., Zhang, X.S., 2007. Overexpression of AeNHX1, a root-specific vacuolar Na1/H1 antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants. Plant Cell Rep. 26, 1663 1672. Saad, R.B., Zouari, N., Ramdhan, W.B., Azaza, J., Meynard, D., Guiderdoni, E., et al., 2010. Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger “AlSAP” gene isolated from the halophyte grass Aeluropus littoralis. Plant Mol. Biol. 72, 171 190. Verma, D., Singla-Pareek, S.L., Rajagopal, D., Reddy, M.K., Sopory, S.K., 2007. Functional validation of a novel isoform of Na1/H1 antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice. J. Biosci. 32, 621 628. Wang, S., Chen, Q., Wang, W., Wang, X., Lu, M., 2005. Salt tolerance conferred by over-expression ofOsNHX1 gene in Poplar 84 K. Chin. Sci. Bull. 50 (3), 225 229. Xianjun, P., Xingyong, M., Weihong, F., Man, S., Liqin, C., Alam, I., et al., 2011. Improved drought and salt tolerance of Arabidopsis thaliana by transgenic expression of a novel DREB gene from Leymus chinensis. Plant Cell Rep. 30 (8), 1493 1502. Xu, J., Duan, X., Yang, J., Beeching, J.R., Zhang, P., 2013. Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots. Plant Physiol. 161, 1517 1528. Zhang, G.H., Su, Q., An, L.J., Wu, S., 2008. Characterization and expression of a vacuolar Na1/H1 antiporter gene from the monocot halophyte Aeluropus littoralis. Plant Physiol. Biochem. 46 (2), 117 126. Zhang, Y.M., Liu, Z.H., Wen, Z.Y., Zhang, H.M., Yang, F., Guo, X.L., 2012. The vacuolar Na1 H1 antiport gene TaNHX2 confers salt tolerance on transgenic alfalfa (Medicago sativa). Funct. Plant Biol. 39 (8), 708 716.

CHAPTER TWO

Plant tissue culture: agriculture and industrial applications Basit Gulzar, A. Mujib, Moien Qadir Malik, Jyoti Mamgain, Rukaya Syeed and Nadia Zafar Cellular Differentiation and Molecular Genetics Section, Department of Botany, Jamia Hamdard, New Delhi, India

2.1 Introduction Plant tissue culture (PTC) is one among the group of biotechnological methods, utilizing in vitro strategies and techniques to culture living plant cells under controlled conditions. The technique is highly dynamic and could be varied to suit the particular type of cell culture, aim, and objective of the work. It has certain limitations too, that is, not all plants could be grown in vitro. PTC has immensely contributed in the field of science for the last 100 years and particularly at the latter half of 20th century after the discovery of somatic embryogenesis (SE) and application of innovative scientific techniques in tissue culture, for example, molecular and proteomic approaches. This includes from simple culturing of cells for study purpose or academic interest to the development of whole plant from a single cell for taping its economical potentials and industrial applications like metabolite productions (Yeole et al., 2016). Together with molecular techniques, PTC significantly contributed in altering genetic traits through gene transfer (Brown and Thorpe, 1995). Despite being at its preliminary stage, currently nanotechnology is being utilized in tissue culture, heralds a new phase in this field. PTC plays a pivotal role in agriculture and plant breeding as it complements crop production through micropropagation, somaclonal variation, hybridization, cybridization, synthetic seed production, haploid culture, hairy root culture, preservation of germplasm, pathogen eradication, etc. (Yeole et al., 2016; Basu et al., 2011; Tazeb, 2017). In India, according to the Department of Biotechnology (DBT), PTC has the potential to revolutionize the agriculture. Thus the DBT created National Certification System for Tissue Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00002-2

© 2020 Elsevier Inc. All rights reserved.

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Culture Raised Plants, a platform in 2006 under the aegis of 1966 Seeds Act. With the growth of agriculture, horticulture, forestry, and plantation crops, the demand for high-yielding, high-quality disease-free planting stocks have been increased in last 20 years. Tissue culture is being used to meet the high demand of these elite stock of plants as it multiplies them at a rapid rate at any given time and irrespective of season. Plants propagated commercially via tissue culture in India include anthurium, apple, bamboo, banana, date palm, gerbera, lilium, orchids, pineapple, potato, pomegranate, strawberry, sugarcane, and teak (Department of Biotechnology, India). The compounds like medicinals, essences, colorants, flavors, and other secondary metabolites, whose production is costly and uneconomical or could not be synthesized by chemical synthesis or microbial cells, could be potentially synthesized by using tissue culture method (Matkowski, 2008; Smetanska, 2008; Kumar and Reddy, 2011). High demand for medicinal plants particularly in developing world (over 80%) has led to the overexploitation of many medicinally important plants (Pant, 2014; Chen et al., 2016). Presence of multiple chiral centers and complex structure of medicinally important secondary metabolite makes their chemical synthesis expensive and commercially unviable (Hussain et al., 2012a, b; Ghirga et al., 2017). These factors have led to direct extraction of the chemicals from plants resulting in overexploitation of plants to meet the demands threatening their existence (Sharma et al., 2010). Thus to boost the production of these economically important secondary metabolites without threatening the survival source plant, scientists are successfully using in vitro cell and PTC. A large number of medicinally important chemicals have been commercially produced via tissue culture methods using cell, tissue, organ, or whole plant culture. The commercial production of the chemicals at mass scale especially in bioreactors has generated the economic interests in this field (Dornenburg and Knorr, 1996). Various elicitors have been shown to augment the production of chemical/secondary metabolites in vitro. PTC also has certain limitations to the commercial production of certain metabolites. These include slow growth, long dividing phase of cells, cell wall damage by industrial equipment, genetic instability, light, wall adhesion, mixing, product secretion, and the varieties of cells present in the culture (Zhong, 2001; Smetanska, 2008). The last one can be addressed by identifying and selecting the cells that are highly active in the metabolite synthesis and subsequently cloning them. This chapter describes the importance of tissue culture for genetic transformation studies and illustrates the viability of transgenic methods for clonally producing valuable germplasm.

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2.2 Micropropagation as a multiplication method Micropropagation, the technique of mass production of propagules and plants, has met the industrial and other demands in several instances and has served as an important tool in propagating the medicinal and crop plants (Gosal et al., 2010; El-Esawi, 2016). The micropropagation of Tylophora indica, a climbing perennial vine, producing tylophorine (alkaloid) is successfully achieved (Fig. 2.1). Micropropagation could be divided into various phases (Torres, 1989).

2.2.1 Stage 0: Preparation of donor plant In this stage, the plant is prepared for in vitro culture. The healthy and vigorous plants are grown in the good and suitable conditions preventing it from pathogens and viruses. The disease-free explant is desirable for the successful plant propagation program through PTC (Janse and Wenneker, 2002; Teixeira da Silva et al., 2015). The explant is inoculated in aseptic conditions and all the subsequent plant clones are obtained from it.

2.2.2 Stage I: Initiation stage Healthy and suitable explants after all preliminary and preparatory steps, that is, isolation, collection, and surface sterilization, are inoculated on the suitable medium containing all the nutritional and plant growth regulator (PGR) requirements. The medium mostly used is MS (Murashige and Skoog, 1962). The medium consists of macronutrients, micronutrients, amino acids, sugar alcohol (inositol), carbohydrate, and iron. Suitable PGR is added and pH is maintained. The gelling agent, that is, agar is added and the whole system is sterilized. The sterilized medium solidifies and is ready for inoculation under aseptic condition.

2.2.3 Stage II: Multiplication stage This phase includes the repeated subcultures of propagules so as to multiply them as per the requirement. The PGR combinations are manipulated or varied as per the requirement. Multiple shoots appear after two three weeks (depending on the type of plant, explants, etc. e.g., in shoot apices and axillary buds, the meristematic areas directly give rise to multiple shoots in less time as compared to other explants). The shoots developed may be further multiplied by repeated sub culturing.

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Figure 2.1 Micropropagated Tylophora indica plant: (A) callus induced from leaf tissue; (B) and (C) single and multiple shoots; and (D) rooted plant (Bar A: 1.5 cm; B D: 0.5 cm).

2.2.4 Stage III: Rooting stage The shoots developed in stage II are inoculated for root development in the same or different medium. It has been shown that the rooting medium mostly consists of higher auxin and lower cytokinin, a combination where roots are developed specifically. Root development takes some time, which differs in different plants, explants, PGR concentration, etc.

2.2.5 Stage IV: Acclimatization stage Plants grown in in-vitro condition are under standard and controlled environment conditions and hence are not exposed to harsh

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environmental vagaries. The propagules as such transferred to the field would not establish into mature plant and need to be weaned and hardened. The propagules are accustomed by carefully reducing the humidity and altering light requirements, transferred to pot containing sand, clay, peat, etc., and transferred to greenhouse and finally to the field (Kumar and Reddy, 2011). This whole phase takes time depending on the type of propagules. Millions of plants could be propagated from any source propagating material in laboratories at commercial scale in less time.

2.3 Organ cultures Organ culture can be defined as the organs or plant parts culturing in an artificial media or a culture from isolated medium. Any part of plant can serve as explants in organ culture-like shoot (for shoot tip culture), root (for root tip culture), leaf (for leaf culture), and flower (for anther, ovary, ovule cultures). Organ culture has been shown to be very much reliable in studies of the dependence on growth regulators and other growth factors. It also helps in broadening the horizon for the developments in agriculture and horticulture. Most important types of organ culture for in vitro plant propagation are meristematic culture, shoot tip, nodal culture of separate lateral bud, isolated root, and embryo culture. In vitro organ culture and metabolic composition of chemicals have been established in several plants like Fritillaria unibracteata. The plant species can be rapidly propagated from small cuttings of the bulb by the technique of organ culture and cultured bulb can be harvested after some period of culture in MS medium where growth rate, alkaloid, and microelements were reported to be many times (30 50) more in cultured bulbs. Hence, organ culture has been proved to be very significant tool for the enrichment of alkaloid in plants.

2.4 Somatic embryogenesis and synthetic seeds Somatic embryogenesis (SE) is the in vitro technique of the production of bipolar embryos from a single or group of somatic cells (Hofmann, 2014). In this technique, the explant is inoculated on medium containing specific PGR with various combinations and concentrations. The cells of explant after few days of inoculation start dedifferentiating into the mass

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of cells, that is, callus or directly give rise to the embryo without forming callus. The stress in microenvironment of the medium (hormonal, nutrient, oxygen, light) (Feher, 2015; Gulzar et al., 2019) triggers the process of the dedifferentiation in which complete transition from nondividing (somatic) to dividing cells occur. The stress imposed by environment triggers the cascade of signals by modulating many molecular switches that activates and deactivates thousands of genes (Feher, 2015). The complex process of SE after initiation passes through many stages including globular, heart, torpedo, and cotyledonary shape (Guan et al., 2016). Development of embryo formed via SE into complete plant is a crucial step because all somatically regenerated embryos do not establish into full plant. It has been shown that many plants fail to establish into a complete plant even though the somatic embryos are normal and healthy. This is a big limitation in the propagation of plants through SE (Isah, 2016). The SEs after development need to be carefully transferred to the field without any damage and viability (i.e., the plant should have the potential to establish itself in the field). The transferred embryos develop into mature plants, process known as conversion. It has been shown that the synthetic seed technology eases the aforesaid processes to a large extent. Synthetic seed is the artificially developed seed in which the embryo or any other plant propagating materials like axillary bud, cell aggregates, and shoot bud are encapsulated into a nutrient medium surround by a thin layer of gelling agent like sodium alginate, carrageenan, potassium alginate, and sodium pectate (Maqsood et al., 2012; Haque and Ghosh, 2014; Rihan et al., 2017). Synthetic seed methodology is a rapid tool for the regeneration of plant specially for those plants, which do not propagated through seeds naturally (Haque and Ghosh, 2014; Maqsood et al., 2012; Rihan et al., 2017). Despite its wide use in conversion and delivery of the tissuecultured plants, there are some limitations to this technology. All plants do not undergo SE and many plants among those that show SE produce limited number of viable embryos. The asynchronous development of somatic embryos that occur due to the variations in the genome generated through somaclonal variations limit the use of the technique in many plants (Ara et al., 2000; Magray et al., 2017). The conversion of somatically generated embryo into a complete plant is very crucial. Different plants have different conversion rates. The plant with high conversion rates is easily propagated by the synthetic seeds as compared to the SE having less conversion rates (Ara et al., 2000; Magray et al., 2017). Other limitation to this technique is the lack of dormancy in synthetic seeds and

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needs to be immediately sown without prolonged storage (Ara et al., 2000; Magray et al., 2017).

2.5 Haploid development via tissue culture Haploids are of much value to the plant breeders as they can be easily doubled when required to make every locus homozygous in a very short period of time (Basu et al., 2011; Tazeb, 2017). In haploids, the recessive genes are expressed and any gene that has significant function to yield or disease resistance (Basavaraju, 2011) or any other trait could be expressed through haploids (Basu et al., 2011; Tazeb, 2017). Haploids have been generated very fast and multiplied through tissue culture and then used for crop improvements. The in vitro haploids were generated through the culture of anther, pollen, ovary, ovule, etc. (Basu et al., 2011; Tazeb, 2017) Rice, potato, barley, wheat, maize, asparagus, tobacco, sunflower, and brassica are some of the plants in which haploidy has been induced and positive results were noted (Bajaj, 1990).

2.6 Pathogen-free plant propagation The healthy propagating materials free from pathogens are a prerequisite for the healthy growth and yield. It has been reported that the yield of the pathogen-free plants has higher yield than the field-grown plants. The most pronounced effect is shown by the plants that are vegetatively propagated (Brown and Thorpe, 1995; Tazeb, 2017). This is because if the initial material is infected by the pathogens, it cannot be eradicated effectively in the subsequent generations and hence reduces the potential yield of the plants. Tissue culture has been very successful in overcoming this problem in several crops and medicinal plants. The PTC has exploited the property of totipotency to regenerate the whole plant from the single or group of cells either by proliferating pathogen free or by regenerating from the disease resistance transformed cells (Brown and Thorpe, 1995; Basavaraju, 2011; Tazeb, 2017). The pathogen-free plants are being regenerated from the pathogen-free meristem cells derived from the plant shoot tips. The plant regenerated is multiplied by micropropagation, organogenesis, or SE and then transferred to the field after hardening.

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The technique has boosted the production of vegetatively propagated plants, for example, strawberry (Brown and Thorpe, 1995).

2.7 Tissue culture and plant breeding Tissue culture has played a very significant role in the field of plant breeding. Plant breeding creates genetic variability and tries to tap the beneficial variations in a population or living organisms. Plant breeding also evaluates the plants for/against different traits and attributes (Hussain et al., 2012a,b; Tazeb, 2017). Many genetically modified plants have been developed for the last two decades by the help of genetic engineering techniques (Bawa and Anilakumar, 2013). These plants were developed by using either a vector for transformation or nonvector means, for example, liposome, biolistic, microinjection, and electroporation methods (Jones, 2005; Bhalla, 2006). The vector used was Agrobacterium tumefaciens in which the Ti (tumor-inducing) plasmid transfers the T-DNA to the host plant. The DNA segment of interest has been inserted into the T-DNA (transfer DNA) by removing the nonessential part (part of plasmid that is not required for the act of transfer) (Gheysen et al., 1998; Gelvin, 2003; Wang et al., 2016). After the successful transformation, the engineered cells have been recovered and regenerated in vitro into complete plant. The most important and critical thing in any crop improvement program is the act of bringing the vital and beneficial traits together in a crop of interest. This is done either by genetic transformation or by hybridization program. Mostly, a single gene is being preferred to be transferred by the techniques of genetic engineering. To transfer more than one gene of interest, hybridization is preferred by plant scientists. Tissue culture has been the key in aiding the process of hybridization if the embryo is aborted and is unable to establish the plant. Embryo rescue via tissue culture has been used successfully to overcome the problem of abortion of embryo or inability of seed to develop (Tazeb, 2017).

2.8 Plant tissue culture and development of transgenic plants The word population is growing at an alarming rate and is estimated to be 9.7 billion by 2050 (FAO, 2017). The vast expansion in population size has created food crises worldwide and pushed the large section of

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human population toward state of destitution. Feeding such a huge population is a matter of concern and a big challenge before the world governments and other stakeholders. Crop improvement programs have led to the increase in production in various crops. The objectives of these program include increased production and improved traits like stress, herbicide tolerance, disease resistance, prolonged shelf life, and biochemical alterations through genetic engineering by transformation and alteration of various genomic segments (Tazeb, 2017). Tissue culture has been playing a very significant role in regeneration, mass multiplication, and propagation of whole plant from transformed or genetically modified cells or tissues. This is one of the most important steps in tissue culture-based genetic engineering program. The entire process of genetic modification via genetic engineering includes various steps: I. Selection of target plant/cell which is to be transformed Plant genetic transformations are targeted toward meeting the food and medicinal requirement of the growing population. The detailed morphology, physiology, and genetic makeup of the plants should be studied before transforming it with particular trait. The inplanta transformation and germline modification may not require regeneration via tissue culture. Apart from these, the other transformed cells are put in culture medium for regeneration, further propagated, and multiplied via tissue culture methodology. The identification, selection, and isolation of genetic segment of interest from particular organism are done according to the desired trait to be incorporated in the plant. II. Gene delivery systems to transfer the genetic segment of interest There are two classes of delivery systems, biological and nonbiological systems. The biological system includes Agrobacterium mediated and transformation through virus; and nonbiological procedure includes microinjection, sonication, particle bombardment, electroporation, lipofection, use of laser and fibers, and foreign DNA uptake by protoplast after stimulated chemically. III. The act of transformation The DNA segment transferred to the target should be stably integrated and expressed without the significant disruption in target genome that would compromise with or dilute the objective of whole process of transformation. Genetic transformation technique yielded the production of transgenic plants with enhanced agronomic traits. Agrobacterium-mediated transformation has been found to be an efficient tool for introducing gene of interest

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in different plants. Since the emergence of Agrobacterium-mediated transformation, this technique has been widely used for production of plants resistant to insects, diseases, and abiotic stresses (Naing et al., 2016). Various explants such as hypocotyls, nodes, leaves, cotyledons, petioles, and peduncle have been used for the recovery of transgenic plants via Agrobacteriummediated transformation (Jin et al., 2000). This method is widely practiced because of its high efficiency, low cost, simplicity, and low copy number of integration (Arockiasamy and Ignacimuthu, 2007). Embryogenic calli and protoplast were the primary tissue sources used for the establishment of Agrobacterium-mediated transformation system in rice. The other transformation methods such as electroporation, polyethylene glycol (PEG), and particle bombardment were also used as transformation system in rice. Agrobacterium-mediated transformation using shoot apex as explants instead of callus was reported to result in transgenic rice plants with superior qualities grains. Transgenic cucumber susceptible to a wide range of pathogens, and fungal and viral diseases showed enhanced viral resistance after transformation (Wako et al., 2001). Cotyledonary explants were used for the production of transgenic cucumber (Cucumis sativus L.) by using SE pathway. An efficient Agrobacterium transformation system using stem nodes as explants was also developed in cucumber. Leaf explants were used for efficient in vitro propagation of transgenic broccoli carrying two different Bacillus thuringiensis (Bt) genes cry1Ac and cry1C (Cao et al., 2002). The development of transgenic broccoli with improved tolerance to dehydration and other types of stresses such as heavy metal, salt, and hydrogen peroxide was reported earlier (Vinocur and Altman, 2005). PTC-based genetic modification provides a platform for incorporation of desirable traits in economically important plants like legumes. Tripathi et al. (2013) successfully demonstrated Agrobacterium-based transformation in chickpea using immature cotyledons as explants, which overcame its narrow genetic base and sexual incompatibility. In Azadirachta indica, hairy roots, cultured in Ohyama and Nitsch’s basal medium, showed enhanced yield of azadirachtin by B5-fold (0.074% dry weight, DW) in the presence of biotic elicitors, whereas jasmonic acid and salicylic acid improved B6 (0.095% DW) and B9-fold (0.14% DW) increases, respectively (Ramesh et al., 2007). Naina et al. (1989) reported A. tumefaciens-mediated genetic transformation in Azadirachta indica with an improved alkaloid composition. Transformation of Atropa belladonna hairy root cultures with rolABC genes showed enhanced tropane alkaloid production in root biomass up to 75 times (Bonhomme et al., 2000).

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Similarly in Artemisia annua L., the concentration of artimisin was increased about 4.8 mg/L by infection with Agrobacterium rhizogenes (Ghosh et al., 1997). Transformed organ cultures of Artemisia annua were established following infection with two wild-type nopaline strains of A. tumefaciens. Tumor-inducing frequency was, however, influenced markedly by strain, explant type, age of plant source, etc. and all transformed and nontransformed clones showed artemisinin but shooty teratomas produced enriched level of artemisinin (0.063 g/100 g DW). Belny et al. (1997) reported A. tumefaciens-mediated transformation in which A. tumefaciens strain GV3101(pMP90) containing binary vector pTHW136 (consisting of uidA reporter gene and nptII selectable gene, encoding enzymes β-glucuronidase, and neomycin phosphotransferase II) or binary vector pO35SSAM (consisting of sam1 gene and nptII gene, encoding the enzyme S-adenosyl-L-methionine (SAM) synthetase) was cultivated together with hypocotyl-derived cell suspension cultures of Papaver somniferum. Out of five transformed cell lines, one expressed a significant overactivity of SAM synthetase. Pradel et al. (1997) reported higher amounts of anthraquinones and flavonoids in transformed hairy roots than in nontransformed opium (P. somniferum L.). Genetic transformation of Eschscholzia californica Cham. (California poppy) through SE was attempted similar to P. somniferum except cocultivation medium and A. tumefaciens strain GV3101 carrying the pBI121 binary vector (Park and Facchini, 2000). Transformation of dwarf pomegranates (Punica granatum L. var. nana) was carried out using A. tumefaciens strains LBA4404 (Clontech) and EHA105 and both these strains possess binary vector pBin19-sgfp (Hood et al., 1993; Chiu et al., 1996; Ghorbel et al., 1999), containing neomycin phosphotransferase (npt II), driven by nos promotor and synthetic green fluorescent protein (gfp) gene driven by cauliflower mosaic virus. The A. tumefaciens strain EHA105 was noted to be the most effective for the transformation of dwarf pomegranate and the regenerated adventitious shoots from leaf explants showed high transformation rate in dark as compared to other fruit trees tested (Terakami et al., 2007). Importantly, the regenerated transformed plants produced fruits in short duration of time (Terakami et al., 2007). Miguel and Oliveira (1999) reported transformation of leaf explants in almond (Prunus dulcis Mill.) by using A. tumefaciens. The strain LBA4404 (Hoekema et al., 1983) carrying the plasmid p35SGUSINT (Vancanneyt et al., 1990) and EHA105 (Hood et al., 1993) carrying p35SGUSINT or the plasmid pFAJ3003 (DeBondt et al., 1996) were used as vector for

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Table 2.1 Some genetically transformed plants. S. No. Plant species Common name of plant Gene of interest

01 02 03 04 05 06 07 08 09

Atropa belladonna L. Vigna mungo L. Hepper Brassica oleracea var. italica Cicer arietinum L. Cucumis sativus L. C. sativus L. Oryza sativa L. Papaver somniferum L. Glycine max L.

Belladonna Black gram Broccoli Chickpea Cucumber Cucumber Indica rice Opium poppy Soybean

rolABC nptII/uidA athsp101/nptII/luc nptII//uidA nptII act/nptII/gfp hptII/gus & gfp SAM synthase & npt hptII/gus; 2 /hptII/gfp; gat/nptII/gus

transformation. The micropropagated shoots showed consistently positive results as histochemical GUS detection and PCR amplification studies indicated (Miguel and Oliveira, 1999). Malus zumi is a dwarfing apple, occurring in semiarid or saline soils; and gene manipulation of M. zumi through transgenic technology successfully modified various plant features. Transformation was attempted with A. tumefaciens strain EHA105 carrying the plasmid pBI121, which is a binary vector containing a kanamycin-resistant gene and an introncontaining β-glucuronidase (gus) reporter gene. Insertion of foreign gene in transgenic plants was confirmed by PCR analysis, and histological gus assay and southern blotting led to the generation of stress tolerant stock of M. zumi. Some genetically transformed plants are listed in Table 2.1.

2.9 Somaclonal variation and its importance in plant improvement Genetic variability has a huge role in the success of any plant breeding program. With the advent of new technologies such as PTC and recombinant DNA technology, a lot of progress has been made in increasing food production. Somaclonal variations are genetic alterations developed under in vitro stressed microenvironment. These could be epigenetic or nonchanges that may be heritable or nonheritable. Somaclonal variations lead to various genetic changes in plants, which can be used to change various plant characters such as plant height, yield, and

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grain quality and provide resistance to plants against diseases, pests, and drought (Patnaik et al., 1999). With change in all these characters, plant production can increase to counter the demand of alarming increase in population. Somaclonal variations have been reported in various varieties of different crop plants such as apple (Rosati and Predieri, 1990), potato (Das et al., 2000), banana (James et al., 2007), Heliconia bihai (Rodrigues, 2008), and sugarcane (Sengar et al., 2009). Somaclonal variations in sugarcane were helpful in increasing its sugar yield and improving the resistance to eye-spot disease (Sengar et al., 2009). Similarly, in potato, it was used to improve its tuber shape and uniformity, and provide resistance against late blight disease (Das et al., 2000). Various new traits were recovered, which were not present in natural gene pool such as glyphosate resistance in tobacco, atrazine resistance in maize, increasing methionine and lysine contents in cereals and fungal resistance in alfa alfa (Sengar et al., 2009).

2.10 Protoplast culture and somatic hybridization In sexually incompatible species, protoplast fusion and somatic hybridization have been effective approach in developing unique hybrid plants (Gosal and Kang, 2012). Cell wall surrounding the cell creates a major obstacle in somatic hybrid formation. However, protoplast, that is, naked cell without cell wall with intact cell membrane can easily be fused to introduce various genetic modifications in plant cell. Protoplast technique involves couple of steps starting with protoplast isolation, fusion of protoplasts, culturing of protoplast, plant regeneration, and at last characterization of regenerated plant. Protoplasts are usually isolated by digesting the outer cell wall with enzymes like cellulases, pectinases, or hemicellulases. Isolation of protoplast is also done by chemical method using various chemical agents. Protoplast fusion is accomplished by addition of PEG with high concentration of calcium at pH of 8 10 and by electrofusion (Olivares-Fuster et al., 2005). Somatic hybrids are formed by the fusion of nuclei and cytoplasm of two species. After the production of first somatic hybrids involving Nicotiana glauca and N. langsdorfi, various intraspecific, interspecific, and intergeneric somatic hybrids have been generated. Biotic and abiotic stresses such as fungal diseases and bacterial diseases greatly affect crop production. Desirable traits/genes such as disease-resistant,

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high-quality, and cytoplasmic male sterility from various intergeneric or intrageneric cultivated crops can be introduced in breeding programs via protoplast fusion, overcoming all sexual barriers (Liu et al., 2005). Application of protoplast in crop improvement has been best depicted in Nicotiana where protoplast fusion was used to change the alkaloid and disease-resistant traits of tobacco. Nouri-Ellouz et al. (2006) reported the use of protoplast fusion in incorporating disease resistance to Solanum tuberosum. Likewise, protoplast fusion in Brassica aided in producing number of somatic hybrids including B. napus 1 Crambe abyssinica (Wang et al., 2003, 2004), B. napus 1 Camelina sativa (Jiang et al., 2009). Lately, protoplast fusion was used to transfer the transformed Lesquerella fendleri chloroplasts into Orychophragmus violaceus to alleviate the effect of transgenic transformation (Ovcharenko et al., 2011).

2.11 Elicitation for enrichment of phytocompounds Elicitors are the chemicals, biotic or abiotic factors that are used to augment the secondary metabolites/phytoallexin accumulation by altering the physiological system of the cell. The use of elicitors in vitro has been tremendously increased since mid-1990s. Elicitors have played a very significant role in the accumulation of secondary metabolite and hence increased their yield. Zafar et al. (2017) reported significant increase in reserpine accumulation in callus of Rauwolfia serpentina. Increase in vincristine and vinblastine synthesis by the use of abiotic elicitor, NaCl was also reported in Catharanthus roseus (Fatima et al., 2015). The treatment of Aspergillus flavus (fungus), a biotic elicitor, improved callus biomass growth and later augmented in accumulation of alkaloids in Catharanthus (Dipti et al., 2016; Maqsood and Mujib, 2017) (Table 2.2). In the hairy roots of Panax ginseng, abiotic elicitors are extensively used to enhance growth and biosynthesis of ginseng saponin. Elicitor treatments inhibited the growth of the hairy roots and improved ginseng saponin biosynthesis instead. Selenium and NiSO4 addition improved ginseng saponin content several times compared to control without inhibiting the growth of the roots (Jeong and Park, 2006). Jeong and Park (2006) reported increased synthesis of ginseng saponin with sodium chloride treatment, an abiotic elicitor; however, chitosan polysaccharide had detrimental effect on anthraquinone production in Rubia akane cell culture (Jin et al., 1999).

Table 2.2 Biotic and abiotic elicitors’ role on alkaloid yield. S. Plant Elicitor Best elicitor No treatment

Tissue exposed

Alkaloid level tested

References

Vinblastine, vincristine Vincristine Vinblastine, vincristine Vinblastine, vincristine Vinblastine, vincristine Reserpine

Fatima et al. (2015)

Reserpine, ajmalicine

Zafar et al. (2017)

NaCl (A)

25 mM

02 03

Catharanthus roseus C. roseus C. roseus

CaCl2 (A) Yeast extract (B)

25 mM 1.5 mg/L

Leaves, shoots, and embryo Embryogenic calli Callus

04

C. roseus

0.15%

Somatic embryo

05

C. roseus

06

Rauwolfia serpentina R. serpentina

Aspergillus flavus (B) Fusarium oxysporum (B) AlCl3 (A)

0.15 mM

Callus

CdCl2 (A)

0.15 mM

Callus

01

07

A, Abiotic elicitor; B, biotic elicitor.

Somatic embryo

Maqsood and Mujib (2017) Dipti et al. (2016) Dipti et al. (2016) Zafar et al. (2017)

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2.12 Precursor addition There are enough evidences in which the precursors have been observed to increase secondary metabolic content in various types of cultures (Smetanska, 2008). Precursor is the compound/constituent, which has the ability to stimulate production of another important compound, if added to the culture media (Karuppusamy, 2009). For instance, Triterpenes enhanced and increased asiaticoside level many folds in leafderived callus and cell suspension of Centella asiatica (Karppinen et al., 2007). T˚umová et al. (2006) reported the effect of coniferyl alcohol, functioning as precursor of flavonolignan and noted improved silymarin and silydianin synthesis in Silybum marianum suspension. Similarly, coniferyl alcohol in the form of complex beta-cyclodextrin was used as precursor for podophyllotoxin accumulation in Podophyllum hexandrum cell suspension cultures (Karuppusamy, 2009).

2.13 Hairy root culture and genetic manipulation Hairy root culture is the culture raised after the infection of explants/cultures by the gram negative soil bacterium A. rhizogenes (Tepfer and Casse-Delbart, 1987). It is also known as transformed root culture, which is used to study plant metabolic processes, producing valuable secondary metabolites or recombinant proteins; it also helps in the analysis of gene (Georgiev et al., 2007). In this process, wounded explants are first inoculated with A. rhizogenes and after two or three days the explants are transferred to solid media with antibiotics, for example, cefotaxime for short period of time. Hairy root culture depicts a remarkable characteristic for a successful high scale-up biomass growth, stable production of secondary metabolites, sensitive to external stimuli and metabolite release in culture medium. The genetically transformed root cultures are basically defined as the involvement of additional bacterial genes in the culture by attachment of bacteria to the roots (Sheludko and Gerasymenko, 2013). This method has its own importance in tissue culture and has proved to be attractive, cost-effective options for mass producing desired plant metabolites and expressing foreign proteins. El-Esawi and coworkers (2017)

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reported that the transgenic hairy roots had increased yield of phenolics, flavonoids, and antioxidants in Lactuca serriola. Transformation of Trifolium pratense with A. rhizogenes was the first report proving to develop a fast-growing hairy-root culture system producing enriched level of pharmaceutically important isoflavones. Genetic stability with understanding of molecular mechanism of T-DNA transfer in transgenic roots showed increased use in metabolic engineering and production of recombinant proteins in investigated plants (Giri and Narasu, 2000; Marisol et al., 2016).

2.14 Endophytes and secondary metabolites Endophytes are bacteria or fungi that live inside plant tissues without causing disease symptoms but often help in the enhancement of secondary metabolites and other important compounds (Wilson, 1995). Plants and endophytic fungi have distinct metabolic pathways for production of secondary metabolites, and this has been clearly observed through biosynthetic studies with radiolabeled precursor of amino acids (Zhang et al., 2009). Study of isolation of enzyme 10-deacetylbaccatin-III-10Oacetyl transferase led to the production of taxol from endophytic fungus Cladosporium cladosporioides MD213, which is isolated from Taxus media, a new species. This gene is involved in the biosynthetic pathway of Taxol and contributes 99% and 97% similarity with the plants T. media and T. wallichiana var. mairei (Chandra, 2012). This shows that plants and endophytic fungi through mutualistic symbiosis produce almost similar secondary metabolites.

2.15 Bioreactor scaling Bioreactors are the vessels used for large-scale production of secondary metabolites (Panda et al., 1989). Initially, bioreactor was employed for Begonia micropropagation and later applied to many species and plant organs like shoots, bulbs, microtubers, corms, and somatic embryos. There are different types of bioreactors; air lift bioreactors are used for the suspension cultivation of Frangula alnus cells to produce anthraquinones

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(Sajc et al., 1995), and plantlet production for Oxalis triangularis. In Lilium Oriental Hybrid, bubble bioreactors have been used for bulblet production. Mujib et al. (2014) used small bioreactor for somatic embryomediated shoot regeneration in Catharanthus roseus, whose regeneration form somatic embryos is not that common. Culture of Dioscorea deltoidea and Panax ginseng in bioreactors for the production of diosgenin and other alkaloids has showed significant increase as compared to control. All the examples indicate that there is enough scope for this technique in commercial production of secondary metabolites.

2.16 Immobilization scaling Immobilization scaling can be defined as a technique, which involves catalytically active enzyme or a cell within a reactor preventing its entry into mobile phase carrying the substrate and product. It is the production of large amount of plant cell culture by fixing the cells, considered to be the most natural method (Murthy et al., 2014). Studies on Plumbagin, which is an important medicinal compound of Plumbago rosea (Binoy et al., 2014), were performed by immobilizing the cells in calcium alginate and cultured in Murashige and Skoog’s basal medium containing 10 mM CaCl2. It was observed that two- to threefold increase in the production of plumbagin was obtained in immobilized, CaCl2-treated cells than the control and nonCaCl2-treated cells (Komaraiah et al., 2003).

2.17 In vitro germplasm storage Germplasm conservation is mainly aimed to ensure the proper preservation and storage of germplasm belonging to economically important plants (Gosal et al., 2010). Germplasm of agricultural crops having seeds are easily stored for longer time by lowering their water content. However, it is very difficult to store the germplasm of vegetatively propagated plants by conventional storage methods. In vitro conservation techniques have lot of pros as compared to in vivo conservation as it lessens the space and time of storage and allows conservation of endangered plant species, and preservation of plants under in vitro conditions (Engelmann, 2011). Cryopreservation is one of the

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cost-effective and long-term storage methods of germplasm, in which biological material is stored at a very low temperature of about 2196 C. At this low temperature, all the metabolic activities of cell cease, which helps in storing the material for longer time without any alterations or modifications. PTC has wide application in collection, multiplication, and storage of germplasm. In various explants of both temperate and tropical plants such as cell suspensions, calluses, somatic, and zygotic embryos, cryopreservation techniques have been successfully employed (Gonzalez-Arnao et al., 2008). Various crop plants have been completely regenerated from their tissues stored at very low temperature from several months to years (Ding et al., 2008). Cryopreservation has also been used for eradication of viruses from affected plants by cryotherapy (Bettoni et al., 2016). Helliot et al. (2002) after cryopreservation noticed the elimination of about 30% and 90% of cucumber mosaic virus (CMV) and banana streak virus (BSV), respectively. Hence, cryopreservation is useful technique not only for long-time preservation of plant samples but also helps in eradicating the pathogens from the sample to a large extent.

2.18 Conclusion and future perspective PTC has been successfully used for the propagation of plants that are not reproducing naturally at a pace with which they could meet the demands of mankind especially to the developing world. The mass production of commercially important compounds is being increased by integrating classical breeding techniques with novel and innovative techniques like micropropagation, SE, and synthetic seed. Genetically transformed cells in almost all cases have been regenerated into full plants via PTC. Developments of distant hybrids and cybrids, which were not possible easily through the conventional breeding methods, have successfully been developed by this same technique. PTC has significantly contributed in almost all fields in improving the plant production whether it is regeneration of plants from single cell, multiplication or propagation of plants, commercial in vitro production of secondary metabolites, or its utilization. There are some limitations of PTC, which need to be addressed in order to make the technique suitable to the plants where the response is very poor. Research needs to be augmented in plants, which are economically very potent and those which are at the verge of the extinction. Integration of PTC novel techniques and other biotechnological advanced

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tools with classical plant breeding, production, and plant improvement will be well within reality.

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CHAPTER THREE

Genome editing technologies for value-added traits in plants Usha Kiran1,2, Malik Zainul Abdin1 and Kamaluddin3 1

Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India Bioinformatics Institute of India, Noida, India 3 Department of Genetics and Plant Breeding, Banda University of Agriculture and Technology, Banda, India 2

3.1 Introduction Demand and consumption of plant produce for food, feed, and fuel is on rise due to growing global population. World crop productivity is faced with challenges to increase the produce, both in terms of quality and quantity, to meet prevailing hunger and malnutrition (Foley et al., 2011). An estimate by United Nations Food and Agriculture Organization (FAO) showed that about one billion people around the globe are suffering from malnutrition. Farmers, on the other hand, are facing decreased productivity. Depletion of soil nutrient due to excessive use of fertilizers and pesticides, loss of biodiversity due to excessive cultivation, and increase in uncertainties in climate due to industrialization and urbanization are major responsible factors for decreased productivity. Conventional breeding is a good and widely used tool for incorporating favorable traits, but is time taking, labor intensive, and cannot cross species barrier. Thus to meet the consumers’ demands, new interventions are required in farming technologies, globally, to increase the production of food, feed, and fuel without the commitment of new area for production (Tilman et al., 2011). The first successful genetic manipulation was done by Herbert Boyer and Stanley Cohen in 1972, which opened the door for more similar experimentation. The transcriptomic and metabolomic studies, using next-generation sequencing (NGS), microarray, GCMS, etc., generated lots of data. Advances in molecular and computational biology led to understanding of cell, its components, and ability to direct and inherit Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00003-4

© 2020 Elsevier Inc. All rights reserved.

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changes in progeny. This enabled scientist to attempt manipulations in genomic organization with unprecedented precision and speed for genome manipulation, using genetic engineering tools. Crossing genetic barriers to incorporate good traits has become possible. Thus the dream of farmers and breeders to match the demands of production to satisfy human hunger could be realized within the realms of time. Transgenic technology has been successful in introducing novel traits by transfer of transgene using physical, chemical, and biological methods of introduction of foreign DNA into the plant cell. The success, however, has always been surrounded by safety concerns and regulations with respect to human health and environment. Governmental concerns and regulations have created significant cost barriers and limited the use of genetically modified crops to limited number of crops (Prado et al., 2014). The ploidy levels in plant genomes are high and contain lots of repetitive sequences, which possess a challenge for heritable genetic manipulations. Genome editing is capable of causing targeted modification at genomic level by introducing double-stranded break (DSB) at specific sequence depending upon the sequence-specific nucleases (SSNs). Every cell has efficient endogenous system of nucleases and ligases to repair these DSBs. Cell repair system uses either homologous recombination (HR)causing gene insertion or replacements or nonhomologous end joining (NHEJ)-causing gene knockouts due to insertion or deletion of nucleotide. The gene manipulation rate using homologous recombination is less (Mengiste and Paszkowski, 1999) and most of the time, plant cell therefore uses NHEJ as principle DSB repair mechanism (Puchta and Fauser, 2014). The NHEJ pathway is, however, error-prone and DNA repair at the break point is often accompanied by some base insertions or deletions (indels). When these DSBs are in coding region of gene, the indels cause frame-shift mutations resulting in loss of function. This feature could be used to study gene function or eliminate undesirable traits for better quality crop produce (Gorbunova and Levy, 1999). HR repair requires homologous sequence upstream and downstream of DSB and therefore, typically error-free method. This pathway could be exploited for targeted gene insertions or gene replacements at predefined genomic location, enabling precise modification of genomes (Shan et al., 2013). Continued investigations had led to the development of genome editing with engineered nucleases (GEEN), a revolutionary tool for

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editing DNA sequences at genomic level, producing stable hybrids with suitable desired traits. This technology has been utilized extensively, in last decade, to understand gene function and improve agronomic and agricultural traits (Osakabe and Osakabe, 2015; Shan et al., 2013). Zinc fingers coupled with Fokl endonuclease domain were shown to be an efficient gene-editing tool (zinc-finger nucleases; ZFNs). These chimeric proteins with modular structure act as site-specific nucleases and hence allow efficient editing at genomic level. Transcription activatorlike effector nucleases (TALENs) have also been used for genome editing efficiently. In this review, we briefly described the principles of different genome editing systems and their application for crop improvements.

3.2 Techniques of genome editing From the start of civilization, the improvements of genetic traits among the cultivated plants were of great interest to the society. The selection of suitable traits is an ongoing process in nature. The understanding of traits and their inheritance pattern were applied in conventional breeding, which aims to select the best-suited complement of genetic material by mixing of desired characteristics from different population of same species. The limitation of this method was that the selected characters were not new to the species and hence were not able to impart any new advantage. This led to the search for new methods to incorporate novel genetic material within the species or outside the species. Genetic engineering involves manipulation of genetic material by insertion, deletion, or replacement of single or multiple nucleotides. Conventional random mutagenesis and transgene integration have been of limited use in agricultural improvements. Insertion of genetic elements at random location leads to disruption of unwanted gene structure and function, creating element of unpredictability. Thus introducing novel traits into plants remained labor-intensive, time-consuming, and unpredictable, before this decade. With the understanding of restriction endonucleases (RE) and ligases’ role in cells repair system, they were seen as new tools for genome editing. Restriction endonuclease cuts DNA molecule at or near a specific nucleotide sequence. RE however, are reported to work

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efficiently for small bacterial genomes as they are specific for short DNA sequence. Further research led to development of engineered nucleases that cleave DNA at specific sequences, which is directed by sequence-specific DNA-binding domain or RNA-guided sequence (Osakabe and Osakabe, 2015). These new tools precisely and efficiently edit large and complex genomes of higher organisms. Sequence-specific nucleases (SSNs) are easily constructed. The SSNs contain recognition domain with short sequence proximal to the DNA break site therefore they cleave genomic DNA by site-specific method triggering the endogenous repair systems that result in targeted genetic modification. Since SSNs are integrated into the genome therefore, are transmitted to progeny. Programmable nucleases such as ZFNs and TALENs are the important contributors in crop improvement for last two decades. Clustered regulatory interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is the newest addition in the designer genome editing tool box. Using these nucleases targeted alteration in genome can be carried out easily by inserting gene of novel trait (knocking in), inactivation of gene by inserting unwanted DNA fragment (knocking out), and gene modification contributing to increased crop yield and without bringing additional area under cultivation. The characteristic features of ZFNs, TALENs, and CRISPR/Cas9 are listed in Table 3.1. These targeted chimeric nucleases are also used to study genome arrangement, gene function, and evolutionary events on the timeline. The tool box is effective in imparting pest resistance, biotic and abiotic stress resistance, pesticide and herbicide resistance, and improved nutritional value to the plants.

3.2.1 Zinc-finger nucleases Hershey and Chase in 1950s showed that DNA is the genetic material and after that all the molecular biologist turned toward DNA to solve the simple and complex problems of living beings. Further, the discovery of double helix structure of DNA by James Watson and Francis Crick opened the doors of molecular understanding of fundamental process of replication, transcription, and translation to control complex chemical process within the cell and organism. The development of recombinant technology came into existence after the discovery of restriction endonuclease by Werner Arber, Hamilton O. Smith, and Daniel Nathans. They

Table 3.1 Characteristic features of targeted chimeric sequence-specific nucleases used for genome editing in plant biotechnology. Protein Mode of Target DNA Target Mutation Multiplexing Structural DNACleaving Length of recognition recognition rates of genes features binding domain recognized engineering action efficiency domain DNA target sequence (bp)

ZFNs

Dimeric

Zinc Nonspecific 9
18 Required fingers FokI nuclease domain Required TALENs Dimeric TALE Nonspecific 30
40 FokI nuclease domain CRISPR/ Monomeric crRNA Cas9 22 and Not Cas9 PAM required sequence

Double- DNA
protein High strand interaction break

High

Double- DNA
protein High interaction strand break

Moderate Difficult

Single- DNA
RNA or interaction doublestrand break

Low

High

Difficult

Very easy

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were jointly awarded Nobel Prize in Physiology or Medicine in 1978. Currently, more than 800 different restriction enzymes from bacteria have been identified and used for primed editing of genomes, which make editing precise, fast, and easy. Zinc finger nucleases (ZNFs) were the first-generation chimerically engineered targetable DNA cleavage proteins designed to recognize unique sequence occurring naturally in target genome and cut at specific site. Each ZFN contains a zinc finger-based DNA-binding domain and a DNA-cleaving domain of the FokI restriction endonuclease. The designed ZF restriction endonucleases are precise genomic cutter due to fusion of two extremely specific domains. Each ZF domain is comprised of around 30 amino acids and has ββα structure with Cys2His2 structure stabilized by chelation of zinc ions (Pavletich and Pabo, 1991). The DNA-binding domain of ZF binds to genomic DNA by inserting its α-helix into major grove of double helix DNA. The two zinc fingers recognize a six base pair unique hexameric sequence of the DNA. Multiple zinc fingers link together to form ZFPs (zinc-finger proteins) and bind to longer DNA sequence. Each zinc finger recognizes a three base pair unique sequence of the DNA in a modular fashion. Thus ZFs can be engineered to bind a large range of DNA site by manipulating the number of fingers and the critical amino acids that contact DNA directly (Desjarlais and Berg, 1992). Once the ZFPs bind to target DNA sequence, a signal is passed to the DNAcleaving domain of the FokI restriction endonuclease. The native FokI recognizes the binding site as monomer but cleaves DNA molecule after dimerization (Bitinaite et al., 1998). The ZFP module recognizes the entire recognition sequence, whereas the cleavage module cleaves only one strand of DNA, thus requiring the dimerization for producing DSB. The binding of two ZFP monomers, each recognizing a 9-base pair inverted site is important for dimerization of catalytic domain (Smith et al., 2000). Thus ZFNs effectively have an 18 base pair-specific recognition site long enough to specify unique sequences in genomes of plants and animals. Phylate biosynthesis in maize was disrupted using ZFNs. Phylate has limited nutritional value and is considered as pollutant in animal waste. Inositol-1,3,4,5,6 pentakisphosphate is an important enzyme in phylate biosynthase pathway and is encoded by IPK1 gene in maize. ZFNs targeted to IPK1 gene in maize resulted in reduction in phytase levels. Shukla et al. (2009) designed 66 ZNFs against 5 target locus of IPK1.

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The disruption of locus was carried out by ZFNs followed by repair (homologous recombination) using short, locus-specific homologous arm and herbicide-resistant gene. Results showed several monoallelic insertions and one biallelic insertion of herbicide cassette at IPK1 locus. Expected segregation frequencies were obtained indicating that the changes were heritable. In the last two decades, customized ZFNs have been successfully used to introduce targeted alterations (point mutations, deletions, insertions, inversions, duplications, and translocations) in Arabidopsis, maize, and soybean (Curtin et al., 2011; Shukla et al., 2009; Zhang et al., 2010).

3.2.2 Transcription activator-like effector nucleases A search for an alternative selective and efficient manipulation of target genomic sequences led to the discovery of unique transcription activatorlike effector (TALE) proteins that recognize specific plant promoter through a set of tandem repeats (Kay et al., 2007; Römer et al., 2007). These unique genome editing tools are constructed by fusing the customizable DNA-binding domain (TALE repeats) and nonspecific catalytic domain of FokI restriction endonuclease (Joung and Sander, 2013). Recent reports show that these TALE-based activators and repressors modulate endogenous gene expression in plants and animals (Cermak et al., 2011; Miller et al., 2011). Originally, TALEs were identified in plant pathogens, and the system is successfully used for genome editing in plants. Xanthomonas bacteria colonize the host plant cell by injecting TALEs, which alters the transcription by binding to the genomic DNA (Boch and Bonas, 2010). The DNA-binding domain consists of up to 30 copies of highly conserved 33
35 amino acid residues with highly variable residues at 12th and 13th positions. These repeat variable diresidues (RVDs) are instrumental in designing TALENs with specific nucleotide recognition. Each repeat unit is specific for single base, and hence, a repertoire of binding sites can be made for any DNA sequence. Recent studies show that TALEs form right-handed superhelix, which wraps around DNA such that the hypervariable RVDs are positioned in the DNA major drove (Mak et al., 2012). The structure of domain is stabilized by interaction of 8th and 12th residues in same repeat and basespecific contacts to DNA is made by amino acid at 13th position (Deng et al., 2012; Mak et al., 2012).

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TALENs were first used for crop improvement in rice. TALEs are very important determinants in inducing host genes for establishment of pathogen. The blight susceptibility gene Os11N3 (also called OsSWEET14) in Oryza sativa was disrupted to develop resistance, using TALENs. Xanthomonas oryzae pv. oryzae (Xoo) activates Os11N3 gene using its TALEs to divert plant sugar to meet pathogen’s energy demand. Successful disruption of Os11N3 resulted in resistance to the pathogen.

3.2.3 Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR associated) The quest for easy programmable nucleases for targeted genome engineering continued even after the discovery of ZFNs and TALENs. In the early days of this decade, several research groups reported new class of genome editing nuclease called RNA-guided engineered nucleases (RGENs). RGENs, similar to earlier ZFNS and TALENs, cleave genomic DNA at specific sites triggering endogenous DNA repair cascade resulting in targeted genome modification. The only difference is that in RGENs, the specificity is guided by a small RNA molecule and not by DNAbinding protein. In bacteria, the adaptive immunity is primed by RNA-guided DNA recognition and cleavage (type II nuclease CRISPR-CAS protein) against invading plasmids and phages (Makarova et al., 2006). The naturally occurring CRISPR system in bacteria often captures small DNA fragments (B20 base pairs) from the DNA of the invading plasmids or phages and inserts them (called protospacers) into their own CRISPR cluster. In Streptococcus pyogenes, type II CRISPR loci are composed of operons of a precursor crRNAs (pre-crRNAs), two noncoding CRISPR RNAs (crRNAs), a trans-activating crRNA (tracrRNAs), and a Cas9 nuclease. In type II CRISPR system, these CRISPR regions are first transcribed into pre-CRISPR RNA (pre-crRNA) and processed to produce targetspecific crRNA (Sander and Joung, 2014). These crRNA (protospacer) are B40 base pair long and contain protospacer adjacent motif (PAM) region complementary to the foreign DNA fragment. Further, targetindependent tracrRNA is also transcribed from CRISPR locus and hybridizes with crRNA to form a guide RNA (gRNA). Processed crRNA and tracrRNA activate Cas9 system and combine with Cas9 to form active DNA nuclease. The endonuclease complex cleaves specific 23 base pair target DNA sequence. The specificity is attributed to 20 base pair guiding sequence at the 50 end of protospacer (crRNA), which directs the

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nuclease to form complementary pair with target DNA (Sander and Joung, 2014). The presence of PAM region after the target DNA sequence is a prerequisite for cleavage by Cas9. Usually, PAM motif contains 50 -NGG-30 or 50 -NAG-30 ; however, other sequences are also being used depending upon the target sequence to be modified. The sequence present at B12 nucleotides upstream of PAM plays an important role in imparting specificity to CRISPR/Cas9 nuclease. RGENs are easy to engineer, affordable, scalable, and specific to the target genome sequence nucleases (Bortesi and Fischer, 2015). The sequence of the target genome is cleaved after recognition by B20 nucleotide gRNA followed by the presence of PAM, which is recognized by Cas9. Thus programmable RGEN with guided sequence (50 X20-NGG-30 or 50 X20-NAG-30 ) is easily synthesized by cloning B20 nucleotides and PAM specific for the target genome (Hsu et al., 2013).

3.3 Application of genome editing systems 3.3.1 Multiplexing and trait stacking The crop breeders and farmers have been introducing functional alterations successfully in the crop plants for quite a few decades to meet the responsibility of producing low cost, safe, and nutritional food. The continual effort to understand the genetic mechanism underlying specific plant responses toward nutritional and environmental stress factors in the last decade resulted in the development of innovative genome editing tools. Meganucleases, TALENs, ZFNs, and CRISPR have revolutionized gene editing in plants and animals. With these powerful gene editing tools, scientists are now inserting or deleting plant gene with great ease and precision within limited time. Crossing genetic barriers to incorporate desired traits have become very easy leading to generation of genetically engineered plants with better nutrient utilization efficiency, disease resistance, and biotic and abiotic stress resistances. Plant responses at cellular levels are complex and regulated by large number of genetic and metabolic networks. To obtain a desired agronomic genotype, a precise engineering of complex metabolic pathway is required such that the manipulation leads to concerted expression of multiple genes. A genome editing tool with ability to modulate

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multiple genes, by altering DNA sequences at two or more loci in a single event, is of great value to plant breeders and scientists. The engineered nucleases such as TALENs, ZFNs, and CRISPR enable easy alteration of two or more target loci simultaneously in cells or whole plant. ZFN and TALENS have been successfully used for multiplex gene editing in plant resulting in targeted chromosomal rearrangements. Ainley et al. (2013) showed ZFN-mediated targeted transgene stacking of Aryloxyalkanoate dioxygenase (AAD1) and phosphinothricin acetyltransferase (PAT) in maize. Aryloxyalkanoate dioxygenase (AAD1) imparts herbicide resistance in maize. Li et al. (2016) used TALENs to knock out four Nicotiana benthamiana genes simultaneously for the production of glycoproteins devoid of plant-specific residues. Both ZFN and TALEN editing require re-engineering of their DNA-binding domains according to the target DNA sequence therefore, require skillful efforts and through understanding of molecular cloning and regulation. Due to dual component nature, separate expression vector, and simplified engineering of target specificity, CRISPR technology facilitates multiplexing of traits very efficiently. More recently, several groups have adapted Golden Gate cloning for assembling multiple gRNAs driven by independent promoters into single Cas9/gRNA expression vector (Sakuma et al., 2014; Lowder et al., 2015). CRISPR/Cas9 using stacked gRNA expression arrays was used to target 14 loci in Arabidopsis thaliana, simultaneously (Peterson et al., 2016). Schiml et al. (2014) were able to insert resistance cassette into ADH1 locus of A. thaliana as a heritable event by exploiting multiplexing feature of CRISPR/Cas9. Steinert et al. (2015) simultaneously used two alternative modified Cas proteins, from Streptococcus thermophilus (St1Cas9) and Staphylococcus aureus (SaCas9) to introduce targeted mutations in A. thaliana. Recent study reported albino phenotype in A. thaliana where two homologs of magnesium-chelatase subunit I (CHLI) were disrupted using CRISPR/Cas system. Magnesium-chelatase subunit I (CHLI) has important role in photosynthesis (Mao et al., 2013). Qi et al. (2016) demonstrated efficient introduction of four genes stacked together, using endogenous tRNA-processing, in maize. This strategy enabled generation of many double-strand breaks (DSB) in genomic DNA and improved the efficiency of the CRISPR/Cas9 editing in maize.

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3.3.2 High-throughput mutant libraries NGS technologies have enhanced the pace of sequencing of genomes. Plants genomes are large and complex; however, with evolution of various NSG platforms, scientists have augmented sequences from crop and medicinal plant genomes. The nucleotide databases are loaded with information on gene sequence and assigning function to all the sequences is a great challenge ahead of scientist in this post genomic-era (Wingender et al., 2000). Mutant libraries are important tools to systematically study genes in the genome and in finding novel genes associated with disease resistance, better nutrient utilization efficiency, and stress resistance. Mutants are generally produced by physical, chemical, and biological agents. Discovery of the mutagenic effect of X-rays on plant genes led to mutation breeding to understand dormancy, flowering, maturity, and plant architecture, and over 3000 novel cultivars have been developed and being used for producing food and feed (Stadler, 1928; Mba, 2013). This method, however, suffers from insertion of mutation at random site leading to labor-intensive and time-consuming screening of mutant libraries. Target-induced local lesions in genomes (TILLING) (Till et al., 2007) and T-DNA insertion (Jeon et al., 2000) are important methods for genomic manipulations done for basic and applied research in plants. TILLING and T-DNA insertion methods for generation of highthroughput mutant libraries are, however, very labor-intensive and timeconsuming. Large mutant clones must be prepared to ensure genomewide coverage. Also, T-DNA may randomly insert mutations in intergenic and noncoding regions. Recently, comparatively simple and highly efficient, SSNs have been used for generating mutant libraries. Genomewide sgRNA libraries have been generated allowing genome-scale loss of function or gain of function to characterize the sequenced genes in genome projects. Rice genome is completely sequenced, relatively small, and has highly efficient transformation protocol. These features make this plant useful to be used as model for construction of genomic mutant library. Baufume et al. (2017) used TALENs integration to construct an allele library of the SWEET14 promoter region of Oryza sativus to screen for susceptibility of rice blight cause by Xanthomonas oryzae pv. oryzae (Xoo). The loss of function caused inability of pathogen-derived effectors to bind to SWEET14 gene and ultimately conferred disease resistance. AvrXa7, TalC, PthXo3, and Tal5 C-EBEs are TALENs found in

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geographically distant rice blight strains and used to construct library for studying rice
Xoo interactions. The CRISPR/Cas9 system produces small indels by introducing double-strand breaks. This causes frameshifts in protein coding regions. In a recent study, 91,004 targeted loss-of-function mutants were generated using 88,541 sgRNAs against 34,234 rice genes, providing a useful resource for rice genome manipulation, research, and breeding (Lu et al., 2017). Meng et al. (2017) searched rice genome and identified 15,35,852 mutation target sites located in the exon regions of 52,916 genes. The sequence information (rice expression profiles database; RED) of 12,802 highly expressed genes from the base tissue of rice shoot and 25,604 corresponding gRNA was taken to generate more than 14,000 mutant lines. The results suggested that CRISPR/Cas9 screening method is robust for identifying mutant phenotype as well as functional genes.

3.3.3 Gene regulation The homeostasis inside the cell is maintained by close concerted expression and repression of genes in complex metabolic network. During the pathogen attack, a major alteration occurs in these networks. The transcriptomic analysis of pathogen and plant can provide a better understanding of virulence and defense mechanism of both pathogen and plant. Variation in expression of proteins, protein
protein interactions, and protein modification are being used to alter the genome of plant resulting in disease tolerance (Barakate and Stephens, 2016). SSNs gene editing tools are being used to study regulation of complex metabolic networks besides introducing gain of function or loss of function. The regulatory regions of endogenous genes in plants are targeted by integrating specific sequence with the DNA-binding domains of SSNs for up- or down-regulation of transcription, blocking the elongation of transcript and inhibiting RNA polymerase or transcription factor. CRISPR/Cas9 system has been successfully used for modulating the regulatory control of gene responsible for quality traits in plants (Barrangou et al., 2007). Rodriguez-Leal et al. (2017) used CRISPR/ Cas9 system to construct eight sgRNA against 2-kbp promoter region immediately upstream of SICLV3 gene coding sequence in tomato. The experiment resulted in large number of mutants. They evaluated the phenotypic impact of numerous promoter variants by studying the heterozygous loss of function for genes regulating plant architecture, fruit size, and

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inflorescence. The results opened the door for directly generating and selecting for most desirable variant with respect to the target quality trait gene. Dissection of transcriptome analyzes suggested important role for upstream open reading frames (uORFs) in regulation of expression of downstream primary ORFs (pORFs). These uORFs are mRNA with a start codon in 50 UTR, which is out of frame with the main coding sequence. Most of the plant mRNAs contain 50 leader sequence and about 20%
50% of these mRNAs contain uORF (Kochetov, 2008). These are potentially very effective targets for gene mutations. A single gRNA against 50 UTR region harboring a uORF initiation codon can produce multiple mutations (Zhang et al., 2018). LsGGP2 encodes key enzyme in vitamin C biosynthesis in lettuce and mutation in uORF of LsGGP2 showed increased ascorbate content (by B150%) and increased oxidative stress tolerance (Zhang et al., 2018).

3.3.4 Targeted structural changes in crop species Molecular studies on genes and genomes have enhanced our basic understanding of plant gene function, inheritance pattern, and their control over plant phenotypic characters. Gene clusters or tandemly arrayed genes (TAGs) are prevalent in plant genomes. In Arabidopsis, TAGs comprise .10% of the genes in the genome (Rizzon et al., 2006). The genesis of TAG creates genetic redundancy and a challenge for functional studies. Gene editing technologies can be efficiently used to delete TAGs. Qi et al. (2013) used ZFNs to target seven genes from three TAGs on two Arabidopsis chromosomes. The result showed deletions (few kb to 55 kb) with frequencies approximating 1% in somatic cells. In addition to small, NHEJ-induced mutations, CRISPR-mediated cleavage can also result in larger DNA sequence deletions. These large segment cleavages can effectively disrupt function of regulatory sequences, gene clusters, or regulatory elements in noncoding regions, which indel mutation fails to achieve. The methodology requires two specific sgRNA targeting two sites within same gene, gene cluster, or on the same chromosome. The process results in easy deletion of large fragment by NHEJ method. Deletion of gene clusters like homoeologs of a target gene which results in observable phenotypic change/s is possible by use nucleases technology. This could serve as basis for chromosome engineering and customized breeding programs.

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Cai et al. (2018) successfully introduced 4.5 kb targeted deletion of soybean flowering locus T (GmFT2a) using CRISPR/Cas9 nuclease. Their results showed that the induced structural alterations were inherited and hence may be applied for obtaining designer soybean crops in future. The marker genes used to assist the selection of transgenics are usually a concern for plant as well as consumers’ health removal of such markers could be easily achieved using nuclease technology. Petolino et al. (2010) removed beta-glucuronidase transgene flanked by ZFNs cleavage sites from a stably transformed tobacco plant. The transgenic was crossed with a plant expressing corresponding ZFN gene leading to progeny with removal of 4.3 kb marker gene used in first plant transformation.

3.4 Conclusion and future perspectives Currently, major advancements are needed in agricultural molecular biotechnology to enhance quantity and quality of the produce required by ever-increasing world population. Introduction of diverse agricultural traits requires necessity to dissect complex metabolic pathways for better understanding of relation between gene-regulatory changes and control of qualitative and quantitative traits. SSNs are the newest genome editing tools, which facilitate precise introduction of novel trait DNA sequence at specific place into genome. The ZFNs, TALENs, and CRISPR/Cas9 are simple, specific, and robust and can be adapted to any kind of DNA molecule. A targeted mutation reduces the undesired side effect of random mutations and is very helpful in breeding programs. The presence of DNA binding and repair using endogenous homologous DNA repair mechanism gives these editing tools specificity and ease of use. These gene-editing molecules are being used to study not only gene function by introducing indels in DNA sequence but also the regulation of gene clusters by altering very large fragments. The indels at specific position in targeted endogenous loci used for understanding genome structural organization and mechanism giving rise to the phenotypic expressions. This would lead to efficient hypothesis driven genome mining for novel trait development. Large targeted deletion experiments are important for reverse genetic approach. The large targeted DNA modifications will have huge impact on agricultural improvement through chromosomal engineering. To support such research, however, large genetic

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dataset describing the sequence specificities of different genome editing is required. Experimentally evaluated as well as in silico predicted reference database of model organism could be of great application.

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898. Available from: https://doi.org/10.1038/nbt.4202.

CHAPTER FOUR

Bioinformatic tools to understand structure and function of plant proteins Ahmad Abu Turab Naqvi1, Usha Kiran2,3, Malik Zainul Abdin2 and Md. Imtaiyaz Hassan1 1

Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India 3 Bioinformatics Institute of India, Noida, India 2

4.1 Introduction Ever-increasing world’s population and changes in the environmental conditions have put lot of stress on the plant kingdom for food and feed. The productivity of plants is greatly reduced by numerous abiotic and biotic factors, such as nutrient limitation, acidity, floods, drought, and salinity. The advances in research have increased our understanding about the interaction between organs, tissues, cells, and subcellular compartments, as well as cellular and metabolic processes. With the publication of number of completed plant genome sequencing projects, the plant research is now ready to leap beyond the threshold of genomics era. Genomic information and proteomic data are currently being used to assign function to annotated plant genes and to study the sequential changes in protein expression in the plants under normal and stressed conditions. This approach enables scientists to develop genotypes by identifying the appropriate genes, which can perform better in harsh environments. Although genome projects generated loads of genomic information; however, proteins have been and still are the center of attraction for plant molecular biologists due to their diversity in structure, function and ubiquitousness (Gutteridge and Thornton, 2005). A large number of experimental strategies have been devised to unravel the underlying mechanism of protein function, and to unearth the structural intricacies of the Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00004-6

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proteins (Conrotto and Souchelnytskyi, 2008). The methods and techniques used have witnessed improvisations that also gave birth to some new approaches during last few decades of experimental biology. Despite all these developments, laboratories have been facing certain limitations that are difficult to overcome. Some of the problems that experimentalists faced and still come across are related to data handling and analysis. Data produced by biological experiments (specifically protein related studies) are massive and have higher values than floating point numbers. Manual handling and analysis of these data is not an easy task. Novel innovation to increase the computing power coincided with the new development processes in life sciences-led sophophile molecular biologists turn toward computers to mine this enormous data deposit. This can be termed as the emergence of so-called “computational biology” or “bioinformatics” that later developed as independent branches of science (Hagen, 2000). The use of computers helped researchers to explore varied aspects of chemical and biological diversity existing in nature, which was difficult using normal experimental process. In the beginning, computers were big, heavy, and enormously costly machines. Only a few laboratories could think of having such giant machines for research purposes. Then began the shrinking process that reduced the size of the computer to average-sized room and then to that of a desk. Computers were still costly, slow, and have memory limitations. But molecular biologists, though not all, were turning to them for data analysis and data-based predictions. Among all the branches of life sciences, molecular biology has been supported mostly by the power and abilities of computers. From visualization to data analysis, sequencing to structure prediction, everything became relatively less exhausting in comparison to experimental approaches. Remarkable developments in the computer during 1960s and 1970s aided molecular biology techniques and analytical approaches in unmatched manner and made their use as a routine process in research. Proteins represent the most dynamic and diverse class of macromolecules. They are involved in the most essential physiological processes that help the origin and survival of the living beings. Hence, understanding the mysteries of their mode of action is crucial to elucidate the mechanisms of biological processes especially in case of stress (Gonzalez and Kann, 2012; Ross and Poirier, 2004). The structural and functional characterization of proteins, carried out particularly to understand the mechanism of their functioning, is known as proteomics and has revolutionized the methods for the functional analysis of the plant genes or proteins. Plant

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proteomic projects include structural proteomics of the whole organism, organs, tissues, cells, and subcellular compartments, as well as comparative proteomics on various processes. The analysis of differential proteome in response to various abiotic and biotic stresses, including ultraviolet radiation (Gu et al. 2018), metals (Chen et al. 2018), salinity (Long et al. 2016; Bandehagh et al. 2011), flooding (Pan et al. 2019), and pathogen infestation in plants (Dong et al. 2017) have unraveled cross-talks in various gene-to-metabolite pathways. The completed genome sequences of model plants, Arabidopsis (Arabidopsis genome initiative, 2000) and rice (Goff et al., 2002) along with the various plant proteomic projects provided insights into genetic makeup and spatial regulation of metabolic networks by genes present. The major challenge, however, in the field of plant biotechnology is a slow integration of new technologies into the breeding programs for crop improvement. The power of genomics, proteomics, and bioinformatics remains underexploited in plants because of big and complex genome, multiple ploidy levels in genome, self-incompatibilities, and overall a long generation time. In this chapter, our aim is to discuss state-of-the-art in silico approaches used for structural and functional characterization of proteins with special attention to various tools, web servers, and databases. Though a lot has been written and reviewed up till now on this topic, novel findings keep adding to this ever-flowing stream of information.

4.2 In silico structural and functional characterization proteins The term in silico refers to the computational methods used for biological problem solving. Pioneers of biological sciences have started using this term to separate computational experiments from those of laboratory. It goes in line with previously used terms like in vitro and in vivo. The area of protein research has tremendously benefitted from computers. From very basic to most intricate biological calculations are being carried out using computers to save time and manual efforts. Last decade saw lots of development in computational approaches for protein research with special focus to “structure and function” prediction. Table 4.1 lists recently developed tools, web servers, and databases related to in silico structural and functional characterization of proteins.

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Table 4.1 List of tools and databases used for structural and functional characterization of proteins. S. Tool/web server/ Description Web address No. database Protein sequence databases

1.

2.

3.

Protein Information Resource Protein Sequence Database (PIR-PSD)

It is the oldest database http://pir.georgetown. of protein sequence edu/ information. It provides comprehensive nonredundant protein sequence data Universal Protein It is highly annotated http://www.uniprot. Knowledge base protein sequence org (UniProtKb) database, which provides comprehensive information on protein annotation NCBI’s Entrez Protein The database contains http://www.ncbi.nlm. sequence data nih.gov/entrez/ translated from the query.fcgi? nucleotide sequences db 5 Protein

Sequence family databases

4.

5.

Phylogenetic classification of proteins encoded in complete genomes (COGs) InterPro

6.

Pfam

7.

PRINTS

8.

PROSITE

Provides information on https://www.ncbi.nlm. phylogenetic nih.gov/COG/ classification of proteins Provides classification of protein families based on domain information It is a database of protein families It provides comprehensive information on protein fingerprints It is database of protein domains, families, and functional sites

http://www.ebi.ac.uk/ interpro/

https://pfam.xfam.org/ http://130.88.97.239/ PRINTS/index.php

https://prosite.expasy. org/ (Continued)

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Bioinformatic tools to understand structure and function of plant proteins

Table 4.1 (Continued) S. Tool/web server/ No. database

Description

Web address

Protein structure databases

9.

10.

11.

12.

13.

14.

15.

16.

RCSB Protein Data Bank (PDB)

Biggest Repository of Protein structures. It contains 129211 protein structures till date Protein Data Bank in European resource for Europe (PDBe) the collection and organization of protein structures Protein Data Bank It provides centralized Japan (PDBj) PDB archive of protein structures along with analysis tools Pictorial database of It is a pictorial database 3D structures in the that provides features Protein Data Bank and overview of (PDBsum) protein structures deposited in PDB Biological Magnetic It is a repository of Resonance Data NMR spectroscopy Bank (BMRB) data of proteins Electron Microscopy It contains electron Data Bank (EMDB) microscopy data of protein structures Molecular Modeling It provides value-added Database (MMDB) features such as explicit chemical graphs, computationally identified 3D domains, literature links for the structures deposited in the PDB Protein Model Portal It is a database of (PMP) computationally predicted protein models

www.rcsb.org

https://www.ebi.ac.uk/ pdbe/

https://pdbj.org/

http://www.ebi.ac.uk/ pdbsum

http://www.bmrb.wisc. edu/ https://www.ebi.ac.uk/ pdbe/emdb/; http:// emdatabank.org/ https://www.ncbi.nlm. nih.gov/Structure/ MMDB/mmdb.shtml

https://www. proteinmodelportal. org/ (Continued)

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Table 4.1 (Continued) S. Tool/web server/ No. database

17.

SWISS-MODEL Repository

18.

Protein Data Bank of Transmembrane Proteins (PDBTM)

Description

Web address

It is a database of https://swissmodel. annotated protein expasy.org/ models generated repository/ using SWISSMODEL homologymodeling pipeline It is a comprehensive http://pdbtm.enzim. curated database of hu/ transmembrane proteins selected from PDB

Protein structure family databases

19.

20.

21.

22.

23.

CATH

It is one of the largest and widely used databases of protein families Conserved Domain It is a database providing Database (CDD) information on conserved domains Structural Classification As the name suggests, it of Proteins (SCOP) is a database of protein structural classification Homologous Structure It provides information Alignment Database on structure-based (HOMSTRAD) alignments of homologous protein families ArchDB It gives information on loop classification based on known protein structures

http://www.cathdb. info/

https://www.ncbi.nlm. nih.gov/cdd/ http://scop.mrc-lmb. cam.ac.uk/scop/

http://mizuguchilab. org/homstrad/

http://sbi.imim.es/ archdb/

Protein functional family databases

24.

25.

Aminoacyl-tRNA It provides sequence and http://www.man. Synthetases Database structural information poznan.pl/aars/ (AARSDB) on aminoacyl-trna synthetases Allosteric Database It gives useful http://mdl.shsmu.edu. (ASD) information on cn/ASD/ allosteric proteins (Continued)

75

Bioinformatic tools to understand structure and function of plant proteins

Table 4.1 (Continued) S. Tool/web server/ No. database

26.

BRENDA

27.

GPCRdb

28.

29.

Transporter Classification Database (TCDB) RNase P Database

30.

KinBase

Description

Web address

It is comprehensive information resource for enzymes. In contains detailed information on G protein-coupled receptors (GPCRs) It is a useful resource for membrane transport proteins It provides detailed functional and structural information on Ribonucleases P It is curated database of protein kinases

http://www.brendaenzymes.org/ http://gpcrdb.org/

http://tcdb.org/

http://www.mbio.ncsu. edu/RNaseP/home. html http://www.kinase. com/kinbase/

Protein interaction and pathway databases

31.

STRINGdb

32.

IntAct

33.

BioGRID

STRING is one of the https://string-db.org/ biggest databases of protein interaction networks. It is highly annotated and provides user-friendly interface for network visualization and analysis Is a curated database of https://www.ebi.ac.uk/ molecular interactions intact/ run by EMBL-EBI. Like String, it also provides easy to use GUI along with helpful analysis tools It provided curated https://thebiogrid.org/ interaction data from experimentally investigated interactions (Continued)

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Table 4.1 (Continued) S. Tool/web server/ No. database

34.

KEGG PATHWAY Database

35.

RECTOME

36.

BindingDB

Description

Web address

KEGG is a database of http://www.genome. manually drawn jp/kegg/pathway. pathways of html molecular interactions It is comprehensive, https://reactome.org/ curated reviewed database of pathways It is database of proteins https://www. considered to be drug bindingdb.org/ targets in interaction with small molecules and drug-like substances

Databases of protein modifications

37.

O-GlycBase

38.

Phospho3D

It is a database of Ohttp://www.cbs.dtu. and C-glycosylated dk/databases/ proteins OGLYCBASE/ It is a database of 3D http://www. structures proteins phospho3d.org/ with phosphorylation sites

Protein tertiary structure prediction tools and servers

39.

SWISS-MODEL

40.

Modeler

41.

Iterative Threading ASSEmbly Refinement (ITasser) Protein Homology/ It uses fold recognition analogY method for structure Recognition Engine prediction (PHYRE) Robetta It is a full chain structure prediction server

42.

43.

It is an automated https://swissmodel. homology modeling expasy.org/ server Stand-alone tool for https://salilab.org/ homology/ modeller/ comparative modeling of proteins It is a threading-based https://zhanglab.ccmb. structure prediction med.umich.edu/Iserver TASSER/ http://www.sbg.bio.ic. ac.uk/phyre2/html/ page.cgi?id 5 index http://robetta.bakerlab. org/ (Continued)

Bioinformatic tools to understand structure and function of plant proteins

Table 4.1 (Continued) S. Tool/web server/ No. database

44.

PSIPRED

45.

SuperLooper

46.

ModLoop

Description

77

Web address

It integrates several http://bioinf.cs.ucl.ac. other structure uk/psipred/ prediction methods to give optimized predicted structure It is a web server for http://proteinformatics. protein loop charite.de/ngl-tools/ modeling sl2/start.php It is also used for loop http://modbase. modeling compbio.ucsf.edu/ modloop/

Protein secondary structure prediction tools and severs

47.

48.

49.

50.

51.

52.

Chou & Fasman It is based on the Chou http://www.biogem. Secondary Structure and Fasman algorithm org/tool/chouPrediction Server of secondary structure fasman/ (CFSSP) prediction COILS It is suitable for the https://embnet.vital-it. prediction of coiled ch/software/ regions in protein. It COILS_form.html predicts the structure of coils using known structural information GOR IV Secondary This server is based on https://npsa-prabi.ibcp. Structure Prediction the GOR method of fr/cgi-bin/ Method secondary structure npsa_automat.pl? prediction page 5 npsa_gor4. html NetSurfP It is a neural networkhttp://www.cbs.dtu. based secondary dk/services/ structure prediction NetSurfP/ server. It also gives the information on surface accessibility NetTurnP It predicts beta turn http://www.cbs.dtu. regions in the query dk/services/ protein NetTurnP/ TMHMM Server This server predicts http://www.cbs.dtu. transmembrane dk/services/ helices in proteins TMHMM-2.0/ (Continued)

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Table 4.1 (Continued) S. Tool/web server/ No. database

53.

HMMTOP

54.

Jpred

Description

Web address

This server is also suitable for the prediction of transmembrane helices and the topology of proteins It is Jnet algorithmbased secondary structure prediction server

http://www.enzim.hu/ hmmtop/

It is a metaserver that incorporates six different structure assessment and analysis programs to validate newly modeled proteins ModEval is a model evaluation server run by Sali Lab

https://servicesn.mbi. ucla.edu/SAVES/

http://www.compbio. dundee.ac.uk/jpred4/ about.shtml

Protein model evaluation tools

56.

Structure Analysis and Validation Server (SAVES)

57.

ModEval

http://modbase. compbio.ucsf.edu/ evaluation/

Active site and binding site prediction tools

58.

CASTp

59.

CAVER

60.

ProBis

It is a web server for the http://sts.bioe.uic.edu/ prediction of active castp/index.html?1ycs site residues in the protein. It is also available as pymol plug in It is useful for predicting http://www.caver.cz/ tunnels and channels in protein structures ProBis predicts the http://probis.cmm.ki.si/ presence of index.php structurally similar binding sites in the query protein. It uses protein structural information to detect the binding sites (Continued)

79

Bioinformatic tools to understand structure and function of plant proteins

Table 4.1 (Continued) S. Tool/web server/ No. database

Description

Web address

Protein function prediction and analysis tools

61.

ProFunc

62.

InterProScan

63.

PfamScan

64.

Sequence Annotated by Structure (SAS)

65.

PredictProtein

66.

RADAR

It predicts protein function using 3D structure It works as interface for the InterPro to predict protein domains using query sequence It uses fasta sequence to search against the library of Pfam HMM profiles and helps with assigning function to the protein It assigns structural information to the given protein sequence based on known protein structures from the PDB database It integrates several prediction strategies such as the prediction of secondary structure, solvent accessibility, and transmembrane helices, which can be helpful in assigning the function to a protein It stands for Rapid Automatic Detection and Alignment of Repeats. It detects repeats in protein sequences

https://www.ebi.ac.uk/ thornton-srv/ databases/profunc/ http://www.ebi.ac.uk/ interpro/search/ sequence-search

https://www.ebi.ac.uk/ Tools/pfa/pfamscan/

https://www.ebi.ac.uk/ thornton-srv/ databases/sas/

https://www. predictprotein.org

https://www.ebi.ac.uk/ Tools/pfa/radar/

(Continued)

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Ahmad Abu Turab Naqvi et al.

Table 4.1 (Continued) S. Tool/web server/ No. database

67.

Simple Modular Architecture Research Tool (SMART)

68.

Conserved Domain Architecture Retrieval Tool (CDART)

Description

Web address

SMART helps in http://smart.emblidentifying the heidelberg.de/ distribution of domains and motifs in the query sequence that facilitates its functional annotation NCBI’s CDART server https://www.ncbi.nlm. predicts the presence nih.gov/Structure/ of conserved domains lexington/lexington. in the proteins of cgi interests

Protein mutational analysis tools

69.

70.

71.

72.

73.

HOPE Server

This server provides easy-to-use web interface to study the effects of point mutations on protein structure CUPSAT It is a structure-based tool to protein stability in response to point mutations PMut PMut server is useful for predicting the pathology of protein mutations I-mutant2.0 Server It uses both sequence and structural information of protein to predict stability in response to mutations Sorting Intolerant from SIFT server predicts the Tolerant (SIFT) effects of mutations on protein function

http://www.cmbi.ru. nl/hope/

http://cupsat.tu-bs.de/

http://mmb. irbbarcelona.org/ PMut/ http://folding.biofold. org/i-mutant/imutant2.0.html

http://sift.bii.a-star.edu. sg/

Protein structure visualization and analysis tools

74.

PyMol

It is the widely used https://pymol.org/2/ python-based visualization program, (Continued)

81

Bioinformatic tools to understand structure and function of plant proteins

Table 4.1 (Continued) S. Tool/web server/ No. database

75.

DeepView Swiss PDB Viewer

76.

Visual Molecular Dynamics (VMD)

77.

LIGPLOT

Description

Web address

which also provides several embed analysis tools and plug-ins It provides several utility https://spdbv.vital-it. ch/ tools like Pymol but it is best suited for introducing Amino Acid mutations, energy minimization, and correcting protein structures. It is also used as a client side platform for SwissModel server Apart from other http://www.ks.uiuc. visualization and edu/Research/vmd/ analysis properties, it is best used for molecular dynamics studies It is used for graphical https://www.ebi.ac.uk/ representation of thornton-srv/ protein ligand software/LIGPLOT/ interactions

Tools for protein property analysis

78.

79.

80.

81.

ProtParam

It predicts various physical and chemical properties of query protein ProtScale It is used to compute various parametersbased amino acid scale SignalP 4.1 Server It predicts the presence of signal peptide cleavage sites in amino acid sequences SecretomeP 2.0 Server It facilitates the prediction of nonclassical protein secretion

https://web.expasy.org/ protparam/

https://web.expasy.org/ protscale/

http://www.cbs.dtu. dk/services/SignalP/

http://www.cbs.dtu. dk/services/ SecretomeP/ (Continued)

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Table 4.1 (Continued) S. Tool/web server/ No. database

82.

AACompIdent tool

83.

TargetP 1.1 Server

84.

PSLPred

85.

PSORTb

86.

VirulentPred

87.

VICMPred

Description

Web address

It allows the identification of a protein from its amino acid composition It predicts the subcellular location of eukaryotic proteins It predicts the subcellular location of prokaryotic proteins It is used for bacterial protein subcellular localization prediction It is web server for predicting virulent factors It is a SVM-based prediction server that used available information of virulent proteins and predicts the virulent factors among set of given queries

https://web.expasy.org/ aacompident/

http://www.cbs.dtu. dk/services/TargetP/ http://crdd.osdd.net/ raghava/pslpred/ http://www.psort.org/ psortb/ http://203.92.44.117/ virulent/ http://crdd.osdd.net/ raghava/vicmpred/

Protein sequence and homology detection tools

88.

Basic Local Alignment Search Tool (BLASTp)

89.

FASTA

90.

HMMER

It is a standard protein https://blast.ncbi.nlm. similarity search tool nih.gov/Blast.cgi? based on BLAST PAGE 5 Proteins algorithm It is FASTA algorithm- https://fasta.bioch. based similarity search virginia.edu/ tool fasta_www2/ fasta_www.cgi? rm 5 select&pgm 5 fa It is a hidden Markov https://www.ebi.ac.uk/ model-based Tools/hmmer/; https://toolkit. homology detection tuebingen.mpg.de/ server #/tools/hmmer (Continued)

83

Bioinformatic tools to understand structure and function of plant proteins

Table 4.1 (Continued) S. Tool/web server/ No. database

91.

HHpred

Description

Web address

It is used for homology detection and structure prediction using hmm comparisons

https://toolkit. tuebingen.mpg.de/ #/tools/hhpred

Protein sequence alignment tools

92.

LALIGN

93.

SIM Sequence Alignment Tool

94.

ClustalOmega

95.

MAFFT

96.

ConSurf

It uses pairwise alignment approach to find similarities between two sequences It finds a user-defined number of best nonintersecting alignments between two protein sequences or within a sequence It uses multiple sequence alignment approach. The program is based on hidden Markov models It is best suitable for short sequences. Other multiple sequence alignment tools like ClustalW or Omega do not give better results in case of relatively short sequences It predicts evolutionary conserved residues in protein sequences based on phylogenetic analysis

https://embnet.vital-it. ch/software/ LALIGN_form.html

https://web.expasy.org/ sim/

https://www.ebi.ac.uk/ Tools/msa/clustalo/

https://mafft.cbrc.jp/ alignment/server/

http://consurf.tau.ac.il/ 2016/index_proteins. php

(Continued)

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Table 4.1 (Continued) S. Tool/web server/ No. database

97.

WebLogo

Description

Web address

It generates sequence https://weblogo. logos for given berkeley.edu/logo.cgi protein sequences based on multiple sequence alignment. Sequence logos are used for graphical representation of MSA results to find to relative positions of conserved residues

Protein information resources

98.

Human Proteinpedia

99.

Protein Information Resource (PIR)

100. GeneCards

It is community portal for protein information It is an integrated centralized platform for protein information It gives comprehensive, user-friendly information on annotated and predicted genes

http://www. humanproteinpedia. org/ https://pir.georgetown. edu/

http://www.genecards. org/

There are mainly two approaches based on the availability of protein information, that is, sequence-based approach and structure-based approach. Fig. 4.1 gives a graphical overview of the in silico structural and functional characterization of the proteins.

4.3 Sequence-based approach In this approach, amino acid sequence of the proteins is used as foundation stone for making structure and function predictions. In recent years, our group has remained active in the field of functional annotation of hypothetical proteins from common Indian bacterial pathogens

Bioinformatic tools to understand structure and function of plant proteins

85

Figure 4.1 Graphical overview of the in silico structural and functional characterization of proteins.

(Shahbaaz et al., 2013; Kumar et al., 2014, 2015; Naqvi et al., 2015a,b; Shahbaaz et al., 2015a,b). For this, we have developed a novel pipeline and improvised the previous ones to get computational predictions as accurate as possible (Naqvi et al., 2015). The in silico structural and functional characterization of proteins using sequence information is comprised of following steps:

4.3.1 Biophysical characterization of proteins Knowledge of physicochemical properties of the protein is useful for deducing its function. Properties such as molecular mass, theoretical pI, stability index, aliphatic index, hydrophobicity index, subcellular localization, amino acid percentage in terms of different types of amino acids, signal peptides, and other such parameters are crucial to overall behavior of the proteins. There is an array of tools and databases developed so far for the biophysical parameterization of proteins. PROTPARAM (Wilkins et al., 1999) and PROTSCALE (Wilkins et al., 1999) are two widely used tools for the prediction of physical and chemical properties of the proteins. SignalP 4.1 Server (Petersen et al., 2011) facilitates the prediction of signal peptide cleavage sites in protein. SecretomeP 2.0 Server (Bendtsen et al., 2004) is used for the prediction of nonclassical protein secretion.

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Subcellular localization of the proteins defined as prediction of local environment of proteins in the cell. Deducing this information helps in understanding the possible function of the protein (Donnes and Hoglund, 2004). Plants have independent membrane-limited organelles. Important metabolic pathways such as photorespiration consist of enzymes residing in multiple organelles. Also, processes like glycolysis are catalyzed by multiforms of enzymes existing in the organelles. Thus, it is important to know the localization of enzyme to understand its function. Furthermore, effector molecules from the invading pathogen colocalize with the host target molecular in particular organelle. An effective prediction of both pathogen effector protein and target host protein is important for manipulating the metabolic pathway for plant protection. There are a number of tools and databases available for predicting subcellular location of proteins. TargetP 1.1 (Emanuelsson et al., 2007) server is widely used for subcellular location of eukaryotic proteins while PSLPred (Bhasin et al., 2005) is used for prokaryotic proteins (Table 4.1). In case of microbial pathogens, identification of virulent protein is helpful in computer-based function prediction of unknown proteins. VirulentPred (Garg and Gupta, 2008) and VICMPred (Saha and Raghava, 2006) are two useful servers for the separation of virulent proteins from the nonvirulent ones.

4.3.2 Structure prediction The primary structure of the protein contains all needful information for its 3D conformation. Therefore in this approach, amino acid sequence of a protein is used to predict its structure. The traditional sequence-based characterization of protein goes from structure prediction to functional annotation. The amino acid sequence of proteins is used to assign structural elements to them using previously available information (i.e., homology or comparative modeling) or predicting the structures (ab initio modeling). The specific organization of protein structural elements is crucial for its functionality. The selection of structure prediction strategy depends on the preliminary information available for the query protein. For example, if the query protein is found homologous to some proteins with known structure, then comparative and homology-based structure prediction is done; otherwise, threading or ab initio approaches are taken into consideration.

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Following is the basic framework that is generally used in structure prediction process. 4.3.2.1 Secondary structure prediction Conventionally, proteins have four structural levels, that is, (1) primary structure or amino acid sequence; (2) secondary structural elements like helices, beta sheets, random coils, and turns; (3) tertiary; and (4) quaternary structure. Structural elements of the protein such as alpha helix, beta sheets, loop or random coil, and turns make the secondary level of structure. Each of these elements has their importance in the overall structural organization of protein. Therefore meticulous analysis of these elements is strongly taken care of during higher level structure predictions. An array of web servers and tools using specific algorithms for secondary structure prediction using amino acid sequence is available. Chou and Fasman (Goodman and Moore, 1977; Chen et al., 2006) and GOR (Garnier et al., 1996) are the most widely known prediction algorithms for protein secondary structure. Apart from these classical models, artificial neural network-based algorithms are also applied for secondary structure prediction. NetSurfP (Petersen et al., 2009) server uses neural networkbased algorithm. COILS (Lupas et al., 1991) program uses information of known protein structures to predict the topology of the random coils. TMHMM (Chen et al., 2003) and HMMTOP (Tusnady and Simon, 2001) servers are useful for identification of transmembrane helices in the protein. NetTurnP (Petersen et al., 2010) is a neural network-based server that predicts beta turn in protein sequence. 4.3.2.2 Tertiary structure prediction The three-dimensional shape of protein having maximum stability and lowest energy is called the tertiary structure. The tertiary structure formation results due to covalent and noncovalent intramolecular interactions among the amino acids. Formation of disulfide bond between cysteine residues is essential for the overall shape and stability of 3D structure of the proteins. Also, hydrogen bonds and other electrostatic interactions play major role in structure formation and stabilization. 3D structure of proteins is predicted using various approaches. The prediction of structure using sequence homology is the widely used method and attains maximum accuracy in comparison to other methods. Structural bioinformaticians are constantly working to reach maximum accuracy in terms of ab initio structure prediction. Still, it remains one of

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the biggest challenges for structural bioinformatics community. Following are the approaches along with some representative tools and servers for tertiary structure prediction: (1) Homology modeling: Proteins originated from common ancestors are said to be homologous proteins. This phenomenon is exploited to predict structures of unknown proteins by comparing the sequences of unknown protein with those of know 3D structures and deducing similar patterns in its organization. Though, there is plethora of computer programs and servers for homology modeling but SWISS-MODEL (Biasini et al., 2014) and Modeller (Webb and Sali, 2016) are two most widely used programs by the bioinformatics community. SWISS-MODEL is a web-based server, whereas Modeller is a python-based standalone program. Apart from these two, Protein Homology/analogY Recognition Engine (PHYRE) (Kelley and Sternberg, 2009) is used for homology modeling. (2) Ab initio or de novo modeling While homology-based methods use previously solved structures to prediction structures of unknown proteins, ab initio model predicts 3D structure of protein directly from the scratch. In the absence of sequence similarity or very less similarity (below 30%), ab initio modeling approach is used. (3) Loop modeling Loop modeling is one of many limitations of computational methods for protein structure prediction. Loops are comparatively difficult to model due to the randomness in their structural organization. SuperLooper (Hildebrand et al., 2009) and ModLoop (Fiser and Sali, 2003) are majorly used programs for loops modeling. Modeler also provides loops optimization tools to reduce the random generation of loops while protein structure prediction. 4.3.2.3 Model validation and evaluation Computer-based structure prediction is completed with model evaluation and validation. The modeled protein should pass through established benchmark validation parameters in order to prove accuracy and correctness. Programs like Ramachandran plot and energy calculations are applied to the modeled protein for the evaluation and validation of predicted structure. Structure Analysis and Verification Server (SAVES) is used for postprediction analysis, evaluation, and validation of predicted

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protein models. Apart from this, ModEval is also used for the validation of, usually, proteins modeled using Modeller program.

4.4 Function prediction During recent years, the amount of sequence data has increased astronomically. This increase in the number of protein sequences has given rise to several annotation strategies to add further information to newly sequenced unknown proteins. To assign functions to unknown protein using sequence-based methods involves a range of analysis strategies that use empirical information (Shahbaaz et al., 2013; Kumar et al., 2014, 2015; Naqvi et al., 2015a,b; Shahbaaz et al., 2015a). Biophysical characterization of protein is done using the available tools. Protein sharing homology to the query protein is searched across the available data using sequence similarity search methods such as BLAST (Altschul et al., 1990), FASTA (Pearson and Lipman, 1988), and HMMER (Finn et al., 2011). These methods use sequence alignment and hidden Markov model profiles to search proteins similar to the query sequence. Sequence-based function prediction methodologies also comprise of protein classification, domain annotation, motif search, and clustering. These methods help us deduce the function of unknown proteins using already available information. Protein family databases like CATH, SCOP, Panther, and Pfam are widely known comprehensive database with user interfaces for database searching using the query sequence. There are many proteins whose structures are known or modeled but no specific function is known for them. The protein structure is used in these cases to assign functions. The topological annotation leads to understanding the functional properties of the protein. Following are the methods that are applied in structure-based characterization of proteins.

4.4.1 Fold recognition/assignment Protein folds are specific topological arrangement of proteins that provide stability and functionality to the proteins. Fold recognition/assignment strategies are widely used to assign folds to novel proteins for the purpose of annotations.

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4.4.2 Structure-based function prediction Structure-based function prediction strategy uses the available information of structure to function relationship and then predicts function of proteins whose structures are known but their functions are not known. The process of structure-based function prediction comprises of identifying domain organization of the protein, motif discovery, identification of repeats specific to certain functional classes, and finding out the active sites and binding site of the proteins. Assigning properties to protein structure help to decide their probable functions. There are number of tools and databases that provide interfaces for structure-based functional characterization of the proteins. Some of them are ProFunc (Laskowski, 2017), InterPro (Apweiler et al., 2000), and SMART.

4.4.3 Active site prediction The active site of protein by convention is known as the group of adjacent residues in the 3D spatial arrangement of protein molecule essential for its function. During the process of structure-based function prediction, the knowledge of active site resides or binding pocket plays critical role. Therefore we usually go for the prediction of protein active site using available tools and databases. CASTp (Binkowski et al., 2003) is one of the most widely used tools for the prediction of active sites in protein.

4.5 Summary The computational structural and functional characterization of proteins has witnessed crucial advancements during last decade in terms of accuracy and cost-effectiveness. The computer-based structure and functional characterization of proteins encompasses several tools, prediction servers, and databases that help to deduce structural and functional properties of the protein of interest. Advancement in in-silico characterization of proteins are going parallel with computational power; as a result, accuracy in the prediction of structure and function of the proteins has increased. Acquisition, storage, and analyses of genome-based information are the major concern in crop science bioinformatics. This chapter has shed light on the pipelines and approaches to study structural and function of unknown proteins. Research community has strong inclination to

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implement integration of standalone and open-source software facilitating sharing and reuse of information for the improvement of crop and medicinal value of the existing varieties.

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CHAPTER FIVE

Transgenic technology for efficient abiotic stress tolerance in plants Arun Gokul1, Lee-Ann Niekerk1, Mogamat Fahiem Carelse1 and Marshall Keyster1,2 1

Environmental Biotechnology Laboratory, Department of Biotechnology, University of the Western Cape, Bellville, South Africa DST-NRF Centre of Excellence in Food Security, University of the Western Cape, Bellville, South Africa

2

5.1 Introduction In the near future, the very rapidly growing world population will reach a plateau where zero growth in population numbers will be observed. For such a growing population, urbanization and industrialization will have to be at close to 100%. Lele et al. (2018) state that in such situations climate change will be the main stressor. To feed this growing population, annual food production needs to be doubled which would require huge agricultural innovations. For decades, the agricultural sector has contributed significantly in feeding the world population especially, in third world countries where it is the backbone of economic development (Sabir and Arshad, 2014). Agricultural practices, however, are underneath severe pressure from urbanization and industrialization, because of overlaps in the usage of fertile lands. Thus the decreasing land will have to be optimally used to grow food and feed crops in order to sustain plant and meat-based diets. Pimentel and Pimentel (2003) reported that approximately 67% of the world’s population relies primarily on plant-based diets. Therefore most of the fertile land available in the future will have to be devoted to crop plant production. The current environmental state brought about by climate change will only get more severe over time. With the growing population, human activities such as emission of greenhouse gases (carbon dioxide and methane) will intensify and result in much more server climate change cycles (Wheeler and Von Braun, 2013). Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00005-8

© 2020 Elsevier Inc. All rights reserved.

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These climate change cycles will directly or indirectly intensify abiotic stresses such as increased heat, more severe colds, increased soil salinity, longer and severe droughts, increased floods, decreased soil nutrient contents, and increased heavy metal contamination. Abiotic stress hampers plant growth and development leading to poor crop yields (Pandey et al., 2017). With limited fertile land and an increase in the severity and time of abiotic stresses, the ever-growing population will outweigh food production in the near future. Transgenic approaches to make plants more tolerant to abiotic stresses, therefore provide a rapid and sustainable solution for increased food and feed crop production (Bhatnagar-Mathur et al., 2008). Various strategies and technologies have been developed for efficient and effective transferring of genes from parent organisms and into plants (Husaini et al., 2010; Rivera et al., 2012; Wani et al., 2016; Khanna and Deo, 2016). However, not many publications focus on providing an overview of the successes of improving plant tolerance to various abiotic stresses. Therefore this chapter highlights a few of the successful studies which used transgenic technologies to improve plant tolerance to abiotic stresses and provide an outlook on future plant transgenic technologies based on the highlighted studies.

5.2 Transgenic approaches for engineering heat and cold tolerance in plants A series of physiological, morphological, molecular, and biochemical changes occur in plants due to the adverse effects of abiotic stresses such as cold and heat (Zhu, 2016). These changes often lead to impaired growth and development as well as a decline in the productivity of crop plants. Conventional breeding methods have been met with limited success (Lasley et al., 1994) in improving the cold and heat tolerance of important crop plants through interspecific and intergeneric hybridization. It was important, therefore to look for alternative strategies to develop cold and heat stress-tolerant plants. Hence, transgenic approaches are being actively pursued to improve traits for cold and heat tolerance in a number of crops. The alterations of genes in response to cold and heat tolerance are followed by an increase in the levels of hundreds of metabolites, of which some are known to have protective roles against the damaging effects of cold and heat stress. Various low- and high-temperature inducible genes

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have been isolated and transformed from plants and other organisms in an effort to induce heat (Table 5.1) and cold (Table 5.2) tolerance. This section highlights some of the transgenic approaches pursued to elucidating both heat and cold tolerance in plants. Lee et al. (2017) performed a study to develop heat-tolerant transgenic Medicago sativa (alfalfa) using an Agrobacterium-mediated transgenic approach by overexpressing a small heat-shock protein 23 (MsHsp23). Alfalfa plants overexpressing the MsHsp23 gene were generated under the control of a CaMV 35S promoter. The resultant chimeric gene cassette was then inserted into the KpnI and Zbal site of the pCAMBIA1300 binary vector and was subsequently introduced into Agrobacterium tumefaciens strain EHA105. The seeds were allowed to culture in a growth chamber at 25
C with a 14 h photoperiod. Transgenic and nontransgenic alfalfa lines were exposed to 42
C for 24 h. Transgenic lines showed less wilting and increased levels of ascorbate peroxidase (APX) and were able to withstand higher temperatures compared to the nontransgenic strains. Table 5.1 Engineering heat stress tolerance in plants using transgenic approaches. Genes Vectors Mode of transformation References

LimHSP16.45 pBI101.2 HSP26

pMDC164

rolB

pGBrolB

HSP100/ ClpB Fld

p35S-sGFP

Hsp21

pCAMBIA 2200 pCGN 1559

Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated

Mu et al. (2013) Chauhan et al. (2012) Shabtai et al. (2007) Yang et al. (2006) Tognetti et al. (2006) Neta-Sharir et al. (2005)

Table 5.2 Engineering cold stress tolerance in plants using transgenic approaches. Genes Vectors Mode of transformation References

CdSAMDC1 AtDREB1A CbCBF MYBS3 OsMYB3R-2

pCAMBIA pBIH pCAMBIA1301 pENTR pBI221

Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated

Luo et al. (2017) Shah et al. (2016) Zhou et al. (2014) Su et al. (2010) Dai et al. (2007)

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Choi et al. (2013) investigated whether the ABF3 gene introduced into Agrostis stolonifera (bentgrass) can lead to the development of heattolerant plants. The ABF3 gene a known stress-responsive gene from Arabidopsis was introduced into bentgrass by using the pCAMBIA3301 vector inserted into Agrobacterium tumefaciens under the CaMV 35S promoter. For heat treatments, bentgrass plants were subjected to heat stress for 2, 4, and 6 h at 48
C and heat tolerance was evaluated after 15 days of the treatments. ABF3 transgenic bentgrass plants maintained relative water content (RWC) after heat treatment by 57%61% as compared to the wild-type bentgrass plants under the same conditions. Collectively, ABF3 transgenic plants showed enhanced heat tolerance stress. Xu et al. (2014a,b,c) examined whether the isolation and cloning of PpEXP1 (expansin 1 gene) found in Poa pratensis (Kentucky bluegrass) can improve heat tolerance in Nicotiana tabacum. Transgenic plants overexpressing PpEXP1 were generated using Agrobacterium tumefaciens-mediated genetic transformation. The construct used in this study consisted of the pEZT-(K)-LC vector containing the PpEXP1 gene of interest under the CaMV 35S promoter and the kanamycin resistance gene NPT11 as the selective marker. Two weeks old seedling of both transgenic and wildtype plants were subjected to heat stress at 42
C or 25
C for a duration of 6 days. Transgenic tobacco plants showed lower electrolyte leakage, membrane damage and hydrogen peroxide content under heat stress at 42
C compared to wild-type plants. Moreover, these results demonstrated positive roles of the PpEXP1 gene in enhancing plant tolerance to heat stress. Montero-Barrientos et al. (2010) investigated the function of the Trichoderma harzianum T34 hsp70 gene in Arabidopsis thaliana. The coding region of hsp70 gene was polymerase chain reaction (PCR) amplified and subcloned into BamHI and SacI double digested pBIN121 vector. The T-DNA region of the pBIHSP construct was transferred into Agrobacterium tumefaciens C58C1 by electroporation. Arabidopsis seeds were sown on MS plates and were administered under three different heat conditions; 38
C for 90 min, 45
C for 2 h or at 38
C for 90 min followed by 2 h at 22
C, and then 45
C for 2 h. After heat treatment, the seeds were incubated at 22
C. The overall results indicate that the T. harzianum hsp70 gene confers tolerance to heat and other abiotic stresses and that the HSP70 protein acts as a negative regulator of the heat-shock transcription factor (HSF) transcriptional activity in Arabidopsis. Furthermore, the transgenic lines also displayed increased transcript levels of Na1/H1

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exchanger 1 (SOS1) and APX1 genes involved in salt and oxidative stress response, respectively. Jamshidnia et al. (2018) performed a study to investigate whether the overexpression of the P5cs gene, which is a bifunctional enzyme required in the biosynthesis of proline can increase cold stress tolerance in an Iranian species of Petunia (Fig. 5.1). The P5sc gene originating from Arabidopsis was inserted into a pBI121 vector driven by the CaMV 35S promoter and further transformed into Petunia with the Agrobacterium strain LB4404. The study compared proline content in the Iranian nontransgenic and transgenic Petunia plants under cold and control conditions administered at 4
C for cold treatment over a period of 3 days. Transformed Petunia plants under cold stress produced more proline (295674 µg/g of fresh weight) compared to the nontransformed Petunia plants under cold stress. They concluded that the increase in proline assisted in reducing the osmotic pressure which helped to maintain cellular integrity, structure, and function, and thus helped the transformed plants to withstand lower temperatures compared to the wild-type plants.

Figure 5.1 Schematic flow chart illustrating the overexpression of the P5cs gene in Iranian Petunia spp. for cold stress tolerance.

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A study done by Byun et al. (2015) investigated the cellular role of constitutive expression of a DaCBF [C-repeat-binding factor (CBF)] homolog from an Antarctic vascular plant, Deschampsia antarctica to improve cold tolerance in rice. The full-length coding region of DaCBF7 was inserted into the pEarleyGate 100 binary vectors. The vector was then transformed into Agrobacterium tumefaciens for subsequent transformation of rice plants. Wild-type plants were grown under a 16 h light/8 h dark photoperiod at 15
C. For cold-stress treatments, plants were transferred to a chamber at 4
C. DaCBF7-overexpressing transgenic rice plants exhibited markedly increased tolerance to cold stress compared to wild-type plants. Byun et al. (2015) concluded that the overexpression of DaCBF7 directly and indirectly induces diverse genes in the transgenic rice lines which conferred enhanced tolerance to cold stress. Pino et al. (2007) examined whether the use of a stress-inducible promoter to drive ectopic AtCBF expression can improve Solanum tuberosum (potato) freezing tolerance without affecting tuber yield. The potato (cv. Umatilla) was transformed with three Arabidopsis CBF genes (AtCBF13) driven by either a constitutive CaMV 35S promoter or a stress-inducible Arabidopsis rd29A promoter using Agrobacterium tumefaciens strains EHA105 or GV3101. Plant growth conditions included growing both transformed and untransformed potato lines at 25
C followed by a 3-day controlled environmental conditioning. Plants were transferred to an environmentally controlled cold room maintained at 2
C (16 h photoperiod) for 24 h (for 2 weeks) before harvesting plant material. AtCBF1 and AtCBF3 overexpression via the CaMV 35S promoter increased freezing tolerance at 2
C, whereas the AtCBF2 overexpression failed to increase freezing tolerance. These results suggest that the use of a stress-inducible promoter to direct CBF-transgene expression can yield significant gains in freezing tolerance without negatively impacting the agronomically important traits in potatoes. Sugie et al. (2006) investigated the effect of overexpression of a wheat alternative oxidase (AOX) gene Waox1a under low temperature in Arabidopsis. To study the wheat AOX function, a chimeric gene construct of the Waox1a cDNA under the control of the CaMV 35S promoter was introduced into the Arabidopsis genome through Agrobacterium-mediated transformation. A beta-glucuronidase (GUS) reporter gene of pBI121 vector was replaced by the Waox1a cDNA to produce the 35S::Waox1a chimeric construct. Seedlings of Arabidopsis

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were grown in a growth cabinet at 25
C with a 16 h photoperiod and the cold treatments were carried out at 4
C over a period of 5 days. Sugie et al. (2006) showed a decrease in reactive oxygen species (ROS) in the transgenic plants under low-temperature stress. Moreover, their results supported the hypothesis that AOX alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under abiotic stress conditions.

5.3 Transgenic approaches for engineering salinity stress tolerance in plants Salinity is a major abiotic stress which inhibits growth of crops and reduces crop yield (Klein et al., 2018; Menzi et al., 2018). Roughly 20% of cultivated land and about 50% of irrigated land, globally, has high salt content (Yokoi et al., 2002). Salinity stress causes ionic stress by increased Na1 and Cl2 levels and osmotic stress through inhibition of water uptake by the roots as well as water loss from plant tissue due to an imbalance in the water concentration gradient (Keyster et al., 2012). Furthermore, salinity stress causes oxidative damage to cellular constituents and ultimately leads to whole plant senescence (Klein et al., 2013). Hence, the importance of understanding the responses of plants to salinity stress can assist in the generation of transgenic plants with enhanced salinity stress tolerance. This section will focus on studies that took transgenic approaches to combat salinity stress in plants and some of the other studies are also highlighted in Table 5.3. In a study conducted by Nakaminami et al. (2018), 17 Arabidopsis thaliana genes coding for small peptides were found to be upregulated by salinity stress. Hence, they generated transgenic Arabidopsis thaliana plants overexpressing these genes to test the peptides for possible roles in salinity stress tolerance. The small coding genes were inserted into a pMDC32 vector with a double CaMV 35S promoter. The results indicated that 4 of the 17 overexpressed genes increased salinity stress tolerance of transgenic plants. Based on these findings, Nakaminami et al. (2018) focused on the AtPROPEP3 gene, which exhibited the highest upregulation under salinity conditions. The AtPROPEP3 gene was cloned into the pMDC32 vector and the recombinant vector was introduced into Agrobacterium tumefaciens followed by transformation

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Table 5.3 Genetic engineering to confer salinity stress tolerance in plants. Genes Vectors Mode of References transformation

OsCDPK7 pMSH1 ZFP252

pCAMBIA1301

Hval

pBI121

SbpAPX SsCBF1

pRT101 and pCAMBIA1301 pCAMBIA1302

StNAC2

pBI121

Tbosm

pCAMBIA1301

HSP23

pCAMBIA1300

PtSOS2

pCAMBIA2301

XERICO

pHB-X

OsMYB3R- pBI221 2

Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated

Saijo et al. (2000) Xu et al. (2008) Checker et al. (2012) Singh et al. (2014) Zhang et al. (2013) Xu et al. (2014a,b,c) Subramanyam et al. (2012) Lee et al. (2012) Yang et al. (2015) Zeng et al. (2015) Dai et al. (2007)

into Arabidopsis thaliana. The Arabidopsis thaliana plants overexpressing AtPROPEP3 exhibited increased survival under salinity stress and possessed longer roots when compared with wild-type plants. Furthermore, the chlorophyll content was significantly higher in the transgenic lines under salinity conditions. Nakaminami et al. (2018) then treated plants with synthetic peptides encoded by AtPROPEP3 and showed that a Cterminal peptide fragment (AtPep3) inhibited salt stress bleaching of chlorophyll. The data collected by them conclusively proved that the AtPep3 is important for salinity stress tolerance in Arabidopsis thaliana. Bouaziz et al. (2013) used a StDREB1 gene to improve the tolerance of Solanum tuberosum (potato) to salinity stress. To generate transgenic potato plants, the StDREB1 cDNA was inserted upstream of the CaMV 35S promoter in the pMDC32 binary vector (Fig. 5.2). This construct was inserted into Agrobacterium tumefaciens for transformation into a potato. The authors recorded that the potato lines overexpressing StDREB1

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Figure 5.2 Studies using transgenic approaches to confer salinity stress tolerance in various plants.

exhibited improved salinity stress tolerance and more induced cyclindependent kinase (CDK) transcription. This result suggested that the StDREB1 transcription factor was involved in the regulation of salinity stress tolerance in potato plants. The study produced transgenic potatoes with increased chlorophyll content under salinity stress. Furthermore, an upregulation in StDREB1 gene expression was observed in response to abscisic acid (ABA) which suggested that the StDREB1 gene is involved in salinity stress responses through ABA-dependent signaling. In an attempt to generate Zea mays (maize) plants with improved salinity tolerance, Chen et al. (2007) examined the Na1/H1 antiporter gene (OsNHX1) from Oryza sativa (rice) as a potential solution to engineer salinity stress tolerance in maize. The OsNHX1 gene subcloned into the pCAMBIA3301 vector under the control of the CaMV 35S promoter. Using the particle bombardment method (gene gun), the expression cassette harboring OsNHX1 gene was integrated into the genome of maize plants. Results obtained indicated that the transgenic lines overexpressing OsNHX1 gene displayed growth that was slightly less inhibited by 200 mM NaCl. The transgenic lines showed higher biomass when grown in the presence of NaCl as compared to the nontransgenic plants. Furthermore, increases in Na1 and K1 content were observed in the leaves of the transgenic plants which were significantly higher than in nontransgenic plants under high NaCl concentrations. The data collected by Chen et al. (2007) signifies that transgenic maize plants overexpressing OsNHX1 were better survived under salinity stress conditions. In a study by Park et al. (2005), the ME-leaN4 gene from Brassica napus was inserted into Lactuca sativa (lettuce) using the Agrobacterium-mediated

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transformation through the use of the binary vector pIG121 (Fig. 5.2). In this vector, the GUS gene was replaced by the LEA gene under the control of the CaMV 35S constitutive promoter. The transgenic lettuce exhibited enhanced growth and an increase in fresh weight as compared to the untransformed control plants under salinity stress. The results suggested that the growth characteristics in the transgenic lettuce plants were improved in response to salinity stress due to the constitutively expressed LEA gene.

5.4 Transgenic approaches for engineering drought stress tolerance in plants Drought is a serious limiting factor for crop production worldwide and prolonged drought has serious effects on crop growth and yield (Zhang et al., 2018). The increasing population worldwide has impacted our food security majorly and thus our food crisis is expected to worsen (Basson et al., 2018). Therefore to ensure a stable food supply, it is crucial to improve drought stress tolerance in plants. Understanding how plants respond to various stresses is a requirement for discovering promising genes because such knowledge could provide useful insight for generating crop plants with improved stress tolerance. The conventional or molecular breeding of crops is time-consuming and thus attention has shifted to the development of genetically engineered plants. Genetic engineering or transgenic technologies provide a faster means to insert beneficial genes in food and feed crops. Fig. 5.3 schematically represents recently used transgenic approaches to confer drought stress tolerance in various plants. Furthermore, some of the studies are also highlighted in Table 5.4. Japonica rice (Oryza sativa japonica) transgenic lines were produced in order to improve drought stress tolerance. El-Esawi and Alayafi (2019) used the vesicle trafficking gene OsRab7 gene in a modified pCU vector under the control of a Zea mays ubiquitin 1 promoter to transform rice. The transgenic rice plants under drought stress were subjected to physiological, biochemical, and transcriptional analyses. The overexpression of OsRab7 increased the growth and survival of rice plants under drought stress when compared to wild-type plants. The transgenic lines exhibited lower levels of ROS accumulation in comparison to the wild-type plants, suggesting that the overexpression of the OsRab7 gene counteracted the toxic ROS effects and reduced oxidative damage, thus conferring greater tolerance to drought

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Figure 5.3 Four studies in which transgenic approaches were used to confer drought stress tolerance in various plants. Two of the approaches used genestacking and the other two used overexpressed single genes.

stress. Furthermore, the gene expression levels of four genes encoding ROS-scavenging enzymes and eight genes conferring abiotic stress tolerance were measured in both transformed and untransformed lines. Transgenic lines exhibited significantly higher expression levels of antioxidant and abiotic stress-related genes. The results suggest that the OsRab7 overexpression reduced oxidative damage via induction of ROS-scavenging proteins thereby improving drought stress tolerance in rice. A study conducted by Kudo et al. (2019) used DREB1A, a drought stress-tolerant gene, to confer drought tolerance in Arabidopsis thaliana. Gene-stacking approach was employed by using the DREB1A in conjunction with either PIF4 or GA5 derived from Arabidopsis thaliana. Each gene was inserted into a binary vector pGKX under the control of the CaMV 35S promoter to produce the relative single overexpressing constructs as per the study of Kudo et al. (2017). The constructs were inserted into Agrobacterium tumefaciens and subsequently used to transform Arabidopsis thaliana. The GA5-DREB1A double overexpressed transgenic lines were generated by using Agrobacterium-mediated transformation with the single DREB1A and the single GA5 overexpressed lines (Kudo et al., 2019). The results observed indicated enhanced biomass, improved drought tolerance, additive regulated primary metabolism, and gene expression in the two overexpressed transgenic lines. Furthermore, the

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Table 5.4 Genetic engineering to confer drought stress tolerance in plants. Genes Vectors Mode of References transformation

TaWRKY2

pMWB112

DREB1 and OsPIL1 GmNFYA3

pGHX vector and pGKX vector pCAMBIA3301

AtTRE1

pCB302

miR319

pZH01

SNAC1

pAHC25

GsZFP1

pFGC1008

TaMYB33

pCAMBIA1300

ZmPIS

pCAMBIA1300

OsMYB2

pUN1301

OsDREB1F

pXQ35S

OsMYB3R-2

pBI221

Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Particle bombardment Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated Agrobacteriummediated

Gao et al. (2018) Kudo et al. (2017) Ni et al. (2013) Van Houtte et al. (2013) Zhou et al. (2013) Saad et al. (2013) Luo et al. (2012) Qin et al. (2012) Liu et al. (2013) Yang et al. (2012) Wang et al. (2008) Dai et al. (2007)

double overexpressed GA5-DREB1A transgenic line showed enhanced drought stress tolerance (Kudo et al., 2019). Xu et al. (2014a,b,c) used small-interfering RNA to suppress the expression of the ESKMO1 (ESK1) gene in Arabidopsis thaliana to improve the drought tolerance. ESKMO1 (ESK1) gene encodes a plantspecific polysaccharide O-acetyltransferase involved in xylan acetylation. In combination with the suppression, Arabidopsis thaliana CBF and ICE genes were also overexpressed in a gene-stacking approach. The CBF and ICE genes under the control of the CaMV 35S promoter were inserted into pGPTV and pSDAL vectors, respectively. The constructs were introduced into Arabidopsis by Agrobacterium-mediated transformation. Analysis of the root growth and leaf formation of all transgenic lines showed increased drought stress tolerance. Xu et al. (2014a,b,c) also indicated that none of the transgenic lines could confer a decrease in abscisic

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acid sensitivity and that the results showed that a gene-stacking approach can indeed improve drought stress tolerance in Arabidopsis. Tang et al. (2012) used the OsbZIP46 gene from Oryza sativa (rice) to assist in producing drought stress-tolerant Japonica rice. The OsbZIP46 gene was cloned into the binary vector pCAMBIA1301U under the control of a maize ubiquitin promoter. The construct was introduced into Oryza sativa japonica by means of Agrobacterium-mediated transformation. As a general rule, positive regulation of abscisic acid signaling may contribute to drought stress tolerance in plants. However, Tang et al. (2012) observed that the native OsbZIP46 gene decreased the germination rate of the wild-type rice under increased abscisic acid application. Hence, the overexpression of the native OsbZIP46 gene increases abscisic acid sensitivity but had no effect on drought tolerance in transgenic Japonica rice. This result suggested that the overexpression of OsbZIP46 in Oryza sativa japonica had a negative effect on drought stress tolerance. Furthermore, no transcriptional activity of the native OsbZIP46 gene was observed. A series of deletions experiments in the OsbZIP46 gene domains by Tang et al. (2012) showed activation only in transgenic with no domain D suggesting that the domain D has a prominent role in the regulation of the transactivation activity of OsbZIP46. A constitutive active form of the OsbZIP46 gene was constructed by inserting OsbZIP46CA1 coding region with a deleted domain D in the pCAMBIA1301U vector and transformed the construct into Oryza sativa japonica. The transgenics with overexpression of OsbZIP46CA1 showed increased drought tolerance at both seedling and reproductive stages. Furthermore, the rate of water loss also significantly decreased in the transgenic lines compared to the wildtype lines. Thus results suggest that the OsbZIP46 gene may require posttranslational modification and the modified domain D in the OsbZIP46CA1 gene mimics posttranslational modification and significantly increased drought stress tolerance in Oryza sativa japonica.

5.5 Transgenic approaches for increased flooding stress tolerance in plants Flooding or submergence stress ranks alongside drought as the most serious of abiotic stresses. Areas in Asia lose entire fields of crops due to flooding (Quimio et al., 2000). One of the reasons for crop reduction

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Table 5.5 Increasing flooding stress tolerance in plants through genetic engineering. Genes Vectors Mode of transformation References

AdPCD1 VHb HaHB11 APX OsEREBP1 VHb VHb

pCAMBIA1301 pBI121 pBI101 pPZP200 Ubi-NC1300-RFA pBI121 pCAMBIA-1201

Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated Agrobacterium-mediated

Zhang et al. (2016) Du et al. (2016) Cabello et al. (2016) Chiang et al. (2015) Jisha et al. (2015) Zelasco et al. (2006) Li et al. (2005)

under flooding stress is due to plants being exposed to anaerobic conditions causing low solubility of gases due to the waterlogging (Miro and Ismail, 2013). Another factor that could impact crop production under flooding is the osmotic pressure being imposed on the plants due to the high water concentration. The use of transgenic approaches allows for the development of plants with enhanced abilities to flooding and submergence stress. Table 5.5 includes candidate genes which were used in transgenic studies to improve flooding tolerance in plants. A study by Lv et al. (2016) used the ethylene response factor BnERF2-like (ERF2.4) to enhance submergence tolerance in Arabidopsis thaliana. The BnERF2.4 open reading frame (ORF) from Brassica napus was cloned into the pBI121 vector under the control of the CaMV 35S promoter and used to transform Arabidopsis by Agrobacterium-mediated transformation. The transgenic lines showed an increase in root length as well as the chlorophyll content as compared to corresponding wild-type plants after 6 days of submergence. The BnERF2.4 transgenic lines displayed no significant increase in malondialdehyde (MDA) content under the treatment period, however, the wild-type plants showed 1.6-fold increase. Antioxidant enzymes [catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)] showed enhanced expression in transgenic lines under the submergence stress. The study concluded that the transgene expression of BnERF2.4 enhances the defense of Arabidopsis thaliana to submergence stress by upregulating the antioxidant enzymes allowing for appropriate ROS scavenging. Liu et al. (2012) performed a study to investigate the overexpression of the RAP2.6L gene in an effort to delay waterlogging induced senescence. The gene was obtained from the leaves of wild-type Arabidopsis thaliana. The construct used in this study consisted of the pBI121 vector and the RAP2.6L gene. The construct was then introduced into Arabidopsis

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thaliana using Agrobacterium tumefaciens (EHA105)-mediated transformation via floral dip method. The plants overexpressing RAP2.6L showed increased growth under waterlogging conditions when compared to the wild-type plants. The decrease in chlorophyll content in the RAP2.6L transgenic line was far lower than in the wild-type plants which exhibited a 40%45% decrease. The transgenic lines displayed lower lipid damage as well as hydrogen peroxide accumulation when compared to the wildtype plants. One interesting observation was that the transgenic lines overexpressing RAP2.6L had lower stomatal conductance when compared to the wild-type plants. The study concluded that stomatal closure was regulated by abscisic acid signaling which then prevented water loss as well as reduced oxidative damage leading to delayed premature senescence. Xu et al. (2006) overexpressed Sub1A, an ethylene-response-factor-like gene from submergence tolerant rice cultivar FR13A, into submergence sensitive rice cultivar to increase submergence tolerance. The full-length Sub1A cDNA sequence was cloned into pUbi-C1300 and mobilized into submergence sensitive rice cultivar using Agrobacterium tumefaciens (EHA105). The results suggested that the overexpression of Sub1A was sufficient to enhance the submergence tolerance of submergence sensitive rice. Quimio et al. (2000) showed that overproducing pyruvate decarboxylase (PDC) in Oryza sativa increases its submergence tolerance. The pdc1gene isolated from rice (cultivar IR54) genomic library was cloned in pDC38 vector under the Act1 promoter and immobilized in 812 days old embryos by particle bombardment method. The transgenic seedlings were transferred to the tank after 14 days and submerged in water to simulate flooding. The transgenic lines, T309 and FR13A, showed tolerance to submergence and thus waterlogging. The FR13A transgenic line showed a significant increase in growth as compared to the nontransformed rice plants. Ethanol production was used as an indicator of survival and the rate of ethanol production was increased due to increased activity of PDC leading to increased survival under submergence stress.

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5.6 Improving plant tolerance to nutrient deficiency through genetic engineering Micronutrients are imperative for maintaining proper plant growth (Tavakoli et al., 2014). The lack of these essential nutrients may lead to impaired growth as well as higher mortality rates (White and Brown, 2010). The problem arises when soils designated for crop production are poor and deficient in essential micronutrients such as iron and zinc. This not only leads to decreased crop yields but also has negative implications on human health. The transgenic approach in conjunction with data mining for new genes could be used to increase the tolerance of plants to micronutrient deficiencies. Zhao et al. (2016) showed that overexpression of MdbHLH104 gene affected iron deficiency tolerance in Malus domestica (apple). The MdbHLH104 gene cloned in pIR vector under the CaMV 35S promoter was used to transform apple calli via Agrobacterium. Phenotypic analysis was performed to evaluate the transgenic lines. Under iron sufficient conditions, both wild-type and transgenic lines showed similar chlorophyll content and growth. The transgenic plants, however, showed lower chlorosis and a higher chlorophyll concentration than wild type under iron deficiency conditions. Higher iron accumulation was observed in transgenic lines as compared to wild-type plants suggesting that the transgenics had higher acidification ability than the wild-type plants. Li et al. (2015) overexpressed a ZIP3 gene from Zea mays (maize) in Arabidopsis to enhance zinc uptake and to improve iron and zinc deficiency tolerance. The pBI121-ZmZIP3 construct was used to transform Arabidopsis plants by Agrobacterium tumefaciens (GV3101)-mediated floral dip method. The ZmZIP3 transgenic lines showed improved tolerance to iron deficiency due to longer roots as compared to wild-type plants. The transgenic lines, however, did not show improved zinc deficiency tolerance. Thus the study showed that overexpressing ZmZIP3 in Arabidopsis enhanced zinc uptake as well as improved iron deficiency tolerance in the transgenic plants. Kobayashi et al. (2007) showed an increase in tolerance of plants to iron deficiency using iron deficiency-responsive cis-acting elementbinding factor 1 (IDEF1) gene.

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Figure 5.4 The role of IDEF1 in iron deficiency stress tolerance.

The construct was made by replacing the GUS gene in the pIG121Hm vector by an iron deficiency-responsive cis-acting elementbinding factor 1 (IDEF1) gene. Leaf discs of tobacco were then transformed with the construct using Agrobacterium-mediated method. Another construct (IDS2-IDEF1) was produced by inserting the IDEF1 gene into the pIG121Hm vector containing the IDS2 promoter (irondeficiency-responsive promoter) from Hordeum vulgare (barley). This construct was also moved into Agrobacterium which facilitated the transformation of Oryza sativa (rice) (summarized in Fig. 5.4). The positive transgenic lines were then used for further experimentation. The transgenic tobacco lines displayed slightly higher chlorophyll content than the control lines. Furthermore, transformed IDS2-IDEF1 rice lines grown in iron-deficient hydroponic media accumulated similar iron concentrations as the untransformed lines, in leaf blades as well as roots. The IDS2IDEF1 lines also showed improved growth, when challenged with iron deficiency compared to the untransformed lines.

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Ramesh et al. (2004) performed a study on Hordeum vulgare (barley) to understand how a zinc transporter from Arabidopsis could increase zinc uptake under zinc deprivation. The AtZIP1 gene from Arabidopsis thaliana was cloned in vector pWVec8 under CaMV 35S promoter. Immature embryos of barley were isolated and transformed by Agrobacteriummediated transformation using the approach from Tingay et al. (1997). An increase in root weight was observed in the plants overexpressing the AtZIP1 gene under zinc-deficient conditions as compared to the nontransgenic plants.

5.7 Improving plant tolerance to heavy metal stress tolerance through transgenic approaches Due to anthropogenic activities such as mining and industrial manufacturing processes, levels of heavy metals are ever-increasing in both the water and soil (Gokul et al., 2018). The increase in heavy metals in the soil has far-reaching implications. It disrupts the ecosystem and also

Figure 5.5 Studies involving overexpression of candidate genes in an effort to improve heavy metal tolerance in crop plants.

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negatively affect crop growth. Physical remediation of soils is often labor intensive as well as very expensive. Therefore new ways to improve the growth of crops under heavy metal stress must be investigated (Gasic and Korban, 2007). One method is to identify crucial genes in plants and to use a transgenic approach to confer and increase heavy metal tolerance in crop plants (Fig. 5.5). A study to improve the tolerance of tobacco plants to cadmium (Cd) toxicity was undertaken by cloning a Cd-induced catalase (CAT) gene from Brassica juncea into the tobacco plants (Guan et al., 2009). The plasmid pBI121 harboring BjCAT3 gene driven by CaMV 35S promoter was used to transform tobacco plants by Agrobacterium tumefaciens (strain EHA105). Biochemical and physiological assays were performed to assess the effect of overexpression of the CAT gene on Cd tolerance in tobacco. The transgenic tobacco lines displayed an overall higher CAT activity as compared to the wild type. Furthermore, transgenic tobacco also displayed increased growth when exposed to cadmium stress. The overexpression of the CAT gene also resulted in a reduction of root hydrogen peroxide content as well as lower lipid peroxidation, which resulted in higher cell viability under Cd stress. A study by Gasic and Korban (2007) used an Arabidopsis phytochelatin synthase gene (AtPCS1) to increase tolerance in Brassica juncea (B. juncea) to arsenic and cadmium toxicity. The gene construct consisted of a C-terminal FLAG (DYKDDDDL)-tagged AtPCS1 gene under the control of an AtPCS1 promoter (Lee et al., 2002). The construct was transformed into B. juncea facilitated by Agrobacterium tumefaciens transformation (Gasic and Korban, 2007). The transgenic lines displayed an increase in tolerance to cadmium and arsenic toxicity. The authors observed an increase in root and shoot growth under heavy metal stress as well as an increase in the heavy metal-binding compounds (phytochelatins and thiols) in the roots of transgenic lines (Gasic and Korban, 2007). Lee et al. (2007) worked on improving Festuca arundinacea (tall fescue) plants to a wide array of abiotic stresses by introducing and overexpressing a copper/zinc (Cu/Zn) superoxide dismutase (SOD) and APX in transgenic lines. The desired construct consisted of the vector pCAMBIA1300, Cu/ Zn SOD from Manihot esculenta (cassava), APX isolated from Pisum sativum (pea), and the stress-inducible promoter sweet potato peroxidase anionic 2 (SWPA2) which controlled the expression of both genes independently. The recombinant constructs were then introduced into Agrobacterium tumefaciens. Calli from the fescue plants were then transformed by Agrobacterium-

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mediated transformation. The transgenic fescue plants were observed to have higher SOD and APX activity (simultaneously) as compared to the nontransgenic plants. The enhanced ROS scavenging observed in the transgenic plants lead to a decrease in lipid peroxidation and an increase in chlorophyll content under high copper, cadmium, and arsenic levels. A Brassica napus (canola) transgenic line was produced by inserting 1aminocyclopropane-1-carboxylate (ACC) deaminase gene from Pseudomonas putida (P. putida) under the direct control of a rolD promoter, which was obtained from the Agrobacterium rhizogenes Ri plasmid (Stearns et al., 2005). The final construct contained one or two copies of the ACC deaminase gene. The resulting constructs were then transformed into Agrobacterium tumefaciens and further used to clone canola calli. The tolerances of these plants to nickel stress was assessed by looking at physiological parameters such as shoot and root length, shoot and root dry weight and chlorophyll content. The transgenic lines were observed to have increased shoot and root length and biomass under nickel stress when compared to their untransformed counterparts (Stearns et al., 2005).

5.8 Conclusion Abiotic stress tolerance (heat stress, cold stress, salinity stress, drought stress, flooding stress, nutrient deficiency stress, and heavy metal stress) are the major cause of crop loss. The transgenic technology is being successfully used to achieve tolerance to abiotic factors. This chapter highlights some of the studies which achieved successes with transgenic technologies in plants using genetic engineering. Various promoters (most studies still used the CaMV 35S promoter) as well as the vectors are cited which are being used efficiently in plant transformation. Furthermore, the chapter highlights the need to shift focus from only one abiotic stress to addressing multiple genes together to achieve desired plant improvement. Literature searches suggest Agrobacterium-mediated transformation method still to be the leading method of choice for gene delivery. Studies suggest Arabidopsis thaliana as the most used plant followed closely by Oryza sativa (rice) for proof of concept. The single-gene approach still dominates the field, but the gene-stacking (using multiple genes) approach, which is often viewed as a more difficult approach, is being used in competent

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laboratories. Lastly, the transgenic plants are still grown in controlled conditions (growth room or greenhouse) rather than the field conditions clearly highlighting a serious gap in engineering plants tolerant to various stresses using transgenic approaches, in the future.

5.9 Future prospects The world population is growing at a rapid speed and therefore the human race will require more fertile land in order to double our food supply. However, more people will require more housing, hence, giving rise to rapid urbanization. This will ultimately reduce the land for food production. The increase in population as well as urbanization will also increase our contribution to climate change. Hence, the already decreased agricultural land will be under severe pressure from abiotic stresses. Relying on the plants’ natural ability to cope with these stresses has proven futile and, therefore transgenic technologies provide an opportunity to increase the speed of improving plant tolerance to stress. However, the technology is far from perfect and much room for improvement still exist in the field. To start with, novel promoters will have to be identified, which get rapidly activated only under a given stress. For this purpose, researchers will have to identify promoters that functions throughout the plant (not only one tissue specific), because during stress onset, the whole plant is under stress. In addition, highthroughput screening can assist in identifying stress-specific transcription factors, which can bind to the novel stress-inducible promoters for more efficient gene expression. Also, there is no doubt that highthroughput technologies (genomics and proteomics) will lead to a huge increase in abiotic stress tolerance genes in plants. But, the bottleneck will be to understand posttranslational modifications of the identified genes. Agrobacterium-mediated plant transformation for genetic engineering of plant stress tolerance remains the gold standard in many laboratories, however, the search for novel Agrobacterium species and strains is required in order to improve transformation efficiency and gene integration into plants. For this purpose, culture-based techniques will have to improve drastically. Novel species and strains can be subjected to wholegenome sequencing and, therefore will increase our knowledge

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about Agrobacterium. Furthermore, the knowledge about genomes can guide genetic engineering efforts to improve Agrobacterium strains for improved plant transformations. Plasmids or vectors should also be synthesized to increase the success of genetic engineering but also to hold and carry multiple genes (as huge constructs) into plants. However, gene-stacking has not yielded much success due to many reasons (such as insufficient transfer to the next generation as well as linkage drag) and therefore minichromosome technology will have to be advanced more rapidly. Furthermore, the minichromosome technology will have to be used in conjunction with vector-based gene-stacking in order to increase plant tolerance to multiple stresses (at once), therefore future research (soil-pot experiments preferably) should focus on multiple stresses only seeing that this will be a more true reflection of field or environmental stress conditions. By focusing on multiple stresses in the growth room or greenhouse, further experiments should move to field trials with much better success.

Acknowledgments Dr. Gokul, Miss. Niekerk, and Mr. Carelse were financially supported by the National Research Foundation (NRF) of South Africa (Grant numbers: 106427, 111871, and 113857). Dr. Keyster’s research was financially supported by the NRF (Grant numbers: 116346 and 109083), DST-NRF Centre of Excellence in Food Security (Project ID: 170202), and the University of the Western Cape (UWC). Therefore the authors would like to express appreciation toward these funding bodies.

References

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CHAPTER SIX

Transgenic technologies for efficient insect pest management in crop plants Faisal Saeed1, Muneeb Hassan Hashmi1, Md. Jakir Hossain1, Muhammad Amjad Ali2 and Allah Bakhsh1 1

Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, Nigde, Turkey Department of Plant Pathology, Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan

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6.1 Introduction To feed the increasing population world around, especially undernourished people of Asia, Africa, and Latin America, it is urgent need to increase the food production by enhancing yields of staple crops. Notorious insect pests from several insect orders are the main cause of damage to agricultural crops that has been estimated around 14% of total agricultural crop production including 52% in wheat, 83% in rice, 59% in maize, 74% in potato, 58% in soybean, and 84% in cotton, respectively (Oerke et al., 1994). For the assurance of higher crop yield, control of these notorious insect pests is prerequisite. Minimization of losses associated with insect pest is essential for the sustainable crop production and maintenance of the economic threshold level. Besides the direct and vector-mediated crop losses, another means of economic damage is the unwanted and excessive application of pesticides aimed to suppress the attack of these insect pests on a crop field. Economic injuries due to pesticide uses and control measures of biotic and abiotic factors in semiarid farming conditions, in recent years, have been valued at 10 billion and 2 billion USD, respectively [ICRISAT (International Crops Research Institute for the Semi-Arid Tropics), 1992]. Although the control of insect pests by the extensive and indiscriminate application of insecticides gives transient good results, the harmful effects of same on the ecological balance and human health are seen after some time. Voluminous application Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00006-X

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of chemical pesticides causes lethal effects on many beneficial microorganisms and natural habitats. The residual remains of these chemical agents cause food poisoning and environmental pollution. Considering these evil effects, prohibition of the extensive use of chemical components needs to be ensured by the law-enforcing authorities, and regulations regarding the frequency of spray, amount of residual part on food stuff, and mode of action of chemicals should be implemented properly. Managing insect pest with the harmony of adequate food production and environmental safety is the major challenge faced by grass root-level farmers and pest control agencies. The current strategies taken for the reduction of crop losses primarily focus on use of chemical pesticides; thus the utility of chemicals for the control of harmful pest population cannot be overlooked. It is therefore essential to develop substitute technology that will allow the logical level usage of pesticide along with the breeding techniques for sustainable crop production and environmental protection. In order to ensure sound soil health and chemical hazard-free environment, the use of specific chemical agents having transient existence and integrated pest management (IPM) depending on plant resistance against insect pest should be practiced. As a substitute to conventional technologies, crop plant possessing inherent resistance to the insect pest opens a new dimension for the crop protection. The great achievement of modern plant biotechnology is the generation and commercialization of several transgenic plants showing elevated resistance against major devastating insect pests. Thus the global evidence of insect resistance transgenic technology has added a new era to pest control strategies and being considered as a vital tool of IPM all over the world. As a gene-silencing strategy, RNAi (highly conserved in eukaryotic organisms)-mediated defense mechanism was first narrated by Fire et al. (1998). The double-stranded RNA (dsRNA), introduced in an organism exogenously, has potentiality to silence the genes posttranscriptionally (Geley and Müller, 2004). Currently, these are classified into four groups including: siRNAs (short interfering RNAs), piRNAs (Piwi-interacting RNAs), endo-siRNAs or esiRNAs (endogenous siRNAs), and miRNAs (microRNAs) (Terenius et al., 2011). The resistance against the notorious insect by these miRNAs is confirmed by the following functions: disruption of sperm release and egg-hatching, inhibitory effect on gene expression, larval abnormalities and high rate of mortality, reduced chitin content in the midgut, delayed development, and reduced fertility. Lately, although the efficacy of RNAi technology greatly differs between the

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insect orders, this technique has shown its extraordinary utility to control and reduce the crop insect pests. Gene knockdown efficacy rate in many RNAi recalcitrant insect species was found near about 60% or lower with the record of temporary action (Huvenne and Smagghe, 2010; Li et al., 2013). Among the coleopteran insects, those are sensitive to RNAi, the efficacy rate was recorded 90% or higher, where the small doses were enough and the effect was durable and transmittable to the next progeny (Baum et al., 2007; Zhu et al., 2011; Bolognesi et al., 2012; Rangasamy and Siegfried, 2012). Integration of gene of interest into nuclear genome to assure the resistance against insect pests usually yields very low altitude of gene expression unless extensive modifications are carried; however, the incorporation of the same gene into the plastome ensures the high level of toxin accumulation (McBride et al., 1995). Leaves of transplastomic plant contain high-level toxic protein and cause maximum damage to the insect larvae which feed on green tissues and injure the plants by chewing leaves and sucking sap. The effect was achieved by incorporation of BT toxin into the genome of plastid, which resulted in the elevated level of toxic protein accumulation (3% 5% of total soluble protein (TSP) by plastid transformation but only .0.2% of TSP through the nuclear genome transformation) (McBride et al., 1995). High levels of toxin accumulation as the cuboidal crystal formation in the stroma of chloroplast were reported when the transgene was inserted together with a putative chaperon (45% 46% of TSP in leaves). On the contrary, lower transgenic protein resulting about 0.4% of TSP and absence of crystals were was seen when the transgene was used alone (De Cosa et al., 2001). In cabbage genome, cry1Ab gene was successfully transferred and the toxic protein was detected between 4.8% and 11.1% of TSP. Despite generating good results in some species, this method has not been widely adopted due to problems in achieving stable transformation. Further, the adaptation of the technique in some species proved to be difficult (Gatehouse, 2008). The financial feasibility is hampered by excessive expense and downcasts output of transplastomic lines. Recently, a new non-BT-type strategy of insect resistance has been developed. To achieve effective RNAi-mediated damage against large number of plant pests, plants should be engineered to express dsRNAs targeting important insect gene (Zhang et al., 2015). A hairpin-structured dsRNA specific against β-actin Colorado potato beetle (CPB) gene proved to be lethal even when it expressed to 0.4% of total cellular RNA

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in the leaves of transplastomic potato plants. Mortality was recorded to be 100% within 5 days, in both of adults and larvae, thus supporting the proof of the above-mentioned concept. The above data depict the great potentiality of transplastomic plants to control the notorious insect pests without the use of chemical inputs. Since the last two decades, many attempts have been taken to develop our knowledge and competency on genome editing tools to edit the genome sequence of several agriculturally important plants and organism. In today’s 21st century, due to availability and simplicity of the process, CRISPR is the leading genome editing tool that is hugely being used to accelerate research and commercialization of agricultural products. Initially, CRISPR was extensively being used for human disease control (Samuel et al., 2016; Alphey, 2016; Windbichler et al., 2011). Of late, through the application of CRISPR, Nikolay and Omar (2019) at University of California devised a novel, environmentally friendly and relatively lowcost method named “precision-guided sterile insect technique” or pgSIT, for altering the key genes that regulate the insect sex determination and fertility. As an alternative to the radiation, Kandul et al. (2019) got 100% efficacy to control the fruit dry fly where through the CRISPR, female viability and male fertility genes were altered and mutated. Recently, a mathematical model of gene drive has been developed to control the agricultural insect pest including Drosophila suzukii, the medfly, the pink bollworm, and the Asian citrus psyllid. This model can be used to determine the optimal CRISPR-Cas9-based gene drive architectures that will help to successfully control their agricultural impacts ensuring biosafety. By considering the above-mentioned technologies, it is mandatory and demand of the time to initiate scientifically sound applications to incorporate the external and plant-originated genes for the reduction of insect-mediated crop losses. Concurrently, emphasis should also be paid regarding the biosafety issues, public concern, social customs, and better presentation of the beneficial aspects of GMO plants. Understanding the biology of insects, behavior, response to the insecticidal proteins, spatial and temporal expression of these proteins in plant tissues, resistance management strategies, and the impact of insecticidal proteins on insect pests and non-target organisms are the main key aspects to achieve the desired goals. Finally, for the utmost success, the transfer of technology to the root-level user needs to be ascertained. This will enhance the deployment of insect-resistant transgenic technology for the reduction of insecticide dependency, suppression of natural predators, and implementation of IPM in agricultural practices.

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6.2 Bt genes One of the widespread agricultural and forestry biocontrol agents is Bacillus thuringiensis (Bt). Basically, it is a soil borne gram positive bacterium, which forms spores in nature. During vegetative growth phase, this gram-positive bacterium secretes many insecticidal proteins such as Sip (Secreted insecticidal proteins) and Vip (Vegetative insecticidal proteins). β-exotoxins and parasporal crystalline δ-endotoxins, Cyt (cytolytic toxin) and Cry (Crystal toxin), are other proteins produced during stationary phase of bacterial growth (Chattopadhyay and Banerjee, 2018). These parasporal proteins basically are nonhemolytic by nature but have potential to kill cancer-causing cells (Xu et al., 2014a,b). The ability to make colonies inside the gut of insect makes it more suitable vector to be used against insect pests of agriculture (Deist et al., 2014). The first reported species is Bt israelensis, which was harmful against dipteran larvae, especially black flies, mosquitoes, and chironomid midges. Due to availability of more effective chemicals against insect pests, the demand of Bt insecticides decreased in mid 1970s. However, within next 10 years, demand again accelerated due to marvelous progress in the field of genetic engineering. In mid 1990s, commercialization of transgenic plants expressing toxins such as Bt gene started and by 1999, different transgenic crops having Bt genes such as Bt potatoes, Bt cotton, and Bt corn were introduced (Tabashnik et al., 2013).

6.2.1 Bt strains and toxins One of the broadly used microbial pest control agents is Bt. Its demand enormously increased all over the world. There are 84 strains of Bt discovered but all the strains are not harmful for insects like a Bt strain known as NTB-88 did not show its harmful effect against any insect (Ibrahim et al., 2010). There are some Bt strains such as Bt israeliensis that have specific toxicity against dipteran pest (Land and Miljand, 2010). Some strains of Bt active against lepidoptera and coleopteran only (Azizoglu et al., 2015).

6.2.2 Applications The major application of Bacillus thuringiensis toxins is development of genetically engineered plants, which show resistance against insect pest.

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Notable achievement of using Bt genes successfully is the decreased lepidopteran population, which are the main pests in United States, India, Pakistan and Canada. The control of this lepidopteran was mainly achieved by the use of Bt strain producing toxins such as Cry1Aa, Cry1Ab, Cry1Ac, and Cry2a. Effective application of Bt depends on appropriate timing and weather conditions (Bauce et al., 2004; Khan et al., 2011; Bakhsh et al., 2012, 2018). Genetically engineered plants having Bt gene brought down the use of chemical insecticides and made genetically engineered crops, favorable to environment. Cry toxin is genetically modified such that it is expressed uninterruptedly in genetically engineered plants without being degraded and hence available for boring and chewing insect pests. Expression of this cry protein has been enhanced by usage of specific plant codon and deletion of splicing signal sequences (Schuler et al., 1998). So, planting of genetically edited Bt crops has reduced the application of pesticide remarkably in planting areas (Qaim and Zilberman, 2003). Fascinatingly, the use of Bt cotton in countries like Mexico, India, and China displayed positive outcome on production and in reducing the use of chemicals (Toenniessen et al., 2003). The first successful endeavor to produce insect-resistance transgenic plants was initiated with single insecticidal Bt genes isolated from Bacillus thuringiensis (Bt) that was capable to exhibited extraordinary achievement in relation to insect pest resistance, majorly in corn and cotton. This was considered as a significant milestone with respect to ecology and economy. As a beneficial aspect of Bt-mediated technology, the application of citable Bt crops (Bt-maize and Bt-cotton) assured the maximum reduction of insecticides, convenience to farmers, positive effect on farmer’s health, and sustainable environment. In 1987 first transgenic cotton with Bt was produced expressing truncated toxin of Bt (Cry1A) with constitutive promoter. The results showed mortality against Manduca sexta (Tobacco hornworm) larvae (Barton et al., 1987). Cotton cultivar Coker312 genetically engineered using modified PM Cry1A (c) under the control of CaMV35s promoter harboring a duplicated enhancer region exhibited complete resistance against Spodoptera exigua, Trichoplusiani, and Heliothis zea. The maximum level of toxin protein accumulation in transformed plants was about 0.1% of the TSP. Tobacco plants transformed with Bacillus thuringiensis var. kurstaki (B.t. k.) HD73 derived truncated crylA(c) gene, encoding only insecticidal domain of the lepidopteran active CrylA(c), cloned under the control of Arabidopsis ribulose-1,5-bisphosphate carboxylase small subunit

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ats1A promoter with transit peptide showed 10- to 20-fold rise in Cry1A(c) messenger RNA and protein in contrast to gene cassette possessing CaMV35s promoter and duplicated enhancer region (Wong et al., 1992). This effort was succeeded to gain the elevated level of toxin protein accumulation near about 1% of the TSP. Hence, BT crop can be a unique choice of interest for the practice of IPM to target the harmful pests of economically important crop plants. To ascertain the stability and sustainability of insect resistance, new and up-to-date strategies should be designed to ensure the long-time efficacy of insect resistance of second- and third-generation BT-mediated crop plants. The alternation of metabolism pattern of amino acid through the inhibition of protein digestion process could be another strategy for insect pest control (Johnson et al., 1989; Hilder et al., 1993). Serine proteases (chymotrypsin, trypsin, and elastase endoproteases) play a vital role as primary enzymes for protein digestion, especially for lepidopteron pest. Genes responsible for the encoding members of different serine protease inhibitors were successfully cloned and incorporated into plant genomes. The expression of trypsin inhibitor gene in tobacco exhibited near about 1% elevated mortality rate. The expression of trypsin inhibitor gene ensured the suppression of physiological growth of insect pests (Heliothis virescens) and the damages associated by them (Hilder et al., 1987). Several transgenic crop plants were produced by the integration of protease inhibitor genes. Jongsma et al. (1995) reported that the caterpillars raised on transgenic plants expressing potato inhibitor II gene showed about 18% gut proteinase activity as compared to 78% gut proteinase activity in caterpillars found in control plants. A heterogeneous group of sugar-binding proteins known as plant lectins are also being used as protective weapons to control the crop pest. It has protective functions against a wide range of organisms that can be classified on the basis of the nature of sugarbinding proteins. Transgenic potato plant developed through the incorporation of rice cystatin-I revealed around 53% mortality in larvae raised on transgenic potato leaves as compared to ,17% mortality in untransformed plants (Lecardonnel et al., 1999). By using this technology, Fitches et al. (1997) achieved 32% and 23% reduction of larval biomass in tomato moth (Lacanobia oleracea); on the other hand, they also showed 48% reduction potentiality in their greenhouse trials. Expression of specific enzymes into transgenic plants can be suggested as another alternative to Bt-mediated technologies in order to treat the insect pests. Transgenic tobacco plant expressing chitinase (an important enzyme of insect integument) has

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shown greater resistance against lepidopteran insect pests (Ding et al., 1998). Proteins from Bacillus cereus (VIP-1, 2, and 3) have similar activity to d-endotoxins and insecticidal proteins that create gut paralysis and finally larval mortality. Although the commercialization of biotechnological strategies to control the insect pests largely depends on the expression of insecticidal Bt proteins however, the recent data on the resistance development capability of few insect pests (Ostrinia nubilalis and Heliothis virescens of Lepidoptera) against this protein has threatened its efficacy (Ferré and Van Rie, 2002; Baum et al., 2007). Due to this limitation of Bt protein, it is essential to develop an alternative and up-to-date technology to control the insect pest that will be ecologically and financially feasible. To fulfill this research gap, gene silencing technology can be an attractive alternative to mitigate the crop damage by the insect pests.

6.3 First-generation genome editing technology 6.3.1 RNA interference RNAi is a biological approach, which causes post-transcriptional gene silencing. This mechanism down-regulates the expression of desired or targeted genes and proteins, which are triggered by dsRNA molecules (Bosher and Labouesse, 2000; Kim and Rossi, 2007). RNA interference mechanism has the potential for crop improvement by identification and functional assessment of many genes within any genome. Lately, RNAi technique to knockdown the targeted genes has become a more reliable and powerful tool. Its analysis is outlined by the loss-of-functional activities of target genes (Tierney and Lamour, 2005). First time, RNA interference technique was applied on Petunia hybrida L. to increase anthocyanin pigment through introducing chalcone synthase gene (chsA) in the plant (Napoli et al., 1990). Due to the overexpression of the chsA gene, new flower color was produced in the transgenic petunia plant (Van der Krol et al., 1990). RNA interference is a vital approach against essential genes silencing or down-regulation of gene expression instead of knocking out the genes. However, sometime silencing effect is temporary because its efficiency mainly depends on delivery methods of dsRNA or uptake mechanism of

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dsRNA in the cells of targeted insect pests (Rajagopal et al., 2002). There are two types of gene suppression through RNA interference, that is “cell-autonomous and noncell-autonomous” (Whangbo and Hunter, 2008). Suppression of the targeted gene in the cell occurs when dsRNAs are delivered to targeted cell; this process is called cell-autonomous RNA interference. However, in entomology, many studies of gene function rely on noncellautonomous RNA interference. In this the suppression of gene of interest is not observed where the dsRNAs are delivered instead suppression is observed in the the targeted cells or tissues. It means in noncell-autonomous RNAi, the dsRNA is not delivered inside the cell but cell has to either absorb it from surroundings or it absorbed by one tissue and transported to the targeted cell (Whangbo and Hunter, 2008; Huvenne and Smagghe, 2010). Two types of noncell-autonomous RNA interference have been classified, which are systemic and environmental friendly. A systemic RNA interference response happens when downregulation of the desired or targeted gene is extant to other cells (Whangbo and Hunter, 2008; Huvenne and Smagghe, 2010). The second type of non cell-autonomous RNAi named environmental RNAi exhibit response when dsRNA are taken up from the environment and results in suppression of targeted gene, simultaneously (Huvenne and Smagghe, 2010; Whangbo and Hunter, 2008). RNA interference is persuaded by many factors, mainly dsRNA delivery method (Rajagopal et al., 2002; Araujo et al., 2006; Yao et al., 2013). The salient examples of successful delivery of dsRNAs via microinjection in insect pests is shown in Table 6.1. RNA interference can be accomplished via feeding, soaking, and microinjection by the insertion of dsRNA to the hemocoel and midgut of insects (Scott et al., 2013).

6.3.2 RNAi pathways and mechanism RNA interference pathways are classified into three main classes according to their biogenesis and associated protein. These noncoding small RNAs are characterized as siRNA, micro RNA, and piwiRNA. Several distinctive but associated core RNAi proteins are engaged to these three RNAi pathways. The two pathways siRNA and miRNA function as downregulation of gene expression or knockdown of the desired gene while the third one, piwiRNA pathway, is to defend against transposable elements (Aravin et al., 2007). RNA interference pathways (siRNA, miRNA, and piRNA) are evolutionarily conserved while between insect’s siRNA pathway core genes

Table 6.1 Applications of microinjection-based RNAi technology to target insect pests. Insect pests Target gene Aim of study Application Results

Rhodnius prolixus CPB

NP2

CYP450 GS CP

Silencing of salivary gland in the triatomine bug Injection

Downregulation of three targeted transcripts in CPB to decreased resistance to insecticide

Injection

CPB

Ldace1

Peregrinus maidis

VATPase Development of RNAi approach to control P. Injection B maidis VATPase D Alucβ-actin PM-RNA interference to protect plants from A. Injection lucorum VTE Investigation of RNAi effects in T. castaneum Injection IAP and dsRNA constancy

Apolygus lucorum Tribolium castaneum

Acyrthosiphon VTE pisum

RNAi-mediated silencing of Ldace1 effect Ache Injection activity in CPB

Investigation of RNAi efficiency in A. pisum using injection method of dsRNA delivery

Injection

References

Decreased gene expression near 75% 6 14% Enhanced susceptibility Significant knockdown efficiency Significance level of mortality around 43% Disturbed growth of nymphs Higher mortality

Araujo et al. (2006) Clements et al. (2017)

Highest mortality

Liu et al. (2019) Cao et al. (2018)

100% mortality Continuous suppression of transcript 65% mortality 40% transient downregulation

Revuelta et al. (2011) Yao et al. (2013)

Cao et al. (2018)

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are more variable. The conservation of core proteins of RNAi among different insect species and C. elegans in the two pathways siRNA and miRNA, function as downregulator of gene expression (Shreve et al., 2013; Swevers et al., 2013). Central RNA interference genes of the small interference RNA pathway among species are less preserved than central RNAi genes involved in the piwiRNA (piRNA) and microRNA pathways. The siRNA functions as a guard against invading nucleic acids. This siRNA pathway is activated through exogenous dsRNA. The dsRNA delivery initiates RNA interference by activating siRNA pathway to persuade downregulation or silencing of a target gene. When a dsRNA is introduced into a cell, it is treated through an RNase III enzyme (Dicer) into 21 23 bp size siRNA (Bernstein et al., 2001). Argonaute (AGO), a member of PPD family of proteins (PAZ and PIWI Domain), is required for RNAi in flies. The RNA-induced silencing complex (RISC) integrates with argonaute-2 protein for functional activation (Hammond et al., 2001). The integration of siRNA into RISC-AGO-2 complex leads to ATP-dependent unwinding of ds-siRNA, which is followed by the degradation of passenger strand. Now, the single strand called as guide strand act as a template for RISC to find complementary messenger RNA (Nykänen et al., 2001). Once targeted or complementary mRNA is found, the RISC complex endonuclease activity is induced, which cleaves mRNA at the site near middle of siRNA complementarity. This activity is mediated by Argonaute (AGO) activates and cleaves the mRNA resulting in loss of expression of targeted gene or protein for which the mRNA is coding (Nykänen et al., 2001). This techniques is now extensively used for successfully silencing the desired genes of insect pest to protect crops.

6.3.3 Oral delivery method of dsRNA Three main categories are sprayable RNAi, nanoparticles-coated RNAi, and plant-mediated RNAi. 6.3.3.1 Sprayable RNA interference approach RNAi-based sprayable products to control insect pests are ready to be commercialized. In fact, some companies have already commercialized RNA interference-based sprays at smaller level and for household pests (Zotti et al., 2018). Indeed, dsRNA by spraying cannot be an effective tool to control insect pests at field level but it can be useful method at

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smaller level or only few pests can be controlled in field. According to Li et al. (2015), spraying methods to deliver dsRNA silencing could be achieved in case of piercing-sucking insect pests but will not be efficient for those insects, which feed on the phloem sap and also for those which feed inside plant stem-like stem-borer pests. Zhu et al. (2011) reported that dsRNA feeding assay of CPB effectively caused the downregulation of five targeted genes, namely, actin, sec 23, COPβ, and vATPase E or B. More importantly, targeting vATPase B gene for silencing in expression level of mRNA, a 93% reduction was recorded in the same way COPβ showed higher mortality as observed. Hence, this study is a first example in which dsRNA produced in bacteria used to feed insects for efficient RNAi. Kong et al. (2014) found that RNA interference using feeding bioassay to silence a targeted gene LdShd at the second instar larvae stage of CPB insect can give desired results. Therefore dsRNA was delivered, by oral feeding method, to silence targeted LdShd gene. After 5 days of feeding dsRNA, expression levels were downregulated to 46.8%. Hence, decrease in messenger RNA (mRNA) level of Ecdysone receptor (LdEcR) gene of CPB, which caused 80.0% deaths, also delayed development. Moreover, decreased mRNA level also affected pupation of CPB larvae according to the report. Spit et al. (2017) reported that in the gut of CPB, two nuclease genes, when expressed exclusively, result in degradation of dsRNA. Subsequently, it was proved that protection of potato crop from damaging insects can be improved by elimination of these nucleases activity in the CPB adult. These observations suggest that RNAi efficiency can be improved by silencing the gene involved in nucleases activity in insect pests. Wan et al. (2014) silenced ryanodine receptor gene (LdRuR) of Colorado potato beetle. dsRNA of LdRyR was introduced through diet and significant level of expression decreased. It was reported that at immature and adult stage of insect, the targeted gene was downregulated by 45% and also mortality percentage associated with chlorantraniliprole reduced. Very recently, Naqqash et al. (2020) successfully knocked down transcripts of three important genes (CP, p450, GSS) that are involved in resistance development mechanism in Colorado potato beetle (CPB) against chemical insecticides using leaf dip method. The lethal and sublethal effects of three dsRNAs were documented on different larval instars of CPB. Targeted genes by dsRNA oral delivery (sprayable) have been summarized in Table 6.2.

Table 6.2 dsRNA delivery via oral feeding (sprayable) targeting vital genes of insects to protect plants. Insects Target Aim of study Application Results gene

Leptinotarsa COPβ Ingestion of dsRNA against targeted decemlineata Sec23 genes to knockdown for control vATPases of CPB population Leptinotarsa Shd Downregulation of LdShd gene in decemlineata L. decemlineata (CPB)via RNAi approach Leptinotarsa dsRNase1 Enhancement of RNAi efficiency decemlineata dsRNase2 through silencing of nuclease genes in the gut of CPB Leptinotarsa RyRs Using RNAi approach knockdown decemlineata of RyRs gene in CPB Aphis gossypii CarE To reduce resistance to insecticides (organophosphorus) in cotton aphid via RNA interference approach

Oral feeding

Oral feeding

References

Significant level of mortality Zhu et al. Reduction in body weight (2011)

Delayed growth Significance level of knockdown Oral feeding Significantly silenced Decreased transcript level 84% 86% Oral feeding Significant level of downregulation of gene Artificial diet 33% silencing efficiency

Kong et al. (2014) Spit et al. (2017) Wan et al. (2014) Gong et al. (2014)

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6.3.3.2 Nanoparticles-coated RNAi Degradation of dsRNA can be decreased by using nanoparticles to deliver at the targeted site without any damage; it also enhances the uptake intact form of dsRNA through cell. Polymeric nanoparticles are degradable so these are safe for use. Due to their stability, these can be used for many purposes including for spray-induced gene silencing (SIGS) (Vauthier et al., 2003; Herrero-Vanrell et al., 2005). According to Zhang et al. (2010), RNAi technology to control mosquitoes was quite effective. The dsRNA was encapsulated in a polymer named chitosan and was reported to be suitable for long dsRNA and siRNA. The larvae were fed chitosan mixed with food through oral ingestion. As nanoparticle chitosan is not toxic and inexpensive, this can be used to deliver dsRNA. The efficacy was studied by assessing phenotype of insect, behavioral evaluation, and other related parameters. The results showed repression of AgCHS1 and AgCHS2 (chitin synthase genes) in Anopheles gambiae larvae by chitosan/AgCHS dsRNA-based nanoparticles. Moreover, Zhang et al. (2015) analyzed that during the development of larva of Anopheles gambiae and Aedes aegypti by using chitosan loaded with interfering RNAs, the targeted genes can be downregulated successfully. He et al. (2013) using RNAi system observed clear gene silencing of Asian corn borer larvae fed on the diet containing the combination of FNP and CHT10 dsRNA. The silencing reduced larvae size, molting failure, and finally resulted in death. Zheng et al. (2019) developed a nanocarrier delivery system that carried dsRNA into insects at targeted site through oral feeding and proficiently downregulated the expression of targeted gene. The aim of this study was to control insect pest at field level by degradation of dsRNA by nucleases (enzymes) in gut of insects. Nanocarrier was developed to deliver dsRNA to silence target gene. Hence, formulation of dsRNA-detergent was applied through dropping on notum of aphid and as perceived within one hour, it extended to different tissues. However, nanocarrier dsRNA delivered successfully and significance level of targeted gene expression decreased around 95.4%, total population of insects (aphid) also repressed around 80%. Thus to control robust insects, this formulation of dsRNA can be speedy tool for gene silencing.

6.3.4 Plant-mediated RNAi Plant-mediated RNAi for the control of agricultural insect pests has become environment-friendly approach. Moreover, it is shown that RNA interference proficiently and stably downregulates the expression of targeted genes (Baum et al., 2007). More specifically, the efficiency of RNAi

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approach seems to vary on target gene (Terenius et al., 2011). Plantmediated RNAi approach has led to the development of new generation of insect-resistant crops by expressing dsRNA in transgenic plants, which inhibit the expression of targeted gene of insect pests (Price and Gatehouse, 2008; Mutti et al., 2006, 2008). Agrobacterium-mediated plant transformation is reported to be the most efficient method for the development of successful plant-mediated RNA interference (Mao et al., 2013). For efficient RNAi, two key steps need to be followed, that is development of effective way of dsRNA in plants, and delivery of dsRNA into the gut cells of insect. Pitino et al. (2011) reported that by using plant-mediated RNAi system, two genes Rack1 and C002 were targeted in green peach aphid. According to this study, silencing of these two genes, which were expressed specifically in gut and salivary gland of aphid, could be helpful to control green peach aphid. Therefore, transformation of Nicotiana benthamiana and Arabidopsis thaliana with dsRNA targeting Rack1 and C002 under CaMV35S promoters was carried out using Agrobacterium tumefaciens. Transgenic plants produced transitorily dsRNA against targeted genes of green peach aphid. When aphids were fed on transgenic plants, it was observed that 60% expression level of both targeted genes (MpC002, Rack1) reduced in those aphids fed on transgenic as compared to control. As Mao and Zeng (2014) demonstrated, the silencing of gap gene (hunchback), which has very important role in insects for tissue differentiation, can cause distortion and death of offspring. Hence, using RNAi technology to silence hunch back gene, transgenic tobacco was developed and varying prototype of integration was perceived. Moreover, neonate aphids were fed on T2 generation of homozygous transgenics for bioassays. According to this study, expression levels of hunchback gene of aphid were downregulated and insect reproduction was reduced after feeding on transgenic plants. Xu et al. (2014a,b) reported CbE E4 gene, which is involved in detoxification of numerous pesticides, could be a vital target for plant-mediated RNAi technology using biolistic method, wheat cultivar Jinghual was co-transformed with RNA interference construct under the control of ribulose-1,5-biphosphate carboxylase small subunit promoter (rbcS). For analysis of these transgenic lines, aphids were raised and fed on them, which caused 30% 60% reduced expression level of carboxylesterase in Sitobion avenae (English grain aphid). So, using the plant-mediated RNAi system, it was observed that total number of aphids fed on transgenic plants were lower compared to those fed on non transgenic plants.

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Thakur et al. (2014) successfully downregulated the expression of vATPaseA gene in Bemisia tabaci (whitefly) by using RNAi system developed in transgenic tobacco plant, which expressed long dsRNA predecessor to generate siRNA. CaMV35S promoter in construct was used to produce dsRNA. 62% decrease in expression level was observed as compared to those insect (whitefly), which fed on a nontransgenic plant. The feeding results on the base of each day were recorded. The mortality rate were 15% 38% after two days of feeding, after four days of feeding mortality rate was 26% 58%, and after sixth day of feeding 34% 83% mortality rate. Results of this study suggested that host plants (transgenic) developed resistance against whiteflies. According to Liu et al. (2015), arginine kinase gene from Helicoverpa armigera that encodes phosphotransferase can be a vital target to silence via RNAi technique because it plays important role in the invertebrate’s metabolism. Transgenic Arabidopsis plant were developed with RNAi technique targeted for gene HaAK. After 3 days of feeding on transgenic plants, 55% mortality rate of first instar was observed and downregulation of targeted gene HaAK more than 52% was recorded. Bally et al. (2016) proposed a study for crop protection against harmful insects via transkingdom RNA interference approach. Transgenic plants were developed expressing dsRNA to knockdown vital genes of insect pests. Genetic construct coding 200 nt hpRNAs was integrated into N. benthamiana plant in both nuclear and chloroplast genomes. Consequently, it was observed that hpRNAs accumulated in Nicotiana benthamiana transplastomic lines showed more resistance against Helicoverpa armigera than nuclear transformed N. benthamiana plants. According to results, it was suggested that in chloroplast high level of RNAi efficiency can be achieved. Hence, trans kingdom RNA interference delivery through chloroplast hp-RNA is more efficient than nuclear-encoded hpRNA. Niu et al. (2017) in their study reported that using RNA interference system, western corn rootworm can be controlled. Transgenic maize was developed expressing dsRNA against two vital genes dvvgr and dvbol of Diabrotica virgifera. Silencing of these genes causes reduction in fertility of insect (Diabrotica virgifera). The adult feeding bioassays conducted using different concentrations of dvvgror dvbol dsRNAs in artificial diet resulted in 46.5% 6 12.5% and 75.4% 6 11.3% lower fecundity. Senthilraja et al. (2018) investigated that by using RNAi technology peanut stem necrosis disease, one of the most important diseases of peanut worldwide, caused by Tobacco streak virus (TSV). Perhaps, they further revealed the

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possibility of making transgenic peanut plant resistant to PSND using RNAi knockdown technology. However, they observed that resistance against TSV through genetic engineering in peanut plants can be persuaded by expressing dsRNA of TSV-CP gene, which was a targeted gene for RNA interference by using TMV-7 peanut cultivar and hpRNA vector. TMV-7 was transformed with a hpRNA vector using Agrobacterium-mediated transformation method, a hairpin RNA vector containing inverted repeats (IR) of coat protein (CP) gene of TSV, a spacer sequence (Pdk) under the control CaMV 35S constitutive promoter. Transgenic plants T0-T1-T2 containing TSV coat protein gene were confirmed using coat protein gene-specific primers, through PCR analysis. Evaluation of T2 transgenic plants for resistance against TSV showed that not a single inoculated transgenic plant of peanut had any symptoms of infection, while non transgenic plants exhibited necrosis. Eakteiman et al. (2018) demonstrated a control strategy for severe pests of agricultural crops, complimentary to chemical pesticides, using plantmediated RNA interference (RNAi) method. More importantly, the purpose of this approach was to downregulate vital “detoxification genes” commonly involved to neutralize defensive ability or toxic plant chemistry of phloem-feeding insects. Hence, they targeted a GST gene “BTGSTs5” in the B. tabaci, a phloem-feeding whitefly. Moreover, three major findings were discovered. First, when phloem-feeding insect (B. tabaci) were fed on transgenic plants of A. thaliana expressing dsRNA against glutathione S-transferase gene under a tissue (phloem)-specific promoter, significant level of down-regulation of the targeted gene BTGSTs5 in the gut of B. tabaci was obtained. Second, in vitro analysis of BTGSTs5 enzyme indicated that this enzyme can accept as substrates and indolic glucosinolates, and create their consequent detoxified conjugates. The third finding suggested that due to downregulation of BTGSTs5, gene development period of whitefly (B. tabaci) nymphs prolonged. Therefore, these three findings suggest that “BTGSTs5” likely plays an important role in allowing B. tabaci to effectively feed on plants producing glucosinolate. Zhao et al. (2018) reported that to control agricultural insect pest, plant-mediated RNA interference is environment friendly technique. chs1 was selected as the target gene of Sitobion avenae (grain aphid) to be knocked down via RNAi technology. Chitin is an important component of insect exoskeleton and; moreover, chs1 gene has an important role in insect molting and development. Chitin synthesis will not occur if chitinsynthase1 enzyme gene is silenced. Therefore, to control aphid, transgenic

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wheat was developed using plant-mediated RNAi. After feeding on transgenic lines chitinsynthase1 expression level decreased in grain aphids as compared to control. Moreover, significant decrease in total numbers of aphid infesting T4 and T5 transgenic lines were observed under the field condition. Comparatively, significant level of increase in grain weight of T5 transgenic lines was reported as compared to the wild plants. Hussain et al. (2019) stated that using plant-mediated RNAi technology, Colorado potato beetle (CPB) which is a damaging pest of potato crop, can be controlled. Ecdysone receptor (Ecr) gene, which has vital role in the development of CPB, was selected as a target gene to be knock-down via RNAi approach, as shown in Fig. 6.1 schemetically. The amplification of Ecr gene with specific primers, in sense and antisense directions, was done and cloned in the pRNAi-GG vector. Two potato cultivars lady Olympia and Agria were selected to be transformed using Agrobacterium strain LBA4404 under the control of CAMV-35S promoter. Transgenic plants of both cultivars showed significant level of resistance against first, second, and third instar Colorado potato beetle. CPB insects fed on transgenic plants

Figure 6.1 An illustration of mechanism of RNAi delivery to insect pest via of transgenic plant. Tijsterman, M., Plasterk, R.H., 2004. Dicers at RISC: the mechanism of RNAi. Cell 117 (1), 1 3.

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showed 15% 80% mortality and also showed lower larval weight change ranging from 0.87- to 4.149-fold as compared to those fed on nontransgenic plant leaves that showed 1.87- to 6.539-fold larval weight change. Moreover, CPB larvae fed on transgenic plants showed decreased Ecdysone receptor gene expression level. The targeted genes by plantmediated dsRNA delivery and crops have been shown in Table 6.3.

6.4 Second-generation genome editing technology Development in editing technologies for the whole genome plays a vital role in understanding the fields of functional genomics and improvement of crops. Clustered regularly interspaced short palindromic repeats (CRISPR), a second-generation editing tool, is used for different purposes in genome engineering. Usually, it consists of two parts, first is singleguided RNA (sgRNA) and the second is known as cas9, which has endonuclease activity. The discovery of CRISPR Cas9 greatly helped agricultural research by developing new plant varieties either by deletion or addition of important characters (Dangol et al., 2019). This guided genome engineering technology is a marvelous breakthrough in the field of plant biology and with the passage of time, this technology is advancing day-byday. Numerous applications such as activation or repression of target genes, generating knockouts and other modifications in the genome are reported (Arora and Narula, 2017). Gene editing technology for crop improvement offers numerous choices from changing only one nucleotide to the whole genome or by adding a new gene in a specific region of genome, and/or alter a full allele. Three types of modifications take place in genome editing such as alteration in a few nucleotides, changing allele with existing one and inserting of new genes in the genome. Editing of genes is more accurate as compared to first-generation editing technologies (ZFN, TALEN) or conventional breeding methods. CRISPR is more specific than previous technologies. In future, this technology will play an important role toward securing the food supply for world. This technique makes crops more resistance to pests, makes crop survival rate high under tough conditions, and helps in improving nutritional value. The creation of genome-edited crops could evade the strict rules and regulation procedures, which are usually associated with transgenic crops. So, most of the scientists believe that crops improved by using genome editing techniques will be more considered and

Table 6.3 Featured examples of plant-mediated RNA interference approach in different crops against harmful insects/pests/viruses. Crop Insect-pest-virus Target gene Aim of study Results

Cotton

Helicoverpa armigera

CYP6AE14

Tobacco

Myzus persicae

hunchback

Tobacco

Myzus persicae

Rack1, C002

A. thaliana

Myzus persicae

Rack1, C002

Tobacco

Bemisia tabaci

v-ATPaseA

A. thaliana

Helicoverpa armigera

AK

Maize

Diabrotica virgifera virgifera

Dvvgr, dvbol

Peanut

Tobacco streak virus

Coat protein (CP)

A. thaliana

Bemisia tabaci

BTGSTs5

Using PMRi approach for reduction of larval tolerance to gossypol To develop transgenic tobacco plant tolerant to M. persicae Knockdown of aphid targeted genes Downregulation of gene through feeding dsRNA from plant Development of transgenic tobacco plant resistant to whitefly via RNAi technique Disturbed larval growth via silencing of targeted gene of cotton bollworm using RNAi approach Protection of maize plant by controlling reproduction of western corn rootworm using plant-mediated RNAi approach Development of transgenic peanut resistant to TSV via RNAi Through RNAi system protection of crops from phloem-feeding insects

Stunted larval growth Inhibited insect reproduction 40% level of gene expression decreased 50% 60% gene expression reduced Significance level of knockdown

References

Mao et al. (2007) Mao and Zeng (2014) Pitino et al. (2011) Pitino et al. (2011) Thakur et al. (2014)

55% mortality

Liu et al. (2015)

Reproduction capability decreased

Niu et al. (2017)

No infection shown

Senthilraja et al. (2018) Eakteiman et al. (2018)

Nymphs development period prolonged

Wheat

Sitobion avenae

CHS1

Potato

Colorado potato beetle

Ecr

Rice

RBSDV

1. S7-2 2. S8

Tobacco

Helicoverpa armigera

EcR

Tobacco

Tobacco

Manduca sexta, Manduca quinquemaculata Cotton bollworm

HaHR3

Rice

Nilaparvata lugens

EcR

Wheat

Sitobion avenae

CbEE4

CYP6B46 BG1

Development of transgenic wheat resistance to grain aphid through Silencing of chs1 gene Plant-mediated RNAi-based transgenic potato lines increased resistance against CPB Using RNA interference approach development of transgenic rice resistant to RBSDV Enhancement of Pest Resistance in Transgenic Tobacco via PMRi Through PMRi knockdown of “lepidopteran” insects’ vital genes Transgenic tobacco plant expressing dsRNA against HaHR3 disrupt cotton bollworm reproduction Development of transgenic rice plant expressing dsRNA of Ecr gene to control insect pests Knockdown of grain aphid gene via PMRi approach make S. avenae susceptible to phoxim insecticides

Reduced gene expression and population of aphids 80% mortality

Zhao et al. (2018)

Showed maximum resistance against RBSDV 1. Molting defects 2. larval lethality 80% 90% knockdown efficiency

Ahmed et al. (2017)

1. Retarded growth 2. larval lethality

Xiong et al. (2013)

66.27% number of offspring reduced

Yu et al. (2014)

60% knockdown efficiency

Xu et al. (2014a,b)

Hussain et al. (2019)

Zhu et al. (2012) Poreddy et al. (2017)

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easily acceptable for commercialization rather than transgenic plants. The new crops developed with newly discovered genome editing technologies show very low risk of effects of off-targets. The technique offers ease of performing genetic manipulation through simple easy laboratory experiments, even for those crops which cannot be bred easily (Abdallah et al., 2015). Though improvement of crops by conventional method such as recombination or mutations in genome randomly, is a laborious method and cannot meet with high food demands. The targeted genome engineering technologies such as CRISPR have important role in helping increase the production of crops under different stresses. This new technology has lower risks of off-target effect and is highly efficient and accurate as compare to conventional mutational methods. The modern editing technonogies have gained populatiry with so many examples of repression, activation and knockouts of selected genes (Abdelrahman et al., 2018).

6.5 CRISPR against insects CRISPR Cas9 editing in insects is studied by conducting a number of experiments in model insects such as Bombyx mori, Drosophila melanogaster, and other crop-damaging insects. Its ease of designing leads toward multifarious experiments to find out the function of genes (Zhang et al., 2014). So, this fascinating technology is not only limited to finding out functions of genes but it is also emerging as a game changer tool for the control of pest insects and diseases (Reid and O’Brochta, 2016). Transgenic crops having genes of BT insecticidal protein are broadly used for the protection of crop against harmful pest (Bravo et al., 2011). However, the resistance against Bt toxins is increasing day-by-day in insects, and it is an alarming situation toward pest protection. A cadherin-like receptor for BT Cry1A toxin is discovered in numerous insects of Lepidoptera (Wu, 2014). Several experiments of RNA interference show that in Lepidoptera, cadherin is engaged in Cry1Ac resistance. Wang et al. (2016) injected a mixture of Cas9 mRNA and sgRNA into eggs of H. armigera and targeted ninth exon of cadherin gene. The mutation of insect cadherin gene led to higher resistance to Cry1Ac as compared to control strain. The results shows that this cad gene is an essential receptor of cry1Ac and also its has role in insect resistance against cry1Ac. With the help of CRISPR Cas9, pigment genes of H. armigera were also mutated and these mutations showed several

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physical phenotypical changes (Khan et al., 2017). Chang et al. (2017) showed in H. armigera a new mechanism to destroy mating of pest with help of CRISPR/Cas9 system for genome editing of insects. In another study, two sgRNAs were used for the deletion of cluster of genes. The whole CYP6AE cluster was edited in H. armigera using dual sgRNAdirected CRISPR-Cas9 system. For this purpose, each of the two sgRNA were designed to target those genes, which found at each end of the CYP6AE cluster. The Cas9 protein and two guided RNAs were injected into the embryos of H. armigera. Altogether, 400 eggs were injected. Out of these 125 eggs hatched and from these hatched eggs, 65 developed into adults. These findings show that genome editing of these genes affects the survival rate of cotton bollworm (Wang et al., 2018). Drosophila melanogaster is a model insect for CRISPR Cas9 and effective use of Cas9 system is achieved in insects by introducing diverse mixtures of CRISPR Cas9 reagents injecting into embryos (Kondo and Ueda, 2013). The Mediterranean fruit fly known as Ceratitis capitata is directly linked with agriculture because this medfly feeds on more than 260 species of crop and is counted among the one of the most dangerous pests of agriculture (Liquido et al., 1991). The wild populations can be controlled by introducing sterile insect technique. This strategy is based on elimination and infertility is acquired by the release of large number of sterile males into pestridden areas (Morrison et al., 2010). Meccariello et al. (2017) demonstrated successful CRISPR cas9 application in medfly. They targeted pigmentation gene of the eye, known as white eye, and disrupted its function by injecting Cas9 ribonucleoprotein complex in early embryo along with sgRNA specific to the gene. Rate of mutation in white eye gene achieved 100% and also demonstrated recovery of large deletions of edited gene with the help of nonhomologous end joining mechanism.

6.6 Nematode resistance in crop plants Plant parasitic nematodes (PPNs) are a serious menace to a variety of crop plants worldwide (Ali et al., 2017). Over 4300 species have been reported as PPNs within 197 genera, which accounts for 7% of the phylum Nematoda (Decraemer and Hunt, 2006). The PPNs infect a variety of economically important crops like rice, wheat, maize, soybean, potato, tomato, and sugar beet. PPNs use various molecular and genetic tools to

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parasitize plants (Ali et al., 2017). Nematode infection results in the formation of root galls as below-ground symptoms; however, leaf necrosis, leaf chlorosis, stunted and patchy growth, possible wilting, and predisposition to various pathogenic fungi can also be observed in above ground parts of plants (Ali et al., 2015, 2019; Webster, 1969). Root-knot nematodes and cyst nematodes develop the giant cells and syncytia, respectively, on the plant roots, which act as the continuous source of nutrition to the sedentary nematode stages (Jones, 1981). Due to substantial host range, PPNs cause a serious decrease in the crop productivity leading to over $150 billion annual crop losses, globally (Abad et al., 2008). This emphasizes the urgent need of nematode resistance in crop plants. Enhancement of nematode resistance in crop plants in combination with higher crop yields has been the prime objective of plant breeders and nematologists (Fuller et al., 2008). Early improvement in this context could be largely attributed to the directed genetic utilizations via classical selection breeding of resistant genotypes and finding quantitative trait loci (QTLs) associated with resistance genes (Ali et al., 2019). Marker-assisted selection (MAS) has been practiced at large scale by the plant breeders for selecting nematode-resistant plants. Similarly, in recent times, genome-wide association studies (GWASs) are extensively being used to associate particular genomic regions of the genomes of crop plants with nematode resistance or even the susceptibility (Pariyar et al., 2016; Zhang et al., 2017). Conventional breeding methods employed for enhancement of nematode resistance usually need a screening of genotypes at large scale followed by the identification of resistance genes and then incorporation of this resistance through multiple filial generations. In the recent times, pyramiding of resistant genes in cultivars using conventional technique followed by screening through marker assisted selection (MAS) has largely been used for this purpose. However, conventional breeding and its allied approaches are timeconsuming and more laborious. Due to the recent advances in biotechnological tools, scientists are able to transform the crop plants using both heterologous and indigenous genes in a shorter period of time. Use of this technology offers innovative epoch of crop improvements, after 1960s green revolution. Plants developed through transgenic technology provide alternate approach to evolve brand new systems of nematode resistance through homologous and/or heterologous transfer of defense-related proteins. A number of transgenic approaches have been worked out and employed producing nematode resistant plants. Utilization of Resistance (R) genes encoding R proteins have been cloned from several plant species

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and could be transferred to other plant species, for instance Mi-1.2 from tomato (Williamson, 1998), Hs1pro-1 from sugar beet (Cai et al., 1997), Gpa-2 and Gro1 4 from potato (Van Der Vossen et al., 2000), and Hero from tomato (Sobczak et al., 2005). Moreover, various Cre and Rhg loci have also been characterized as R genes to confer resistance against cereal and soybean cyst nematodes, respectively. Transgenic expression of most of these R genes has resulted in hypersensitive response just after the initiation of at the feeding sites on the roots, which led to the development of abnormal and stagnated female nematodes. Very recently, a comprehensive review has been carried out on different R genes utilized in the resistance against PPNs (Ali et al., 2017). The heterologous expression of different proteinase inhibitors such as cowpea trypsin inhibitor (CpTI) and sweet potato trypsin inhibitor (SpTI1) into potato has improved nematode resistance in potato (Cai et al., 2003). Similarly, a proteinase inhibitor (PIN2) was transformed into wheat from potato improved resistance against cereal cyst nematode (Heterodera avenae) (Vishnudasan and Khurana, 2005). Moreover, a rice proteinase inhibitor, cystatin (Oc-IΔD86) has been expressed in number of plant species like Arabidopsis, potato, rice, cavendish dessert bananas (Musa acuminata), lily (Lilium longiflorum), and eggplant (Solanum melongena) to enhance resistance against the most dangerous PPN species including Globodera spp., Heterodera spp., Meloidogyne spp., Radopholus spp., and Pratylenchus spp. (reviewed by Ali et al., 2017; Fuller et al., 2008). The incorporation of these proteinase inhibitors improved the anti-feeding ability of the plants and the nematodes were not able to establish well on the plants harboring these inhibitors. Transgenic potato plants harboring a chemo-disruptive peptide led to 52% reduction of females of Globodera pallida (Liu et al., 2005). Recently, different synthetic chemodisruptive peptides like ACHE-I-7.1, LEV-I-7.1, and nAChRbp have been employed for increasing resistance against different species of PPNs in potato and plantain (Musa spp.) (reviewed by Ali et al., 2017). The anti-feeding approach using proteinase inhibitors and anti-invading approach by incorporating synthetic chemo-disruptive peptides have been combined in a number of recent reports to augment nematode resistance in various plant species including potato, tomato, plantain, and Arabidopsis (Ali et al., 2019). By combining these two approaches, University of Leeds, United Kingdom, in collaboration with The International Institute of Tropical Agriculture (IITA), has transformed a synthetic nematode repellent peptide and a maize digestive cystatin in plantain (Roderick

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et al., 2012; Tripathi et al., 2015). Additionally, pyramiding of chemodisruptive peptide and cystatins into various crops elevated resistance against PPNs coupled with enhanced crop productivity (Ali et al., 2019; Tripathi et al., 2017). Host-induced gene silencing of vital nematode effectors and parasitism genes has been one of the most interesting research topics for scientists in the recent times (Gheysen et al., 2010; Gheysen and Vanholme, 2007). Similarly, modification of gene expression in the feeding sites of sedentary nematode species has shown good promise to enhance nematode resistance in plants (Ali et al., 2013, 2014).

6.7 Conclusions Transgenic crops with in-built trait of insect resistance (expressing genes from Bacillus thuringiensis and cp4-epsps from Agrobacterium CP) have performed remarkably, since commercialization, in terms of farm productivity. The plant chloroplastic genome of crops has been engineered to produce foreign proteins in abundant quantity to encode resistance against insects and weeds, and also to confirm gene expression in green parts of the plants. Recently, RNAi has proved itself as a robust technology to study functional genomics. Based on scientific evidences that show efficiency of RNAi against insect pests, scientists trust potential of this technology in IPM. More recently, CRISPR technology has opened new avenues to mutate or introduce trait of interest in transgene-free crops or plants. The literature suggests that up to 30% 40% crop yield losses are incurred by insect pests. The integration of all these new technologies along with classical approaches can help to reduce such yield losses from insect pests, ultimately leading to increased agricultural produce.

Acknowledgment The research work related to RNA interference technology in laboratory of corresponding author (Dr. Allah Bakhsh) was supported by the scientific and technological research council of Turkey (Tübitak), Project No 215O520. We would like to apologize from the fellow scientists whose work could not be cited in this chapter due to page limitations.

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CHAPTER SEVEN

Transgenic plants with improved nutrient use efficiency Sadia Iqrar1, Kudsiya Ashrafi1, Usha Kiran1,2, Saman Fatima3, Kamaluddin4 and Malik Zainul Abdin1 1

Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, New Delhi, India 2 Bioinformatics Institute of India, Noida, India 3 Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India 4 Department of Genetics and Plant Breeding, Banda University of Agriculture and Technology, Banda, India

7.1 Nitrogen It is estimated by the United Nations that the world population will increase to approximately 11 billion, by 2050. Rapid increasing population is a global problem. With this increase, comes the responsibility to feed the population using environmentally and economically responsible practices. Shirking cultivable land and sudden climate changes are big challenges in front of scientists and farmers. Good agricultural practices with less utilization of herbicides and fertilizers are the need of hour to save the depleting nutrients of the soil. Nitrogen (N) is a key limiting nutrient in the growth of staple crops. It is absorbed from soil mainly in the form of nitrate (NO3−), urea [CO (NH2)2], or ammonia/ammonium (NH3/NH41). The vital stage of N‐ use in the plant life cycle is the vegetative stage, when N assimilation is most required for plant growth and development. The members of nitrate transporter (NRT), ammonium transporter (AMT), and high‐affinity urea transporter (DUR) families take up NO3−, NH41, and urea in roots, respectively (Masclaux-Daubresse et al., 2010; Wang et al., 2012). NO3− is reduced to NH41 by the action of nitrate and nitrite reductases (NR and NiR, respectively) while urea is converted to NH41 by the action of urease. Amino acids can be transported into roots, or can be utilized directly or catabolically degraded to increase the cellular NH41 pool Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00007-1

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(McAllister et al., 2012). The glutamine synthetase/glutamate synthase (GS/GOGAT) system assimilates NH41 into glutamine and glutamate. N is transported to different tissues for utilization in the form of glutamate, aspartate, glutamine, and asparagine through amino acid permeases (AAP) (Masclaux-Daubresse et al., 2010). It is predominantly stored in photosynthetic tissue as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPc), and GS (McAllister et al., 2012). These proteins are degraded and N is remobilized for use during grain filling, senescence, or N starvation (Feller et al, 2007). Insufficient N critically affects the yield of crops, whereas over-supply has no effect on yield and contributes crucially to N pollution (Liao et al., 2012; Amiour et al., 2012). High amount of inorganic nitrogen in freshwater can cause algal blooms, which result in eutrophication of aquatic ecosystems (Vitousek et al., 2009). In addition, the emission of greenhouse gas mainly nitrous oxide (300 times more potent than CO2) has increased 5%–7% per decade, since 1979 (Montzka et al., 2011). It was recently estimated that the environmental cost of excess N application round off to a minimum of 44% of the total cost of the applied N. The most cereal crops use roughly 40% of applied N and the remaining 60% is lost to the environment; approximately 20% is leached into groundwater, 21% is volatilized into N2O, and 19% of the applied N is lost due to denitrification or bacterial uptake (Good and Beatty, 2011). Through the Haber–Bosch process, the production of ammonia fertilizer requires approximately 1% of the world’s annual energy supply (Smith, 2002), reflecting a notable worldwide economic cost. Considering the environmental and economic cost of global fertilizer use, addressing the difference between utilized and lost N is, therefore of great importance.

7.1.1 Nitrogen use efficiency The efficiency of N uptake and assimilation by a plant is revealed in its nitrogen use efficiency (NUE). NUE can be estimated in various ways (Good et al., 2004), but is mainly defined as the mass of grain produced relative to the amount of supplied N (McAllister et al., 2012). Generally, NUE can be further segregated into two parts: assimilation efficiency and utilization efficiency. At the cost of current agricultural practices, there is a need to improve production practices. Better management practices including crop rotation and timing of the fertilizer application represent agreeable methods to reduce N application. The improvement of crops

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with elevated NUE presents another means to attenuate environmental consequences and lower the crop production costs. Plant responses to N starvation provide effective insight into the underlying processes involved in N metabolism. A significant increase in lateral root development enhances exploration for new N sources and increases root surface area for N uptake under N starvation. N uptake, assimilation, and remobilization are three main features of nitrogen metabolism that may be changed to improve NUE (Masclaux-Daubresse et al., 2010). The comparison of rice and soybean cultivars with differing NUEs indicated that more efficient N storage and remobilization as well as altered expression of certain key genes involved in N metabolism may be considered as physiological markers for improved NUE (Fan et al., 2007: Hao et al., 2011; Minglin et al., 2005). While one way to increase NUE is the improved agricultural practices and the other is use of enhanced understanding of the genetics of NUE (Coque et al., 2008; Sylvester-Bradley and Kindred, 2009). Enhancing the NUE of crops through biotechnological intervention can end the asynchrony between N supply and demand. By ectopic regulation of key enzymes involved in N metabolism, attempts have been made to improve NUE in transgenic crops (Good et al., 2004, 2007; Shrawat et al., 2008). In addition, efforts have been focused towards deciphering the molecular basis of plant responses to N and the identification of N-responsive genes (Crawford and Forde, 2002).

7.1.2 Transgenic crops with elevated nitrogen use efficiency Increasing the efficiency of either N uptake or N use could lead to elevate crop NUE. There are plant-bred crop cultivars with improved NUE. For example, modern maize hybrids can produce a higher yield and grow better than older lines under limited-N conditions. Similarly, other crop varieties like rice, wheat, and barley are also known to possess enhanced NUE (Ortiz-Monasterio et al., 1997; Le Gouis et al., 1999; Anbessa et al., 2009; Beatty et al., 2009; De Carvalho et al., 2012; Chen et al., 2013). Many of the genes targeted to increase NUE are the primary N uptake and assimilation genes that include ammonia and nitrate transporters, nitrate reductase, glutamine synthase (GS), and glutamate synthase (GOGAT). The overexpressed genes that have shown an elevated NUE phenotype in a crop plant are the genes involved in N metabolism further downstream of GS and GOGAT, such as alanine aminotransferase

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(alaAT), or are transcriptional regulators, such as Dof1 (Kurai et al., 2011), but not the genes involved in primary N assimilation. Alanine aminotransferase [E.C. 2.6.1.2] is a pyridoxal-5-phosphatedependent (PLP) enzyme. It catalyzes the reversible transfer of an amino group from glutamate to pyruvate to form alanine and 2-oxoglutarate (McAllister et al., 2013). Homozygous single-insert, alaAT overexpressing transgenic (TG) rice lines AGR-1/7 and AGR-3/8 were developed using the aldehyde dehydrogenase 1 and alanine aminotransferase (OsANT1:: alaAT) gene construct from Hordeum vulgare (Shrawat et al., 2008). The transgenic lines showed more biomass that WT (wild type). Especially in the TG lines sampled at 44 DAG (days after germination), both root and shoot dry mass showed enhancement when compared with the WT. There was however, more biomass increase in the shoots as compared to the roots in the TG lines (Fig. 7.1). This coincided with the higher NUpE (nitrogen uptake efficiency) values seen in the shoots than in the roots, in these lines. In general, there were more changes in N amount, N uptake, biomass, and usage in the TG shoots compared with the roots, and these changes were greater at the later developmental stage of maximum tillering as contrary to active tillering. During the early stages of growth, both the TG lines and WT react to N availability and assimilate in similar fashion. However, at some point between active and maximum tillering, the TG lines begin to take up more N in the roots and assimilate as well as translocate it more efficiently to the shoots as compared to the WT. The plants, when not under N stress, do not show high N uptake efficiency, even when the supplied N level is high. Under medium and high

Figure 7.1 Impact on increasing nitrogen uptake efficiency in transgenic Hordeum vulgare.

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N availability, TG lines appeared to have an N uptake advantage over the WT at active tillering, and at all levels of N availability at maximum tillering. The elevated N uptake efficiency decoupled from external N availability levels, especially at the later stage of vegetative growth, when N remobilization to the sink tissues occurs, may allow the TG plants to enhance their biomass and their internal N levels. When the usage efficiency ratio (UI) of the TG versus WT lines was considered for each tissue, developmental stage, and N regime, the shoots had high UI at both active and maximum tillering over all three (high, low, and moderate) N levels supplied, while the roots only showed high UI at maximum tillering over the three N regimes. Even though N uptake from the soil occurs in the roots, it is interesting that the usage efficiency is significantly higher in transgenic shoots than roots. This suggests that in the alaAT overexpressing plants, the additional N taken up by the transgenic plant roots is then rapidly translocated to the shoots, although the transcriptome profile of the TG lines versus the WT does not show increased gene expression of known transport proteins. Some of the transgenic lines that are significantly higher in NUpE than the WT have a more percentage of N in the tissues. All of the TG lines, however, have substantial dry mass than the WT and, therefore more total N in the tissues as compared to the WT (Beatty et al., 2013). There was significantly more alaAT-specific activity in the roots at the excessive N levels at both active and maximum tillering. The TG lines had at most eightfold greater alaAT activity (maximum tillering shoots) and minimum, a twofold greater (seedling roots) than the WT. It can be seen that members of the glutamate family require 2-oxoglutarate, aspartate family require oxaloacetate, Ala–Val–Leu family require pyruvate, and the Ser–Gly family require glyceraldehyde-3-phosphate when the amino acids, grouped according to their alpha-keto acid starting compounds, were generally lower in concentration in the TG roots when compared with WT during active tillering, regardless of N supply. Many of the key N primary assimilatory N storage and transport amino acids, such as aspartate, glutamate, glutamine, asparagine, arginine, and alanine were lesser in the TG roots, especially at low N concentrations. Expression levels of the key genes involved in amino acid synthesis were not differentially regulated in the TG shoots or roots except upregulation of the genes encoding branched-chain amino acid degradation enzymes and downregulation of the genes encoding glutamate family synthesis enzymes, at high N supply. OsANT1::alaAT overexpressing plants grown

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with low N supply did not have NUE phenotype, while the same overexpressing plant grown under medium and high levels showed NUE phenotype. The medium to high N/NUE phenotype plants were, however, not significantly different in amino acid concentrations from the WT. The other detected metabolites were products of TCA cycle and glycolysis. Under low N supply (measured in 44 DAG plants only), these metabolites showed the same levels in the TG shoots and roots as in the WT, except sucrose and phosphate, which were more abundant in the TG roots and for fructose and glucose, which were lower in the TG roots. There was very lesser change in these metabolite levels in the TG when compared with the WT shoots. Most of the transcripts related to the TCA cycle, such as the transcripts encoding citrate synthase, 2oxoglutarate dehydrogenase, and malate dehydrogenase, were not distinctively regulated at 44 DAG in the TG lines versus the WT at any N level, although the transcript encoding pyruvate dehydrogenase was downregulated in TG roots at high N. At any N level, glycolysis and fermentation-related transcripts were not differentially regulated in TG plants; however, some gluconeogenesis–glyoxylate cycle transcripts, in particular pyruvate dikinase, were upregulated in TG roots at high and medium N supply. Chen et al. (2009) found that after simulation by grazing, the genes associated with pyruvate anaerobic metabolism were upregulated, which include lactate dehydrogenase, aldehyde dehydrogenase, and isocitrate lyase. These transcripts in roots of alaAT overexpressing rice plants were also upregulated when grown in medium to high levels of N. This is interesting as alaAT has been involved in recovery from hypoxia along with anaerobic pyruvate metabolism in rice and soybeans (Miyashita et al., 2007; Rocha et al., 2010). Chen et al. (2009) showed the upregulation of oxidation–reduction associated cytochrome P450s (known to be involved in wounding and stress responses), and secondary metabolism genes such as flavonoids. Other NUE studies on Arabidopsis grown under N-limited conditions have shown upregulated phenylpropanoid biosynthesis genes and secondary metabolism genes such as anthocyanin (Bi et al., 2007). It is feasible that flavonoid synthesis genes and the cytochrome p450 were upregulated either in response to alaAT overexpression and that their increased gene levels help enhance NUE, or in response to a perceived pathogen attack, during the rice transformation procedure. The upregulation of the E3 ubiquitin ligases and receptor-like kinases in the alaAT overexpressing rice suggests that these

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NUE plants may benefit from a more active immune system against rice pathogens. In the roots of alaAT overexpressing rice plants grown under the medium levels of N, an early nodulin (ENOD93) gene (Os06g0141600) was highly upregulated. ENOD93-1 was also identified by Bi et al. (2009) in a transcriptome profiling experiment with the N-limited rice in response to both, N reduction and N induction conditions. Bi et al. (2009) overexpressed ENOD93-1 in rice and found that the gene product promoted the accumulation of amino acids in the roots and xylem sap resulting in improved NUE, and increased seed yield. In the roots of the alaAT TG lines grown in high levels of N, the nonmelavonate pathway for terpene synthesis produces terpenes, carotenoids, and the phytol chain for chlorophyll, and so may be important for the production of photosynthetic compounds (Dubey et al., 2003; Eisenreich et al., 2004). It is possible that alaAT overexpression in the NUE rice induces high levels of pyruvate that stimulates the production of terpenes via the nonmelavonate pathway, which in turn benefits C metabolism and photosynthesis with increased chlorophyll production. An essential cofactor for AlaAT, that is Pyridoxal-5-phosphate, is also synthesized from a precursor (1-deoxy-D-xylulose-5-phosphate) produced in the nonmelavonate pathway. The alaAT overexpression resulted in increased alanine aminotransferase activity in the roots (and to a lesser extent the shoots), which produced amino acids and alpha-keto acids by the active-maximum tillering at vegetative stage, which mediates enhanced N uptake. Although plants regulate N transport and N metabolism by sensing the cellular glutamate levels but they do not appear to sense the requirement of transport of N as a response to other amino acid pool, like alanine pool. The NUE alaAT-ox rice may be producing alanine that is quickly transported to the shoots and used in N storage and protein synthesis; however, this form of N transport cannot be perceived by the plants. This activates, the positive feedback loop where the plant improves N uptake, which in turn increases C metabolism. More carbon is sent to the roots to provide C skeletons by this positive feedback loop. The N is incorporated in C skeletons to produce nitrogenous compounds and transported to the shoots. The increased pool of nitrogenous compounds leads to increased biosynthetic activity resulting in increased root and shoot biomass in the TG plants compared to the WT when grown on the same N supply.

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Evidence shows that the overexpression (OX) of N metabolizing enzymes such as glutamine synthetase (GS), glutamate synthase, and alanine aminotransferase enhances the remobilization of N into biomass production (Brauer and Shelp, 2010; Good et al., 2004) in monocots. There are two GS isoforms, GS1 (cytosol) and GS2 (chloroplast), and their expression changes throughout the development as well as in relation to the environmental cues (Miflin and Habash, 2002). GS1 is involved in the reassimilation of ammonia released during catabolism and is of greater interest (Miflin and Habash, 2002). Also, an increase in GS1 activity is noticed during spikelet filling (Kamachi et al., 1991; Ladha et al., 1998). Reduced spikelet filling is found in GS1 knockout lines (Tabuchi et al., 2005). The rate of leaf discoloration and quantitative trait loci for spikelet weight have been mapped to GS1 (Obara et al., 2001; Yamaya et al., 2002). Three OsGS1;2 OX lines of rice derived from individual transformation events possessed 5–15 times more than the levels of gene expression in the untransformed rice plants. The GS activity was also found to be 23 times more than levels found in untransformed rice plants Wt compared with 2–3 times the GS activity. It provides evidence for posttranscriptional control of the overexpressed gene in rice, which is constant with previous studies of GS OX in pea, rice, tobacco, and alfalfa (Fei et al., 2003; Obara et al., 2000; Oliveira et al., 2002; Ortega et al., 2001). It was shown in a recent research with Medicago truncatula that GS1 activity is controlled by protein turnover, a process affected by stabilization and phosphorylation of the enzyme (Lima et al., 2006). Phosphorylation of the enzyme is affected by light and N fixation, which suggests that this form of regulation is important for correlation of N assimilation and external stimulus (Lima et al., 2006). It is also feasible that there is a limit for the increase in GS activity in rice by an unknown mechanism, though studies on tobacco, poplar, lotus, wheat, pea, and maize reported enhancement of 15%–400% (Brauer and Shelp, 2010). The studies with single insert results in better activity levels (Fei et al., 2003; Fuentes et al., 2001; Martin et al., 2006). An early nodulin gene (OsENOD93-1) was one of the strong NUE candidate genes selected because of its extreme response to both N induction and N reduction. With the nitrate level being changed significantly in response to either N induction or N reduction, it appears that the difference in nitrate concentration must reach a threshold before transcription is affected. Lian et al. (2006) reported expression profiles for 10,422

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genes at an early stage of low N stress in rice seedling. Transgenic plants overexpressing this OsENOD93-1 gene showed higher seed yield and yield-contributing factors than WT plants. The early nodulin (ENOD) genes are expressed at early stages of nodule development in leguminous plants, and may conciliate rhizobial infection and/or nodule organogenesis (Oldroyd and Downie, 2008). Furthermore, differential functions of ENOD genes have been postulated. As an example, ENOD40 has been shown to be expressed during the early stage of nodule initiation (Kouchi and Hata, 1993; Yang et al., 1993), differentiation of vascular bundles (Kouchi et al., 1999), or photosynthate transport (Kouchi and Hata, 1993; Yang et al., 1993) but its protein product may be involved in cortical cell division (Charon et al., 1997). A number of homologs of ENOD genes have been deduced in nonleguminous plants (Trevaskis et al., 1997; Reddy et al., 1998), but most of them are of unknown function. In the sieve element plasma membrane (SEPM) of Arabidopsis, an early nodulinlike protein accumulates (Khan et al., 2007). Some sucrose transporters restrained in the SEPM are involved in sucrose loading, recovery during transport, and unloading (Kuhn et al., 2003). In addition to that, other transporters including those for amino acids are also localized in the SEPM (Hirner et al., 1998). In WT plants, the OsENOD93-1 gene was expressed at high levels in roots, and its protein product was localized in mitochondria. Further the in situ hybridization showed that expression of OsENOD93-1 occurred in vascular bundles, as well as in epidermis and endodermis. Overexpression of the OsENOD93-1 gene stimulated the accumulation of amino acids in roots and in xylem sap. The first NH41 transporters to be isolated were two related highaffinity NH41 transporters from Arabidopsis (Ninnemann et al., 1994) and yeast (Marini et al., 1994). After this, NH4 1 transporter proteins have been found in several species of plants including rice (Von Wiren, 1997). In rice, ammonium uptake in roots and transport in shoots is mediated by NH41 transporters of OsAMT family containing 10 OsAMT and OsAMT-like genes (Suenaga et al., 2003). Some of the genes in the OsAMT family have been partially characterized (Hoque et al., 2006). The OsAMT1 subfamily comprises of three members: OsAMT1;1, OsAMT1;2, and OsAMT1;3. These three members show high sequence similarity to each other and also to other AMT1-type NH41 transporters, previously identified from other plant species. Since these members have different affinities for NH41, it is likely that these transporter proteins enable plants to make use of a wide range of soil NH41 concentrations.

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OsAMT1;1, a prominent member in this subgroup, is known to show constitutive expression pattern in shoots and roots (Sonoda et al., 2003). It has been suggested that OsAMT1;1 is present in the plasma membrane and its expression is modulated by NH41 cellular concentration (Hoque et al., 2006; Sonoda et al., 2003). In rice, it was shown that overexpression of the OsAMT1;1 gene results in the accumulation of maximum levels of NH41. The higher NH41 permeability in roots enhances NH41 uptake in OsAMT1;1 transgenic plants resulting in improved ammonium assimilation in shoot under suboptimal, optimal, and high levels of NH41 in the medium (Ranathunge et al., 2014). Enhanced overall plant growth and yield under suboptimal and optimal levels was observed. Higher levels of NH41 result in toxicity. In the absence of high-affinity transporters in wild type plants, transport of NH41 to shoots is not so effective leading to its accumulation. This results in lower growth in wild-type plants (Ranathunge et al., 2014). The changes in NH41 pool also resulted in change in expression of a number of nitrogen assimilatory genes. The roots of transgenic and WT plants grown under an optimum NH41 concentration show similar gene expression levels of OsAMT1;2 and OsAMT1;3. Thus the greater NH41 utilization in OsAMT1;1 transgenic lines is most probably due to higher NH41 transport mediated by the plasma membrane OsAMT1;1 protein. Improved NH41 uptake in transgenic plants under suboptimal and optimal levels of (NH4)2SO4 showed significant yield augmentation in grain filling and total grain yield per plant suggesting that OsAMT1;1 transgenic rice has the ability to increase NUE. This means the crop would possibly require lesser applications of nitrogen fertilizer which would result in the reduction in the crop production cost. Also the application of very high concentrations of NH41 can cause toxicity and would have detrimental effects on the grain filling process resulting in immensely low seed setting (Ranathunge et al., 2014).

7.2 Phosphorus Phosphorus (P) is a crucial macronutrient for plant growth, development, and reproduction. It is also an integral part of biomolecules

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including phospholipids, nucleic acids, ATP, and other biologically active compounds. P is considered to be the second most important nutrient after nitrogen in limiting agricultural production. Phosphorus is highly reactive and is available for uptake by plant root cells over a narrow range of neutral soil pH values. P forms low-solubility molecules with iron (Fe) and aluminum (Al) in acidic soils, but reacts with magnesium (Mg) and calcium (Ca) in alkaline soils to form sparingly soluble phosphate compounds (Bar-Yosef, 1991). This narrow range reactivity makes phosphorus unavailable for plant uptake even though the total amount of phosphorus in the soil may be high. To ensure plant productivity and reduce P deficiencies, nearly 30 million tons of P fertilizer is applied worldwide every year. However, up to 80% of the applied P is lost as it becomes immobile and unavailable for plant uptake due to adsorption, precipitation, or conversion to organic forms (Holford, 1997). In recent years, attention has been paid to the environmental pollution caused by the presence of this immobile P in the soil as a result of the uncontrolled use of fertilizers (Sharpley, 1995).

7.2.1 Phosphorus utilization efficiency There are several definitions and calculation methods applicable for the determination of crop P efficiency. P efficiency can be interpreted as the yield or biomass of a crop under certain available P supply conditions (Holford, 1997; Sharpley, 1995). Phosphorus efficiency can be partitioned into phosphorus acquisition efficiency (PAE) and phosphorus utilization efficiency (PUE). PAE is considered as the ability of crops to take up P from soils, and PUE is the ability to produce biomass, or yield using the acquired P. Improving P efficiency in plants can be achieved through improving P acquisition and/or utilization (Loneragan, 1997; Schachtman et al., 1998; Hoffland et al., 1989). However, the contribution from PAE or PUE to crop P efficiency alters with crop species and environmental conditions. A higher PUE is mainly ascribe to efficient re-translocation and reuse of the stored P in plants.

7.2.2 Transgenic with elevated phosphorus utilization efficiency Phosphorus efficient plants may play a major role in improving food production as there is a shortage of inorganic P fertilizer resources, limited water resources and land, and increasing environmental concerns.

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Research in the enhancement of P efficiency often ascribes more to PAE under limited P supply and to PUE under high P conditions. Therefore, the enhancement of both P acquisition and P utilization efficiency of the plant species under different P supply conditions in the different soil types seems to be an appropriate breeding approach. However, the recent conventional breeding strategies are mainly focused on the selection of high yield or biomass producing varieties under high fertility soils, such as super hybrid rice varieties, which require high P fertilizer than other rice strains, to achieve an economically favorable yield. This is not only costly but also subject to environmental effects. Therefore PUE enhancement will be a significant bottleneck for further enhancement in crop P efficiency, but surely is a potentially strong strategy for increasing P efficiency in modern crops grown in rigorous cropping systems. With the rapid improvement in molecular techniques, more and more QTLs and/or genes are being discovered for involvement in P acquisition and internal utilization. Therefore the development of novel plant varieties, which are more productive in the use of P, represents a way to achieve more sustainable agriculture that could help to satisfy the growing world demand for food. Furthermore, plant varieties with an increased P acquisition ability should be of great importance for subsistence farmers, who cannot afford P fertilizer. In response to P-limiting conditions, some plants undergo developmental and physiological adaptations to scavenge limited phosphate from the environment. These adaptations include the changes in root architecture, rhizosphere acidification, induction of genes encoding high-affinity P transporters, and exudation of organic acids (Schachtman et al., 1998). An increase in the excretion of organic acids, particularly citrate, has been explained in Brassica napus, Lupinus albus, and the Proteaceae family of plants as a potential mechanism to increase P uptake (Hoffland et al., 1989; Dinkelaker et al., 1989, 1995). It has been proposed that because of its high affinity for divalent and trivalent cations, citrate can remove P from insoluble complexes in the soil and make, it more available for plant uptake. In fact, it has been evaluated that citrate exudation can produce a two- to three-fold increase in the concentration of soluble P in the rhizosphere (Zhang et al., 1997). The exudation of organic acids has been proposed to play an important role in enhancing P uptake and facilitating the use of sparingly soluble Ca-P for certain plant species adapted to alkaline soils (calcicoles). Alkaline soils are usually infertile because of their high

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content of CaCO3 and low P availability and they cover more than 25% of the earth’s surface. When grown in alkaline soils with P-limiting conditions, insoluble P compounds can be mainly enhanced by engineering plants to produce more organic acids and that citrate overproducing plants yield more leaf and fruit biomass. Transgenic tobacco plants (CSb lines) were developed to express the citrate synthase coding sequence from Pseudomonas aeruginosa under the control of 35 S CaMV promoter using Agrobacterium-mediated transformation. In limited P, the transgenic CSb plants were able to grow and reproduce, whereas the growth of wild type plants was acutely restricted, and after six months, they failed to produce seeds and flowers. The inability of wild type plants to complete their life cycle under the conditions used for the test was probably a reflection of a calcifuges behavior (inability of the plant to grow in alkaline soils), in which the inability to dissolve P seems to be of central importance (Tyler, 1992). In fact, the growth of control plants was regained by the addition of soluble phosphate. The transgenic CSb plants also produced 23%–35% more fruit dry weight than control plants. The difference in dry fruit weight was due to their size and the number of seeds they contained at the flowering stage and not because of the number of fruits produced per plant. The transgenic CSb lines accumulated higher amounts of P than the controls (Fig. 7.2). This higher level of P in the CSb TG plants correlated with a shorter time to anthesis (10 days). The observed differences in biomass accumulation between the controls and the CSb transgenic lines were due to the enhanced capacity of CSb lines to utilize insoluble sources of P, by a mechanism involving

Figure 7.2 Impact of increasing phosphorus uptake efficiency in transgenic tobacco.

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citrate exudation as citrate is able to dissolve insoluble P compounds. The increase in P uptake by organic acid excretion can be governed by two mechanisms: (1) rhizosphere acidification that leads to the higher P solubilization and (2) reduction in the concentration of free calcium by the formation of calcium citrate. The formation of calcium citrate in the rhizosphere of the CSb lines was easily detectable. This suggests that, at least in this case, the most important mode of action of citrate is the removal of P from the sparingly soluble calcium–phosphorus complexes, by the formation of calcium citrate. Besides organic acid excretion, plants make use of different approaches to improve their P acquisition capacity. Establishment of the symbiotic association of plant with mycorrhizal fungi has been considered as one of the most important strategies to improve P acquisition (Pate, 1994). When the plants inoculated with Glomus fasciculatum were compared with wild type plants, it was found that under all P treatments, CSb lines had a statistically significant (P , .05) higher fruit yield than the wild type plants. The effect of enhanced citrate excretion and mycorrhizal symbiosis on productivity appeared to be additive. These results suggest that the ability of mycorrhizae to improve P acquisition might not be by dissolving P soil sources, but rather by acting as an addition of the plant root system, which allows more efficient utilization of the soluble P present from the soil (Bolan, 1991). Transgenic plants with higher levels of citrate exudation have the highest capacity to use insoluble forms of P, and this characteristic is not abolished but rather enhanced when the plant coupled with mycorrhizae. The citrate overproduction has been enhanced in limited plant species (De la Fuente et al., 1997). It is therefore, quite possible that the ability to use P insoluble compounds can be improved in most of economically and medicinal important plant species as well. The two advantages evident in plants that have a high capacity to extract P from soils are: (1) better utilization of natural soil P reserves and (2) ability to meet P demands with a smaller application of P fertilizer. There has been increasing interest in overexpressing phytase genes in plants roots to promote P uptake from soil. Most of the work has been carried on genes cloned from microorganisms, such as the BPP phytase gene from Bacillus subtilis (168phyA) and the HAP phytase gene (phyA) from Aspergillus niger. Phytate is the major reservoir of organic P in soil and phytase enzymes hydrolyze these phytates to make P available for uptake by plants. Transgenic expression of phytase gene (phyA) from Aspergillus niger

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in subterranean clover (Trifolium subterraneum L.), Arabidopsis and tobacco (Nicotiana tabacum L.) resulted in enhancement in biomass and total P accumulation when phytate was supplied as sole P source (Mudge et al., 2003; George et al., 2004, 2005; Richardson et al., 2001). Similarly, transgenic expression of the 168phyA gene in Arabidopsis and tobacco and a synthetic phytase gene (PHY) in potato (Solanum tuberosum L.) also showed increased P acquisition and growth performance when organic phosphate (Po), instead of inorganic phosphate (Pi), was supplied (Lung et al., 2005; Yip et al., 2003; Zimmermann et al., 2003). However, to date, there is no report showing that the plants are able to increase P acquisition and plant growth by hydrolyzing soil organic P under natural soil conditions without the expression of any of phytase genes. White clover (Trifolium repens L.) is almost always grown as a nitrogen-fixing species with grasses to supply nitrogen and improve the feed value of pastures. This excellent nitrogen-fixing perennial pasture legume is widely grown in temperate regions of the world. However, it demands adequate phosphorus for establishment, persistence, and growth. Therefore, increasing P uptake ability of white clover may have a great effect on the productivity of pasture lands, which usually receive much less fertilizers than the croplands. Endogenous phytase or acid phosphatase (APase) expression can be secreted from plant roots, under low phosphorus conditions. However, activities of these released endogeneous enzymes are inadequate for effective acquisition of organic P. The overexpression of phytase and/or APase genes enhances organic P utilization however, the expense of overexpression effect depends on the transgenes and species used. Transgenic expression of MtPHY1 and MtPAP1 in Arabidopsis affirmed that both enzymes have phytase activity and leads to significantly improved utilization of organic P (phytate) when the transgenics were studied in culture media (Xiao et al., 2005, 2006a,b). The effects of the transgenes MtPHY1 and MtPAP1 introduced in white clover showed diminished effect than those in Arabidopsis (Xiao et al., 2005, 2006a,b). This observation suggests that although model species is convenient for rapid functional characterization of transgenes, the usefulness of the transgenes has to be verified in crop species of agronomic importance. In general, plants with higher levels of transgene (phytase and/or acid phosphatase) expression were shown to have higher enzyme activities, higher total P accumulation, and more biomass production. Enzymatic activity in apoplasts of the transgenic white clover was higher than that in

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whole roots, which indicates the accumulation of phytase and APase in root apoplasts. The bacterial phytase (168phyA), fungal phytase (phyA), and the synthetic phytase (PHY) have been affluently expressed in plant roots, resulting in the improved ability to hydrolyze Po in the rhizosphere (Mudge et al., 2003; George et al., 2004, 2005; Lung et al., 2005; Yip et al., 2003; Zimmermann et al., 2003). The phytase and APase genes of plant origin can also be used to demonstrate the importance of Po for plant P nutrition in a commercially important forage crop. Genes of plant origin have advantages over bacterial or fungal genes with consideration to biosafety and regulatory issues (Nielsen, 2003; König, 2003). One of the major concerns causing objections to GMOs is the introduction of genetic material in plants from organisms other than the plant kingdom (Nielsen, 2003). Because MtPHY1 and MtPAP1 have been cloned from the model legume M. truncatula, expression of these transgenes in white clover provides important information for their usefulness in other plant species, at least in legumes. The plant acid phosphatase and phytase genes can be used to enhance organic phosphorus utilization, and thus provide the potential of maintaining agricultural productivity while limiting the input of P fertilizer. These fertilizers are one of the major sources of water contamination and overgrowth of algae in many water bodies.

7.3 Sulfur Sulfur (S) is a vital macronutrient for crops, used by the plants in the form of sulfate. It plays an important role in plant defense mechanisms and redox control of cellular processes. Generally, plants acquire sulfate from the soil solution by their roots and then it is distributed to different plant parts for assimilation. S is mainly taken by the plant in the form of sulfate from the soil. Within the plant, sulfate is transported via the xylem to the leaves, the main sites of assimilation and reduction or remains in the roots (Rennenberg, 1984). The sulfate from the leaves is remobilized back to roots via the phloem (Hartmann et al., 2000; Herschbach and Rennenberg, 1991). Plant vacuoles are the main storage site for the sulfate (Leustek and Saito, 1999) and if required can be mobilized from these storage compartments (Bell et al., 1995). The sulfate transport processes

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within the plant are controlled by sulfate transporters (SULTRs) (Buchner et al., 2004; Hawkesford and De Kok, 2006). In recent years, Sdeficiency has become a global problem, which is resulting in a declined crop yield. This has lead to over-utilization of fertilizers however, the application of fertilizers is not a remedy for this problem. There is an urgent need to develop varieties with improved sulfur utilization efficiency even under limited supply of sulfur.

7.3.1 Transgenic with elevated SUE Plants use inorganic sulfate as the major source of sulfur for the synthesis of sulfur-containing amino acids. The incorporation of sulfate is not only essential for the synthesis of coenzymes but also for the biosynthesis of cysteine and methionine, and components of plant membranes. Most of the sulfur-containing biomolecules present in plants are generally synthesized directly or indirectly from cysteine. Consequently, the formation of cysteine is the main step for the assimilation of reduced sulfur into organic compounds. The main targets to improve S utilization efficiency may be divided into two levels: the first is aimed at increasing resource capture by enhancing S-uptake and assimilation (efficient transporters) and the second one is aimed at efficient use of the accumulated S (mobilizable reserves and sinks). The legumes used for feeding animals are deficient in the sulfurous amino acids (methionine and cysteine) and to overcome this deficiency supplement of the amino acids is often added. Langlands (1970) showed 23% greater wool production when a daily supplement of 360 mg methionine added to the abomasum of grazing sheep. The dietary requirements of animals could be met by developing transgenic plants with high sulfurcontaining amino acids. Sunflower seed albumin (SSA expressed from the gene sfa8), rich in sulfurous amino acids, was isolated and characterized from seeds of Helianthus annuus (Kortt et al., 1991). It is a seed storage protein and normally accumulates in the vacuole until seed germination. The sunflower albumin SFA8 has 141 residues when synthesized as a precursor, which is then cleaved to generate the mature single-chain molecule of 103 residues with molecular mass of 12,133 Da (Kortt et al., 1991). It contains methionine (15.5 %) as well as cysteine (7.5%) (Kortt et al., 1991) and resistant to in vitro degradation by rumen fluid (McNabb et al., 1994). Homozygous transgenic line of Lupinus angustifolius

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accumulating high SSA in the seeds was used in animal feeding trials. The sheep fed with transgenic L. angustifolius seeds showed a significant increase in wool growth rates and weight gain as compared with animals fed with nontransgenic seeds of the same cultivar (Molvig et al., 1997). These results clearly demonstrate that transgenic expression of SSA in grain legumes results in the nutritional increment of the resulting feed. If expressed in forage species, similar benefits might be expected. Data have shown that SSA appears to be degraded shortly after its translation when expressed in leaves of transgenic N. tabacum. For the accumulation of SSA in leaves of N. tabacum, the coding sequence was modified by introducing a short sequence encoding the endoplasmic reticulum (ER) retention signal TSEKDEL (Munro and Pelham, 1987) upstream of the stop codon. It was demonstrated that the modified SSA accumulates in the ER. Further, results suggested that the ER in leaves was more suitable for SSA accumulation as compared to the vacuole. The ingestion of forage, containing relatively rumen-stable proteins, which are rich in S-amino acids, would increase the availability of limiting essential amino acids for ruminant nutrition and, thus results in increased animal productivity, especially wool growth (Higgins et al., 1988; Rogers, 1990). Forage grasses expressing chimeric sunflower albumin gene accumulated sulfur-rich SFA8 protein in leaves, resulting in increased productivity. The sfa8 gene was cloned under the control of the CaMV 35 S and wheat cab promoters to obtain chimeric constructs for expression in lucerne (alfalfa; Medicago sativa) and tobacco (Nicotiana tabacum). Constitutive expression as well as light-regulated expression of the transgene led to the accumulation of SFA8 protein. An ER retention signal (KDEL) was added to the coding region of the transgene to direct the gene product directly to the ER, where the protein is more stable (Tabe et al., 1995). The presence of a KDEL-targeting signal led to improvement in the accumulation of heterologous protein in transgenic lucerne and tobacco leaves (Wandelt et al., 1992). The chimeric sfa8 gene used to generate transgenic tall fescue plants encodes altered SFA8 protein with seven extra residues (TSEKDEL). The chimeric protein from extracts of transgenic tall fescue plants migrated differently in the western analyses when compared to the pure SFA8 protein. Nutritionally, useful levels of SFA8 for a significant influence on the ruminant diet would be approximately 4% of the total leaf protein. Seed protein in legumes like chickpeas is relatively deficient in sulfur amino acids, methionine and cysteine. The seed sulfur amino

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acid concentration has been increased by addition of a sulfur-rich sink protein (Jung et al., 1997; Tabe and Higgins, 1998; Tabe and Droux, 2002). The addition of sulfur-rich sinks, in some cases were perceived by plants as the sulfur deficiency and responded in altered endogenous protein composition. In rice (Oryza sativa L.) the insertion of a foreign, sulfur-rich protein resulted in a dramatic change of endogenous storage protein composition and resulted in no net increase in seed sulfur amino acids (Hagan et al., 2003). On the other hand, transformation of lupin (L. angustifolius L.) with the seed-specific gene encoding the sulfur-rich SSA successfully enhanced the methionine concentration in narrow-leaf lupin seeds (Molvig et al., 1997). Total methionine concentration in the seed protein was increased in the transgenic lupin seeds that accumulated high levels of SSA but total seed cysteine was not enhanced when compared with nontransgenic controls. No appreciable enhancement in the total seed cysteine in transgenic plants was attributed to down-regulation of expression of genes encoding endogenous cysteine-rich seed-storage proteins, particularly conglutin delta (a sulphur-rich protein in lupin seeds) (Tabe and Droux, 2002). A chimeric gene encoding SSA with carboxy ER retention signal under the control of the strong, endosperm‐specific high molecular weight glutenin promoter from wheat, was expressed in Japonica rice (cultivar Taipei). The transgenic rice line expressing highest SSA showed maximum accumulation of approximately 7% of total protein in the mature grain. There was no significant change in the total sulfur seed content in transgenic rice plants with respect to non-transformed plants. The results obtained for transgenic SSA rice were, therefore, different from the transgenic SSA lupin. It was suggested SSA accumulation in transgenic rice leads to diversion of the limited cellular S pool away from the synthesis of endogenous proteins (Hagan et al., 2003). The potato plants transformed with E.coli cysE gene encoding serine acetyltransferase (SAT), an enzyme of the cysteine biosynthetic pathway, showed accumulation of SAT mainly in chloroplast. Leaves from these plants consisted of significantly high amounts of cysteine and glutathione. Under normal conditions, where the sulfur supply is not limiting, one of the factors limiting the cysteine biosynthetic pathway is the low endogenous activity of SAT. Interestingly, overexpression of SAT and accumulation of the enzyme in plastids showed increased levels of cysteine and glutathione in leaves, but no change in thiol content of tuber. Possibly there was no effect of overexpression of E.coli cysE gene on the expression

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of O‐acetylserine(thiol)‐lyase, the enzyme which converts O‐acetylserine, the product of SAT, to cysteine. Indian mustard, Brassica juncea L. is one of the world’s most predominant crops of vegetable oil and protein-rich meal. It is also used in condiments, food industry, hair industry, lubricants, bio-diesel, and, in some countries, as a substitute for olive oil and the seed residues are utilized as cattle feed and in fertilizers (Aoun et al., 2008). Due to a number of advantages, researchers have worked toward the development of canolaquality B. juncea since the early 1980s. The advantages of B. juncea over B. napus include early maturing, high yield, quicker ground covering ability, more vigorous seedling growth, and greater tolerance to heat and drought (Woods et al., 1991; Burton et al., 1999), as well as beneficial for phytoremediation project, as it can effectively accumulate heavy metals (Clemente et al., 2005; Qadir et al., 2004). The sulfate transport system in Brassica categories into two parts: nutritionally regulated and nonregulated. The regulated system is represented by the group 1, 2, and 4 sulfate transporters and the nonregulated system by the group 3 sulfate transporter. During sulfate deprivation in the soil, the group 1, 2, and 4 sulfate transporters get upregulated to increase uptake, vacuole efflux, and vacuole transport of sulfate to the growing shoot (Buchner et al., 2004). The main problem with B. juncea L. cv. Pusa Jai Kisan, however, is having low-affinity sulfate transporters; therefore its requirement of sulfur for growth and yield is very high (Heiss et al., 1999). So, it required a high-affinity sulfate transporter (HAST), which can work along with other sulfate transporters during various growth stages to enhance sulfur uptake and assimilation. The first step of sulfur assimilation involves the uptake of sulfate from the soil; therefore transgenic Indian mustard expressing the high-affinity sulfate transporter gene (LeST 1.1) of tomato was developed. The sulfate uptake in B. juncea L. cv. Pusa Jai Kisan was enhanced by cloning high-affinity sulfate transporter gene (LeST 1.1) of tomato (Abdin et al., 2011). All the transgenic lines except T1 showed higher uptake of sulfate than untransformed control. This could be due to the incorporation of functional copy of LeST 1.1 gene in transgenic lines. The transgenic lines of B. juncea cv. Pusa Jai Kisan showed increase in sulfur uptake under both S-insufficient (25 and 50 μM SO42−) and Ssufficient conditions when it was compared with untransformed plants (Fig. 7.3). However, the magnitude of elevation in these values was higher under S-insufficient conditions. This may be due to S-insufficient

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Figure 7.3 Transgenic Indian mustard expressing high-affinity sulfur transporter from Solanum lycopersicum results in increasing sulfur uptake.

conditions resulting in higher levels of its mRNA in the transgenic plants under upregulation of LeST 1.1 gene. The uptake rate was gradually declined from first to seventh days in all sulfur-supplied conditions, which could be due to feedback inhibition caused by accumulation of metabolites or end product like cysteine and glutathione (Lappartient and Touraine, 1996). It has been reported that improved sulfate uptake had resulted in the accumulation of sufficient amounts of sulfate in the system, its transport and distribution into the aerial parts of the plant for assimilation (Takahashi et al., 2000; Shibagaki et al., 2002; Yoshimoto et al., 2002, 2007). This observation is further supported by higher total sulfur contents in shoots of transgenic lines under both S-insufficient and S-sufficient conditions, compared to untransformed plants. The leaves are regarded as the primary site of sulfate assimilation and there is a shoot/root recycling of the sulfur (Bell et al., 1995). The transgenic lines showed improved growth and higher accumulation of biomass than the untransformed plants. It has been reported that the growth and yield of crop plants limited by the sulfur deficiency in the agricultural field mainly because of impairment in the nitrogen uptake and assimilation (Zhao et al., 1993; Sexton et al., 1998; Ingenbleek and Young, 2004).

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The nitrogen availability has a role in regulating ATP sulfurylase, as well as nitrate reductase. The synthesis of cysteine, which results from the incorporation of the sulfide moiety into O-acetylserine, appears to be the meeting point between N- and S-metabolism. The enhancement in nitrogen and protein contents in the transgenic lines thus may be due to a metabolic coupling between S and N assimilation. A positive role of sulfate in regulating nitrate reductases, which is a key enzyme that catalyzes the rate-limiting step of nitrate assimilatory pathway, was found by several workers (Ahmad et al., 1999a,b; Beevers and Hageman, 1969). Efficient sulfate uptake not only improves the N uptake but also increases NUE of the transgenic plants by enhancing protein synthesis, as S is the constituent of the initial amino acid, methionine (Ahmad and Abdin, 2000). The enhanced protein and chlorophyll accumulation in transgenic lines could be the result of better S and N metabolism. It can be concluded that the similar approach therefore, can be used in other crops to improve their nutrient uptake and, consequently, leading to higher yields and better quality can be developed to limit the use of fertilizers as well as cultivate them in limited supply of nutrients. These crops with better nutrient uptake and assimilation potentials can be profitably grown in soil with low nutrient levels.

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Vitousek, P.M., Naylor, R., Crews, T., David, M.B., Drinkwater, L.E., Holland, E., et al., 2009. Nutrient imbalances in agricultural development. Science 324 (5934), 1519 1520. Von Wiren, N., 1997. OsAMT1-1: a high-affinity ammonium transporter from rice (Oryza sativa L. cv. Nipponbare). Plant. Mol. Biol. 3, 681. Wandelt, C.I., Khan, M.R.I., Craig, S., Schroeder, H.E., Spencer, D., Higgins, T.J., 1992. Vicilin with carboxy‐terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant. J. 2 (2), 181 192. Wang, W.H., Köhler, B., Cao, F.Q., Liu, G.W., Gong, Y.Y., Sheng, S., et al., 2012. Rice DUR3 mediates high‐affinity urea transport and plays an effective role in improvement of urea acquisition and utilization when expressed in Arabidopsis. N. Phytol. 193 (2), 432 444. Woods, D.L., Capcara, J.J., Downey, R.K., 1991. The potential of mustard (Brassica juncea (L.) Coss) as an edible oil crop on the Canadian Prairies. Can. J. Plant. Sci. 71 (1), 195 198. Xiao, K., Harrison, M.J., Wang, Z.Y., 2005. Transgenic expression of a novel M. truncatula phytase gene results in improved acquisition of organic phosphorus by Arabidopsis. Planta 222 (1), 27 36. Xiao, K., Katagi, H., Harrison, M., Wang, Z.Y., 2006a. Improved phosphorus acquisition and biomass production in Arabidopsis by transgenic expression of a purple acid phosphatase gene from M. truncatula. Plant. Sci. 170 (2), 191 202. Xiao, K., Liu, J., Dewbre, G., Harrison, M., Wang, Z.Y., 2006b. Isolation and characterization of root‐specific phosphate transporter promoters from Medicago truncatula. Plant. Biol. 8 (4), 439 449. Yamaya, T., Obara, M., Nakajima, H., Sasaki, S., Hayakawa, T., Sato, T., 2002. Genetic manipulation and quantitative‐trait loci mapping for nitrogen recycling in rice. J. Exp. Bot. 53 (370), 917 925. Yang, W.C., Katinakis, P., Hendriks, P., Smolders, A., Vries, F., Spee, J., et al., 1993. Characterization of GmENOD40, a gene showing novel patterns of cell‐specific expression during soybean nodule development. Plant. J. 3 (4), 573 585. Yip, W., Wang, L., Cheng, C., Wu, W., Lung, S., Lim, B.L., 2003. The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco. Biochem. Biophys. Res. Commun. 310 (4), 1148 1154. Yoshimoto, N., Takahashi, H., Smith, F.W., Yamaya, T., Saito, K., 2002. Two distinct high‐affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots. Plant. J. 29 (4), 465 473. Yoshimoto, N., Inoue, E., Watanabe-Takahashi, A., Saito, K., Takahashi, H., 2007. Posttranscriptional regulation of high-affinity sulfate transporters in Arabidopsis by sulfur nutrition. Plant. Physiol. 145 (2), 378 388. Zhang, F.S., Ma, J., Cao, Y.P., 1997. Phosphorus deficiency enhances root exudation of low-molecular weight organic acids and utilization of sparingly soluble inorganic phosphates by radish (Raghanus satiuvs L.) and rape (Brassica napus L.) plants. Plant. Soil. 196 (2), 261 264. Zhao, F., Evans, E.J., Bilsborrow, P.E., Syers, J.K., 1993. Influence of sulphur and nitrogen on seed yield and quality of low glucosinolate oilseed rape (Brassica napus L). J. Sci. Food Agric. 63 (1), 29 37. Zimmermann, P., Zardi, G., Lehmann, M., Zeder, C., Amrhein, N., Frossard, E., et al., 2003. Engineering the root–soil interface via targeted expression of a synthetic phytase gene in trichoblasts. Plant. Biotechnol. J. 1 (5), 353 360.

CHAPTER EIGHT

Genome editing of staple crop plants to combat iron deficiency Mather A. Khan
and Nga T. Nguyen
Division of Plant Sciences, C.S. Bond Life Sciences Center, University of Missouri, Columbia, MO, United States

8.1 Introduction Iron is essential for all forms of life. It is a micronutrient because plants and humans only need a small amount in a range of micrograms. Unlike macronutrients (e.g., carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur), micronutrients do not comprise the main components of energy-rich compounds, but they are required to activate many enzymatic reactions and enable chemical pathways. An excessive amount of micronutrient, including iron, is toxic for biological systems. Iron can form hydroxyl radicals, which means an excessive amount of iron enhances the production of reactive oxygen species and induces oxidative damage. Thus in biological systems, cells need to sequester the extra iron and store it safely. In many parts of the world, people rely on plant-based food as the main source of micronutrients including iron. However, most of the staple crops have low iron value compared to the iron daily intake required. Therefore it is important to increase iron concentration in staple crop plants without disturbing the iron homeostasis of the plants. In this chapter, we will cover genes, proteins, enzymes, and compounds that are important for iron uptake and transport within the plants. In the end, we will discuss studies and strategies on genome editing to enhance the iron content of crop plants. Imagine living in an autonomous metropolitan that was designed to be self-sufficient. However, one day, the city administration announced its energy department is undergoing malfunction and the food department


Equal contribution.

Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00008-3

© 2020 Elsevier Inc. All rights reserved.

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can no longer maintain its production to meet the city demand. A plant cell under iron-deficient condition is like a city threatened by the potential of running out of food and energy. Indeed, plants, as autotrophs, synthesize their food from carbon dioxide and water using energy from the sunlight—a process known as photosynthesis which takes place in the chloroplast. A plant cell, without iron, cannot synthesize chlorophylls for functional chloroplasts. Iron is required for mitochondria, the house of cell respiration. Ironsulfur clusters are essential for the proper functions of many respiratory chain protein complexes. Without iron, plant cells cannot synthesize enough food and energy compounds. In addition to photosynthesis and respiration, iron is required for DNA replication and repair. These are a few examples to demonstrate that iron-limiting condition gradually leads to plant cell starvation with functional impaired organelles.

8.2 Iron uptake and transport Iron is abundant in the soils, but mostly in the form of precipitation as ferric hydroxides (Fe(III)OH3) or ferric oxides (Fe2O3). In arid calcareous soils, where the average pH is 8.0, iron deficiency is one of the problems causing yield reduction. When iron deficiency chlorosis occurs, it can cause up to 30% yield losses. According to USDA data of 2013, in the North Central United States, iron chlorosis showed up in more than 4.7 million acres of soybean fields and it was estimated to cause 375,000 tons of yield losses, equal to $120 million US dollars (NDSC, 2016). Iron is not a well-mobile micronutrient, which means when an iron deficiency occurs, chlorosis symptom first appears in young tissues such as immature leaves or flower buds. For the mature tissues, because transmembrane proteins on the tonoplast release the iron stored from the vacuole, it takes more time for these tissues to turn yellowish since when roots start experiencing iron deficiency. One problem when dealing with iron deficiency on the field is that iron-deficient status can happen way before the chlorosis symptom becomes visible on plants. By the time when we see a plant with pale, yellowish tissues, iron deficiency already affected the plant development and thus will affect the yield. Though iron deficiency chlorosis affecting yield were observed in different crop plants, we have

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limited knowledge about the molecular mechanism that plants use to take up iron and respond to iron deficiency. The current knowledge identified two strategies that plants evolved to take up iron: the reduction strategy (Strategy I) and the chelation strategy (Strategy II). In this chapter, the discussion will focus on Arabidopsis thaliana (Arabidopsis), Zea mays (maize), and Oryza sativa (rice). Arabidopsis is the model plant that is well studied for iron uptake. Arabidopsis represents for nongrass plants that use the iron uptake Strategy I. Although it is not a crop plant, information from studies based on Arabidopsis can be translated to crop plants. On the other hand, maize is categorized as a grass plant and evolves Strategy II of iron uptake. Lastly, rice is an exception because rice can use both Strategies I and II of iron uptake even though it is a grass.

8.2.1 Root uptake: iron uptake Strategy I and Strategy II In Arabidopsis, three plasma-membrane proteins expressed in root epidermal cells are involved in the reduction strategy of iron uptake. In the first step, the PLASMA MEMBRANE PROTON ATPase 2, also known as AtAHA2, acidifies the rhizosphere by releasing protons from the roots. Soil particles have negative charges and therefore absorb cationic mineral nutrients including Fe31 to their surface. With the positive charge, the protons released by AtAHA2 can replace Fe31 bound on the soil particles and thus make more Fe31 available to the rhizosphere. For every pH unit drop, the solubility of iron increased by 1000-fold (Guerinot and Yi, 1994). There are several AtAHA proteins in Arabidopsis. However, AtAHA2 was found to have the strongest expression in the root epidermis and the loss of AtAHA2 significantly reduces the rhizosphere acidification ability during Fe limiting condition (Morrissey and Guerinot, 2009). In the second step, a membrane-bound protein called FERRIC REDUCTION OXIDASE 2 (AtFRO2) reduces Fe31 to Fe21 by transferring an electron from NADH 1 to the iron ion. In Strategy I plant like Arabidopsis, Fe21 is the soluble form of iron that can be transported across the plasma membrane (Morrissey and Guerinot, 2009). Finally, the IRON-REGULATED TRANSPORTER 1 (AtIRT1) transport Fe21 across the plasma membrane of root cells (Nishida et al., 2011; Vert et al., 2002). IRT1 is a member of the Zrt/Irt-like protein (ZIP) family, the divalent metal transporter family that is conserved in many species (yeast, plants, and animals). In sum, this strategy with three steps of acidification, reduction, and transportation is employed by nongraminaceous (nongrass)

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plants, and it has been extensively characterized in the model plant Arabidopsis. Maize is a representative of C4 plants that tolerate drought stress better as compared to C3 plants. Maize reproductive system evolved to favor outcrossing: the male flowers (tassels) and female flowers (ears) develop separately on the same plant. This feature allows crossing to increase the phenotype range, which is important in breeding programs to develop plant lines that can adapt to a wide range of environmental stress. A large portion of the world agriculture has been familiar with this staple crop; therefore if we can increase iron concentration in maize, it will benefit a big part of the world population. Maize and other grass plants like barley and sorghum (also known as graminaceous species) use the chelation strategy (Strategy II) to take up iron from soils. Plants using this strategy release small organic compounds having a high affinity for Fe31 called phytosiderophores (PSs). PSs mean “plant iron-carrier” because they are iron-chelating compounds secreted by the plants. Mugineic acid (MA) and 20 -deoxymugineic acid (DMA) are known to be released from maize roots playing the role of Fe carriers (Von Wirén et al., 1998). In barley, it was identified that TRANSPORTERS OF MUGINEIC ACID 1 (HvTOM1) responsible for secreting MA into the rhizosphere (Nozoye et al., 2011). Later, there was evidence suggesting that maize homolog of HvTOM1 is the protein responsible for the yellow-striped phenotype observed in yellow striped 3 (zmys3) maize mutant and this mutant was unable to produce a sufficient level of PS (Chan-Rodriguez and Walker, 2018). Once Fe31 is chelated by a PS, the whole Fe(III)PS complex is transported across the root plasma membrane via the proton-coupled symporter YELLOW STRIPE 1 (ZmYS1) (Schaaf et al., 2004). To date, rice is the only grass plant found to evolve both the reduction strategy and the chelation strategy for iron uptake. While maize and other grass plants lack the Fe(II)-reduction transport system, the expression of OsIRT1 and OsIRT2 allow the rice to import Fe21 under anaerobic conditions (Ishimaru et al., 2006). In human history, wet-rice farming is the most prevalent method for producing grains in southern and eastern Asia. In the paddy fields, water flooded in replaces the air to fill up pores in the soil. This condition reduces the oxygen concentration in the soil, which results in decreased oxidationreduction or redox potential. Therefore Fe21 is the more abundant form of iron in flooded fields. Under iron-limiting conditions, rice can activate OsIRT1 and OsIRT2 to take up Fe21, and OsTOM1 and OsYSL15 to take up Fe31 (Ishimaru

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et al., 2006). OsTOM1 is the homolog of HvTOM1 that plays a role in extruding PS compounds into the rhizosphere. OsYSL15 (YELLOW STRIPED LIKE 15) in rice acts as ZmYS1 in maize, transporting Fe (III)PS complexes into the root cells. Although rice produces less PS than maize and barley, PS synthesis and secretion of PS are necessary for the rice to survive in calcareous soils. Mutant rice lacking the ability to produce PS to chelate metal ions was observed to be unable to survive in aerobic soils or hydroponic supplemented with only Fe31 (Kobayashi et al., 2014).

8.2.2 Chelators and long-distance transport of iron In plants, one phloem vessel and one xylem vessel develop side by side to form a vascular bundle. One plant can have multiple vascular bundles, from roots to tip as continuous pipe structures to serve as the transport routes for water, mineral nutrients, photosynthetic products, and signaling compounds. While xylem vessels constitute of death cells (tracheids and vessel elements) to transport water and mineral nutrients toward the aerial tissues based on evapo-transpiration, phloem vessel delivery photosynthates including iron-containing compounds from mature tissue to premature tissues such as young leaves, buds, flowers, and emerging roots. Phloem vessels are made of sieve elements, which are modified, elongated cells connected by sieve plates to form the sieve tubes. Because phloem is living tissue, it allows bidirectional long-distance transport based on active transport. Despite its vast function in biological systems, free Fe21 can participate in the Fenton’s reaction in which iron and hydrogen peroxide react to generate hydroxyl radicals (Winterbourn, 1995). Hydroxyl radicals can damage macromolecules of the cell, such as DNAs, proteins, and lipids. To prevent cellular damage, free ions noncovalently bind to organic compounds—also called an iron ligand. An iron ion and its ligand form complex based on the donation of one or more electron pairs of the ligand to the iron. Iron complexes are soluble and safe for storage or long-distance transport (Álvarez-Fernández et al., 2014). Various techniques have been applied to identify the nature of iron ligand in long-distance transport in plants. A range of molecules was proposed: organic acids such as citrate and malate, amino acids including nicotianamine (NA), histidine, and cysteine, PSs derived from NA such as MA and DMA, peptides and proteins such as ferredoxins and histidine-rich proteins (Álvarez-Fernández et al.,

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2014; Krüger et al., 2002; Schuler et al., 2012). Based on the metabolic profiling of xylem sap and phloem sap, it was proposed that Fe(III)citrate is the predominant form of iron in the xylem sap and apoplast (Rellán-Álvarez et al., 2010). Tri-iron(III) (Fe3Cit3) and dinuclear iron(III) (Fe2Cit2) were found in xylem sap of tomato, castor bean, and soybean (Ariga et al., 2014; Rellán-Álvarez et al., 2010). Meanwhile, in the pH condition of phloem sap and cytosol, it is thought that Fe(II)nicotinamide (NA) is the predominant form of iron (Von Wirén et al., 1999). The pH of the cell environment determines which ionic form of iron is more stable. In general, at the slightly acidic environment (pHB5.5) of the xylem sap and apoplast, citrate binds more stable to the Fe31 while at a higher pH (B7.5) like in the phloem sap and cytosol, an abundant amount of NA was found (Ariga et al., 2014; Schuler et al., 2012). In Arabidopsis, several genes that are important for iron transport into xylem were characterized. FERROPORTIN1 or IRON REGULATED1 (AtFPN1/AtIREG1) and FERROPORTIN2 or IRON REGULATED2 (AtFPN2/AtIREG2) localize to the tissue layers where they are important for translocation into the xylem vasculature. AtFPN1 localizes to the plasma membrane and expresses at the root stele where vasculature tissues develop from the procambium. Meanwhile, AtFPN2 localized to the tonoplast of cells at the two outermost layers of the root in response to iron deficiency. It was suggested that the loss of AtFPN2 expression resulted in the mislocalization of iron that should be sequestered in the vacuoles, and therefore a significant decrease in ferric chelate reductase activity in fpn2 single mutant was recorded when compared to wild-type plants. A single mutant of AtFPN1 was found to display a chlorotic phenotype, although no significant increase in ferric chelate reductase activity of fpn1 single mutant was found; fpn1fpn2 double mutant displayed an elevated iron deficiency response—significantly increased levels of reductase activity and IRT1 expression (Morrissey et al., 2009). However, homologs of FERROPORTIN in rice and maize have not been identified yet. In terms of long-distance transport in Arabidopsis, citrate is fluxed into the xylem via FERRIC REDUCTASE DEFECTIVE 3 (AtFRD3) to ensure iron transport from the outer layers of the root into the xylem. Loss of AtFRD3 expression in Arabidopsis results in a higher level of iron accumulation in roots but shoots remained chlorotic because without citrate in the xylem, iron cannot be mobilized to the aerial part of the plant (Durrett et al., 2007; Roschzttardtz et al., 2011). In rice, the FERRIC REDUCTASE DEFECTIVE-LIKE 1 (OsFRDL1) was suggested to be a

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homolog of AtFRD3 (Yokosho et al., 2009). As it was found in Arabidopsis frd3 mutant, the loss of OsFRDL1 function in rice results in plants with chlorotic symptoms in the shoot, reduced citrate level in the xylem sap, and iron precipitation in xylem vessels due to the lack of citrate. Both AtFRD3 and OsFRDL1 proteins, when expressed on the heterologous system to test for substrate specificity, were found to transport citrate across the plasma membrane. Under iron-sufficient condition, atfrd3 and osfrdl1 loss-of-function mutants were found to have a constitutive iron-deficient response (Durrett et al., 2007; Roschzttardtz et al., 2011; Yokosho et al., 2009). In maize, however, the homologs of AtFRD3 and OsFRDL1 have not been found. Citrate is the major chelator that keeps iron soluble in the xylem sap. In Arabidopsis, five citrate synthases were identified, but the single-gene knockout mutants of these genes were not described to be associated with the iron-deficient phenotype. This is probably because citrate is part of the citric acid cycle, the central driver of cellular respiration. Therefore if there is a knockout of single citrate synthase, the activities of other citrate synthases compensate. However, if there is a knockout of all five citrate synthases genes, the mutant will not survive due to energy deficiency. There are studies describing the iron-deficient phenotype in plants that lack nicotinamide. Although the chelation strategy mediated by PSs is not adopted for iron uptake in roots of Arabidopsis and nongrass plants, NA—a precursor of PSs—is required for iron transport within tissues. NA production in Arabidopsis depends on four NICOTIANAMINE SYNTHASEs: AtNAS1, AtNAS2, AtNAS3, and AtNAS4 (Schuler et al., 2012). Eliminating or diminishing NA production by mutating these four AtNAS genes can result in phenotypes ranging from early interveinal chlorosis to severe chlorosis at the reproductive stage and sterile flowers (Schuler et al., 2012). In maize, nine ZmNAS genes have been identified. ZmNAS1 and ZmNAS2 expression are upregulated in roots during iron deficiency (Mizuno et al., 2003). Three rice NA synthase genes, OsNAS1, OsNAS2, and OsNAS3, were found to be involved in long-distance transport of iron (Inoue et al., 2003). In grass plants like maize and rice, as mentioned before, NA is not only important for long-distance transport of iron but also it can be modified to 2-deoxymugineic acid (DMA) by the enzyme nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) for iron uptake in roots (Takahashi et al., 1999). Members of the YELLOW STRIPE family have been identified as transporters involved in long-distance transport of iron and, in many cases,

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were suggested to serve in the phloem unloading and translocation. For example, in Arabidopsis, phloem unloading and translocation of iron to sink tissues are thought to be mediated by two FeNA transporters, YELLOW STRIPE LIKE 1/3 (YSL1 and YSL3). AtYSL1 and AtYSL3— both encode for plasma membrane proteins expressed in the vasculature and flowers. The Arabidopsis double mutant ysl1ysl3 displays an interveinal chlorotic phenotype and has reduced iron concentration in the seeds. The elemental profile of ysl1ysl3 leaves suggests that AtYSL1 and AtYSL3 also transport Mn, Zn, and Cu. However, only the generous application of iron can alleviate the defective phenotype of ysl1ysl3 mutants (Chu et al., 2010; Waters et al., 2006). In rice, besides OsYSL15 (the rice ortholog of the maize YS1), OsYSL16, OsYSL9, and OsYSL18 are suggested to transport metalPSs complexes (Inoue et al., 2003; Senoura et al., 2017). During iron deficiency, OsYSL9 is upregulated and expressed in the root vasculature cylinder. OsYSL9 is also found to express in the endosperm and the seed coat. OsYSL9 knockdown plants showed lower iron in leaves than in nontransgenic plants. Embryos of OsYSL9 knockdown plants also have lower iron concentrations than the embryos of wild type (Senoura et al., 2017). OsYSL18 is another candidate for a phloem transporter of iron (III)PSs because it was found to express in flowers and companion cells (Aoyama et al., 2009). In maize, as mentioned earlier, YS1 and YS3 are essential for iron transport. These genes, however, have been known for iron uptake into roots cells rather than for long-distance transport of iron. Recently, an Arabidopsis gene named OLIGOPEPTIDE TRANSPORTER 3 (AtOPT3) has been identified to encode for a plasma membrane transporter mediating iron transport into the phloem. AtOPT3 localizes to the plasma membrane and preferentially expressed in phloem tissues (Mendoza-Cózatl et al., 2014; Zhai et al., 2014). atopt3 knockout mutant (atopt3-1) is embryo-lethal while the atopt3 knock-down mutants (atopt3-2 and atopt3-3) have a constitutive upregulation of iron-uptake genes in roots. Before atopt3 knock-down mutants, several Arabidopsis mutants were previously found to have constitutive iron-deficient responses (Chu et al., 2010; Durrett et al., 2007; Roschzttardtz et al., 2011; Schuler et al., 2012). However, in these mutants, the roots were unable to take up iron, or iron was not effectively delivered to the shoots. In contrast, atopt3 knock-down mutants overaccumulate iron in both roots and shoots but the roots keep taking up iron even under iron-sufficient conditions (MendozaCózatl et al., 2014; Stacey et al., 2008; Zhai et al., 2014). The specific

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iron complex that AtOPT3 transport has not been identified and the gene function has not been fully understood. However, the phenotype of atopt3 mutants is suggesting that plants require a suppression signal that comes from the shoots to turn off the iron-uptake machinery when iron is abundant in the cells (Khan et al., 2018; Mendoza-Cózatl et al., 2014). This unknown signal was somehow missing or not perceived in atopt3 knock-down mutants, thus resulted in the misregulation of iron uptake. In rice, similar to the AtOPT3, OsOPT7 was identified to localize in the plasma membrane. The knockout mutant of OsOPT7 (osopt7-1) displays iron-deficient responses even when plants were grown under iron-sufficient condition. The specific substrate of OsOPT7 is also unknown (Bashir et al., 2015).

8.2.3 Iron storage and vacuole sequestration To avoid cell damage, cellular iron must exist in chelation forms or bound to proteins. In the cytosol, Fe(II)NA is the most abundant form, meanwhile, in chloroplast and mitochondria, ironsulfur clusters and heme are predominant (Álvarez-Fernández et al., 2014; López-Millán et al., 2016; Morrissey and Guerinot, 2009; Von Wirén et al., 1999). Ferritin is a universal iron-storage protein among species and is found abundant in the chloroplast. Each ferritin molecule stores about 4500 Fe31 ions, mostly in the form of Fe(III)-hydroxides (Andrews et al., 1992; Theil, 1987). Ferritin proteins are encoded by FERRITIN genes, such as AtFER1 and AtFER2 in Arabidopsis, ZmFER1 and ZmFER2 in maize, and OsFER1 and OsFER2 in rice (Petit et al., 2001). In Arabidopsis, Fe31 in the chloroplast is first reduced to Fe21 by FERRIC REDUCTION OXIDASE 7 (AtFRO7) activity, and it is thought to be transported in and out by PERMEASE IN CHLOROPLASTS 1 (AtPIC1) (Duy et al., 2011; Jeong et al., 2008). To date, not many players involved in iron transport in mitochondria and chloroplast in maize and rice are identified. Zea maize FeDEFICIENCY-RELATED (ZmFDR3), which localized in maize chloroplasts, is proposed to be involved in iron transport (Han et al., 2009). In Arabidopsis mitochondria, iron is imported by FRATAXIN (AtFH) and exported by ABC TRANSPORTER OF THE MITOCHONDRION 3 (AtATM3) (Busi et al., 2004; Chen et al., 2007). Recently, the Arabidopsis MITOCHONDRIAL IRON TRANSPORTER 1 and 2 (AtMIT1 and AtMIT2), which are members of the mitochondrial carrier family of transport proteins, were identified as essential iron transports. Loss of MIT1 and MIT2

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functions resulted in the mislocalization of iron while the double mutant mit1mit2 was embryonic lethal (Jain et al., 2019). OsMIT1 in rice was identified as orthologs of AtMIT1 and AtMIT2 with a similar function (Bashir et al., 2011). Arabidopsis FERROPORTIN 2 (AtFPN2) and VACUOLAR IRON TRANSPORTER 1 (AtVIT1) are two important iron importers localized in the tonoplast (Kim et al., 2006; Schaaf et al., 2006). The NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEINS 3 and 4 (AtNRAMP3 and AtNRAMP4) are responsible for releasing iron from the vacuole upon iron depletion (Lanquar et al., 2005). In rice, OsVIT1 and OsVIT2—two orthologs of the Arabidopsis AtVIT1—localize in the tonoplast and facilitate the transport of iron into the vacuole. Rice plants with functional disruption of OsVIT1 or OsVIT2 showed an increase of iron and zinc accumulation in the embryo (Zhang et al., 2012). In the grains, myo-inositol hexaphosphate, also known as phytic acid (PA), is the most abundant and strong chelator of metal cations, including iron. Though phytates that are formed between iron cations and PA are abundant in rice grains, they are not in the form of bioavailable iron. Thus selecting rice varieties with low PA concentration or developing low-PA rice using transgenic techniques is among the approaches to increase bioavailable iron in rice (Perera et al., 2018). Reverse genetics approach using genome-wide association study (GWAS) and quantitative trait locus (QTL) mapping tools identified chromosomal regions associated with high iron accumulation in crops. In rice, few QTLs linked to iron concentration have been identified on chromosomes 1 and 5 (Anuradha et al., 2012). In maize, there is one QTL on chromosome 5 that is proposed to account for 16.89% of phenotypic variation in grain iron concentration (Jin et al., 2013). Thus it is promising that more genes related to iron storage and regulation will be identified in the future. It will increase the number of potential genes that can be selected for crop genome editing in biofortification studies.

8.3 Genetic engineering to improve iron content in crops Globally, Fe deficiency is one of the most prevalent and widespread nutritional disorder affecting an estimated 2 billion people. Young children and women (mainly pregnant and post-partum women) are severely affected by iron deficiency anemia (WHO, 2019). It is the only nutritional deficiency which is significantly prevalent in many of the developed

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Figure 8.1 Global estimates of the prevalence of anemia in infants and children aged 6-59 months. Reproduced with permission from WHO. The Global Anemia Prevalence in 2011. Geneva: World Health Organization; 2015.

countries (WHO, 2019). In developing countries, every second pregnant woman and about 40% of preschool children (Fig. 8.1) are estimated to be anemic (WHO, 2019). To combat iron deficiency, consumption of iron-rich food in a balanced proportion is the best option. Plants are the main source of iron to humans, either directly by consuming plant-based food or indirectly from animal-based food. While planning and designing strategies to increase the Fe content in crop plants, the approach should be properly studied so that the transgenic plants should not result in (1) loss of yield, (2) reduction in the content of other essential micronutrients (Zn, Mn, Cu), and (3) should not favor the uptake of nonessential elements (cadmium, arsenic, etc.). To develop Fe-biofortified cereal crops using transgenic approaches, five steps can be targeted: (1) enhanced uptake, (2) increase translocation to grains, (3) enhancement of Fe accumulation in endosperm, (4) reduction of “antinutrients” (such as PA and polyphenols), and (5) increase of bioavailability (Mulualem, 2015).

8.3.1 Enhancing iron storage Targeting FERRITIN genes under the control of endosperm specific promoters is one of the initial approaches to increase iron content in seeds. As discussed earlier, ferritin is an Fe storage protein found in most of the organism, including plants and animals. It consists of 24 subunits,

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Table 8.1 Selected examples of genetic engineering approaches to increase iron content in rice and maize. Gene(s) Promoter Crop/cultivar Fold References increase in Fe Iron uptake and translocation

B1.5

ZmZIP5

ZmLegumin1

Maize/B73

OsIRT1

ZmUBQ

Rice

1.1

HvNAS1

OsACTIN1

Rice/Japonica cv. Tsukinohikari

2.5

OsNAS2

CaMV 35S

Rice/Japonica cv. Nipponbare

4

OsNAS3

CaMV 35S

3

OsYSL2

OsSUT1

Rice/Japonica cv. Dongjin Rice/Japonica cv. Tsukinohikari

OsYSL9

RNAi line

Rice/Japonica cv. Tsukinohikari

2.5

OsYSL13

OsYSL13

Rice/cv. Nipponbare

GmFERH1

Zm27gZEIN

Maize/B73

GmFERH1

OsGLUB1

GmFERH1

OsGLUB1

OsFER2

OsGLUA2

Rice/Japonica cv. Kitaake Rice/Japonica cv. Taipei 309 Rice/Indica cv. Pusa-sugandhi II

4

B1.2

Li et al. (2019) Lee and An (2009) Masuda et al. (2009) Johnson et al. (2011) Lee et al. (2009) Ishimaru et al. (2010) Senoura et al. (2017) Zhang et al. (2018)

Iron storage

Intercellular/intracellular transport and storage

OsVIT1

T-DNA insertion line

Rice/Japonica cv. Zhonghua 11

1.2

3 2.2 2.1

B1.3

Kanobe et al. (2013) Goto et al. (1999) Lucca et al. (2002) Paul et al. (2012)

Zhang et al. (2012) (Continued)

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Table 8.1 (Continued) Gene(s) Promoter

OsVIT2

T-DNA insertion line

Crop/cultivar

Rice/Japonica cv. Dongjin

Fold increase in Fe

B1.4

References

Zhang et al. (2012)

Multigene overexpression

OsNAS1 HvNAATb

ZmUBQ1

Rice/EYI 105

AtNAS1, PvFER, AfPHY

CaMV 35S, OsGLUB

Rice/Japonica cv. Taipei 309

.6

CaMV 35S, OsGLUB, OsGLU CaMV 35S, OsGLB, ZmUbi AtNAS1, AtFRD3, CaMV 35S, PvFer ZmUbi, OsGLB

Rice/Japonica cv. Nipponbare

3.3

AtNAS1, PvFER, ZmPSY, PaCRT1 AtNAS1, PvFer, AtNRAMP3

Rice/Indica cv. IR64 Rice/Japonica cv. Nipponbare

4

Banakar et al. (2017) Wirth et al. (2009) Singh et al. (2017)

5

Wu et al. (2019)

5.4

Wu et al. (2018)

The first two letters denote the organism of origin: Af, aspergillus fumigatus; At, arabidopsis thaliana; CaMV, cauliflower mosaic virus; Gm, glycine max; Hv, hordeum vulgare; Os, oryza sativa; Pa, pantoea ananatis; Pv, phaseolus vulgaris; Zm, zea mays.

assembles to form a large complex which holds up to 4500 Fe atoms in its cavity (Goto et al., 1999). To improve the Fe content of rice, Goto et al. (1999) developed a transgenic rice plant (Table 8.1) that expressed the soybean ferritin GmFERH1 under the control of endosperm specific 1.3 kb GluB1 rice promoter. The seeds from the transgenic rice accumulated up to threefold more iron (38.1 6 4.5 μg/g DW) than normal seeds (11.2 6 0.9 μg/g DW). Later on, there were a number of studies of expressing ferritin gene under different promoter and genetic background (Table 8.1). Instead of expressing soybean ferritin, Paul et al. (2012) used rice ferritin (OsFER2) to generate transgenic rice under the control of endosperm specific GlutelinA2 (OsGluA2) promoter. The resulting T3 transgenic seeds showed about 2.1-fold higher iron in milled rice grain as compared to the control Indica cv. Pusa-Sugandhi II seeds. However, as compared to rice, very little work has been reported to improve the Fe content of maize. In one of the studies in maize, Kanobe et al. (2013)

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analyzed the expression of soybean ferritin GmFERH1 under the control of the 27 kDa zein promoter. In this experiment, they reported that transgene was successfully introduced and expressed in the maize seed endosperm but resulted in a very slight increment in Fe content. It will be interesting to analyze the expression of the endogenous ferritin gene instead of exogenous ferritin in maize. Targeting vacuolar iron storage is another option to enhance Fe content in seeds. Overexpression of VIT genes in rice and maize has not been fully explored, but T-DNA insertion lines of VIT1 or VIT2 increased the total iron content in brown rice by about 1.3- to1.4-fold (Zhang et al., 2012).

8.3.2 Increasing iron translocation Enhancing the Fe transport within the plant by overexpressing genes involved in metal-chelator transport (e.g., NAS, YSLs) is another approach to increase the Fe content in seeds. Several research groups reported the overexpression of NAS genes in rice under the control of different promoters, and increasing iron uptake into the plant via chelation-based strategy (Table 8.1). It has been demonstrated that overexpression of rice OsNAS1, OsNAS2, and OsNAS3 (Johnson et al., 2011), OsNAS2 (Lee et al., 2012), OsNAS3 (Lee et al., 2009), and barley HvNAS1 (Masuda et al., 2009) genes are able to increase the iron content up to fourfold in rice grain. Enhancing iron transport from the phloem into the developing seeds by overexpressing YSL2 is another promising approach in iron biofortification in grains. Ishimaru et al. (2010) developed transgenic rice (cv. Tsukinohikari) by overexpressing OsYSL2 driven by a sucrose transporter. By this approach, they were able to increase the Fe content up to fourfold in polished rice grains. Ishimaru et al. (2010) also described that it was important to use the OsSUT1 promoter to increase iron levels in polished rice, as no effect was seen with the 35S promoter.

8.3.3 Improving iron uptake Iron uptake by Strategies I and II involves a specific gene set of which were described in Section 8.1. Maize is a grass plant that utilizes Strategy II, meanwhile rice can use both Strategies I and II of iron uptake. Manipulating genes that are major players of the iron-uptake step is an approach that has not been fully explored. However, at the same time,

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this approach should be dealing with extra precaution. A number of genes involves in iron uptake is also involved in the uptake of other metals, including nonessential metals, such as cadmium (Khan et al., 2014). Lee and An (2009) overexpressed OsIRT1 driven by maize ubiquitin promoter in rice. However, this approach increases the iron concentration only by 1.1-fold in grains.

8.3.4 Multigene expression The multigene expression is a more promising approach in increasing Fe content compared to overexpressing a single gene. Wirth et al. (2009) demonstrate that the combined and targeted expression of Pvferritin, AtNAS1, and Afphytase led to a more than sixfold increase in iron content in transgenic rice polished grains. The presence of a phytase does not prevent this iron accumulation, but may be useful to reduce the iron antinutrient phytate. Using the power of simultaneous expression of several genes, Banakar et al. (2017) reported that the coexpression of OsNAS1 and HvNAATb in rice increased the abundance of NA and DMA and allowed the accumulation of higher levels of Fe (fourfold) in the endosperm. Likewise, a transgenic line expressing AtIRT1, Pvferritin, AtNAS1, and Afphytase was shown to have a fourfold increase of iron accumulation in polished grain (Boonyaves et al., 2016; Masuda et al., 2013). Recently, Wu et al. (2019) developed transgenic rice lines expressing AtNRAMP3, AtNAS1, and PvFER, resulting in a fivefold increase in Fe content in polished grains. Overall, this multigene approach has the potential to increase Fe content in staple crops, but a more in-depth investigation is required to understand transgene stability over multiple generations.

8.4 Conclusion Rice and maize are the most consumed staple foods across the globe. Notably, they serve as an affordable source of calories in most of the developing countries. However, both rice and maize are very poor in micronutrient content, including iron. Iron biofortification by genetic engineering is an ideal method to increase Fe levels in a more effective manner by targeting a specific gene(s). In the last two decades, we see significant progress in the development of iron-rich rice. However, there is

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very little progress in developing iron biofortified maize. The lack of a complete picture of iron uptake and transport mechanism in rice and maize is also a limiting factor to research studies on this subject. Thus more rice and maize genes that are parts of the iron regulation networks are awaited to be revealed. On the other hand, genetically modified crops are still not acceptable in many countries. Lastly, with the advent of the new genome-editing method based on CRISPR/Cas9, the future of crop biofortification is more promising.

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Kobayashi, T., Nakanishi Itai, R., Nishizawa, N.K., 2014. Iron deficiency responses in rice roots. Rice 7, 27. Krüger, C., Berkowitz, O., Stephan, U.W., Hell, R., 2002. A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L. J. Biol. Chem. 277, 2506225069. Lanquar, V., Lelièvre, F., Bolte, S., Hamès, C., Alcon, C., Neumann, D., et al., 2005. Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. EMBO J. 24, 40414051. Lee, S., An, G., 2009. Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell Environ. 32, 408416. Lee, S., Jeon, U.S., Lee, S.J., Kim, Y.K., Persson, D.P., Husted, S., et al., 2009. Iron fortification of rice seeds through activation of the nicotianamine synthase gene. Proc. Natl. Acad. Sci. U. S. A. 106, 2201422019. Lee, S., Kim, Y.S., Jeon, U.S., Kim, Y.K., Schjoerring, J.K., An, G., 2012. Activation of rice nicotianamine synthase 2 (OsNAS2) enhances iron availability for biofortification. Mol. Cell 33, 269275. Li, S., Liu, X., Zhou, X., Li, Y., Yang, W., Chen, R., 2019. Improving zinc and iron accumulation in maize grains using the zinc and iron transporter ZmZIP5. Plant Cell Physiol. 60, 20772085. López-Millán, A.F., Duy, D., Philippar, K., 2016. Chloroplast iron transport proteins— function and impact on plant physiology. Front. Plant Sci. 7, 178. Lucca, P., Hurrell, R., Potrykus, I., 2002. Fighting iron deficiency anemia with iron-rich rice. J. Am. Coll. Nutr. 21, 184S190S. Masuda, H., Usuda, K., Kobayashi, T., Ishimaru, Y., Kakei, Y., Takahashi, M., et al., 2009. Overexpression of the barley nicotianamine synthase gene HvNAS1 increases iron and zinc concentrations in rice grains. Rice 2, 155166. Masuda, H., Aung, M.S., Nishizawa, N.K., 2013. Iron biofortification of rice using different transgenic approaches. Rice. 6, 112. Mendoza-Cózatl, D.G., Xie, Q., Akmakjian, G.Z., Jobe, T.O., Patel, A., Stacey, M.G., et al., 2014. OPT3 is a component of the iron-signaling network between leaves and roots and misregulation of OPT3 leads to an over-accumulation of cadmium in seeds. Mol. Plant 7, 14551469. Mizuno, D., Higuchi, K., Sakamoto, T., Nakanishi, H., Mori, S., Nishizawa, N.K., 2003. Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status. Plant Physiol. 132, 19891997. Morrissey, J., Guerinot, M.L., 2009. Iron uptake and transport in plants: the good, the bad, and the ionome. Chem. Rev. 109, 45534567. Morrissey, J., Baxter, I.R., Lee, J., Li, L., Lahner, B., Grotz, N., et al., 2009. The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis. Plant Cell 21, 33263338. Mulualem, T., 2015. Application of bio-fortification through plant breeding to improve the value of staple crops. Biomed. Biotechnol. 3, 1119. NDSC, 2016. Growing a Healthy Industry. Nishida, S., Tsuzuki, C., Kato, A., Aisu, A., Yoshida, J., Mizuno, T., 2011. AtIRT1, the primary iron uptake transporter in the root, mediates excess nickel accumulation in Arabidopsis thaliana. Plant Cell Physiol. 52, 14331442. Nozoye, T., Nagasaka, S., Kobayashi, T., Takahashi, M., Sato, Y., Sato, Y., et al., 2011. Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants. J. Biol. Chem. 286, 54465454. Paul, S., Ali, N., Gayen, D., Datta, S.K., Datta, K., 2012. Molecular breeding of Osfer 2 gene to increase iron nutrition in rice grain. GM Crops Food 3, 310316.

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Perera, I., Seneweera, S., Hirotsu, N., 2018. Manipulating the phytic acid content of rice grain toward improving micronutrient bioavailability. Rice 11, 4. Petit, J.M., Briat, J.F., Lobréaux, S., 2001. Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family. Biochem. J. 359, 575582. Rellán-Álvarez, R., Giner-Martínez-Sierra, J., Orduna, J., Orera, I., Rodríguez-Castrilln, J.Á., García-Alonso, J.I., et al., 2010. Identification of a tri-iron(III), tri-citrate complex in the xylem sap of iron-deficient tomato resupplied with iron: new insights into plant iron long-distance transport. Plant Cell Physiol. 51, 91102. Roschzttardtz, H., Séguéla-Arnaud, M., Briat, J.F., Vert, G., Curie, C., 2011. The FRD3 citrate effluxer promotes iron nutrition between symplastically disconnected tissues throughout Arabidopsis development. Plant Cell 23, 27252737. Schaaf, G., Ludewig, U., Erenoglu, B.E., Mori, S., Kitahara, T., Von Wirén, N., 2004. ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals. J. Biol. Chem. 279, 90919096. Schaaf, G., Honsbein, A., Meda, A.R., Kirchner, S., Wipf, D., Von Wirén, N., 2006. AtIREG2 encodes a tonoplast transport protein involved in iron-dependent nickel detoxification in Arabidopsis thaliana roots. J. Biol. Chem. 281, 2553225540. Schuler, M., Rellán-Álvarez, R., Fink-Straube, C., Abadía, J., Bauer, P., 2012. Nicotianamine functions in the phloem-based transport of iron to sink organs, in pollen development and pollen tube growth in Arabidopsis. Plant Cell 24, 23802400. Senoura, T., Sakashita, E., Kobayashi, T., Takahashi, M., Aung, M.S., Masuda, H., et al., 2017. The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains. Plant Mol. Biol. 95, 375387. Singh, S.P., Gruissem, W., Bhullar, N.K., 2017. Single genetic locus improvement of iron, zinc and β-carotene content in rice grains. Sci. Rep. 7. Stacey, M.G., Patel, A., McClain, W.E., Mathieu, M., Remley, M., Rogers, E.E., et al., 2008. The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds. Plant Physiol. 146, 589601. Takahashi, M., Yamaguchi, H., Nakanishi, H., Shioiri, T., Nishizawa, N.K., Mori, S., 1999. Cloning two genes for nicotianamine aminotransferase, a critical enzyme in iron acquisition (strategy II) in graminaceous plants. Plant Physiol. 121, 947956. Theil, E.C., 1987. Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu. Rev. Biochem. 56, 289315. Vert, G., Grotz, N., Dédaldéchamp, F., Gaymard, F., Guerinot, L., Briat, J., et al., 2002. IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. Plant Cell 14, 12231233. Von Wirén, N., Gibrat, R., Briat, J.F., 1998. In vitro characterization of ironphytosiderophore interaction with maize root plasma membranes: evidences for slow association kinetics. Biochim. Biophys. Acta Biomembr. 1371, 143155. Von Wirén, N., Klair, S., Bansal, S., Briat, J.F., Khodr, H., Shioiri, T., et al., 1999. Nicotianamine chelates both Fe(III) and Fe(II) implications for metal transport in plants. Plant Physiol. 119, 11071114. Waters, B.M., Chu, H.H., DiDonato, R.J., Roberts, L.A., Eisley, R.B., Lahner, B., et al., 2006. Mutations in Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds. Plant Physiol. 141, 14461458. WHO, 2019. Malnutrition. ,www.who.int/nutrition/topics/ida/en/.. Winterbourn, C.C., 1995. Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol. Lett. 8283, 969974. Wirth, J., Poletti, S., Aeschlimann, B., Yakandawala, N., Drosse, B., Osorio, S., et al., 2009. Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin. Plant Biotechnol. J. 7, 631644.

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CHAPTER NINE

Transgenic technology to improve therapeutic efficacy of medicinal plants Monica Saifi1, Shazia Khan1, Usha Kiran2, Saman Fatima3 and Malik Zainul Abdin1 1

Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India Bioinformatics Institute of India, Noida, India 3 Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India 2

9.1 History of medicinal plants and natural products The use of medicinal plants is old as man itself. Even though not any written information available from the beginning of civilization, the information on medicinal plants and their use is supposed to be known and followed by mankind over many centuries. One of the oldest and diverse of all medicine systems belong to African traditional medicine; however, it is poorly recorded. The oldest written record for the usage of medicinal plants dates back to 5000 years written on clay slabs from Mesopotamian dynasty including the description of around 250 plants (Kelly, 2009). Veda, an ancient text from India, the earliest of which date back to 2000 years BCE is the basis of Ayurveda medicine system in India. The document contains thousands of poetic hymns describing the uses of medicinal plants (Tucakov, 1971). The ancient system of Chinese medicine is believed to be more than 5000 years old. Publication of The Neijing (The Yellow Emperor’s Canon of Internal Medicine) is the oldest record of Traditional Chinese Medicine which dates back to 2698 2598 BCE (Liu et al., 2015). The modern day encyclopedia of Chinese materia medica was published in 1977 and is regarded as one of the comprehensive text containing almost complete references to Chinese herbal prescription. It lists nearly 6000 drugs out of which 4800 are of plant origin (Wu, 2005). The history of medicinal plants from Europe includes the Ebers Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00009-5

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Papyrus’s work from 1550 BCE who wrote circa describing 700 plants for the treatment of various ailments (Glesinger, 1954). In 800 BCE, two epic poems, Iliad and Odyssey, known as Homer’s epic, were written during the war. Homer’s epic includes 63 plant species from Egypt, Mycenae, and Minoa. The names were given to plants after their mythological role in these epics. Artemisia, which was believed to restore health is derived from Greek word “artemis” meaning “healthy.” Theophrastus (371 287 BCE) in his two books “Enquiry into plants” and “On causes of plants” classified 500 plant species on the basis of origin, size and their uses as food, juices, and herbs (Dimitrova, 1999). Another scientist from Rome, Celsus (25 BCE CE 50) in his work, De-medicina, described 250 medicinal plants. In CE 77, the “Father of pharmacognosy” Pedanius Dioscorides, a military physician, pharmacologist, and botanist, wrote a five-volume Greek encyclopedia De-materia medica on herbal medicine and related medicinal substances. He included nearly 800 items from plants, minerals, and animals origin, from which drugs could be prepared. Out of these, around 600 were of plant origin. One of his contemporary, Pliny the Elder, traveled throughout the Spain and Germany and documented about 100 medicinal plants (Touwaide et al., 1997). Apart from these ancient works, holy books; the Bible and the Talmud (Jewish) also mentioned various aromatic plants (Toplak, 2005). In the middle age, Arabs introduced various medicinal plants from India in their pharmacotherapy. Middle age European medical practitioner consulted Arab work “De Re Medica” by John Mesue (CE 850), Liber Magnae Collectionis Simplicum Alimentorum Et Medicamentorum by Ibn-Baitar (Tucakov, 1964), and Canon Medicinae by Avicenna (980 1037), which documented the description and use of over 1000 medicinal plants for prevention and cure. Marco polo’s journey from Europe to different parts of the world, discovery of America and Vasco de Gama’s journey to India resulted in exchange of medicinal plants across the globe. Early 19th century was a turning point in the knowledge and usage of medicinal plants. Isolation of pure compounds from plants like alkaloids and glycosides, laid the foundation of molecular pharmacy. New methods for the isolation of active compounds such as tannins and saponins were identified. In 20th century, stabilization methods for cultivation of medicinal plants were proposed. New research and development in cultivation and isolation of active compound restored the use of numerous forgotten plants for example, Aconitum, Punica granatum, Hyosciamus, and Stramonium etc. to be used as a drug source in modern pharmacy,

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(Saad et al., 2017). Today, almost all pharmacopoeias in the world prescribe the plant-based drugs. In fact, some countries like the United Kingdom and Europe have separate herbal pharmacopoeias (British Pharmacopoeia, 2013; European Pharmacopoeia Commission, 2010).

9.2 Natural products: biosynthesis and classification Natural products are the secondary metabolites present in plants. They are derived from nitrogen metabolism. The term secondary metabolite was given to these compounds by biochemist Albrecht Kossel, who described them as compounds other than those required in primary metabolism. Secondary metabolites are organic molecules and are necessary for adaption and defense. They contribute to plant fitness by acting as antifungal, antimicrobial and substances detrimental to other organisms, thus preventing plant from being infected or eaten. Also, some secondary metabolites absorb UV light and thus protect leaves from serious damage by light. There are three large families of secondary metabolites classified on the basis of their biosynthesis pathway in plants, namely terpenes, alkaloids, and phenolics. Fig. 9.1 is showing involvement of different primary metabolic pathways in the biosynthesis of secondary metabolites.

9.2.1 Terpenes This largest class of secondary metabolites is biosynthesized by two basic metabolic pathways: The mevalonic acid (MVA) pathway and the methylerythritol pathway (MEP). All terpenes are made up of multiple 5-carbon isoprene units, which serve as the basis of their classification in different groups. Ten carbon compounds having two isoprene units and are known as monoterpenes. Fifteen carbon compounds with three isoprene units are sesquiterpene and twenty carbon compounds having four isoprene units are diterpenes. Larger terpenes include triterpenes, tetraterpenes, and polyterpenoids. This large terpene family includes essential oils, carotenoids, gibberellic acid, and saponins. Terpenes have broad-spectrum pharmacological activities and thus used in various disease treatments. The insect repellent properties and toxic nature of some terpenes provide plants defense against microbes and insects and led to their use as fungicides and pesticides. Some terpenes like carotenoids; accessory pigments in

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CO2 Photosynthesis Primary carbon metabolism Erythrose-4-phosphate Phosphoenolpyruvate

Tricarboxylic acid cycle

3-Phosphoglycerate (3-PGA)

Pyruvate

Acetyl CoA

Aliphatic amino acids

Malonic acid pathway

Shikimic acid pathway

Mevalonic acid pathway

MEP pathway

Aromatic amino acids Nitrogen-containing secondary products

Phenolic compounds

Terpenes

Secondary carbon metabolism

Figure 9.1 Schematic view of biosynthesis of secondary metabolites in plants. Sourced from: Taiz, L., Zeiger, E., 2010. Plant Physiology, fifth ed. Sinauer Associates, Sunderland, MA.

photosynthesis, gibberellic acid; a plant hormone and sterols; essential components of cell membrane play important role in plant physiology and development. Such terpenes may sometimes are be considered as primary rather than secondary metabolite (Gershenzon and Croteau, 1993; Taiz and Zeiger, 2010).

9.2.2 Alkaloids The alkaloids are the large family of nitrogen-containing secondary metabolites. The nitrogen atom in these compounds is usually part of a heterocyclic ring, a ring that contains both nitrogen and carbon atoms. However, some plant secondary metabolites like ephedrine and colchicine having nonheterocyclic nitrogen atom are also considered as alkaloids (Schläger and Dräger, 2016). In contrast to other classes of secondary metabolites, alkaloids are characterized by a large structural diversity, and

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therefore, no uniform classification adopted for them. In the biosynthesis of alkaloids, the sources of nitrogen are usually the common amino acids like lysine, tyrosine, and tryptophan. However, the carbon skeleton of some alkaloids contains a component derived from the terpene pathway. As the name suggests, these are alkaline in nature. The nitrogen atom present in the alkaloids plays an important role in plants defense against the microbial infections and minimizing the damages caused by other factors like herbivores attacks, and ecological and climatic disturbances (Kutchan, 1995; Taiz and Zeiger, 2010). Alkaloids are toxic to humans when taken in higher quantities but at lower doses, many are useful pharmacologically. Around 12,000 natural alkaloids are recognized from existing plant species and are of great medicinal properties. Morphine, codeine, and scopolamine are few of the plant alkaloids currently used in medicine. Other alkaloids, like cocaine, nicotine, and caffeine, are well known for their use as stimulants or sedatives (Velu et al., 2018).

9.2.3 Phenolics Plants produce a large variety of secondary products containing a phenyl group known as phenolic compounds. The plant phenolics are biosynthesized by two basic pathways: the shikimic acid pathway and the mevalonic acid pathway. The shikimic acid pathway is used for the biosynthesis of most plant phenolics. While the mevalonic acid pathway is an important source of phenolic secondary products in fungi and bacteria, it is less significant in higher plants (Taiz and Zeiger, 2010). The plant phenolics are a highly diverse group of compounds having different structures. Several classifications of phenolics have been categorized on the basis of (1) Number of hydroxyl groups present in the compound. There may be 1-, 2-, or polyatomic phenols. (2) The chemical composition: mono, di, oligo, and polyphenols. (3) Substitutes in their basic skeleton, there the number of aromatic rings and the carbon atom in the side chains. They are classified as phenolic compounds with one aromatic ring, with two aromatic rings, quinones, and polymers. Although polyphenols are structurally similar, due to some distinctive differences, they are divided into two groups: flavonoids and nonflavonoids (Dai and Mumper, 2010). Phenolics exhibit anti-inflammatory, antioxidant, anticarcinogenic and bactericidal activities (Saad et al., 2017). Recent reports on animal models and clinical trials showed that plant-derived polyphenols and polyphenolrich diet can regulate carbohydrate and lipid metabolism. However

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extensive research on possible adverse effects of high polyphenols intake is required and till then its level in the daily diet should not be increased beyond the recommended dietary level (Kabera et al., 2014).

9.3 Use of medicinal plants and secondary metabolites in traditional and modern medicine WHO has estimated that more than 80% of the world’s population relies for their primary health care needs on traditional medicines, in the form of plant parts, extracts or their active principles. All the famous pharmacopeias like American Herbal Pharmacopoeia (AHP), British Herbal Pharmacopoeia (BHP), and Korean Herbal Pharmacopoeia (KHP) include herbal medicine from natural origin as the medicament. The detailed description of the drug and its formulations in pharmacopoeia is called monograph. BHP contains a total of 169 herbal raw material descriptions while KHP included 384 monographs (British Pharmacopoeia, 2013; European Pharmacopoeia Commission, 2010; Korean Food and Drug Administration, 2012). The Indian Herbal Pharmacopoeia has 40 monographs published (Indian Herbal Pharmacopeia, 2002). WHO published four volumes of monographs on medicinal plants from 1999 to 2010 that include 150 monographs (World Health Organization, 1999). Plant-based drugs contribute remarkably to modern medicine also. Drugs like vinblastine, vincristine, deserpidine, rescinnamine, and reserpine were introduced in the market during 1950 70 in the United States. These are the secondary metabolites, which were either extracted in pure form from plants or chemically modified from plant-derived compounds. Since 1970, ginkgolides, paclitaxel, topotecan, artemisinin, Z-guggulsterone, lectin, gomisin, irinotecan, and many more plantbased drugs were introduced in the market (Verma and Singh, 2008). India is one of the richest countries in having varieties of medicinal plant species in the world. There are over 1.5 million practitioners who are using the traditional medicine system in preventive and curative therapy. Indian Council for Medical Research (ICMR) has taken the initiatives to research on medicinal plants from India and published 13 volumes of the quality standards, consisting 449 medicinal plants, 3 volumes of reference standards book comprising 90 phytochemical standards, and 13 volumes of review monographs on 3679 medicinal plant species

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advocating the role of medicinal plants in modern therapy (Tandon and Yadav, 2017). Medicinal plants played a vital role in the discovery of many potent drugs. Modern techniques in chemistry enhance the discovery and identification of phytochemical compounds in pure form. Around 25% of the drugs in current use worldwide, come from the plants. It is estimated that 60% of anti-tumor and anti-infectious drugs already on the market or under clinical trials are of natural origin (Shu, 1998). From 2000, more than 300 natural products, and around 100 plants were studied for antidiabetic activities (Jung et al., 2006). Apart from pure natural compounds, many potent drugs are the derivative of the compounds obtained from plants. Arteether and artemether, derivatives of artemisinin, from Artemisia annua are used for the treatment of malaria, tuberculosis, cancer and diabetes. Nitisinone derivative of Leptospermone from Callistemon citrinus is used in the cure of tyrosinemia. Apomorphine, the therapeutic agent used in the treatment of Parkinson’s disease, is a derivative of morphine. Ttiotropium, a derivative of atropine, from Atropa belladonna is used in the treatment of chronic pulmonary disease (Frankel et al., 1990; Tashkin et al., 2008; Ali et al., 2017). The majority of these cannot be produced ex-Plantae. Their complex chemical nature make the synthesis process uneconomical and therefore, they are still obtained from wild or cultivated plants.

9.4 Technologies for enhancement of secondary metabolites 9.4.1 Elicitors Secondary metabolite production in plant cell and organ cultures is enhanced by the elicitation process. The effects of biotic and abiotic elicitors on stimulation of secondary metabolite production in plant tissue cultures are dependent on the specific secondary metabolites. Scientists have been working on the exploration of various plant cell and tissue cultures of medicinal plants as production factories for useful secondary metabolites through modulation of biosynthetic pathways. The process of elicitation augments secondary metabolism in plant cells in vitro; however, the exact mechanism is still not known. Elicitors have incredible application as an agent to enhance large-scale production of secondary metabolites in the

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plant tissue culture. These are the chemicals from biotic and abiotic sources that can stimulate plant stress responses resulting in increased biosynthesis and accumulation of secondary metabolites or the induction of introduced novel secondary metabolites in cell culture. The modulation of secondary metabolite production pathways depends on the elicitor type, dose, application method, and duration of exposure. Cell line, age and stage of cell culture, nutrient composition of media are the other important determinants, which influence the production and accumulation of secondary metabolites. Elicitors are classified as abiotic and biotic according to their nature (Fig. 9.2). Abiotic elicitors are nonbiological origin substances. These are grouped as physical, chemical, and hormonal factors. Biotic elicitors are the compounds of biological origin such as polysaccharides originated from plant cell walls (e.g., chitin, pectin, and cellulose) and microorganisms. 9.4.1.1 Abiotic elicitors Abiotic elicitors include chemical, physical, and hormonal elicitors. They show a wide range of effects on plants especially on the production of secondary metabolites. Abiotic stress such as light is known to stimulate secondary metabolites. Reports showed that light stimulates gingerol and zingiber production in Zingiber officinale callus culture (Anasori and Asghari, 2009). The effect of light irradiation on anthocyanin production in Perilla frutescens cell suspension cultures was reported (Zhong et al., 1991). Ultraviolet (UV) exposure also stimulates secondary metabolite production. Reports have suggested that increase in UV-B exposure in field-grown

Figure 9.2 Types of elicitors based on their nature.

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plants results in increase in the total essential oil and phenolic content and decreases the amount of toxic β-asarone (Kumari et al., 2009). In the hairy root culture of A. annua, the light irradiation modulated the artemisinin biosynthesis (Liu et al., 2002). The abiotic elicitors (proline and PEG) stimulated the production of steviol glycoside content in both Stevia rebaudiana callus and suspension cultures (Gupta et al., 2015). PEG elicited hypericin and pseudohypericin, in Hypericum adenotrichum (Yamaner & Erdag, 2013). Exposure to salinity is known to induce terpenes and alkaloids biosynthesis (Winkel-Shirley, 2002; Selmar, 2008; Haghighi et al., 2012). Salinity increased the polyamine and diamine contents in Oryza sativa (Krishnamurthy and Bhagwat, 1989). Enhancement in the synthesis of vinblastine and vincristine was observed in embryogenic tissue culture of Catharanthus roseus under the salt stress. Mild water stress increases saikosaponin anti-inflammatory molecule content in Bupleurum chinense (Zhu et al., 2009). Temperature also influences the metabolic activity and plant ontology (Morison and Lawlor, 1999). Reports suggest that high temperatures result in enhanced leaf senescence and increased accumulation of root secondary metabolites Panax quinquefolius (Jochum et al., 2007). Temperature increment as small as 5 C significantly resulted in increased roots ginsenoside content in herb P. quinquefolius (Jochum et al., 2007). 9.4.1.2 Biotic elicitors Biotic elicitors are compounds derived from either pathogen or host, which can induce defense responses in plant tissue. Biological preparations such as microbial cell-wall preparations and yeast extract, where the molecular structure of active ingredient is not defined, are also used as elicitors. Efforts are being made to elucidate and classify the complex structure of these elicitors. Oligosaccharides, polysaccharides, proteins, glycoproteins, and fatty acids are some of the elicitors being used to increase therapeutic molecules/secondary metabolites in medicinal plants. The cell wall-derived elicitor oligogalacturonic acid significantly increased the ginseng saponin content in Panax ginseng cell suspension (Hu et al., 2003). The application of chitin or chitosan induced the production of fluoroquinolone, coumarins, and alkaloids in shoot cultures of Ruta graveolens (Orlita et al., 2008). Yeast extracts used as biotic elicitor, stimulated bacterial resistance genes in bean (Phaseolus vulgaris) (Stangarlin et al., 2011) and ethylene biosynthesis in tomato (Felix et al., 1991). The production of tanshinone was enhanced using yeast extract in the root culture of Perovskia abrotanoides (Zaker et al., 2015). Secondary metabolism is also enhanced using fungal origin elicitors. Fungal spores

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induced eightfold increase in codeine, sanguinarine, and morphine expression in Papaver somniferum (Balazova et al., 2002; Heinstein, 1985). Cell suspensions of C. roseus resulted in enhanced the production of the indole alkaloids serpentine, ajmalicine, and catharanthine up to five times with fungal cell wall components (Zhao et al., 2001; Namdeo et al., 2002). The content of antimicrobial alkaloid acridone epoxide was increased up to 100-fold in cultures of R. graveolens using a mixture of fungal polysaccharides (Eilert et al., 1984). Fungal elicitor increased the production of raucaffrincine and 12-oxo-phytodienoic acid in Rauwolfia canescens (Parchmann et al., 1997). Diosgenin content in Dioscorea deltoidea cells was increased using fungal mycelia (Rokem et al., 1984). Endophytic fungus found in the bark of Taxus chinensis tree elicited up to three times as much taxol in T. chinensis cells as compared to nonelicited cells (Wang et al., 2001). The adventitious hairy root cultures of Scopolia parviflora were stimulated using bacterial elicitors to increase biosynthesis of scopolamine via inhibition of hyoscyamine 6β-hydroxylase (H6H) expression (Jung et al., 2003).

9.4.2 Homologous overexpression of therapeutic molecule/ secondary metabolite biosynthesis key genes The regulation of the expression of genes involved in biosynthetic pathways highly governs the production of pharmaceutical terpenoids (De Geyter et al., 2012). P. ginseng squalene synthase 1 (PgSQS1), a key gene in ginsenosides biosynthetic pathway, is reported to upregulate the expression of squalene epoxidase (SE), β-amyrin synthase (β-AS), and cycloartenol synthase (CAS). The overexpression of PgSQS1 gene resulted in a twofold increase of phytosterols and 1.6- to 3-fold increase of total ginsenosides in transgenic P. ginseng adventitious root cultures. However, the growth rate of transgenic P. ginseng roots were slower than that of nontransgenic adventitious roots (Shim et al., 2010). In P. ginseng plants, overexpression of CYP716A52v2, another key enzyme of ginsenoside biosynthesis, resulted in increased oleanane-ginsenoside (ginsenoside Ro) content, while no change was reported for the levels of dammarene-type ginsenosides with respect to untransformed lines (Han et al., 2013). Overexpression of artemisinin biosynthesis genes, Amorpha-4,11-dienesynthase gene (ADS), cytochrome P450-dependenthydroxylasegene (CYP71AV1), and NADPH:cytochrome P450 oxidoreductase gene (CPR), enhanced the accumulation of artemisinin in A. annua. The artemisinin content could reach upto 15.1 mg/g dry weight (DW), which was 2.4-fold higher than the untransformed plants (Lu et al., 2013).

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9.4.3 Ectopic expression of genes to produce therapeutic molecule/secondary metabolite Genetic transformation of most medicinal plants is not easy especially those producing low amounts of pharmaceutical terpenoids. Ectopic expression of terpenoid synthases/cyclases (TPSs) to produce pharmaceutical terpenoids is now considered as an efficient way to produce pharmaceutically active compounds in different plants. Geraniol is used in fragrance industries due to its pleasant rose-like odor. It is also used in the pharmaceutical industry to make anticancer drugs and antimicrobial reagents (Unlu et al., 2010; Polo et al., 2011). Twenty tobacco hairy root cultures transformed with the geraniol synthase gene from Valeriana officinalis (VoGES) produced geraniol in range of 13.7 31.3 mg/g DW (Ritala et al., 2014). Ectopic expression of Arabidopsis transcription factor MYB12 in tomato activated the expression of plastidial enolase and 3deoxy-D-arabino-heptulosonate-7-phosphate synthase (Zhang et al., 2015). Ectopic expression of P. ginseng HMGR1 (PgHMGR1) in Platycodon grandiflorum hairy root cultures resulted in 1.5- to 2.5-fold higher platycoside and 1.1- to 1.6-fold increase of phytosterols contents (Kim et al., 2013). Protopanaxatriol (PPT) is a high medicinal value aglycone of ginsenoside and its production from natural source requires artificial deglycosylation of ginsenosides. Ectopic expression of dammarenediol-II synthase (PgDDS) and two cytochrome P450 enzymes (CYP716A47 and CYP716A53v2) in tobacco cell lines showed accumulation of 2.8, 7.3, and 11.6 μg/g DW of PPT, protopanaxadiol (PPD), and dammarenediol (DD) in leaves, respectively (Chun et al., 2015). The treatment of transgenic tobacco suspension culture with 2,4-D increased the content of PPT by 37.25-fold as compared to the content of PPT present in leaves of pot grown transgenic tobacco (Gwak et al., 2018).

9.4.4 Role of miRNAs in increasing the production of secondary metabolites Plant microRNAs play an important role in modulating secondary metabolite production (Bulgakov and Avramenko, 2015). The secondary metabolite production can be enhanced by either overexpressing miRNA or transcription factor. The knocking down of miRNA/TF interfering in the secondary metabolite production is another approach to modulate secondary metabolite synthesis and accumulation.

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The interaction between squamosa promoter binding protein-like (SPL) and miR156 in Arabidopsis resulted in the negative regulation of anthocyanin biosynthesis and accumulation of methyl farnesoate (Gou et al., 2011). Recent reports suggested that miR163 also regulate mRNA encoding S-adenosyl methionine-dependent methyl transferase responsible for methylation of secondary metabolites and other signaling molecules (Ng et al., 2011). In Papaver somniferum, miR2161, miR408, and miR13 were reported to be involved in indole alkaloid biosynthesis (Boke et al., 2015). Vashisht et al. (2015) reported that miR4995 in Picrorhiza kurroa was involved in the regulation of terpenoid biosynthesis. Samad et al. (2016) showed that the SPL and AP2 (targets of miR156 and miR172) were downregulated, whereas MYB and WRKY (targets of miR858 and miR894) were upregulated under elicitation by Fusarium oxysporum in Persicaria minor plant. The results of investigation of interaction among different miRNAs and TFs showed that under stress conditions, the TFs having a role in defense mechanisms are upregulated and TFs involved in plant development is downregulated by miRNA.

9.4.5 Artificial miRNAs for secondary metabolites enhancement The extensive studies reporting efficient application of miRNA resulted in development of different RNA-based gene-altering techniques. Artificially transformed miRNA (amiRNA) was developed to manipulate one or several genes (Carbonell et al., 2014; Shriram et al., 2016). Single-stranded amiRNA are approximately 21 nucleotide long, similar to miRNAs. These are designed by utilizing the unique stem-loop structure of endogenous primiRNAs, but replacing the mature miRNAs sequence in duplex by amiRNA aimed to silence the target gene, with high efficiency (Eamens et al., 2014). The amiRNA is useful for targeting closely related genes, including tandem arrayed as compared to conventional RNA interference (RNAi). Also modulation of gene targeted by amiRNA is more precise (Schwab et al., 2006; Ossowski et al., 2008; Carbonell et al., 2015). The downregulation of chalcone synthase genes in Arabidopsis was achieved using amiRNA technique (Niemeier et al., 2010; Kamthan et al., 2015).

9.4.6 Regulating the expression of transcription factors Transcription factors are known to play a key role in regulating flux through secondary metabolism pathways by modulating the gene expression. The

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overexpression of terpenoid indole alkaloid (TIA) pathway activator ORCA3 in Catharanthus cells showed for the first time that transcription factor can controls the biosynthetic pathway gene expression (van der Fits et al., 2000). In addition to TIA pathway genes, analysis of transcript levels also showed induction of DXS gene. No induction was noticed in Geraniol 10-hydroxylase gene (G10H), another gene in the monoterpene branch of the pathway. Thus the results suggested that ORCA3 regulates TIA biosynthesis, with or without other factors (van der Fits et al., 2000). Xu et al. in 2004 analyzed the promoter of delta (1) cadinene synthase, and noticed the presence of a putative binding motif for WRKY transcription factors. This finding led them to look for genes predicted to encode WRKY-type transcription factors in Gossypium arboreum (cotton). The analysis of the transcription factor and its interactions with the delta (1) cadinene synthase promoter provided strong evidence that GaWRKY1 is involved in regulating sesquiterpene biosynthesis and possibly other terpenoid pathway genes in cotton (Xu et al., 2004). Confirmation of its biological role in cotton plants will provide the final piece of evidence that GaWRKY1 is a regulator of sesquiterpene biosynthesis.

9.4.7 Regulating the endogenous levels of phytohormones involved in terpenoid biosynthesis Pathogen attack, herbivore feeding, and other biotic stress induce the production of volatile compounds mainly belonging to terpenoid class (Vranová et al., 2012). Jasmonate (JAs), a phytohormone produced during stress, plays a crucial role in controlling the complex signaling cascade (Moses et al., 2013; Ahmad et al., 2016). Overexpression of the key enzyme of the jasmonate biosynthetic pathway, allene oxide cyclase gene (SmAOC), results in increased expression levels of diterpenoids biosynthetic pathway genes and increased rosmarinic acid, lithospermic acid B, and tanshinone II A contents in S. miltiorrhiza hairy root cultures (Gu et al., 2012). Jasmonates also play critical role in the artemisinin biosynthesis regulation. The allene oxide cyclase gene from A. annua (AaAOC) was cloned and overexpressed in A. annua plants. The levels of endogenous JA showed increased 2- to 4.7-fold as compared to the control. The increased endogenous JA induced the expression of DBR2, CYP71AV1, and FDS, which resulted in a significant increased production of artemisinic acid, DHAA, and artemisinin (Lu et al., 2014). The high concentrations of JA in plants may sometime inhibit plant growth, hence limiting the use of jasmonate as elicitor.

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9.4.8 Regulating interrelated primary metabolic pathways Productivity of pharmaceutically important terpenoids in plants can also be enhanced by modulating the related primary metabolic pathways. Carbohydrates from primary metabolism provide the required carbon flux needed for biosynthesis of secondary metabolites. Carbohydrates are reported to play a major role in determining the geraniol production in transgenic tobacco cell suspension cultures. The use of sucrose resulted in higher geraniol production and biomass accumulation in the cell cultures as compared to cultures elicited by glucose and D-mannitol (Vasilev et al., 2014). The neutral/alkaline invertase gene (NINV) is a key gene of sucrose hydrolysis. The overexpression of NINV gene in T. chinensis cells significantly enhanced the expression and accumulation of taxadiene synthase gene (TAS). Dong et al. (2015) showed that the average contents of seven individual taxanes including 10-deatetyl baccatin III, baccatin III, 10-deacetyl taxol, cephalomanine,7-epi-10-deatetyl taxol, taxol, and 7-epitaxol were 2.1, 3.3, 2.2, 3.7, 3.5, 1.9, and 1.8 times higher than the controls, respectively. The results suggest that modulating the TcNINV-mediated sucrose metabolism can lead to augmented biosynthesis of taxanes in T. chinensis cells.

9.5 New approaches of engineering plant metabolic pathways to enhance secondary metabolites Engineering of plant metabolic pathways has been proved to be a promising approach for increasing the concentration of economically important secondary metabolites. During the last decade, substantial efforts are being made to develop new technologies to manipulate the same. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) genome editing system has emerged as an important genome engineering tool for manipulating big and bulky plant genomes (Schaeffer and Nakata, 2015). Due high efficiency and specificity of this approach, it has been used to manipulate gene expression in large number of economically important crop plants such as rice (Zhou et al., 2014), tomato (Brooks et al., 2014), wheat (Wang et al., 2014), soybean (Jacobs et al., 2015), maize (Liang et al., 2014), poplar (Fan et al., 2015), and potato (Wang et al., 2015). A combination of CRISPR/Cas9 technologies and transcription activator-like effector nuclease (TALEN) was used to mutate the hexaploid wheat mildew-resistance locus (MLO) proteins to confer resistance to powdery mildew (Wang et al., 2014).

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CRISPR/Cas9 technologies have become choice of tool for manipulating recalcitrant plants genomes as well. Although these studies are still in the early stages, the prospective role of CRISPR/Cas9 in dramatically impacting the trait improvement of crop and medicinal plant is certain.

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CHAPTER TEN

Application of transgenic technologies in biofuel production through photosynthetic chassis—new paradigms from gene mining to genome editing Kashif M. Shaikh1,2, Iqra Mariam1, Asha A. Nesamma1, Malik Zainul Abdin2 and Pannaga P. Jutur1 1

Omics of Algae Group, Integrative Biology, International Centre for Genetic Engineering and Biotechnology, New Delhi, India 2 Centre for Transgenic Plant Development, Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard University, New Delhi, India

10.1 Introduction The industrial developments occurring in the world are heavily dependent on fossil fuels, predominantly as a source of energy in the form of fuels, along with the production of other oil-based commodities. As the population is increasing, the demand for energy will also increase. With the current scenario of utilization of nonregenerable conventional fuels, soon the demand may surpass the requirements. Along with limited supply, the conventional fuels are a threat to the environmental sustainability due to its impact on global warming (Razzak et al., 2013). Biofuels seem to be a great alternative for the environmental sustainability as they are regenerable and can reduce the greenhouse gas emissions substantially (Henry, 2010). Biofuels are energy-rich chemicals that are obtained through biological process and/or are derived from the biomass of plants and other algae. Microbial biofuels are being rapidly established due to huge demands but the issue of climate change can only be addressed using organisms that can fix CO2 released in the environment, in particular, photosynthetic organisms such as plants and algae. Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00010-1

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Agriculture has been the best source for food and energy to cater to the needs of the growing population, even though the recent industrial revolution has shifted the regime toward cheaper sources like fossil fuels for energy consumption. Consequently, a huge implication on climate change and environmental sustainability due to excessive utilization of fossil fuels has led to a deleterious effect on society. Photosynthetic organisms can use sunlight and atmospheric CO2 as a source of energy and carbon to produce a number of biological compounds. Unlike heterotrophs, photosynthesis drives the central carbon metabolism in these organisms. Hence, they can be used as a potential chassis for the production of various biological compounds along with fuels for the sustenance of the environment. Billions of tonnes of oil is being consumed annually worldwide, almost all of which is derived from fossil fuel while only a little percentage (B3%) comes from plant biomass (Carlsson, 2009), although plant biomass production is in hundreds of billions of tons per year (Kircher, 2012). However, more than half of the CO2 fixed by plants goes to cellulose and lignin production which are less significant in terms of consumption or industrial purpose, while less than 10% is utilized in agriculture. This illustrates the need for rechannelizing the carbon assimilation toward more suitable compounds for food and feedstock. Since the green revolution, traditional breeding and employment of chemicals have led to substantial improvement in yield and stress resistance in plants, particularly in the agriculture sector, satisfying the demands of increasing population. With the advent of genetic engineering approaches, a number of crops were genetically modified and tested in the field (Christou, 1998). However, most of the newly engineered features covered herbicide/pest and stress tolerance toward yield improvement (Ohlrogge, 1999; Brookes and Barfoot, 2018a,b). These simple genetic alterations have proved to be fruitful in terms of yield, providing food security to a number of nations. However, similar approaches are much less utilized in terms of production of other valuable commodities such as fuel and green chemicals. One example that has been worked extensively to counter dependence on fossil fuels in this decade is the production of cellulose-based fuels using plant biomass. Although this approach is restricted to the use of plants as a source of carbohydrates which is then converted to fuels using engineered microbes, the situation demands much better utilization of plants for the efficient production of required commodities within themselves, converting plants into biorefineries. With

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the dawn of metabolic engineering and synthetic biology, redesigning, refining, and modifying photosynthetic organisms for the production of fuels as well as food are the future of sustenance to meet the social and industrial needs. This chapter will highlight recent developments in the field of systems and synthetic biology that decipher applications using photosynthetic organisms for the production of biofuel molecules such as sugars, fatty acids, terpenes, and organic acids.

10.2 Metabolic engineering and synthetic biology Plant biotechnology has been progressing in the past few decades showing greater potential in applications concerned in improving bioeconomy as a sustainable and environment-friendly platform for the production of high-value fine chemicals and biofuels (Stöger, 2013). The traditional genetic engineering approaches employed one or two simple targets (Ohlrogge, 1999; Brookes and Barfoot, 2018a,b), whereas advances in analytical instrumentation have refined the blueprint of molecular pathways through multiomics approach that has clearly led to alterations at multiple levels, from single-gene mutation to insertion of whole new complex pathways, which will eventually require metabolic engineering and/or systems biology approach. Metabolic engineering refers to the modification of one or several enzymatic pathways of an organism in order to improve and/or implement the production of certain required compounds (Dellapenna, 2001). Synthetic biology, although not clearly defined, perhaps involved in the designing and/or generation of new biological pathways, that is, gene parts, synthetic circuits (Ellington, 2009). Various examples of plant genetic engineering are available which have been successful both at the controlled environmental chamber and even as in field trails. One illustration of such success is the golden rice project, wherein a new rice variety was generated that can produce β-carotene, the precursor of vitamin A by introducing three transgenes (Ye et al., 2000; Paine et al., 2005). Another example is the transient insertion of pathway for synthesis of omega-3 long-chain polyunsaturated fatty acids (Ω3 LC-PUFAs) from linoleic acid in plants using seven enzymes from microalgae that produce Ω3 LC-PUFAs naturally (Petrie et al., 2010). A stable insertion in Arabidopsis thaliana (Petrie et al., 2012), followed by similar engineering in other oilseed

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crops such as Camelina sativa (Petrie et al., 2014) and Brassica napus (Walsh et al., 2016), has demonstrated the concept to produce biomolecules and other nonconventional commodities that are essential in industries such as pharmaceuticals, bioplastics, and pesticides (Craik et al., 2010; Somleva et al., 2013). Systems biology, though nascent in plants, has already established its presence with some studies proving the possibility of integrating synthetic genetic circuits in plants (De Lange et al., 2018). Using synthetic abscisic acid receptor, a semisynthetic line of A. thaliana has been generated that can tolerate water scarcity upon chemical induction (Park et al., 2015). Detector plants have been created through the introduction of a completely synthetic pathway, which bleach in the presence of trinitrotoluene (TNT) in soil (Antunes et al., 2011). Another example is the insertion of a light-sensitive switch in plants that controls the gene expression in response to red light (Muller et al., 2014). The genetic engineering approaches for industrial production of biofuels and other associated coproducts will imply a combinatorial approach of metabolic engineering and synthetic biology in near future, more appropriately termed as synthetic metabolic engineering (Pouvreau et al., 2018). Henceforth, our focus in this chapter is to discuss a few strategies adopted to improve photosynthetic organisms, as the cost-effective roadmap toward establishing biofuel refineries employing advance transgenic technologies for improving their productivities.

10.3 Improving photosynthesis Photosynthesis provides the direct conversion of solar energy into chemical energy which is an attractive source of renewable energy reserves, although it requires an efficient utilization and conversion at the large scale production facility. Many approaches were applied to increase the photosynthetic efficiency of higher plants that include improving Rubisco kinetic properties (Parry et al., 2013), introducing the C4 pathway into C3 crops which require the transfer of over 20 transgenes (Furbank, 2016), more rapid relaxation from photoprotection (Murchie and Niyogi, 2011), increasing the activity of sedo-heptulose bis-phosphatase (Raines, 2011), and improving canopy architecture. Another approach to capture extra light is by transferring cyanobacterial chlorophyll d and f into higher plant pigment
protein

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complexes, thereby extending the waveband of sunlight available for photosynthesis (Chen and Blankenship, 2011). Similarly, increasing cytochrome f content was able to increase electron transport chain flux, which led to enhanced activity of ATP synthase (Von Caemmerer and Evans, 2010). Recent studies demonstrated that introduction of the algal-based carbon concentrating mechanisms and their compartments has improved the efficiency of photosynthesis in land plants (Atkinson et al., 2016; Hanson et al., 2016). Exposure to higher light intensities can lead to almost 70% dissipation of energy in the form of heat or fluorescence due to saturation of photosynthetic electron transfer (Melis, 2009; Ort and Melis, 2011). Reducing antenna size to optimize light utilization efficiency is one potential way to improve the photosynthetic chassis. The reduced antenna size enables microalgae to absorb less light per cell; the probability of saturating electron transfer at full sunlight intensities is reduced giving them the ability to tolerate high light intensities, thus increasing the light penetration into cells (Perrine et al., 2012). Mussgnug et al. (2007) were successful in downregulated multiple genes involved in LHCI and the LHCII antenna complex of Chlamydomonas reinhardtii employing RNAi technology. These transgenics showed reduced photochemical quenching, higher photosynthetic quantum yield, and lower pigment content when compared to the normal cells.

10.4 Formation of essential products via photosynthetic chassis As discussed previously, the basis for sustainable model using photosynthetic organisms is investigating the possibilities of enhancing the versatility of metabolism in these systems that may potentially transform the platform for determining what compounds can be obtained from these factories in an economical way. Recent studies in the plant systems have been substantially improved via synthetic and systems biology (Patron et al., 2015; Shih et al., 2016a,b). The main focus in genetic approach has been the introduction of complex pathways (Galanie et al., 2015), but the challenge to produce the scale of relatively simpler molecules relevant to industrial production is yet to be exploited (Shih, 2018). Some of the

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industrially relevant molecules needed for sustainable production using transgenic technologies via photosynthetic chassis are as follows.

10.4.1 Sugars Photosynthetic organisms utilize sunlight to fix CO2 in the form of sugars, which are then converted to polymers (cellulose, hemicellulose) or storage molecules (starch, lipids). The sugar-based polymers are amongst the most abundant materials present on earth that can be processed to get simple monosaccharides, the precursors of bioethanol in terms of bioenergy production. Brazil is the best example of a bioethanol-based economy, producing ethanol through yeast fermentation using sugar extracted from sugarcane (Shih, 2018), with a production of B24 billion liters of ethanol per year (Moraes, 2011). However, this success may not be expected in other regions of the world, mainly due to geological and climatic factors (Shih, 2018). Hence, the search for alternative plant sources and the ability to utilize highly abundant plant cell wall material as sugar feedstock has been the main focus in biofuel research. In this context, various approaches have been investigated such as alteration of cell wall sugar composition (Gondolf et al., 2014), reduction of lignin in cell wall (Yang et al., 2013), production of various other bioproducts (Costa et al., 2013), improvisation in chemical degradation of feedstock for efficient breakdown into monosaccharides (Socha et al., 2014) and increasing chilling tolerance in sugarcane (Głowacka et al., 2016). Carbohydrates derived from microalgae, that is, cellulose in the cell wall and starch in plastids can be readily converted to fermentable sugars. However, the content and composition of carbohydrate vary from species to species and are also influenced by various abiotic factors such as light irradiance, nutrient starvation, and CO2 supply. For example, Chlorella vulgaris can accumulate carbohydrates up to 55% of its dry biomass after 14 days of cultivation in a low-nitrogen medium with a biomass concentration of 0.52 g/L (dcw) (Illman et al., 2000). One of the advantages of coupling microalgaederived sugars for bioethanol production is the utilization of CO2 generated during this process for the generation of carbohydrate-rich microalgal biomass. This coupling process can efficiently achieve the goal of CO2 mitigation and reutilization. The simple sugars obtained from such feedstocks will encompass the major input for any sustainable economy, but the coproduction of other molecules using photosynthetic chassis may have a huge impact in transforming the bio-based economy (Lee and Loeblich, 1971).

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10.4.2 Lipids Lipids are one of the most valuable commodities obtained from plants (such as palm, soybean, canola, sunflower) and algae. Research shows that with the help of genetic engineering, the content and variety of lipids can be further improved (Slocombe et al., 2009; Kelly et al., 2013; Wu et al., 2005; Walsh et al., 2016). In fact, the basic knowledge of lipid biosynthesis and metabolism has been the best studies in these systems and has been applied in genetic engineering approaches to enhance lipid yields. Various strategies have been put forward to enhance lipid productivities by diverting the carbon flux from starch synthesis toward oil content (Sanjaya et al., 2011). In the case of plants, recent advances have been made through metabolic engineering for the production of lipids in vegetative tissues such as leaves, giving rise to the leaf oil platform technology in a variety of crops (Vanhercke et al., 2013, 2014, 2017; Reynolds et al., 2017; Mitchell et al., 2017; Zale et al., 2016; Alameldin et al., 2017). Successful efforts have been made to enhance the triacylglycerol production in the vegetative biomass of sugarcane (Zale et al., 2016). Unlike higher plants, TAG accumulation occurs in the form of lipid bodies in the cytoplasm of algal cells. Under unfavorable conditions, certain microalgal strains can accumulate TAG up to 20%
50% of their dry cell weight cells. Xin et al. (2010) reported that the microalgae, Scenedesmus sp. when introduced with lower initial nitrogen and phosphorus concentrations could accumulate higher lipid content. Similarly, studies found that the lipid content in Chlorella vulgaris could go up to 35.6% 6 8% under complete nitrogen starvation when compared to 12.29% 6 3% in the control (Mutlu, 2011). The two simple metabolic engineering approaches to increase TAG accumulation in microalgae are either overexpressing DGAT gene (diacylglycerol acyltransferase) or blocking the competitive pathways such as β-oxidation pathway for lipid breakdown. For example, heterologous expression of DGAT2 in both C. reinhardtii and Phaeodactylum tricornutum resulted in 1.5 times and 35% increase in neutral lipid content, respectively (Ahmad et al., 2015; Niu et al., 2013) and overexpression of the same resulted in 128% increase in lipid content of Scenedesmus obliquus (Chen et al., 2016). Similarly, downregulating PEPC1 and PEPCK in C. reinhardtii and P. tricornutum using RNAi technology that resulted in enhanced TAG accumulation (Deng et al., 2014; Yang et al., 2016). These efforts in lipid engineering have been the first to redefine the advantages offered by photosynthetic hosts and extending the concepts of metabolic engineering in a broader sense.

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10.5 Terpenes Terpenes are basically hydrocarbons produced by almost all plants, which include a diverse set of compounds with various biological activities, in the form of secondary metabolites. These molecules have significant applications in food, pharmaceutical, and biofuel industries (Ro et al., 2006; Peralta-Yahya et al., 2011; Augustin et al., 2011). Although terpenes are considered as secondary metabolites, the modulation of their production would require alterations through primary metabolic pathways (Shih, 2018). One such example to illustrate the increase in terpene production is regulating an upstream enzyme HMG-CoA (Polakowski et al., 1998; Martin et al., 2003; Kumar et al., 2012; Brown et al., 2015) and improving the levels of glyceraldehyde-3-phosphate and pyruvate by slight modifications (Liu et al., 2013). Other studies have demonstrated the incorporated production of isoprenoids in nonnative organelles (Wu et al., 2006), or heterologous introduction of alternate pathways in a different host (Martin et al., 2003; Kumar et al., 2012). Terpenes are found in algae at quantities generally ,5% DCW (Lee and Loeblich, 1971), however, colonial green alga, Botryococcus braunii, has been shown to produce, under adverse environmental conditions, large quantities (up to 80% DCW) of very-long-chain (C23
C40) hydrocarbons, similar to those found in petroleum, and thus has been explored over the decades as a feedstock for biofuels and biomaterials. Another class of terpenes, that is, squalene which acts as an antioxidant was accumulated in C. reinhardtii by overexpressing the squalene synthase gene and knockdown of squalene epoxidase resulting in enhanced production of squalene content up to 1.1 mg/g (dcw) (Kajikawa et al., 2015).

10.6 Muconic acid Although most of the conventional energy resource is directed toward the generation of fuels, around 8% of crude oil is utilized for the production of plastic (Koninckx, 2016). Interestingly, since most of the research has been focused upon obtaining fuel molecules, little focus has been given to the production of organic acids within biomass that can be used for the production of petrochemical alternatives such as bioplastics. Muconic acid is a precursor of adipic acid that is used for the synthesis of nylon and

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polyurethane (Weber et al., 2012). Petrochemical processes release a huge amount of greenhouse gases and other toxic byproducts (Thiemens and Trogler, 1991), hence biological production of muconic acid would be greater initiative. One of the microalgal strains Scenedesmus obliquus was also found to contain this acid in the form of fatty acid methyl esters (RasoulAmini et al., 2009). Muconic acid has been produced in engineered Escherichia coli with high titer via engineering approaches (Niu et al., 2002; Draths and Frost, 1994). In the future, transfer of metabolic genes in photosynthetic organisms for the production of such relevant biomolecules using CO2 from the atmosphere, will be an asset for the biorefinery industry.

10.7 Gene mining to genome editing Increasing demand of photosynthetically derived biofuel precursors has led to the exploration and genetic modification of metabolic pathways in plants and algae. Also, there is a substantial increase in the genomic datasets available for metabolic pathways. Gene mining and comparative genomics will help us to identify the genes involved in the production of these precursors. For example, Sharma and Chauhan (2012) identified set of 261 putative genes involved in lipid biosynthesis in oil-based plants such as B. napa, G. max, and R. communis. Similarly, a total of 1003 lipid-related genes were cloned and annotated to enhance the productivity in maize kernel oil (Li et al., 2010). Studies in microalgae on lipid pathways identified nearly 398 orthologous protein-encoding genes, which were involved in the synthesis of phospholipids, glycerolipids, and neutral lipids, that is, triacylglycerols (Misra et al., 2012). Recently, identification and phylogenomic analysis of 221 putative orthologous genes (in Chlamydomonas reinhardtii, Volvox carteri, Coccomyxa subellipsoidea, Ostreococcus tauri, Nannochloropsis gaditana, Cyanidioschyzon merolae, Phaeodactylum tricornutum and Thalassiosira pseudonana) involved in the biosynthesis of omega fatty acids using Arabidopsis thaliana and Schizochytrium aggregatum as reference models (Kapase et al., 2018). However, the structure
function relationship of these genes are confined to a certain model organism only, and lack of adequate knowledge regarding the metabolic pathways in other photosynthetic organisms is a major drawback in the engineering of these organisms for the overproduction of biofuel precursors. Metabolic engineering of crop plants has been primarily focused on improving crop yield and nutritional characteristics for decades. But in the

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past few years, the focus has been shifted to the exploitation of crop plants like Nicotiana tabacum based on their ability to accumulate TAG in seeds as well as vegetative tissues such as stems and leaves (Vanhercke et al., 2017, 2019), that harbors functional TAG biosynthesis pathway. Engineering regulatory molecules such as transcription factor involved in fatty acid synthesis have led to an increase in storage lipid in Nicotiana benthamiana leaf tissue (Vanhercke et al., 2013). Subsequently, cotransformation of the oil droplet protein oleosin, which helps in packaging of oil droplets, a phenomenon unique to oilseeds, yielded more than 15% TAG in tobacco leaves (Vanhercke et al., 2014). Further refinements, like silencing of lipase and engineering of specific high-value fatty acids like medium chain length have led to a remarkable increase in oil content of tobacco leaves when compared with any oil-based crops. Recently, crop plants that produce enormous biomass are nowadays engineered to accumulate oilseed levels of TAG to produce a sustainable supply of food, fuel, and other oleo-chemical properties (Rahman et al., 2016). The most commonly used stable transformation method in plants is Agrobacterium-mediated transformation or biolistic and is pertinent in a large variety of cultivated crops (Barampuram and Zhang, 2011). However, there are certain problems associated with these methods such as limitation of construct size, multiple copy number of inserts, random insertion, and presence of selectable marker for selection of transformants, making it highly inconvenient to introduce multiple genes (Taylor and Fauquet, 2002; Tzfira and Citovsky, 2006). The recent use of nucleases as genome editing tools has revolutionized plant transformation techniques. These include meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) that use their protein motif to recognize DNA target (Epinat et al., 2003; Bibikova et al., 2003; Joung and Sander, 2013). This was demonstrated in rice, where resistance to bacterial blight was obtained by the use of TALENs to introduce mutations in the gene Os11N3 responsible for bacterial blight susceptibility (Li et al., 2012). In addition, the use of genome editing systems allows simultaneous targeting of multiples genes. This was used to confer resistance to powdery mildew in wheat (Wang et al., 2014). The most popular editing system is clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR associated (CAS) double-stranded DNA nuclease, or CRISPR/CAS that use nucleases guided by RNA (ribonucleic acid). These techniques allow the generation of exogenous DNA-free mutant crop plants. The CRISPR-Cas9 technology originates from type II CRISPR-Cas systems that have four endonuclease genes (Cas9, Cas1, Cas2, Csn2) and

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transactivating RNA genes (tracrRNA), which provide bacteria with adaptive immunity to viruses and plasmids. The CRISPR array has a set of clustered repeats (direct/palindromic) separated by variable (spacer) sequences which is similar to the foreign DNA sequence (protospacer). The CRISPR loci are transcribed and processed into smaller, CRISPR RNA (crRNA) that contains “spacer” or guide sequences, this crRNA base pairs with tracrRNA leaving the “guide” sequence free to interact with the protospacer elements. This RNA duplex together, called the “guide RNA” (gRNA), now recruits and activates a Cas9 nuclease, forming an active site-directed endonuclease complex. Once the target sequence in the invading DNA is detected by gRNA, a protospacer::spacer hybrid is formed by base pairing, and Cas9 makes DSBs in the target DNA complementary to those of the gRNA spacer. Cleavage of the target sequence requires a conserved protospacer adjacent motif (PAM) sequence, usually a 50 -NGG-30 downstream of the target DNA, where the precise cut is made three base pairs upstream of the PAM site (Doudna and Charpentier, 2014; Gasiunas et al., 2012). This simple, robust, and unique DNA cleaving mechanism with the capacity of multiple target recognition and the existence of many natural type II CRISPR-Cas system has enabled this cost-effective and easy-to-use technology to precisely and efficiently target and regulate multiple genomic loci within a cell. Jiang et al. (2017) utilized this approach to target the FAD2 gene in A. thaliana and in the closely related emerging oil plant, C. sativa, with the goal of improving seed oil composition. The Camelina seeds showed increased content of oleic acid up to 50% of the fatty acid composition with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e., a decrease from B16% to ,4%) and linolenic acid (a decrease from B35% to ,10%). These changes result in oils that are healthier, more oxidatively stable, and better suited for the production of biofuels. In Camelina, guide RNAs were designed in such a way, that they simultaneously target all three homologous FAD2 genes (Jiang et al., 2017). Application of the CRISPR/ Cas system on microalgae has been limited to the nonoleaginous model algae C. reinhardtii (Jiang et al., 2014), model diatom P. tricornutum (Nymark et al., 2016), and oleaginous model Nannochloropsis sp. (Wang et al., 2016). To reduce the toxicity, Chlamydomonas cells were transformed with ribonucleoproteins (RNP) consisting of Cas9 proteins and sgRNAs to avoid the vectorassisted expression of Cas9. It was observed that the efficiency was also increased 100-fold as compared to the work by Jiang et al. (2014) and the off-targets were less. Quite recently, success has also been achieved using CRISPR/Cas9-mediated precise gene knockout in the oleaginous model

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N. oceanica IMET1 (Wang et al., 2016). The Cas9, sgRNA, and hygromycin resistance genes were all incorporated into a single vector and transformed in IMET1 cells, which naturally increased the chances of transformation. The ability of generated mutants of a nitrate reductase gene; NR g7988, to grow in ammonium (NH4Cl) but not in nitrate (NaNO3) medium as a nitrogen source, enabled researchers to isolate transgenics using this mutant without using antibiotic resistance markers, making it an environmentally friendly chassis strain. Employing the successful targeted gene modifications using CRISPR/Cas9 in photosynthetic hosts such as plants and algae can turn out as a robust platform for biotechnological benefits related to specialty biofuels coupled with reduced cytotoxicity and better transformation efficiency.

10.8 Challenges and future opportunities Currently, there is a need to develop molecular tools for improving the photosynthetic yield to meet the demand of food and fuel component of the ever-increasing world population. Consequently, gene editing strategies and system biology approach advancements have enabled to understand these biological systems in a global context (Pouvreau et al., 2018). There are a lot of intricate and various complex regulatory aspects of metabolism that are essential and can be confirmed to carbon and nitrogen refinements at various levels which are attributed in transcriptomics, proteomics, metabolomics, and lipidomics of large members of biomolecules along with transcription factors (TFs), and/or transcription regulators (TRs), which control, regulate, and determine overall mechanisms of the cells (Banerjee et al., 2018). Genetically modified and improved plants for biofuels production with higher biomass could be converted to valuable coproducts (Furtado et al., 2014). Presently, the understanding of the model microbial systems, like E. coli and Saccharomyces cerevisiae, to evaluate their biological system, metabolism, and regulatory networks has been improved drastically (Pouvreau et al., 2018) even though plants and algae are potential cell factories for the scaleup and commercialization of coproducts along with primary biomolecules. A roadmap for commercialization might lead to exploring new insights using high throughput omics approach leading to a better understanding of the basic biology and fundamentals of these cell factories. Employing synthetic biology combining with the traditional breeding applications

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could lead to the improvement of transgenics that may hold a foresightedness for the production of sustainable biofuels (Mewalal et al., 2017). Few successful attempts such as the production of Ω3 LC-PUFA in canola with reengineering of seven genes employing both synthetic and systems biology have resulted in crop improvement (Pouvreau et al., 2018). Similarly, another tangible approach for crop improvement using photosynthetic chassis demonstrated better photosynthesis efficiency by incorporation of algae-based carbon concentrating components by genetic engineering (Atkinson et al., 2016; Yang et al., 2017). Also few studies have demonstrated that modifying the D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a major rate-limiting carbon-fixating enzyme in photosynthesis, either by reengineering or generating lowefficiency Rubisco will favor crop improvements (Eisenstein, 2014; Lin et al., 2014). A technique known as multiplex automated genome engineering (MAGE) technology has shown that manipulating 1-deoxy-Dxylulose-5-phosphate (DXP) biosynthetic pathway by overexpressing lycopene-based genes resulted in the production of industrially relevant isoprenoids in E. coli (Wang et al., 2009, 2015). The bottleneck for commercialization of biofuels is yet to be resolved, but basic biology studies to characterize plants and algae using biotechnology studies for coproducing high value-added biomolecules along with biofuels would be maximized in the near future. However, advancements in synthetic and systems biology may provide insights for the production of economically viable biofuel and bioproducts in these cell factories (Chen et al., 2019). Transgenic technologies to improve the productivity of biofuels in plants and algae are yet to be exploited and have to overcome several challenges in order to be developed as plant-based solutions to bioindustrial requirements. However, studies have demonstrated that synthetic biology employing genetic circuits or/and reengineering metabolic pathways will be able to significantly accelerate the trends in crop improvement. Employing the pattern of design/build/test/learn cycle may ultimately refine and provide solutions to facilitate the rapid development of biofuels and bioproducts as sustainable solutions in plants and algae.

Acknowledgments Funding from the Department of Biotechnology, Government of India, to PPJ [Sanction No. BT/PR16512/BID/7/647/2016] and AAN [BioCARe No. BT/PR18491/BIC/ 101/759/2016]. Senior Research Fellowship to KMS, IM from University Grants Commission (UGC), Government of India, is duly acknowledged.

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669.

CHAPTER ELEVEN

Genetic engineering of horticultural crops contributes to the improvement of crop nutritional quality and shelf life Saber Delpasand Khabbazi1, Afsaneh Delpasand Khabbazi2, Volkan Cevik3 and Ali Ergül1 1

Ankara University, Biotechnology Institute, Ankara, Turkey Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran 3 Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom 2

11.1 Introduction Human in parallel with civilization has been improving the quality and yield of crops including vegetables and fruits. To this end, many efforts from conventional plant breeding practices to modern genome editing studies have been made. Fruits and vegetables are the vital sources of vitamin, carbohydrate, antioxidant, and fiber contributing substantially to human health. Since the early era of farming, efforts have been made to domesticate the crop plants to fulfill the food requirements of humans. Selection of superior genotypes and hybridization has led to creation of many valuable crops comprising the food table. During the recent decades, with the emergence of recombinant DNA technology, the transfer of genes corresponding to desired properties has facilitated the breeding process particularly in the case of interspecific crossing attempts. In contrast to conventional breeding methods, biotechnological tools effectively tackle the existing breeding barriers to incorporate the desired characteristics to target plants. Crop production that meets the needs and the demands of the growing global population is not likely to rely on conventional plant breeding approaches only. The advent of recombinant DNA technology and discovery of RNA interference system and genome editing tools such as zinc-finger nucleases (ZNFs), transcription Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00011-3

© 2020 Elsevier Inc. All rights reserved.

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activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, facilitate the creation of plant cultivars with desired traits. Intensive breeding attempts for yield have led to reductions in flavor and nutrients of fruit crops. It is commonly claimed by consumers that most of the modern fruits have lost their flavor. Despite the improved crop yield, an early survey of the food composition of 43 vegetables and fruits done during the period between 1950 and 1999 showed reduction in protein content, minerals, and vitamins (Davis et al., 2004). Later studies verified the claim that intensive breeding attempts affect the crop nutrients (Murphy et al., 2008; Garvin et al., 2006; White and Broadley, 2005). Most of the studies have been implemented to confer resistance to biotic and abiotic stresses, fruit size, and phenotypic characteristics of crops. Investigation for flavor is complicated due to the lack of fundamental knowledge of associated metabolites and pathways. The advent of modern next-generation genome sequencing technologies has eased access to nucleotide sequences and expression profile of corresponding genes, the basic step to engineer flavor and nutrient content of crops. Identification of the metabolites involved in fruit ripening is another important prerequisite in improvement of quality and shelf life of fruits and vegetables. Fruit economic value is influenced by postharvest shelf life. Short shelf life causes crucial losses after harvest and during crop transportation and marketing. Enhancement of the fruit and vegetable shelf life could contribute to the reduction of crop losses up to 50% worldwide (FAO, 2011). Preharvest biotic and abiotic stressors can reduce not only the crop yield but also postharvest quality and shelf life (Cerda, 2017; Arah et al., 2015; Toivonen and Hodges, 2011). Transgenic studies have been carried out to increase resistance to biotic and abiotic stress factors in horticulture crops (Sharma et al., 2016; Gaur et al., 2018). In this chapter, we review the transgenic attempts made for improvement of nutritional quality and shelf life of the fruits and vegetables.

11.2 Conventional strategies to prolong the shelf life Commercially increased shelf life is accomplished by different strategies such as early harvest, control of storage atmosphere, and selection of late ripening genotypes. These methods are, however, not invariably

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applicable to all and results are sometimes unsatisfactory (Klee, 2013). Ethylene is the determining agent to make climacteric fruits palatable albeit it also encourages senescence. Control of the internal ethylene induction contributes to the improvement of fruit quality. Therefore climacteric fruits such as tomato, are picked when they are still firm and green and stored under controlled conditions. In case of tomato, the storage temperature is set between 10 C and 15 C to prolong the storage time. Higher or lower temperatures increase the risk of tomato fruit deterioration and chilling damages, respectively (Saltveit, 2003; Saltveit and Morris, 1990). Modification and control of the atmosphere surrounding crops effectively reduce the product loss. Composition of gasses such as carbon dioxide, oxygen, and nitrogen affects the overall quality of the crop during the postharvest period. Manipulation of these gasses reduces the ethylene rate and subsequently delays senescence (Beckles, 2012). Moreover, treatment of fruits with methylcyclopropene delays the ripening through inhibiting metabolic activities like respiration, cell-wall degradation, and fruit color change (Watkins, 2006). Likewise, usage of the ozone gas in storage rooms delays the senescence and reduces the pathogen attacks (Aguayo et al., 2006; Skog and Chu, 2000). Preirradiation and heat treatments of fruits before cold storage (37 C 42 C) could delay the softening process (Beckles, 2012).

11.3 The metabolic basis underlying fruit ripening and shelf life Although ripening is considered as a destructive stage of development in fruits, the process is a precisely orchestrated act of development in plants (Klee and Giovannoni, 2011). Softening of fruit tissue ensures the dispersal of seeds inside and causes a successful spread of genetic alleles in nature. Model plants have been a proper platform to study the plant metabolisms during different developmental stages. Tomato is one of the model plants to study the mechanism of fruit development and ripening. Scientists have deciphered the genetic mechanisms regulating the ripening process in tomato fruit (Seymour et al., 2013). Fruit development and ripening are regulated by the interaction of different hormones and metabolites. Changes in the levels of hormones such as ethylene, abscisic acid (ABA), auxin, jasmonic acid (A), and methyl jasmonate (MeJA) affect the fruit ripening by altering the metabolites composition of cell. Such

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alterations lead to further changes like the accumulation of carotenoids, chlorophyll loss, and cell-wall disassembly (Barry and Giovannoni, 2007). In addition, tomato as a perishable and climacteric fruit is a suitable model plant for identification of the ripening metabolites and process. Ethylene along with environmental conditions such as temperature, light, and water availability stimulates the fruit ripening basically in climacteric fruits. The ethylene control delays the fruit ripening. Thus fresh-from-the-garden quality could be maintained between harvests and reaching to consumers if ethylene production is delayed. Ripening in nonclimacteric fruits is independent of ethylene. Application of exogenous ethylene, however, affects the ripening in these fruits likewise (Giovannoni, 2004, 2007). Key genes controlling the ethylene biosynthesis have been previously characterized in Arabidopsis and tomato plants. Characterization of the homologous genes involved in ripening process suggests that the underlying genetic mechanism is conserved in different fleshy fruits (Adams-Phillips et al., 2004). Earlier studies have well defined the role of ethylene in fruit ripening (Seymour et al., 2008; Barry and Giovannoni, 2007; Giovannoni, 2004, 2001). 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase is the key enzyme catalyzing the conversion of ACC to plant hormone ethylene. ACC oxidase (ACO) is encoded by the multigene family in plants and the occurrence of mutations in these genes inhibits ethylene production (Lin et al., 2009; Alexander and Grierson, 2002). Mutations applied to ACO1, for instance, affected the ethylene production in melon (Dahmani-Mardas et al., 2010). In addition, mutations occurred on ethylene receptor Nr (Never-ripe) gene, and ripening-associated Cnr (colorless nonripening), Nor (nonripening), and Rin (ripening inhibitor) genes acting upstream of the ethylene signaling network verified the effects of these genes in fruit maturation and softening (Giovannoni, 2007; Manning et al., 2006; Vrebalov et al., 2002; Wilkinson et al., 1995).

11.4 Metabolic alterations incorporating the increased shelf life An enormous amount of fruit loss occurs during the postharvest period. To cope with the postharvest losses, conventional methods, such as harvesting the fruits while green and firm, have been applied widely. This, however, negatively affects the aroma, flavor, and tissue of the fruit (Baldwin et al., 2011). Investigations for efficient approaches to overcome

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this problem have been worked upon for more than two decades. Attenuation of the respiration and reduction in the pace of overall cell metabolism contributed to the extended storage time of fruits. Decreasing the turnover rate of macromolecules such as proteins and polysaccharides helps to keep the fruit fresh after separated from the support of the main plant. Engineering plants for modified cell-wall-degrading enzymes (Meli et al., 2010; Powell et al., 2003; Smith et al., 1988, 2002) and metabolite fortification (Centeno et al., 2011; Nambeesan et al., 2010) have successfully contributed to the management of postharvest fruit losses. Enrichment of water-soluble anthocyanin pigments in tomato fruit provided purple color fruits with extended shelf life independent of ethylene production, cell-wall composition, and cuticle thickness (Butelli et al., 2008). In order to extend the postharvest storage time, studies have attempted to attenuate the impact of ethylene on ripening by suppressing the hormone synthesis at different stages. Tomato ripening has been delayed by manipulating ethylene perception (Tieman et al., 2000), silencing ethylene biosynthesis (Xie et al., 2006), enhancing polyamine biosynthesis (Dibble et al., 1988), suppressing ABA biosynthesis (Sun et al., 2012a), and downregulation of enzymes responsible for cell wall disruption (Uluisik et al., 2016). Downregulation of the genes coding for enzymes taking part in cellwall modifications has also led to the prolonged shelf life (Vicente et al., 2007), thus confirming the role of cell wall as another element in retention of fruit rigidity. Employment of reverse genetic approaches such as TILLING has been used to improve the crop shelf life. Using the ethyl methane sulfonate (EMS) agent, CmACO1 gene was mutated and caused late ripening and enhanced the storage span of melon (Ayub et al., 1996). Extension of postharvest storage period has been extensively investigated (Pech, et al., 2013). Some of the identified transcription factors involved in fruit ripening and spoilage belong to the NAC, SBP box, and MADS-box family of transcription factors. For example, mutant alleles of transcription factors encoded by NOR, CNR, and RIN genes result in prolonged shelf life. Based on investigations, NOR gene regulates the majority of the genes associated with ripening process (Casals et al., 2015; Osorio et al., 2011; Kosma et al., 2010). The mutant alleles of nor were identified either as a result of single nucleotide substitution or protein truncation corresponding to alc (alcobaca) and nor alleles, respectively (Casals et al., 2012; Barry and Giovannoni, 2007). The presence of such alleles causes a denser cutin matrix (Kosma et al., 2010) and therefore, resistance to infections (Garg et al., 2008). Genetic background and size of fruits however, influence

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the shelf life as well (Casals et al., 2012; Garg et al., 2008). It is found that mutations in NOR gene strongly associate with fruit storage life. Later studies emphasized the association of other NAC family transcription factors such as NAC1 and NAC4 in the ripening process of tomato fruit (Zhu et al., 2014; Ma et al., 2014). Some of the tomato accessions naturally demonstrate a prolonged shelf life. Kumar et al. (2018) identified a novel allele in tomato Penjar-1 accession with two mutations in NOR gene. Substitution of nucleotides caused truncated protein in both of the mutants and led to extended storage time of tomato fruits after harvest. The phenotype of the mutants bearing novel alleles was consistent with the nor/alc mutants. Furthermore, rin and nor mutant alleles prolonged the shelf life of tomato fruit even when they were heterozygous (Garg et al., 2008; Barry and Giovannoni, 2007). Metabolic profiling of Penjar-1 accession unveiled that the respiratory rate was attenuated during the postharvest period. Moreover, the expression of genes encoding enzymes associated with cell-wall disassembly was downregulated. Abscisic acid and sucrose contents were increased at the beginning of the senescence stage, which could be inferred as a result of reduced water loss (Kumar et al., 2018). Expression of the snapdragon Delila and Rosea1 transcription factors in tomato resulted in the production of purple tomato fruits enriched with anthocyanins and antioxidants. Transgenic tomatoes (Del/Ros1) were also resistant to the fungal pathogen Botrytis cineria and the spoilage was attenuated significantly (Zhang et al., 2013). In addition, the shelf life of Del/Ros1 tomatoes was expanded; thus they could be stored for an extended period. Del/Ros1 expression did not inhibit the ripening process. The firmness of tomato fruits was, however, twice as compared to nontransgenics, when ripe. The firmness was due to the higher levels of anthocyanins accumulated in fruit. During the ripening stage, no difference in the cuticle thickness or cellwall composition was observed. During the overripening stage, however, Del/Ros1 tomatoes expressed lower levels of enzymes responsible for cellwall disassembly such as polygalacturonase (Klee, 2013).

11.5 Transgenic technology as a promising tool for crop nutritional quality and shelf life improvements Genetic engineering of crops is a promising tool for introducing plants with novel traits. Recombinant DNA technology enables the

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genetic modification of crops mainly with the aim of improving yield and quality of crops and resistance against biotic and abiotic stress factors. Through this system, many studies have reported the transfer of genes between different species including plants, insects, and bacteria. Unlike the conventional breeding approaches, transgenic technology is not curtailed with interspecies genetic barriers. Therefore the genes of interest could be transferred between plant species with different ploidy levels or genetic structures. Fruits and vegetables are dietary plants accounted for a crucial supply of nutrients for human health. Many biotic and abiotic factors threaten the production of these crops worldwide. Resistance to insect pests and pathogens, tolerance to herbicides and abiotic factors, prolonged shelf life, and nutrition quality are the main objectives of the genetic modifications in plants. Although many transgenic studies on various plant species have been carried out, only a few GM fruit and vegetable crops including common bean, eggplant, squash, apple, papaya, and pineapple are currently in production (Baranski et al., 2019). The number of transgenic research involving fruit crops is fewer when compared to vegetables and field crops, which is due to the perennial nature of these crops as well as difficulties encountered in genetic transformation and in vitro regeneration procedures. Production of transgenics for prolonged shelf life contributes to the food supply by reducing crop losses in preharvest or postharvest stages. Considering the fact that the global population will double in the next 50 years, the importance of the issue is emphasized. Therefore the introduction of crop plants as the products of the modern technologies will play a vital role in providing food for the growing population worldwide.

11.6 Resistance to biotic stress factors It is reported that almost 67,000 pest species damage agricultural products and approximately 13% of these species are mites and insects (Ross and Lembi, 1985). A published report suggests 37% of global crop loss is due to insect pests and diseases (Gatehouse et al., 1992). Insects and pathogens like viruses and fungi are the most devastating biotic factors forcing the farmers to spend millions of dollars to protect their produce each year. Besides the imposed costs and being unfriendly to the environment, application of chemicals like insecticides threatens the farmer and consumer’s health (Curry, 2002), particularly for fruits and vegetables as

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they are consumed raw. According to a report by Environmental Justice Foundation, 1% 3% of the field workers suffered from severe chemical poisoning representing 25 million to 77 million workers globally (EJF, 2007). To control crop losses caused by biotic factors and to reduce production costs, genetic manipulation of crops has been recognized as a promising tool for developing insect and pathogen resistant cultivars. Till date, fruits such as apple, banana, papaya, grapevine, plum, guava, and orange have been genetically engineered for resistance against viral, fungal, and bacterial diseases (Table 11.1). Resistance to viruses has been generally conferred by targeting the virus coat protein (CP) gene. Fruits resistant to the various viruses including BBT, PRSV, GFLV, GLRAV, CTF, and PPV have been introduced through targeting the coat protein expression in host plants. Furthermore, resistance to fungal and bacterial diseases such as wilt, scab, mildew, brown rot, and fire blight has been achieved in fruits through transgenic approach (Table 11.1).

11.7 Resistance to abiotic stress factors Exposure of crops to abiotic factors in the field reduces the plant growth and productivity. Factors such as salinity, drought, high temperatures, freezing, metal toxicity, and nutrient deficiencies have been integrated to agriculture and crop production. Transformation of horticultural crops with genes involved in signaling and regulatory pathways as well as osmoprotective features have successfully improved the tolerance against abiotic stresses (Wang et al., 2003; Bhatnagar-Mathur et al., 2008). Overexpression of transcription factors involved in signaling and regulatory pathways has successfully improved the tolerance in horticultural crops to abiotic stress factors. Introduction of the bacterial BetA gene involved in biosynthesis of glycine betaine-induced tolerance to salinity in cabbage (Bhattacharya et al., 2004). Overexpression of MusaWRKY71 gene isolated from Musa spp. cv. Karibale Monthan enhanced the tolerance of banana to abiotic stress factors (Shekhawat et al., 2011). Expression of rice Osmyb4 gene in Arabidopsis activates the stress-inducible pathways and accumulation of solutes such as proline, sucrose, and glycine betaine. Overexpression of the Osmyb4 gene in apple improved crop tolerance to cold and drought conditions (Pasquali et al., 2008). Transformation of lettuce with a mutated P5CS gene encoding delta-1pyrroline-5-carboxylate synthase enhanced the proline level, therefore

Table 11.1 Fruit crops resistant to biotic stress factors. Pathogen Disease type Plant species

Viruses

Fungi

Bunchy top

Banana

Papaya ringspot virus

Papaya

Grapevine fan leaf virus and grapevine leaf roll associated virus

Grape Rootstock

Citrus tristeza virus

Grapefruit

Sharka (Plum pox virus)

Plum

Scab

Apple

Transferred genes

References

BBTV-cp: viral coat protein (cp) PRSV CP: untranslatable coat protein gene of PRSV/ PRSV-RP: PRSV replicase

Ismail et al. (2011)

Coat protein genes from GFLV/GLRaV-3: a truncated HSP90-related gene of GLRaV-3/virE2 del B gene: from A. tumefaciens CP/3END/RdRp (RNAdependent RNA polymerase) PPV-CP: coat protein

ech42: fungal cell-wall degrading endochitinase enzyme

Fermin et al. (2004), Magdalita et al. (2004), Lines et al. (2002), Cai et al. (1999), Tennant et al. (1994), and Fitch et al. (1992) Xue et al. (1999)

Febres et al. (2008)

Malinowski et al. (2006) and Hily et al. (2004) Faize et al. (2003)

(Continued)

Table 11.1 (Continued) Pathogen Disease type

Bacteria

Plant species

Transferred genes

References

Sigatoka leaf spot

Banana

Vishnevetsky et al. (2011)

Fusarium wilt

Banana

Powdery mildew

Grapevine

ThEn-42: endochitinase gene/ StSy: stilbene synthase/ Cu, Zn-SOD: chloroplastic/ Cu, Zn-superoxide dismutase gene Atfd3: Arabidopsis root-type ferredoxin gene /Pflp: plant ferredoxin-like protein RCC2: rice chitinase gene RIP: ribosomeinactivating protein

Guava wilt Brown rot

Guava Orange

Fire blight

Apple

Xanthomonas wilt

Banana

Endochitinase Tomato P23 (PR-5) chimeric gene att E: Antibacterial attacin protein Hrap: Hypersensitive Response/ Assisting Protein Pflp: plant ferredoxin like protein

Yip et al. (2011)

Nirala et al. (2010), Bornhoff et al. (2005), and Yamamoto et al. (2000) Mishra et al. (2014) Fagoaga et al. (2001) Ko et al. (2000) Tripathi (2012)

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causing crop tolerance to salinity, chilling, and drought-related osmotic stress (Pileggi et al., 2001). Grapevine tolerance to cold conditions was increased by overexpression of the Arabidopsis cold-inducible transcription factor (Jin et al., 2009). Arabidopsis gene AVP1, which encodes pyrophosphateenergized vacuolar membrane proton pump 1 and regulates auxin mediated organ development, was transferred to tomato. Expression of AVP1 gene caused a strong root system in tomato plants, so the plant could efficiently use limited moisture under water deficiency (Park et al., 2005). Expression of bacterial mtlD gene encoding mannitol-1-phosphodehydrogenase in eggplants improved its tolerance when confronted with cold, drought, and salinity stress conditions (Prabhavathi et al., 2002).

11.8 Biofortification of fruits and vegetables Bio-fortification is a promising approach to sustainably deliver sufficient nutrients to the populations suffering from limited food resources (Saltzman et al., 2014). Various methods such as agronomic and conventional breeding and novel biotechnological tools have been integrated to enhance the nutritional value of crops. Using the fertilizers enriched with micronutrients is the simplest method of bio-fortification. However, the results of this approach are highly variable and depends on the soil composition, mineral mobility, and availability in soil and nutrient utilization efficiency of plants (Wissuwa and Ae, 2001; Frossard et al., 2000). This approach is also labor intensive and hence not very cost-effective. Therefore alternative approaches are considered. Conventional Plant breeding is another approach used to enhance nutritional value of crops. Although a cost-effective and sustainable method, traditional breeding confronts limitations concerning the genetic variability in plant gene pools, multigene control of desired traits, and the genetic barriers existing in intercrossing of different species. The advent of recombinant DNA technology has revolutionized the crop genetic improvements in a variety of aspects including crop biofortification. Nevertheless, studies to improve the nutrition quality of crops are in progress. Fruits and vegetables are among the staple crops improved for nutritional content. Crops such as apple, banana, tomato, cassava, potato, sweet potato, cauliflower, lettuce, and carrot have been successfully biofortified for vitamins, minerals, secondary metabolites, antioxidants, etc., through transgenic approaches (Garg et al., 2018) (Table 11.2). Regulatory mechanisms of gene expression associated with fruit taste and

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Table 11.2 Biofortification of fruits and vegetable crops through transgenic approaches. Crops Type of biofortification Source

Fruits

Apple Banana Tomato

Vegetables Potato

Sweet potato Cassava Cauliflower Lettuce Carrot

Stilbenes β-carotene Folate, phytoene, β-carotene, lycopene, provitamin A, isoprenoids carotenoid 1 flavonoid, ascorbate, folate, antioxidant, anthocyanin β-carotene, zeaxanthin, ascorbate, methionine, amino acid composition, cyclodextrins, anthocyanins, fructan and inulin, reduced amylose and increased amylopectin in starch granules β-carotene and antioxidants

Garg et al. (2018)

β-carotene, provitamin A, iron, protein β-carotene Iron Calcium

aroma have been studied. Identification of candidate genes corresponding to fruit ripening and flavor in apricot contributed to the understanding of gene transcriptional networks, which also paves the way for functional genomic studies (Zhang et al., 2019). Anthocyanins harbor pivotal roles to protect plants and animals against pathogens and diseases. It is also used as bio-active molecule in traditional medicine and neutraceutics. These metabolites are water-soluble pigments giving blue to red colors to plant organs including fruit and leaf (Mazza and Miniati, 2018; Onslow, 2014). Most of the fruits and vegetables contain only limited amount of anthocyanins located in epidermis of plant organs. Anthocyanin biosynthesis is affected by environmental stress factors such as nitrogen and phosphate deficiencies (Lea and Azevedo, 2007) and exogenous application of sucrose (Teng et al., 2005). These factors induce the anthocyanin biosynthesis through expression of MYB, bHLH, and NAC transcription factors (Wu et al., 2012; Morishita et al., 2009; Dubos et al., 2008). Genetic modification attempts have been made for biofortification of horticultural crops. Introduction of purple crops such as carrot (Xu et al., 2017), potato (Liu et al., 2015), cauliflower (Chiu and Li, 2012; Chiu et al., 2010), and tomato (Butelli et al., 2008) has attracted the consumer interest. The transgenic product legislations, however, have limited the commercial production.

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Carotenoids, particularly β-carotene, are the major source of vitamin A. Vitamin A takes a tremendous role in human vision health care (Wiseman et al., 2017). Expression of daffodil phytoene synthase gene (PSY) along with Erwinia uredovora carotene desaturase (CRT) in rice endosperm led to the creation of Golden Rice, rich in carotenoids (Ye et al., 2000). In later studies, however, maize ortholog of PSY gene was introduced to rice endosperm to enhance the enzymatic activity and β-carotene levels (Paine et al., 2005). Overexpression of PSY gene in canola, cassava, and recently in banana resulted in produce rich in β-carotene (Paul et al., 2017; Sayre et al., 2011; Shewmaker et al., 1999). Other genes involved in carotenoid biosynthesis pathway such as lycopene β-cyclase (β-LCY) gene and 1-deoxyxylulose-5-phosphate synthase (DXS) gene have also been used to increase the β-carotene levels in Arabidopsis, tomato, and cassava plants (Sayre et al., 2011; Enfissi et al., 2005; Estevez et al., 2001; Rosati et al., 2000; Cunningham et al., 1996). Likewise, RNA interference-mediated suppression of epoxycarotenoid deoxygenase gene enhanced the level of lycopene and β-carotene (Sun et al., 2012b). L-Ascorbic acid (vitamin C) functions as a vital factor in defense systems as an antioxidant. Therefore it plays a substantial role in human health. Metabolic engineering of crops for ascorbic acid is primarily considered by altering the GDP-L-galactose phosphorylase (GGP), which is a key enzyme in ascorbic acid biosynthesis pathway (Macknight et al., 2017; Bulley and Laing, 2016). Biofortifying plants for ascorbic acid also confer them with tolerance to abiotic stress factors such as salinity, drought, and cold (Strobbe et al., 2018). Introduction of kiwi and potato GGP gene to tomato and potato plants increased the ascorbic acid by six and three times, respectively (Bulley et al., 2012). In addition, recycling or inhibition of ascorbic acid degradation has also increased the ascorbic acid content (Li et al., 2012; Zhang et al., 2011).

11.9 Genome editing as an efficient approach to develop crops with better nutritional qualities Genetic engineering has a pivotal role in today’s and future’s agriculture and creation of crops with desired traits. Identification of gene functions through reverse genetics has contributed to genetic improvement of crops. Traditional approaches of functional analyses of desired

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genes were based on chemical or irradiation-based mutagenesis, transposon mutagenesis, and transfer DNA (T-DNA) technology. In some cases, however, plant genome size is large or genome wide information is lacking; furthermore, the T-DNA-mediated modification is a method with lower success. The employment of sequence-specific nucleases (SSNs) such as ZNFs, TALENs, and recently CRISPR)/Cas9 system has provided a promising horizon to reverse genetics to improve the quality and yield of crops and confer disease resistance (Klümper et al., 2014). The SSNs induced double-strand breaks (DSBs) at specific genomic sites and exposed the broken DNA to error-prone nonhomologous end joining (NHEJ) repair or the accurate homologous recombination (HR) repair mechanism. ZNFs and TALENs have been used to generate successful mutants. These tools are, however, technically complex and low efficient. To date, there is no study report on genome editing of horticultural crops using ZNFs. TALENs have successfully contributed to the genetic modification of tomato (Lor et al., 2014) and potato (Clasen et al., 2016; Sawai et al., 2014) plants. Due to the existing limitations, however, this method has not been further used for related studies of horticultural plants (Kumar et al., 2018). Recently, CRISPR/Cas9 genome editing tool has been successful in genetic engineering of vegetables and fruits. The effectiveness of the CRISPR/Cas9-based genome editing in plants was first demonstrated in the model plants, Arabidopsis and Nicotiana benthamiana (Li et al., 2013; Nekrasov et al., 2013). Since then, many reports support the use of this tool for targeted mutagenesis in different plant species, including vegetables and fruits (Koltun et al., 2018). As an ideal candidate, tomato was among the first horticultural species to investigate the effectiveness of the CRISPR/Cas9 system in crop plants. Disruption of the tomato homolog of Arabidopsis SlAGO7 gene (ARGONAUTE7) resulted in plants with wiry or needle-like leaves (Brooks et al., 2014). In later studies, CRISPR/Cas9-mediated genome editing was used in tomato with the aim of influencing the ripening and increasing the fruit shelf life by targeting the RIN, SLALC, and lncRNA1459 genes (Li et al., 2018; Yu et al., 2017; Ito et al., 2015), resistance to powdery mildew by targeting the SlMlo1 gene (Nekrasov et al., 2017), and production of seedless fruits by targeting SlIAA9 and SlAGL6 genes (Ueta et al., 2017; Klap et al., 2016). The universality of CRISPR/Cas9 system has been investigated by targeting the phytoene desaturase gene (PDS) in different horticultural crops including sweet orange, watermelon, and grape. Knocking out the

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PDS gene causes albino phenotype in mutants, thus confirming the occurrence of targeted mutagenesis (Nakajima et al., 2017; Tian et al., 2017; Jia and Wang, 2014). In another study, disruption of PDS gene in grape demonstrated that the proportion of mutated cells was intriguingly correlated with the cell age. Nakajima et al. (2017) observed higher rates of the mutation with the lower leaves of the plant and suggested that this was due to the less efficient repair mechanism of old cells or the repeated induction of DSBs. The induced mutagenesis in watermelon PDS gene indicated the expected albino phenotype either as a clear or mosaic pattern with no off-target mutation (Tian et al., 2017). Likewise, transient expression of CRISPR/Cas9-gRNA-PDS reagents in sweet orange leaves successfully knocked out the PDS gene without detection of any offtarget mutagenesis. To facilitate the agro-infiltration, sweet orange leaves were pretreated with a culture of Xanthomonas citri ssp. citri (Xcc). Results indicated that Xcc-facilitated agro-infiltration enhanced the protein expression level in treated leaves (Jia and Wang, 2014). Codon optimization of the Cas9 gene has been investigated to increase the efficiency of genome editing. Comparison of three codon optimized Cas9 enzymes, zCas9 (Xing et al., 2014), AteCas9 (Schiml et al., 2014; Fauser et al., 2014), and Cas9p (Ma et al., 2015) showed AteCas9 as most efficient enzyme in disrupting the flavanone-3-hydroxylase (F3H) gene in carrot cells. Knocking out the F3H gene also validated the role of this gene in anthocyanin biosynthesis, as it led to the discoloration of the produced calli (Klimek-Chodacka et al., 2018). CRISPR/Cas9 genome editing technology was also used to induce mutagenesis in potato acetolactate synthase1 (StALS1) gene. The StALS1 function is related to herbicide tolerance in plants; therefore the generated mutants could be screened. Genome editing was implemented on potato plants with diploid and tetraploid genetic backgrounds. Results indicated that the occurrence of somatic mutations was higher in the diploid background (Butler et al., 2015). The quality of potato starch was altered by site-directed mutagenesis in the GBSS gene encoding the granule-bound starch synthase. Multiallelic mutagenesis in tetraploid potato crop led to the creation of individuals producing starch with reduced amylose and increased amylopectin contents (Andersson et al., 2018, 2017). To accelerate the plant growth, TAA1 and ARF8 genes of wild strawberry (Fragaria vesca) were mutated by using of CRISPR/Cas9 tool. Knocking out these genes increased the auxin biosynthesis and subsequently caused a faster growth. Furthermore, this study tested the germline inheritance of the generated mutation in the T1 progeny, and. when the

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mutation rate compared to T0, it was higher in T1 generation (Zhou et al., 2018). Cabbage BolC.GA4.a gene was targeted to investigate the mutagenesis efficiency on the plant phenotypic characteristics. The BolC.GA4.a gene is the ortholog of Arabidopsis GA4 encoding the last enzyme involved in gibberellins biosynthesis (Lawrenson et al., 2015). Manipulation of the BolC.GA4.a gene led to a dwarf phenotype of 10% of the first generation. The off-target mutagenesis was also recorded. Mutations generated to the recessive eIF4E gene of cucumber plant conferred resistance against cucumber vein yellowing virus, potyviruses, zucchini yellow mosaic virus, and papaya ring spot mosaic virus-W (Chandrasekaran et al., 2016).

11.10 Commercialization of GM fruits and vegetables Crop losses either during preharvest or postharvest period threaten the supply of food for the growing population, and is estimated to reach 9.78 billion$ in 2050. Various plant breeding efforts contributed to the improvement of plant quality along with the product yield. Introduction of modern approaches such as recombinant DNA technology paved the way to accelerate the tedious breeding processes. The limited water sources as well as plant pests and diseases are the major threats for crop production. Transmission of genes with different origins into target plants provides resistance to biotic and abiotic stress factors. Many studies have been carried out proposing the suitability of different gene for improving yield, nutritional quality, and tolerance to stress by plants (Baranski et al., 2019; Bakhsh et al., 2015; Xiong et al., 2015). The first transgenic horticultural crop, Flavr Savr tomato, was authorized 25 years ago. Since 1994, genetically modified crops were adopted so widely that in some countries, several crops almost completely replaced the conventional varieties and the cultivation area of GM crops increased to 185 million hectares (ISAAA, 2016a). Despite the substantial incorporation of transgenics in agricultural productions, the public concerns are challenging the production of GM crops. Biosafety regulations have been set to ensure the safety of GM products and reduce the consumer concerns about the introgression of foreign genes into plant genomes. In some cases, although GM crops passed the regulation policies, the permission of entrance to the market never issued. Transgenic horticultural crops are currently cultivated in only 10 countries including USA, Brazil, Australia, Bangladesh,

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China, Japan, Hong Kong, Colombia, Costa Rica, and Ecuador (Baranski et al., 2019). In USA, the main GM crop producer of the world, 90% 100% of soybean, cotton, canola, maize, and sugar beet crops are genetically modified (ISAAA, 2016a). The number of GM horticultural crops being cultivated in commercial scale is, however, low when compared to field crops. Introduction of novel transgenic approaches has substantially improved the pest and disease management strategies. Confining the foreign gene expression to desired organs, tissues, and developmental stages as well as the utilization of genes with plant origin (Khabbazi et al., 2016; Bakhsh et al., 2015) could play a crucial role in biosafety regulations (Khabbazi et al., 2018; Anayol et al., 2016; Bakhsh et al., 2016). The use of CRISPR/Cas9 system may reduce the public hesitation of using transgenic crops. The biggest advantage of the CRISPR/Cas9 is to create genetically engineered nontransgenic products (Sánchez-León et al., 2018) due to the segregation of foreign genes in the sexual reproduction process.

11.11 Conclusion Control of polygenic traits such as fruit flavor and nutritional quality in hybrids is difficult through conventional breeding methods. Transgenic approaches have successfully tackled limitations of conventional methods. The biggest issue challenging the genetically modified crop production is, however, the public acceptance. Although many studies have been carried out to reduce the community concerns, the commercialization of the GM crops is still restricted by different regulatory processes. There have been many successful reports confirming the importance of plant genetic engineering approach in crop improvements. Particularly with the advent of novel genetic editing tools, accurate mutagenesis could be induced in plant genome. The latest genome editing tool, CRISPR/Cas9 system, has been successfully used in various field crops. Assessment of the reported studies had shown the potential of the CRISPR/Cas9 system in genetic improvements of crops by conferring desired traits. Especially considering the increased pressure from the public regarding the safety of genetically modified crops, this novel system can reduce the concerns. It is expected that modern crops modified through CRISPR/Cas9 system will meet the safety expectations of the public, as they will not be harboring foreign genes.

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CHAPTER TWELVE

Transgenic food crops: public acceptance and IPR Usha Kiran1,2 and Nalini Kant Pandey3 1

Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India Bioinformatics Institute of India, Noida, India Mitakshara IP Services, Ghaziabad, India

2 3

12.1 Transgenic technology for genetic modification of plants Food has been fundamental to the existence of mankind. Humans have been altering the genetic makeup of plants to adapt them to particular climate, region, and societal need. In 1970s effective breeding technique along with use of pesticides and fertilizers, enabled scientists to address the issues of hunger and malnutrition. Green revolution made the world experience a gigantic agricultural production. The effects of green revolution have, however, started to fade away in last two decades due to exponential growth of world population. Currently, large proportion of world population is under the realms of hunger and nutritional deficiencies. According to a WHO report, 23% of the world’s children under the age of 5 are affected by stunting and around 815 million people all over the world sleep hungry (UNICEF report). The reversal in progress toward food insufficiency clearly indicates that more efforts are required urgently to attain the United Nation supported Sustainable Development Goal of Zero Hunger by 2030. The increase in global temperature is already affecting the production of major crops especially maize, rice, and wheat in temperate and tropical regions (Zhao et al., 2017). Thus new routes should be adapted to rapidly increase the yield and nutritive value of the agricultural products to meet the greatest global challenge of providing nutrition to all. Genetic modification of food crops offers immediate solution to alleviate the problem of hunger and malnutrition (Oliver, 2014). Advancement in molecular biology and biotechnology had led to the understanding of gene Transgenic Technology Based Value Addition in Plant Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-818632-9.00012-5

© 2020 Elsevier Inc. All rights reserved.

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organization and its control over phenotypic and biochemical expression. High throughput sequencing and molecular screening had led to identification and correlation of gene to their function and controlled expression. Thus identification of genetic material related to economically important traits such as increase in quantity and quality, fortification of crops with vitamins and minerals, expression of pharmaceutics, and neutraceutics has become easy and efficient (Pfeiffer and McClafferty, 2007; Schafer et al., 2011; Kumar et al., 2019). The improvement in delivery of trangenes to target tissue has made genetic material delivery and integration a success story (Sanford, 1990; Schmidt et al., 2008; Barampuram and Zhang, 2011). Genetic manipulations enabled to produce crop varieties with better nutrient utilization efficiencies, resistant to pest and pathogen attack and tolerant environmental imbalances (cold, drought, and salinity) (Dennis et al., 2008). Plants with enhanced nutrition efficiencies for nitrogen, sulfur, and phosphorus are shown to give better quality and quantity produce and reduce the application of fertilizers (Tian et al., 2012; Chen and Liao, 2017). Resistance to pest and pathogen means less dependence on pesticides and hence less crop failure. This further increases the food security. To maximize economic gains, scientists can selectively incorporate genetic traits depending on climate, location, soil nutritional status and texture, microflora and fauna of soil, prevalent pest, and pathogens.

12.2 Adoption and commercial benefits of biotech crops Transgenic technology for enhancement of desirable trait has been considered as fastest adopted crop improvement technology in history of agriculture. The first approval for the commercial release was granted in 1994 to Californian Company Calgene, for genetically modified (GM) tomato (Flavr Savr) with delayed ripening. In 1996 around 1.66 million hectares were used to plant the GM tomato. After two decades of adoption of commercial transgenic/biotech/GM crops, 17 million farmers from 26 countries have planted GM crops (ISAAA, 2018). During last 6 years, area under cultivation of GM crops increased in developing countries. During 2017, 100.6 million hectares (53% of global biotech hectares) were used for cultivation by 19 developed countries, whereas industrial countries cultivated GM crops on 89.2 million hectares (47% of global biotech hectares). In 2017, globally United States was the top producers

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of GM crops production with planting of 75 million hectares (40% of global GM crop planting). Brazil remained at second place with 51.3 million hectares (26% of global GM crop planting). By 2018, 70 countries have adopted transgenic crops, of which 26 countries grew transgenic while 44 countries imported transgenic crop produce for food and feed (Table 12.1). Table 12.1 Global area of biotech crops in 2018: by country (million hectares).a Rank Country Area (million Biotech crops hectares)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

United Statesb Brazilb Argentinab Canadab

75.0

Indiab Paraguayb Chinab Pakistanb South Africab Uruguayb Boliviab Australiab Philippinesb Myanmarb Sudanb Mexicob Spainb Colombiab Vietnam Honduras Chile Portugal Bangladesh Costa Rica Indonesia eSwatini Total

11.6 3.8 2.9 2.8 2.7

51.3 23.9 12.7

1.3 1.3 0.8 0.6 0.3 0.2 0.2 0.1 0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 191.7

Maize, soybeans, cotton, canola, sugar beets, alfalfa, papaya, squash, potatoes, apples Soybeans, maize, cotton, sugarcane Soybeans, maize, cotton Canola, maize, soybeans, sugar beets, alfalfa, apples Cotton Soybeans, maize, cotton Cotton, papaya Cotton Maize, soybeans, cotton Soybeans, maize Soybeans Cotton, canola Maize Cotton Cotton Cotton Maize Cotton, maize Maize Maize Maize, soybeans, canola Maize Brinjal/eggplant Cotton, soybeans Sugarcane Cotton

Data taken from Global Status of Commercialized Biotech/GM Crops: 2018. ISAAA Brief No. 54. ISAAA, Ithaca, NY. a Rounded off to the nearest hundred thousand. b 18 biotech mega-countries growing 50,000 ha, or more, of biotech crops.

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Soybean, cotton, maize, and canola were major transgenic crops adopted for cultivation in 2018, with transgenic soybean being the most planted crop world over (ISAAA, 2018). Transgenic soybean was planted on 95.9 million hectares, occupying 78% of the total area under soybean production worldwide. Maize was the second most planted crop with 58.9 million hectares coverage. This is, however, 30% of the global area for maize production worldwide. Transgenic cotton was cultivated on 24.9 million hectares, followed by transgenic canola on 10.1 million hectares. Apart from these, transgenic of alfalfa, squash, sugar beet, eggplant, potato, papaya, and apple were also planted by different countries. More and more countries are adopting the transgenic technology and the trend will continue because newly developed biotech crops are benefiting farmers and consumers. High oleic acid canola, high oleic acid and herbicide tolerant soybean, salt and HT tolerant soybean, cotton with isoxaflutole herbicide tolerance (HT cotton), insect-resistant (IR) sugarcane are some of the new traits combinations approved for cultivation. The first IR sugarcane was planted by Brazil and first drought-tolerant sugarcane was planted by Indonesia. The research for incorporation of various economically important traits in rice, potatoes, wheat, chickpea, pigeon pea, mustard, and banana is being conducted by public and private groups; thus area under cultivation of transgenic crop is expected to increase further in coming years. Transgenic crops are being adopted because of their contribution towards improvement of socio-economic conditions of farmers, and benefits to human and animal health. More than 17 million farmers are benefited and 95% of them belong to developing countries. The Cropnosis reported the global market value of transgenics to be US$ 17.2 billion for year 2017 which was 9% increase from 2016 (US$ 15.8 billion). Total transgenic crop productivity from 1996 to 2016 was estimated to be of value US$ 186.1 billion, US$ 18.2 billion alone in 2016. Transgenic hybrids saved 22.5 million hectare of land in 2016 alone, by reducing the pesticide application by 48.5 million kg and thus stopping its release in the environment by 18.3%. The biotech crops contribute effectively to mitigate the damages by extensive use of pesticide. The transgenic technology is therefore not only benefiting the farmers and consumers locally but also affecting agri-biotechnology business and environment, globally. The US agency leads the development, adoption, and commercialization of biotech crops. In 2018, United States tops the transgenic planted crop area with 75 million hectares, covering 39% of the global transgenic

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planted area, followed by Brazil, Argentina, Canada, and India. The plantation of biotech crops was favored by small countries as well. Public acceptance of clean technology motivated farmers from Bangladesh to cultivate transgenic eggplant. In Indonesia, drought-tolerant sugarcane was planted, which gave 20% 30% higher yield than parental varieties. The new variety could be planted by farmers even under low rainfall period. Maize, soybean, and cotton are the major biotech crops adopted for commercialization; however, new crops like high oleic acid canola, sugar beet, apples, papaya, HT cotton, and low gossypol cotton are also approved for environmental release. Biotech crops are approved by 70 countries for either human and animal consumption or commercial cultivation. Regulatory approvals are granted for 387 individual events in 27 crops. Of 4349 approvals, 2063 were granted for crop produce to be used as food, either directly or after processing, 1461 for crop produce to be used as feed, either directly or after processing, and 825 for environmental release or cultivation. United States had the most number of approvals for GM events (Table 12.2). Table 12.2 Top ten countries which granted food, feed, and cultivation/environment approvals.a Rank Country Number of approvals

1 2 3 4 5 6 7 8 9 10 11

b

United States Japana Canada South Korea Brazil Mexico Argentina Philippines European Union Australia Others Total

Food

Feed

Cultivation

Total

190 185 147 156 89 188 76 103 99 118 712 2063

180 177 138 148 89 29 68 102 100 19 411 1461

174 130c 144 0 85 15 74 13 3 39 148 825

544 492 429 304 263 232 218 218 202 176 1271 4349

Data taken from Global Status of Commercialized Biotech/GM Crops: 2018. ISAAA Brief No. 54. ISAAA, Ithaca, NY. a For Japan, data are collected from Japan Biosafety Clearing House (JBCH, English, and Japanese) as well as the website of the Ministry of Health, Labor and Welfare (MHLW). However, intermediate events derived from an approved pyramided event recorded in JBCH are not included in our database if they do not appear in MHLW. Also, expired approvals are included in our database from 1992 while JBCH’s record starts in 2004. b United States only approves individual events. c While cultivation approvals are granted in Japan, there are no current GM planting done.

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Maize had highest number of approval (137 in 35 countries) and HT maize event NK603 has maximum number of approval (61 approvals in 28 countries 1 EU 28).

12.3 Transgenic hybrids in India India has made remarkable progress in agriculture. With the translation of idea of Norman Borlaug (awarded Nobel Prize in 1970 for higher yielding varieties of wheat) green revolution, India moved toward selfreliance in cereal production. Indian green revolution started in 1965. High yielding variety (HYV) seeds, correct use of fertilizers, and improved and efficient irrigation methods contributed to increased crop production. India, however, has fastest growing population, estimated to reach 1.7 billion in 2050. The impressive effect is now fading up and pressure is building up on land, water, and biodiversity to meet the nutrition requirements of this increasing population. Food security is further challenged by rapidly shrinking land recourses, drastic climate change, rapid urbanization, and migration for less labor-insensitive jobs (Kumar and Joshi, 2016). The self-sufficiency in cereal production in India, unfortunately, did not translate into decreased malnutrition. Moreover, concentration of efforts on cereal crops like wheat and rice has resulted in selective depletion of nutrients from soil and loss of agro-diversity (indigenous land races and breeds). To harness the real potential of agriculture aimed to mitigate the effect of malnutrition and hunger, India needs to develop technologies for increasing crop yield potential and quality. The adoption of transgenic crops with beneficial traits specific for region, along with integrated pest and nutrient management and efficient irrigation system, is required to usher a second green revolution. India adopted GM cotton as first transgenic crop to be cultivated in 2002. The GM cotton contains gene from Bacillus thuringiensis (Bt), which produces proteins that protect the plant from pink bollworm. The transgenic technology was rapidly adopted by around 6 million farmers of Punjab, Madhya Pradesh, Maharashtra, and Andhra Pradesh and insectresistant Bt cotton planted area increased from 3.8 million hectare in 2006 to over 11.6 million hectares by 2018. The favorable global cotton price and adherence to good farming practices lead to high adoption rate of Bt

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cotton in India. Effects were further augmented by efficient implementation of pink bollworm management in cotton-producing states. India is now the world’s fifth biggest producer of genetically engineered crops and more than 90% of cotton cultivated area is covered by Bt cotton (Kumar, 2015). To fight hunger and malnutrition, India, however, needs to embrace biotech crops. In recent years, devastating infestation of fall armyworm was reported from major maize-producing states making the food status of country worst. Awareness on control management of fall armyworm along with insect resistance maize could improve nutritional status in India. Edible oil is another area where the production needs to be increased and hence high yielding GM Oil seed crop varieties could be used.

12.4 Perceived risks of genetically modified crops Food is inseparable part of existence of human life. The cultivation of crop for food and feed started with human civilization. Soon, crop varieties were selected according to regional, environmental, and population needs, naturally or by human interventions. These changes, however, were very slow and accepted unknowingly. With the exponential increase in population, small changes in agriculture were not sufficient to provide food security. In 1990s scientists experimentally proved that genetic modification of crops for higher yield, better nutrient content, and remedial for environment could be easily achieved. Genetic modification of crop for better traits resulted in greater productivity, reduction of pesticides and fertilizers usage, giving economical benefits to farmers and consumers. A recent meta-analysis of 147 agronomical studies suggested that adoption of GM increased farmer’s profit by 68% on average, crop yield rose by 22%, and the pesticide expenses were reduced by 39% (Klümper and Qaim, 2014). The easy use, time saving, and more flexibility in planting time are associated benefits of GM technology to farmers (Fernandez-Cornejo et al., 2014; Carpenter, 2013). The adoption of GM crops increased rapidly in the last two decades, making this technology the fastest adopted one in history of agriculture. The adoption rate is more than 90% for soybean, maize, and cotton in United States, cotton in China and India, soybeans in Argentina and

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Brazil, and rape oilseed in Canada. Many countries have now approved GM crops and their produce for animal feed as well. In United States alone, GM feed is consumed by more than 95% of food-producing animals. The global demand for GM feed is rising as no detrimental effects were recorded when compared with non-GM feed. Even in EU, majority of soybean-based animal feed contains GM products (Van Eenennaam and Young, 2014). Thus transgenic products constitute a significant proportion of globally traded commodities. The consumer’s perception toward acceptance of genetic modification for incorporation of beneficial traits in food and feed has been subject of continual study (Connor and Siegrist, 2010; Siegrist, 2008). People’s attitude toward food technologies are often based on facts and perception outside the realms of scientific evidences. The use of GM crops benefited farmers with increased production per hectareand reduced use of fertilizers, pesticides, and herbicide. The large-scale production of GM crops reduces carbon and nitrogen (greenhouse gases) emission, environmental footprint of production process, and the pressure for expansion of cultivable land. Less crop failure, cost saving in agricultural inputs, and less requirement of time and efforts provides economic benefits to farmers (Qaim, 2009). These benefits are passed down to consumer without them being aware of it. Studies suggest that the prices would have been higher for soybean, maize, corn, and other food product in the absence of GM crops (Barrows et al., 2014; Brookes and Barfoot, 2014). These are the benefits not considered by the consumers while making their decision to oppose the GM technology. Consumers are more apprehensive about the probable losses, which are based on likelihood of happening of unforeseen results of an involuntary event. The debate sparked off with the declaration by Árpád Pusztai about the damage to rat gastrointestinal tract when they fed with potatoes expressing lectin (GNA), a sugar binding protein from snowdrop (Ewen and Putzatai, 1999). Both, media and nongovernmental organizations (NGOs) broadcasted this to such an extent that it became a societal concern for Europe. Study on toxic effects of pollens from Bt corn on monarch butterfly (Losey et al., 1999) further influenced consumers, environmental activists, and NGOs about the probable loss to humans, animals, biodiversity, soil, and ecosystem, due to the use of GM food, feed, and products. Although the speculated damages were not found in subsequent studies (Batista and Oliveira, 2009), this developed a doubt about the GM technology and GM food.

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12.4.1 Consumption of foreign DNA The people are very apprehensive about consuming foreign DNA. The opposing consumer believes that the presence of foreign DNA may bring about the unexpected change in the nutritional quality or may make it toxic. The integration of transgenes may change the metabolic profile of the plant due to their potential pleiotropic effect or properties of transgene may change in new chemical environment (Bawa and Anilakumar, 2013); however, so far, no conclusive reports have been published showing significant health hazards directly linked to consumption of GM food (DeFrancesco, 2013; Nicolia et al., 2013). The trans- and cis-integration of foreign DNA is natural in the environment and natural selection of favorable events in agriculture has led incorporation of favorable traits.

12.4.2 Allergenicity GM food technology is also labeled as imperfect technology by opponents, especially, after StarLink controversy. Apprehension that GM technology apart from improving nutritional value of food may also introduce allergens as well. In 1998, the US Environmental Protection Agency (EPS) approved genetically engineered yellow corn variety with Bt toxin (Cry9C) called StarLink, developed by Syngenta (then Aventis Crop Science) for animal feed. Bt toxin selectively kills European corn borer and hence GM crop would have advantage from infection. Heat-resistant Bt toxin was found to be resistant to human gastrointestinal digestion suggesting that it may cause allergies, when ingested. Although conclusive studies were not conducted, it was prohibited for human consumption. Syngenta was forced to withdraw its maize-based products from United States, EU, Japan, and South Korea when genes from StarLink corns were detected in food residues. Syngenta discontinued the cultivation of this variety but many other types of Bt corn, after clearing the stringent allergenicity and toxicity tests, are currently cultivated. All this had, however, led to some mistrust in the transgenic technology and GM foods and its adoption.

12.4.3 Horizontal transfer of genetic material Another perceived risk factor associated with GM technology is horizontal transfer of genetic material. The transfer of novel gene to GM crop confers a new trait to the crop and seen as source of potential harm to consumer or environment by opponents. The potential transfer of transgene

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whole or part, especially traits like antibiotic resistance markers, may compromise human or animal immune system (Bennett et al., 2004). According to therapeutic use to animals and humans, antibiotics have been divided into three groups (Van den Eede et al., 2004). The two commonly used antibiotic markers for the selection of transformants, nptII gene conferring resistance to kanamycin, and hpt conferring resistance to hygromycin B belong to group 1 antibiotics, which have no or limited therapeutic use to humans and animals. The probable transfer of gene from plant to bacteria could also lead to the transfer of other bacterial genes, especially the antibiotic resistance gene, used for selection for transformants (Pontiroli et al., 2007). Such genes are, however, already present in abundance in environment and are readily transferred by conjugation and transduction rather than horizontal transfer. Opponents fear that the transfer of viral gene to another virus or bacteria may result in new disease (Falk and Bruening, 1994), or transfer to plant may result in super plant or super weed or transfer of viral gene to human or animals may induce oncogenes (Ho et al., 2000). PetersonBurch and Voytas (2002) reported the presence of 276 retrovirus-related elements related to the Pseudoviridae in Arabidopsis genome. Thus risk posed by these endogenous sequences is more than from the risk of transfer from other sources. The fear of transfer of DNA from GM crops to animals and humans is another concern by the critics of GM technology. Multiple copies of DNA fragments survived after the intestinal digestion were detected in some chicken tissues but not in eggs (Einspanier et al., 2001). Even in animal, small fragments could rarely be detected in other tissues except for milk or blood (Beagle et al., 2006; Deaville and Maddison, 2005; Phipps et al., 2003; Rossi et al., 2005; Wiedemann et al., 2006). The reports therefore, suggest transient uptake of bacterial and viral DNA by somatic cell in animals but no demonstration of stable integration in germ line cells (Van den Eede et al., 2004). Thus fear of horizontal transfer of antibiotics or antiviral traits from GM food to microbial flora of gut or mammalian tissue is unwarranted. Although the chances of transfer of antibiotic resistance gene from transgenic plant are very low, precautions should be taken to make it more unlikely. To gain public acceptance of GM technologies, transparent and targeted communications regarding the use of genetic material for transformation and method for selection of transformants should be done. Development of transgenic plants using genes and protein sequences

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having representation in bacterial genome should be avoided. Antibiotics that are not commonly used for human drug therapy should be used to curb the acquired antibiotic resistance in case of horizontal transfer of genetic trait to microorganism inside the body. Removal of marker gene either through bred out or using guided nucleases should be done before the commercial release ( Rukavtsova et al., 2013; Ebinuma et al., 2001) or use nonantibiotic markers like mannose, green florescence protein (GFP), or GUS (Joersboand Okkels, 1996).

12.4.4 Super plants an environmental risk Cross-pollination among the cultivated and wild varieties of a crop is a natural phenomenon (Snow et al., 2000). This caused concern among the critics of the transgenic technology. Cross-pollination of the transgenic crops with related weeds in nearby area may assist the transfer of new traits probably making them super weeds, posing a risk to environment. Also, wild relative may pick up the transgene, which is real threat to natural gene pool. The probability of spreading of transgene is different for different area. The chance of transfer of transgene from GM wheat to wild variety is more in United States because of the natural occurrence of wild relatives. The wheat, however, is self-pollinated; hence, the risk of transfer of transgene to nearby weed or wild relative is minimal. The transfer of transgene from transgenic corn is also less likely in Europe or United States due to the absence of naturally occurring wild relatives. Refuge areas are mandatory with planting of GM crop, to further reduce the risk of transfer. These areas provide habitat to wild-type insects to feed, mate, and reproduce with possible resistant insects thriving on GM crops, to produce susceptible insects.

12.4.5 Effect on nontarget organism Other major concern of critics of transgenic technology is probable impact on nontarget organism. The decrease in monarch butterfly population in and around Bt corn fields attracted considerable public attention. The decline was linked to the destruction of natural habitat (milkweed) of butterfly, which grows with the corn and ingestion of toxin present in pollens from Bt corn deposited on milkweed leaves. Spraying of herbicides kills all nonresistant plants causing the destruction of habitat. Losey et al. (1999) reported high mortality in larvae of monarch butterfly due to ingestion of toxic pollen dusted on milkweed leaves. Other studies,

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however, showed negligible toxic effects of pollens from Bt corn on butterfly (Sears et al., 2001; Wraight et al., 2000). Hence, there is no clear explanation for decline in butterfly population and GM crops could not be solely blamed for the decline. Studies with Bt corn root exudates showed no detrimental effect on soil micro fauna and hence safe for soil bacteria, fungi, protozoa, and other insects (Saxena and Stotzky, 2001). Detrimental effects of insect-resistant transgenic crops were, however, seen on beneficial insect such as lacewings (Hilbeck et al., 1998) and ladybird (Birch et al., 1997). The changes in agricultural practices and land use could be other unrelated factors affecting the distribution of beneficial organism. However, more comprehensive studies are required to assess the effect of GM technology of other organism.

12.4.6 Contamination of environment with genetically modified proteins The leaking of active proteins and chemical compound from GM crops into the soil is a potential environment concern proposed by public. The losses due unintentional releases are difficult to assess. Bt toxin residue is released in soil by the plant and is stable for long time (B200 days) after the harvest. The Bt toxin was found in the gut and cast of earthworm; however, no difference in their weight or mortality was found. Further, comparison of soil rhizosphere of Bt and nontransgenic crop showed similar number and composition of other soil organism (including protozoa, bacteria, and fungi) (Saxena and Stotzky, 2000). Genetic manipulation leading to incorporation of desired traits has been a key technology to ensure food security especially in countries where majority of population depends on agriculture for livelihood. However, no technology is free from errors. Genetically engineered crops not only provide advantage of high yield, better quality, and reduced environmental risks but also possess new challenges. The arguments and beliefs for and against the use of GM food should be discussed case by case.

12.5 Safety assessment of genetically modified technology GM crops have shown substantial benefits in terms of food security and economic growth. Since 18 years of first introduction of transgenic

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crop in agriculture, the coverage acreage is increasing steadily. More and more farmers are readily adopting the technology and changing the farming landscape from convention plants to transgenic crops with enhanced yield, better nutrient quality, and environment friendly. The GM technology, however, has to face opposition due to apprehensions about the probable damage risk posed to humans, animals, and environment. Further organic crop production is now being advocated by opponents of GM technology as safe and better practice. Safety assessment of GM food therefore, would not only ensure that no compromises are made with consumer’s health but checks for fair practices in food trade as well. The safety of GM food has been matter of concern and risk assessment, at regional level on case-to-case basis, and is also critical for public acceptance. Local population, regional beliefs, and geographies are also critical for evaluation of suitability of GM crops for cultivation and consumption. According to the European Commission and FAO, genetically modified organism (GMO) is defined as organisms produced by altering the genetic makeup, which otherwise not happens naturally by mating and/or natural recombination. This definition also covers several crops that have been accepted as conventional but are otherwise man made. Triticale wheat was made by duplication of genetic pool of sterile hybrids obtained by crossing wheat with rye to develop more nutritious food source (low gluten and high protein). According to the Cartagena Protocol, genetic modified organism is defined as living modified organism possessing a novel genetic material by the use of modern biotechnology tools.

12.5.1 Codex Alimentarius and Codex Alimentarius Commission Risk assessment, risk management, and risk communication are the analytical tools used to assess risk associated with the GM produce used for human consumption. The results of these analyses are intended to be used in making decisions and policies. The coordinated efforts of World Health Organization (WHO) and Food and Agriculture Organization (FAO) ensure safety, quality, and fair trade associated with GMO through Codex Alimentarius International food standards, guidelines, and codes of practice. The 11th FAO conference established the Codex Alimentarius Commission (CAC) in 1961 and was later on supported by WHO to implement a joint FAO/WHO Food Standards Program. It is an intergovernmental body ensuring safe consumable food and products to

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consumer, fair practices in food trade, and coordinate international food standardization work. Currently, the CAC has 189 Codex members (188 member countries and 1 member organization (The European Union)). India signed to become a member of CAC in 1964. The Latin term “Codex Alimentarius” means food code. Codex food standards are based on scientific proofs provided by independent international risk assessment bodies or ad hoc consultations organized by FAO and WHO. Codex Alimentarius includes standards for all principal food (raw, semiprocessed, or processed) intended for distribution to the consumer. The Codex Alimentarius also includes standards for food hygiene, residues of pesticides and veterinary drugs, food additives, contaminants, methods of analysis, labeling and presentation, and import and export inspection and certification. Codex Alimentarius standards and related text are intended to provide consumers a correctly labeled safe, wholesome food product, which is free from adulteration. Every country’s laws and administrative procedures contain provisions in accordance with for Codex standards for food and derived product and needs to be complied absolutely.

12.5.2 Framework for safety assessment Safety assessment is structured analysis to identify, if any, associated hazard, nutritional, or other safety concern as a result of intended uses under the anticipated conditions of processing and human consumption. The nature and severity of concern are assessed following a structured and integrated approach to collect and analyze related information on case-by-case basis. If required, the food or processed food may be subjected to risk management before it is considered for commercial distribution. In the absence of existence of conventional counterpart data for comparison, the safety assessment must be done with the data derived directly from experimental studies or historic references, on case-by-case basis. According to Codex Alimentarius, the safety assessment of a food derived from a recombinant-DNA plant follows a stepwise process of addressing relevant factors that include: 1. Description of the recombinant-DNA plant ( GM plant): An elaborate description of the GM plant along with the transformation event(s) leading to modification, is important. The pedigree map of each transformation event, the type, and purpose of the modification should be submitted in detail to assess the safe use of said plant for food.

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2. Description of the nontransgenic host plant and its use as food: A comprehensive description of the native host plant is essential for safety assessment comparison and should include: a. Common/usual name, botanical name, and taxonomic classification; b. Origin center, cultivation history, and development through breeding, in particular identifying traits that may adversely impact on human health; c. Complete information on the host plant’s genotype and phenotype relevant to its safety, including any known toxicity or allergenicity; and d. History of safe use for consumption as food. The relevant phenotypic information should also be provided for related species and for plants having significant contribution to the genetic background of the host plant. A food may be considered to have safe history, if it has been used for a long time, for a number of generations, by diverse human population. Detailed history of plant cultivation, transportation, and storage is also essential for safety assessments. Plant part used as food source, contribution of important micro and/or micronutrients to the diet, and requirement of special processing to make plant safe to eat are also important factors. The information should be from referenced sources. 3. Description of the donor organism(s): Detailed information should be provided on the donor organism(s) or the other related family member especially, where the donor organism(s) or other closely related family members naturally exhibit pathogenicity or produces toxin, or have other traits that affect human health (e.g., presence of antinutrients). The description of the donor organism(s) should include: its usual or common name, scientific name, taxonomic classification, information about the natural history as concerns food safety. The information should also be provided on naturally occurring toxins, antinutrients, and allergens produced by the donor organism and for microorganisms, additional information on pathogenicity, and their relationship to known pathogens is required. 4. Description of the genetic modification(s): The elaborate details should be listed about all the genetic material delivered to the host plant including information on characterization of DNA inserted in the plant and mediatory vector used. The details about method of transformation should be included in description along with the DNA used

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to modify the plant (vector or helper plasmid), its source, and its expected role in plant. The host to produce DNA for transformation of plant and any other intermediate host organism, should be described in detail. The detailed information about the genetic component including coding region, marker gene, regulatory element and other character affecting the function of introduced DNA, is required. The function, size, and identity along with the location and orientation of DNA sequence in the final construct/vector should be described in detail. 5. Characterization of the genetic modification(s): To assess the impact of inserted DNA on the composition and safety of foods derived from such plants, a comprehensive molecular and biochemical characterization of the genetic event carried out should be provided. Comprehensive information regarding the inserted DNA including sequence of DNA, number of insertion sites, number of insertion carried out, the organization of inserted DNA at the site of insertion, genomic organization around inserted site should be provided to identify any substance expressed as an outcome of inserted DNA. Details about open reading frame(s), if any, within the inserted genetic material or created by insertion into the plant genome including those that results in fusion proteins. The explicit information should be provided on any expressed substances due to recombinant DNA in plant including the gene product (s) and their function, the phenotypic description of the new trait(s), the level and site of expression of inserted gene product in plant particularly in edible portions. 6. Safety assessment: Safety assessment should take into account the biochemical nature and function of newly expressed substance and estimate the concentration of the same in edible parts of the host plant. Current dietary exposure and possible effects of human sub groups should be consider while doing the safety assessments. If the expressed substance is a protein, assessment of potential toxicity based on amino acid sequence similarity to known toxins and antinutrients (lectins, protease inhibitors, etc.) should be done. The substance should be evaluated for stability to processing, heat, and degradation in appropriate representative intestinal and gastric model systems. Potential allergenicity of newly expressed protein should be done by case-by-case approach and essentially use combination of criteria as no single criteria sufficiently predicts allergenicity or nonallergenicity.

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12.6 Assessment of possible allergenicity Allergy is a very serious condition and the whole body can be at risk especially during severe allergic reaction such as anaphylaxis. It has become a major concern worldwide in the last few decades (Ladics and Selgrade, 2009). Food allergies are affecting as many as 3% 4% of adults and 6% 8% of children. According to FAO, the newly introduced gene should be evaluated with respect to already know allergens. Further, if any unintended event results in production of new substance, then that substance should be tested for possible allergenicity using similar approach. In 1996 test results of GM soybean showed 27% higher levels of trypsininhibitor than the nontransgenic soybean. In 2008, GM Bt maize showed the presence of altered form of allergen, zein protein. Thus the genetic manipulation may alter the existing protein profile and may become a major health concern. The testing for allergenicity of transgene must be done before the introduction of GM for commercial use. A rigorous protocol has been developed by internal scientific agencies to assess the potential allergenicity of new developed product. The assessment ensures that consumers get GM crop that do not pose allergic risks unless the allergens are present naturally in conventional counterpart. The first guideline for food safety assessment was released in 1996, jointly by the International Food Biotechnology Council (IFBC) and the Allergy and Immunology Institute of International Life Sciences Institute (ILSI). FAO and WHO together published a refined guideline in 2001 followed by guidelines by Codex Alimentarius Commission (2003).

12.6.1 Source of the gene First, the source of transgene is evaluated for the allergenicity in terms of allergenic, moderately allergenic, or of unknown potential to cause allergic reactions. Rigorous assessments are done to conclude that novel gene to be transferred does not code for an allergen. Usually, the transgene selected for crop modification is taken from source with unknown allergenic potential.

12.6.2 Sequence homology studies Sequence of transgene is compared with the amino acid sequences of all the known allergens. The information for the active tertiary and

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quaternary structure is directed by the primary sequence of protein. Therefore, if two proteins have linear sequence homology, they potentially might share similar three-dimensional structural domains and motifs and may show allergenic cross-reactive epitopes. The guideline by FAO/WHO states a six amino acid match between the two sequences indicates the likelihood of cross reactive epitopes with allergen. If the sequence similarity between the known allergen and transgene is .30% in an 80 amino acid sliding window, then the transgene is considered to be an allergen. FAO/WHO 2001 and Codex (2003) guidelines suggest further safety assessment with IgE serum screening. Limited information is available about food allergen and screening with such small window may lead to high false positive.

12.6.3 Physiochemical stability No specific set of features are defined, which confers allergenicity to certain proteins. Food allergens are generally found to be thermal and pepsin digestion resistant. These thermal resistant proteins tend to resist breakdown due to the heat treatment used during food processing procedure. Depending upon the nature of protein, the allergic potential may be increased or decrease by heat treatment. Pepsin resistance is listed by Codex 2003 guideline as an important safety assessment for allergenicity testing. Simulated digestive fluids are used to carry out in vitro pepsin digestion assay. The nonallergenic foods are digested by these enzymes, whereas many food allergens are resistant to degradation.

12.6.4 Serum screening The newly expressed product should be subject to targeted and specific serum screening to assess the allergenicity. Serum screening evaluates IgE binding and cross reactivity of the transgenes. Targeted screening for allergenicity uses sera from samples sensitive to allergen source, starting from source of gene to the broad taxonomic groups (monocots and dicots). In specific serum screening, the allergenicity is assessed by testing against the sera samples that are allergenic to or sensitized to the source of genetic material or to the source of sequence-matched allergen. Codex Alimentarius recommends specific serum screening for identification of potential antigens.

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12.6.5 Testing models Testing models mimicking the human disease conditions present a useful platform to study the onset of disease, nature of disease triggering molecules, diagnosis, and evolving therapies. Animal model is recommended by FAO/WHO 2001 guidelines. The animal model must have similar allergenic response as of humans and should be able to elicit immune response upon allergen administration, using typical routes of exposure. No adjuvant should be required to elicit immune response as it may influence the antigen-specific response. The model should show an IgE response as well as other Th2 associates immune response to allergen. The models should produce reproducible, sensitive and specific responses, and results. Use of animal models from two different species and use of two different route of administration in single species are recommended. The manifestation of allergic reaction is different for different allergen and is different in different individual; therefore recommending single animal model to assess the allergenic potential of introduced novel food allergen is difficult. Codex Alimentarius abandoned the use of animal model for testing of allergenic potential. Allergenic potential of novel transgene has been evaluated using the guidelines for safety assessment of GM food. Allergenic cross-reactivity of routinely used six transgenes (Triticum aestivum beta-1,3-glucanase, Bacillus subtilis glycine betaine aldehyde dehydrogenase (gbs A), Medicago sativa beta-1,3-glucanase, Oryza sativa chitinase, Nicotiana tabacum osmotin, and Nicotiana plumbaginifolia mitochondrial manganese superoxide dismutase) for genetic modifications of crops was tested using in silico methods (Mishra et al., 2012). No enhancement of allergenicity was recorded for mustard expressing bacterial cytosine deaminase (CodA) gene as compared to nontransgenic mustard (Singh et al., 2006). Osmotin, a protein belonging to pathogenesis-related protein used to develop biotic and abiotic stress tolerant plant, shows potential allergenicity. Osmotin was resistant to heat treatment and pepsin digestion. Cross-reactivity with tomato and apple allergens was also recorded (Sharma et al., 2011). Bacterial choline oxidase (Cox) gene has been used to develop tolerance against abiotic stress in plants. No significant allergenicity and toxicity was recorded against Cox gene in mice (Singh et al., 2008). The human population is challenged by variety of food allergens. Codex Alimentarius provide a guideline to submit information necessary to assess the toxicity and allergenicity of novel genetic material introduced

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and product there off. All the guidelines may not be relevant in every case. The interpretation and explanation may vary case-to-case basis according to new scientific developments.

12.7 Potential accumulation of substances significant to human health The safety assessment should be done for indirect induction or accumulation of potentially hazardous substances like pesticide residues, contaminant, toxic metabolite, or other substance, which may be relevant to human health. The food from transgenics containing antibiotic resistance marker gene should be evaluated for the clinical and veterinary use of marker antibiotic. Marker genes encoding antibiotics, which are used in the treatment of humans and animals, should not be used. In case of nontherapeutic antibiotics, the assessment should comprise of the effects of antibiotic in gastro-intestinal tract when ingested per say, effect of dosage and effect of exposure to digestive enzymes on antibiotic gene product.

12.8 Intellectual property rights in transgenic agriculture biotechnology Intellectual property rights (IPRs) are rights granted to creators of intellectual property by governments for their creations of mind, which are novel and original in the global context. IPRs treated as an intangible property, originated or produced through the efforts of the inventor involving human skill, intelligence, labor, and efforts. These rights largely are territorial rights except copyright, which is a global right and given for fixed period of time. IPRs are therefore rights developed to give creative people an ownership right over their creation in return to disclosure of their work for the benefit of mankind. These rights protect the work of innovators from being copied or imitated. Once the exclusive ownership is granted, property cannot be used without the consent or permission of awardee. After the expiry of protection time, it is available to be used freely, without the permission of owner. Like any other form of property, IPRs can also be used, sold, loaned, gifted, and even stolen.

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Existence of IPRs in its precursor form can be traced back to as early as ancient Roman societies. The modern concept of intellectual property mostly developed in England during 17th and 18th centuries. The Statute of Monopolies (1624) and the British Statute of Anne (1710) are considered as the statutes giving rise to the patent law and copyright law, respectively (Brad and Lionel, 1999). The first use of term “Intellectual property” can be traced back to a monthly review published in 1769. The world economy is now gradually shifting from a labor and capital intensive economy to knowledge-based technological developed economy. Technological innovations are reshaping world’s economy faster than ever. Economic prospects of a country depend on innovative knowledge and technological developments. The protection under IPRs is granted to the technology developed, research made, or invention done in a country and in turn an exclusive right of commercializing the same. This competitive advantage granted to IP in commercial and industrial levels ensures earning of foreign currency and increases export. The economic gain linked to IP inspires the industries to invest in development of new knowledge. Country and its people thus progress both economically and intellectually. Therefore the role of IPRs has become very important in furtherance of economic interests of a country. Presently, the legal mechanism of copyright, trademarks, trade secrets, geographical indications, industrial design, patents, lay-out design of integrated circuits, protection of undisclosed information, and protection of new plant variety and farmers’ rights are defined categories of IPRs used to protect newly developed intangible property, world over. Thus understanding of legal aspect of acquisition and deployment of research leading to invention is very important for both researcher and research administrator to achieve greater globalization of trade. Research and development in the plant biotechnology and agricultural sector is unique among the industries. The scientific intervention in agriculture using biotechnology brought Green Revolution in late 20th century. End of starvation and alleviation of nutrient status especially in developing countries was regarded as one of the important achievements of the century. Over the years, due to urbanization and indiscriminate use of natural resources resulted in sudden climate changes, which has become a large and complex problem for agricultural productivity. The impact of increase population and drastic climate changes has limited the effect of Green Revolution. New approaches to genetically engineer plants with

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desired traits are required for continual improvement in agriculture to achieve same effect. Biotechnology has been successfully used for crop improvement. Tissue culture has been used to produce clonal population, virus-free strains, and shortening the regeneration period. The technique has been successfully used for banana, palm, potato, cassava, yam, etc. Various crops have been genetically engineered for biotic and abiotic stress tolerance, pest and disease resistance, and better nutrient utilization efficiencies. DNA typing and profiling have been used to understand the pathogen diversity, host pest interaction, and plant stress response mechanism. Molecular-assisted classification has been useful in identification, collection, storage, and maintenance of germplasm banks. The biotechnology is also instrumental in value addition of agricultural produce. The plants and tissue culture is being used for expression of pharmaceutics and neutraceutics. So, the impact of biotechnology on agriculture is far from the expectation, with coming up of new application every day. The key issues with the research and innovation in plant biotechnology and agriculture are that innovations and information sources are far more geographically dispersed than in any other industry. Research and development in plant biotechnology is dominated by public research institutions and nearly two third of share in research and development in these areas are contributed by these institutions. Private sector involvement in innovation in these areas is a recent phenomenon. Presently, both public and private sectors are involved in generation of IPRs in agricultural biotechnology. Moreover, there is an urgent need to understand and promote the importance of sharing the information and invest in instruments that support sharing of information with giving due credit to originator or creator. The area of research and development in agriculture and plant biotechnology are very vast. So, presently it is not possible to protect everything under a single IPR category. A number of IPR categories are used to protect the different aspects and inventions in area of plant biotechnology and agriculture. Some important categories of IPRs used in providing protection to inventions and new developments in plant biotechnology and agriculture are:

12.8.1 Trade secrets Trade secrets are proprietary information (such as formula, business plans, device, tool, manufacturing processes, inbred lines required for hybrid

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seed production) that have commercial value and that the firm possessing the information makes an effort to physically conceal from its competitors to prevent them from duplicating or using it. There is no property right to the secret information per se, but a common law right to maintain secrecy, if feasible. Secrecy is often hard to maintain with regard to plants and seeds because if these are once distributed, then the competitors may reverse engineer the trade secret. Trade secrets may be more practical, where secrecy issue is of lesser value, for example, innovations in parental lines for breeding programs or seeds, which are never themselves sold. This form of protection is very weak protection. If a trade secret is kept secret and misappropriation can be proven, then trade secret protection can be effective. The owner of a trade secret about the genetic makeup of corn seed recovered $46 million in damages for trade secret misappropriation by proving the misappropriation done by the other company.

12.8.2 Geographical indications A geographical indication (GI) indicates a specific geographical origin of a product and specific qualities or a reputation attached with that product are due to the specific geographical origin. To function as a GI, a sign must identify a product as originating in a particular geographical region and the qualities, characteristics, or reputation of the product should be essentially due to the place of origin (Kailasm and Vedaraman, 2003). Examples of some GIs are Roquefort cheese, Georgian wine, Scotch Whisky, Pinggu peaches, Darjeeling tea, Agra petha, Kanjivaram silk, and Basmati rice. Although GI as IPR is not involved with protection of new plant varieties, it helps in the protection of an existing plant variety, which has specific qualities or a reputation due to that particular geographical origin. A GI can be registered by any association of persons, producers, organization established by or under the law, representing and protecting the interests of the producers. Any person, manufacturer, or producer residing in that particular geographical region may use the registered GI by taking permission from GI holder. A manufacturer or producer may use GI as well as his trademark together for a GI product. The term of protection for a GI is 10 years, which can be extended for indefinite period by renewing after every 10 years.

12.8.3 Trademarks Trademarks are any sign, symbol, or marking, which help recognize and differentiate a good or service of one entity from the others. They also

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provide assurance of certain qualities associated with these names (McCarthy, 1996). According to Article 15.1 of the Trade-Related Aspects of Intellectual Property Rights (TRIPS) Agreement, “any sign, or any combination of signs, capable of distinguishing the goods and services of one undertaking from those of other undertakings must be eligible for trademark protection.” These signs could be words including personal names, letters, numerals, figurative elements, and combinations of colors, as well as any combination of signs. These signs play a critical role in marketing of a service or product as a brand. Branding has been extensively used in agricultural sector by food and drink manufacture, agricultural tools and machines, seed companies, etc. This legal right enables the owner to use the registered mark in relation to particular good and exclude others from commercial use of mark (Narayan, 2000). The TRIPS agreement provides for minimum term of protection for a trademark for 7 years, which can be renewed after every 7 years to extend the protection. Most of the countries, including India, today provide protection for trademark for 10 years, which can be extended for unlimited period of time by renewal after every 10 years.

12.8.4 Copyright and related rights The legal rights granted by government for a limited period of time to protect the particular form, way, or manner in which an idea or information is expressed are defined as copyrights. These rights do not protect the idea per se but the expression of idea. An idea can be expressed in number of ways and each unique way of expression of idea generates a new copyright. Only condition is that each way of expression of that idea should be sufficiently different from each other. Copyrights are a bundle of rights, which include rights of reproduction, communication to the public, adaptation, and translation of the work. Thus these are exclusive rights, which are given when an idea is translated into a tangible property. It is very relevant and important to include copyright notice especially to the published work although no more mandatory by law. The copyright law balances the private and public interest by the fair use doctrine, which allows limited use of copyrighted material for educational purpose, comments, criticism, news reporting, or research without infringing. In the last decade, there have been a lot of issues regarding the fair use. The copyright protects the rights of creator and ensures that the creator gets his dues. In agricultural biotechnology, protection by copyright is

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given to some areas such as genome and proteome database, geographic information system images. In the absence of clear boundaries, it is difficult to assess the impact this restriction on research and development. As for now, there is no global agreement on enforcement of copyright; therefore a common global platform needs to be developed. Also, the extent of modification is required to ascertain that the expression of idea is novel, should be defined. These are some issues, which need to be addressed on the global platform.

12.8.5 International organization and agreements for intellectual property rights protection The Paris Convention, one of the initial treaties on intellectual property, was signed on March 1883 in Paris, France. Paris Convention through Patent Cooperation Treaty (PCT) sets the basic statutory principles to harmonize the mechanism of filing patent application in the member states. The treaty is administered by World Intellectual Property Organization (WIPO) among its 170 member countries. Apart from patents, the Paris Convention extends protection to trade names, trademarks, and services marks. Filing patent application with Paris Convention establishes a right of priority and the applicant can use this first filing date as an effective filing date in other member countries, provided the application is file within a year of the filing date. The members also have privilege to joint PCT, which allows member countries to file international applications. WIPO, established in 1967, is a self-funding agency of the United Nation. It is a global forum administering intellectual property information, policies, services, and cooperation with 192 member countries. Patent Cooperation Treaty (PCT) facilitates filing of international patent application by providing a standard application format and centralized filing procedures. An applicant can file for national patent at patent office of country according to his residency or nationality or file an international patent application with WIPO, Geneva. The detailed information provided in PCT format not only reduces the burden on applicant to file same information multiple time but also lessens the burden on the local patent offices in the presence of international search report and written opinion. The PCT report also helps third party to decide about patentability of the invention. Trade-Related Aspects of Intellectual Property Rights (TRIPS) is an international agreement adopted by World Trade Organization (WTO) in 1994

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under General Agreement on Tariffs and Trade (GATT). It was enforced from 1 January, 1995 to all the members (157 countries) of WTO. The TRIPS agreement states that how IP should be protected when trade is involved. Article 27.3(b) of the TRIPS Agreement provides the provisions for the protection of new plant varieties. These provisions provide that member states shall provide for protection of plant varieties either by patents or by an effective sui generis (Latin term: of its own kind) protection or both. The International Union for the Protection of New Varieties of Plants or UPOV (French: Union Internationale pour la protection des obtentions végétales) was established in 1961 to provide protection to new plant varieties and farmers’ rights. The convention was adopted in Paris and came into force by 1968 and revised thereafter (1972, 1978, and 1991) to adjust new developments in plant breeding and improvement. The objective of this convention is to protect the new varieties of plants as an intellectual property. All the provisions of this convention were adopted in TRIPS for protection of new plant varieties and farmers’ rights. According to the act, for a new plant variety to be protected, it must be novel for the region where rights are applied for, must have distinct characteristics from other available varieties, should exhibit homogeneity. The distinctive trait/s in the new variety and must be stable and uniformly distributed in progeny such that the plant remains true to type even after repeated cycles of propagation. Protection of Plant Variety and Farmers Right Act, 2001 (PPVFR Act) ensures the establishment of an effective system for protection of plant varieties and rights of farmers and plant breeders. Such protection by law stimulates investment in research and development to facilitate growth of the seed industry and agricultural biotechnology. It also ensures the availability of high-quality seeds and planting materials of improved varieties to farmers.

12.8.6 Patents WIPO has defined patent as “an exclusive right granted for an invention, which is a product or a process that provides, in general, a new way of doing something, or offers a new technical solution to a problem.” Patent is a legal instrument, which gives its owner the right to exclude others from making, using, selling, and importing the invention for a limited period of time. It further emphasizes that to get a patent, technical information about the invention must be disclosed to the public in a patent

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application. The information about the invention could be used by others to make more new invention, thus using the technology for further benefit of society. The disclosed information could only be used for research and can be commercially exploited only after the consent or license agreement with the patent holder. The patent is granted on the basis of novelty of the invention, the inventive step, and its commercial application and cannot be granted for mere discovery, idea, or principle. The subject matter for patent should be a material or process and could be manufactured or leads to manufacturing a product ultimately used by the society. The term patent has originated from the Latin word “patere,” which means “to lay open” or to make available for public inspection. A patent is provided for a new, nonobvious, and useful process, machine, article of manufacture, composition of matter, and/or improvement of any of the above. A patent cannot be granted for laws of nature, abstract ideas, physical phenomena as well as literary, dramatic, musical, and artistic works and inventions, which are not useful or are offensive to public morality. Design patents, utility patents, and plant patents are the three major types of patents granted by different countries. Design Patents: The design patents protect the esthetic appearance of a product or object. Design patents can be issued for the appearance, design, shape, or general ornamentation, pattern and color combination of an invention. The term of protection for a design patent is 14 years from the date of patent granted. India does not grant a design patent but certain countries like United States do. The industrial designs, however, are protected in India by a separate statute of Indian Designs Act. Utility Patents: The most common type of patent recognized by countries world over. These patents protect the utility or functional aspects of an invention, including machines, processes, methods, compositions, formulations, and anything manufactured that has a useful and specific function. The term of protection for these patents is 20 years all over the world. Plants are also protected under utility patent by some countries. Countries like United States have started allowing utility patents for plants since late 1980s. Utility patents have been issued for man-made or genetically engineered plants or elements of plants. These plants can be propagated either sexually (seeds) or asexually (clonal propagation). The patents can be sought for components of plant such as DNA, genes, proteins, pollens, buds, fruit, plant-based chemicals, and the procedures used in the production or control of production of these plant products. The utility patents protection is now extended to cover innovations like research

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tools, transformation processes, vectors, and components of vectors such as markers and promoters. To obtain a utility patent for plants, the plant must be made or GM by the act of inventor and must satisfy the statutory requirements of nonobviousness, novelty, and utility. The patent must describe the modification and claim protection for specific characteristics of the plant, which resulted from the modification. Utility patent is a strong form of protection but is difficult and time-consuming to acquire. The right is considered to be infringed if the plant protected by a utility patent is reproduced either sexually or asexually by anyone, without the consent of patent holder. Plant Patents: The Plant Patent Act established in 1930 by US Government grants the inventor (or the inventor’s heirs or assignee) who asexually reproduced a distinct and new variety of plant, other than a tuber propagated plant or a plant found in an uncultivated state. The plant discovered in uncultivated or wild state cannot be patented; however, a plant discovered in a cultivated area can be patented. The first plant patent was issued to Henry Bosenberg for his climbing, ever-blooming rose in 1931. The term of protection under plant patents is 20 years from the date of filing the application. This legal document protects the inventor’s right to exclude others from asexually reproducing, selling, or using the plant. According to the PPA, 1930, the protection is limited to a plant in its ordinary meaning: • A living plant organism, which expresses a set of characteristics determined by its single, genetic makeup or genotype, which can be duplicated through asexual reproduction, but which cannot otherwise be “made” or “manufactured.” • Sports, mutants, hybrids, and transformed plants are comprehended; sports or mutants may be spontaneous or induced. Hybrids may be natural, from a planned breeding program, or somatic in source. While natural plant mutants might have naturally occurred, they must have been discovered in a cultivated area. • Algae and macro fungi are regarded as plants, but bacteria are not. The plant patent must also satisfy the general requirements of patentability. To be patentable, it should comply with following requirements: • The plant was invented or discovered and, if discovered, the discovery was made in a cultivated area. • The plant is not a plant, which is excluded by statute, that is, the part of the plant used for asexual reproduction is not a tuber.

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•

The person or persons filing the application are those who actually invented the claimed plant, that is, discovered or developed and identified or isolated the plant, and asexually reproduced the plant. • The plant has not been sold or released in the United States of America more than one year prior to the date of the application. • The plant has not been enabled to the public, that is, by description in a printed publication in this country more than one year before the application for patent with an offer to sale; or by release or sale of the plant more than one year prior to application for patent. • The plant is shown to differ from known, related plants by at least one distinguishing characteristic, which is more than a difference caused by growing conditions or fertility levels, etc. • The invention would not have been obvious to one skilled in the art at the time of invention by applicant. Patentability of plants and plant varieties varies in different countries. These are territorial rights, that is, patent granted in one country has no relevance in another country. Individual governments set the statuary standards for patents. Thus a creator who wants to protect his innovation should file individual application in the country he needs the protection or can file international patent application. Patent term also varies from country to country. In United States, new plant varieties are protected by plant patents, utility patents, as well as Plant Variety Protection Act (PVPA). Plant patents protect only asexually reproduced plants while utility patent covers all GM plants and seeds as well. Sexually reproduced plants are protected by PVPA. In India, no plant and utility patents are allowed. India has a separate IPR category known as protection of new plant varieties, which is governed by the Protection of Plant Variety and Farmers Right Act, 2001 (PPVFR Act).

12.8.7 Indian legislation on Protection of Plant Varieties and Farmers’ Rights The extension of intellectual property protection to agriculture led to consideration of farmers’ rights especially in countries where economy is driven by agriculture. New IPR regimes are now being implemented simultaneously with rights of breeders and farmers. India is one among the countries over the world, who have passed a legislation in 2001, granting legal protection to farmers and breeders under the Protection of Plant Varieties and Farmers’ Right Act (PPVFR Act). The law

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protects the interest of stakeholders including farmers, NGOs, public sector institution, and private sector researchers and breeders, within the property right framework. PPVFR Act also encourages the development and cultivation of new varieties of plants, stimulates investments in agricultural development especially facilitating the growth of seed industry. Farmers are also entitled to use, save, sow, and resow the seeds of a registered variety in an unbranded form. They can sell or exchange the farm produce. The act allows the registration under new variety, extant variety, essentially derived variety, and farmers’ variety. Essential requirements to secure registration for new plant variety under Protection of Plant Variety and Farmers Right Act, 2001 are: 1. Novelty: Novelty of plant variety seeking protection under this act is most essential requirement. If at the date of filing of the application for registration of plant variety, the propagating or harvested material of such a variety has not been sold or otherwise disposed of by or with the consent of its breeder or his successor for the purposes of exploitation of such variety: a. in India, earlier than one year b. or outside India, in the case of trees or vines earlier than 6 years, or, in any other case, earlier than 4 years, before the date of filing such applications, then plant variety is considered novel. 2. Distinctiveness: Should be clearly distinguished by at least one essential characteristic from any other variety whose existence is a matter of common knowledge in any country at the time of filing of the application. 3. Uniformity: The new plant variety should be uniform in its essential characteristic in all plants, subject to the variation that may be expected from the particular features of its propagation. 4. Stability: The essential characteristics should remain unchanged even after repeated propagation or, in the case of a particular cycle of propagation, at the end of each such cycle. The period of protection for a new plant variety under this act is 9 years in the case of trees and vines and 6 years in the case of other crops, and may be renewed for the further period on payment of renewal fees, subject to the conditions that the total period of validity shall not exceed: 1. in the case of trees and vines, 18 years from the date of registration of the variety;

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2. in the case of extant varieties, 15 years from the date of the notification of that variety by the Central Government under Section 5 of the Seed Act, 1996; and 3. in the other case, 15 years from the date of registration of the variety.

12.9 Conclusion and future prospects The production of safe, low-cost, and nutritive food by adopting sustainable agricultural practices will be a gigantic task. The world needs technology that can effectively deliver good quality and yield crops, which are able to grow in hostile conditions created by increase in population and urbanization. Transgenic technology is a simple yet effective way to produce GM crops to fulfill the nutritional needs of human beings and farm animals. GM technology, however, is a highly controversial topic for today’s global food consumer. Potential damage caused to environment due to release of unwanted chemicals, disturbing ecological niche, and development of super weeds and bugs is also contributing factor for opposing the use of technology. Apprehension about possible negative effects of GM food on health and unexpected changes in nutritional quality or allergenicity due to introduction of foreign DNA in plants are some doubts on the technology. Although these perceived risks are not corroborated scientifically with the use of GM crops but without clear understanding may bend the consumer’s decisions against the use of GM food. The assessment of allergenicity should take place on a case-by-case basis before GM food is brought to the market. These assessments should be done by government or an independent credible regulatory authority or private agencies and these should not be driven by any commercial interests. Moreover, consumers must be made aware of benefits with complete information. The progress in transgenic technology has resulted in novel invention. The right to use these inventions needs to be protected. Patent is a legal instrument, which gives its owner the exclusive right over the invention. The patented invention cannot be commercially made, used, distributed, imported, or sold or offered for sale by others without the patent owner’s consent. Thus patents are seen as incentives for the investments by companies especially drug manufactures. In the absence of fear of infringement

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or duplication of the products, the companies invest into R&D, in terms of capital, intellect, and time, to continue developing new and innovative products and services.

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Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A AAD1. See Aryloxyalkanoate dioxygenase (AAD1) AAP. See Amino acid permeases (AAP) ABF3 gene, 98 Ab initio/de novo modeling, 88 Abiotic elicitors, secondary metabolites production, 213
215 Abiotic stress, 1
2, 8
15, 37
38, 114
116, 213
216 hampers plant, 95
96 in horticulture crops, 247
248 resistance to, 254
257 tolerance, transgenic technologies for, 114
116 drought, 104
107, 105f, 106t flooding, 107
109, 108t heat and cold, 96
101, 97t heavy metal stress, 112
114, 112f nutrient deficiency through genetic engineering, 109
111 salinity, 101
104, 102t, 103f Abscisic acid, 252 ACC. See 1-aminocyclopropane-1carboxylic acid (ACC) ACC oxidase (ACO), 249
250 ACO. See ACC oxidase (ACO) Active compounds, 208
209 Active site prediction of protein, 90 Adults feeding bioassay, 138
139 Aedes aegypti, 136 Afphytase, 201 Agriculture, 228 intellectual property rights in, 292
303 sector, 95
96 Agrobacterium-mediated plant transformation, 33
34, 137 hpRNA vector using, 138
139 Agrobacterium rhizogenes, 35, 40
41 Agrobacterium species, 114
116

Agrobacterium tumefaciens, 32, 34
35, 98, 100
102, 105
106 Arabidopsis plants by, 110 Brassica juncea by, 98 C58C1, 98
99 PpEXP1 using, 98 strain EHA105, 35
36, 97, 100, 109 strain GV3101, 35 strains LBA4404, 35, 139
141 for transformation, 102
103 transform tobacco plants by, 112
113 Agronomic traits, 16 of taxa, 1
2 Agrostis stolonifera (bentgrass), 98 AHP. See American Herbal Pharmacopoeia (AHP) AlaAT, 161
162 alaAT overexpressing rice plants, 161
163 Alanine aminotransferase, 160 Alkaloids, 210
211 Allene oxide cyclase gene, 219 Allergenicity, 281 assessment of, 289
292 physiochemical stability, 290 sequence of, 289
290 serum screening, 290 source of transgene, 289 targeted screening for, 290 testing models, 291
292 Allergens, 289
290 Alternative oxidase (AOX), 100
101 American Herbal Pharmacopoeia (AHP), 212 Amino acid permeases (AAP), 157
158 Amino acids, transport of, 157
158, 161
162 Amino acid sequence, of proteins, 84
87 1-Aminocyclopropane-1-carboxylic acid (ACC), 113
114, 249
250 amiRNA. See Artificially transformed miRNA (amiRNA)

309

310 Anaphylaxis, 289 Anemia in infants and children, prevalence of, 197f Anopheles gambiae, 136 Anthocyanin biosynthesis, 218, 258 Antinutrients, 288 AOX. See Alternative oxidase (AOX) APase expression, 171 Apocynum venetum DEAD-box helicase 1 (AvDH1) gene, 2
3, 8
10 Apomorphine, 213 Arabidopsis. See Arabidopsis thaliana (Arabidopsis) Arabidopsis CBF genes (AtCBF1
3), 100 Arabidopsis OLIGOPEPTIDE TRANSPORTER 3 (AtOPT3), 194
195 Arabidopsis seeds, 98
99 Arabidopsis thaliana (Arabidopsis), 3, 8
12, 60, 98, 188
189, 229
230, 237
238 AtAHA proteins in, 189
190 AtDREB2A in, 12
13 AtFPN2, 196 AtFRD3 expression in, 192
193 AtVIT1, 196 chalcone synthase genes in, 218 drought tolerance in, 105
106 ESKMO1 (ESK1) gene in, 106
107 genome, 10
11 mitochondria, 195
196 MtPHY1 and MtPAP1 in, 171 Nicotiana benthamiana and, 137 OLIGOPEPTIDE TRANSPORTER 3 (AtOPT3), 194
195 Osmyb4 gene in, 254
257 overexpressing AtPROPEP3, 101
102 168phyA gene in, 170
171 plants, 101
102 salinity stress tolerance in, 101
102 SPL and miR156 in, 218 submergence tolerance in, 108 transcription factor, 217 transgenic plants of, 139 Trichoderma harzianum T34 hsp70 gene in, 98
99 Zea mays (maize) in, 110

Index

ZmZIP3 in, 110 Artemisia annua, 35, 213, 219 Artemisinin biosynthesis genes, 216 Artificially transformed miRNA (amiRNA), 218 for secondary metabolite, 218 Artificial neural network-based algorithms, 87 Aryloxyalkanoate dioxygenase (AAD1), 60 Ascorbate peroxidase (APX), 97 Ascorbic acid, 258
259 Aspergillus flavus (fungus), 38 AtABF3, 12
13 AtAHA2, 189
190 proteins in Arabidopsis, 189
190 AtDREB2A in Arabidopsis, 12
13 AtFRD3, 192
193 AtFRO2, 189
190 AtHB7, 12
13 ATPaseA gene, 138 AtPROPEP3 gene, 101
102 Atropa belladonna, 34, 213 AtZIP1 gene, 111 AvDH1 gene. See Apocynum venetum DEAD-box helicase 1 (AvDH1) gene Azadirachta indica, 34

B Bacillus cereus, 129
130 Bacillus subtilis, 170
171 Bacillus thuringiensis (Bt) genes, 33
34, 127
130, 144
145 applications, 127
130 strains, 127 toxin, 127, 278
279, 284 Bacterial choline oxidase (Cox) gene, 291 Banana streak virus (BSV), 42
43 Begonia micropropagation, 41
42 Bemisia tabaci, 138
139 Beneficial traits, transgenic crops with, 278 BHP. See British Herbal Pharmacopoeia (BHP) Biofortification of fruits and vegetables, 257
259, 258t simplest method, 257 Biofuel production, 227

Index

gene mining to genome editing, 235
238 metabolic engineering, 229
230 muconic acid, 234
235 photosynthesis, 230
231 photosynthetic chassis. See Photosynthetic chassis synthetic biology, 229
230 terpenes, 234 Bioinformatics, 70 Biological delivery systems, 33 Biomass of plants, 227
228, 232 Biophysical characterization, of proteins, 85
86 Bioplastics, 234
235 Bioreactor scaling, 41
42 Biotech crops adoption and commercial benefits, 274
278 global area of, 275t plantation of, 276
277 Biotic elicitors, 213
216 secondary metabolites production, 213
214 Biotic stress, 1
2, 14
15, 37
38 in horticulture crops, 247
248 resistance to, 253
254, 255t BnaC.IGMT5.a gene, 19
20 BnERF2.4 transgene expression, 108 Botryococcus braunii, 234 Botrytis cineria, 252 Brassica juncea, 112
113, 175
176 by Agrobacterium tumefaciens, 98 Brassica napus, 19
20, 103
104, 108, 113
114, 175
176, 229
230 British Herbal Pharmacopoeia (BHP), 212 BSV. See Banana streak virus (BSV)

C CAC. See Codex Alimentarius Commission (CAC) Calcium-dependent protein kinases (CPKs), 11
12 Callistemon citrinus, 213 Camelina sativa, 229
230 CaMV 35S promoter, 99
103, 106
108, 128
129, 137
139, 174

311 Canola, transgenic, 276 Caragana jubata, 15
16 Carbohydrates, 220, 228
229, 232 Carotenoids, 258
259 CAS. See Cycloartenol synthase (CAS) CASTp, 90 Catalase (CAT) gene, 112
113 Catharanthus roseus, 38, 41
42, 214
215 Cell-autonomous RNA interference, 130
131 Cell repair system, 52 Cellular tolerance (CT), 12
13 Cellulose-based fuels, 228
229 Centella asiatica, 40 Ceratitis capitata, 144
145 Cereal production in India, 278 Chelation strategy, iron uptake, 188
191, 200 and long-distance transport of, 191
195 Chemical pesticides, voluminous application of, 123
124 Chickpeas, 174
175 Chinese medicine, 207
208 Chitin synthesis, 139
141 Chlamydomonas, 237
238 Chlamydomonas reinhardtii, 231, 233
234 CHLI. See Magnesium-chelatase subunit I (CHLI) Chlorella vulgaris, 233 Chloroplasts, 175 Citrate, 192
193 CjQM expression, 15
16 Cladosporium cladosporioides MD213, 41 Cleistogenes songorica, 11
12 Climacteric fruits, 248
250 Clustered regulatory interspaced short palindromic repeats/CRISPRassociated 9 (CRISPR/Cas9), 54, 58
60, 62, 126, 141
145, 148, 220
221, 259
260, 262
263 genome editing tool, 260
261 against insects, 144
145 screening method, 62 technology, 235
237 on microalgae, application, 237
238 in photosynthetic hosts, 237
238 CMV. See Cucumber mosaic virus (CMV)

312 Codex Alimentarius, 285
286 Codex Alimentarius Commission (CAC), 285
286 Coding sequence, 173
174 COILS, 87 Cold stress tolerance, in plants, 96
101, 97t Petunia spp. for, 99, 99f Colorado potato beetle (CPB) gene, 125
126, 134, 139
141 Computational biology, 70 Conventional breeding methods, 51, 96
97, 146 Conventional plant breeding, 257 Conventional random mutagenesis, 53
54 Conventional storage methods, 42
43 Conversion process, 29
31 Copyright and related rights, 296
297 Cotton cultivar Coker312, 128
129 Cotton, transgenic, 276 Cotyledonary explants, 33
34 Cowpea trypsin inhibitor (CpTI), 147 CPKs. See Calcium-dependent protein kinases (CPKs) CRISPR RNAs (crRNAs), 58
59, 236
237 Crop improvement programs, 32
33 breeding programs for, 71 by conventional mehod, 141
144 genome editing technology for, 141
144 nitrogen use efficiency (NUE), 158
159 protoplast in, 37
38 Crop losses, 145
146 Crop plant, 1
2, 42
43 disease tolerance, 17
20 drought, 10
13 metabolic engineering of, 235
236 nematode resistance in, 145
148 production, 51, 95
96, 247
248 quantitative trait loci, 16
17 salinity, 2
10 salt-tolerant genes in, 4
8, 9f Cross-pollination of transgenic crops, 283 crRNAs. See CRISPR RNAs (crRNAs) Cryopreservation, 42
43 Crystal toxin (Cry), 127
128

Index

CSb transgenic plants, 167
168 Cucumber mosaic virus (CMV), 42
43 Cycloartenol synthase (CAS), 216 CYP716A52v2, 216 Cysteine, 173
174 biosynthetic pathway, 175 synthesis of, 177
178 Cyst nematodes, 145
146 Cytolytic toxin (Cyt), 127 Cytosine deaminase (CodA) gene, 291

D DaCBF7 overexpression, 100 Dammarenediol-II synthase (PgDDS), 217 Databases, 72t, 85
86, 89
90 DBT. See Department of Biotechnology (DBT) Del/Ros1. See Transgenic tomatoes (Del/ Ros1) De-materia medica, 207
208 20 -Deoxymugineic acid (DMA), 190, 193 Deoxymugineic acid synthase (DMAS), 193 Department of Biotechnology (DBT), 25
26 Deschampsia antarctica, 100 Design patents, 299 Desirable traits/genes, 37
38 Detoxification genes, 139 DGAT gene. See Diacylglycerol acyltransferase (DGAT) gene Diabrotica virgifera, 138
139 Diacylglycerol acyltransferase (DGAT) gene, 233 Dicot and monocot plants, 3
4 Differential display (DD) of mRNA, 15
16 Dinuclear iron(III), 191
192 Dioscorea deltoidea, 41
42, 215
216 Disease-free explant, 27 Disease tolerance, 17
20 DMA. See 20 -Deoxymugineic acid (DMA) DMAS. See Deoxymugineic acid synthase (DMAS) DNA-binding domain of SSNs, 62 of ZF, 56
57

313

Index

DNA segment, 32 Donor plant preparation, 27 Double-stranded break (DSB), 52 Double-stranded RNA (dsRNA), 124
126, 130, 133, 135t degradation, 134, 136 delivery methods, 130
131 feeding assay of CPB, 134 FNP and CHT10, 136 insect-resistant crops by, 136
137 oral delivery method of, 133
136 Drosophila melanogaster, 144
145 Drosophila suzukii, 126 Drought stress tolerance, 10
13, 190 in Arabidopsis thaliana, 105
106 in oryza sativa japonica, 107 transgenic approaches for, 104
107, 105f, 106t Drugs, 212
213 DSB. See Double-stranded break (DSB) dsRNA. See Double-stranded RNA (dsRNA)

E Early nodulin (ENOD) genes, 164
165 Ecdysone receptor gene, 134, 139
141 Ectopic expression of Arabidopsis transcription factor, 217 of dammarenediol-II synthase (PgDDS), 217 of P. ginseng HMGR1, 217 Elicitors abiotic, 39t, 213
215 biotic, 39t, 213
216 for enrichment of phytocompounds, 38
39 oligogalacturonic acid, 215
216 secondary metabolites production, 213
216 types, 214f Embryogenic calli and protoplast, 33
34 EMS. See Ethyl methane sulfonate (EMS) Endogenous phytase, 171 Endophytes and secondary metabolites, 41 Endoplasmic reticulum (ER), 173
174 ENOD93 gene, 163

ENOD genes. See Early nodulin (ENOD) genes Environmental RNA interference, 131 ER. See Endoplasmic reticulum (ER) ER retention signal (KDEL), 174
175 Error-free method, 52 Erwinia uredovora, 258
259 Escherichia coli, 234
235 Eschscholzia californica Cham, 35 ESKMO1 (ESK1) gene, in Arabidopsis thaliana, 106
107 ESTs. See Expressed sequence tags (ESTs) Ethanol production, 109 Ethylene, 248
250 Ethylene biosynthesis, 251 Ethylene glycol, 8
10 Ethyl methane sulfonate (EMS), 251
252 Expressed sequence tags (ESTs), 11
12

F FAO. See Food and Agriculture Organization (FAO) Fe(II)-reduction transport system, 190
191 Ferredoxins, 191
192 Ferric hydroxides, in iron chlorosis, 188
189 Ferric oxides, in iron chlorosis, 188
189 FERRIC REDUCTASE DEFECTIVE 3 (AtFRD3), 192
193 FERRIC REDUCTASE DEFECTIVELIKE 1 (OsFRDL1), 192
193 FERRIC REDUCTION OXIDASE 2 (AtFRO2), 189
190 Ferritin, 195 FERROPORTIN, in rice and maize, 192 Festuca arundinacea, 113 First-generation genome editing technology, 130
141 oral delivery method of dsRNA, 133
136 RNA interference. See RNA interference (RNAi) Flavanone-3-hydroxylase (F3H) gene, 261 Flooding stress tolerance, transgenic approaches for, 107
109, 108t FokI restriction endonuclease, 56 Fold recognition/assignment strategies, 89

314 Food and Agriculture Organization (FAO), 285
286, 289 guideline by, 289
290 Food crops, genetic modification of, 273
274 Food production, and environmental safety, 123
124 Foreign DNA, 303 consumption of, 281 Fossil fuels, for energy consumption, 228 Frangula alnus, 41
42 Fritillaria unibracteata, 29 Fruit ripening, 249
250 Fruits and vegetables biofortification, 257
259, 258t commercialization of GM, 262
263 Function prediction methodologies active site prediction, 90 fold recognition/assignment, 89 sequence-based, 89 structure-based, 90 Fusarium oxysporum, 218

G Gene knockdown efficacy rate, 124
125 Gene mining, genome editing tools, 235
238 Gene regulation, 62
63 Gene-silencing strategy, 124
125 Genetically modified (GM) crops, perceived risks of, 279
284 allergenicity, 281 assessment of possible allergenicity, 289
292 consumption of foreign DNA, 281 contamination of environment with proteins, 284 effect on nontarget organism, 283
284 horizontal transfer of genetic material, 281
283 large-scale production, 280 super plants an environmental risk, 283 Genetically modified cotton, 278
279 Genetically modified fruits and vegetables, 262
263 Genetically modified organism (GMO), 285

Index

Genetically modified technology, safety assessment of, 284
288 Codex Alimentarius and Codex Alimentarius Commission, 285
286 framework for, 286
288 Genetic engineering, 104 approaches for industrial production, 229
230 of crops, 252
253 crops with nutritional qualities, 259
262 program, tissue culture based, 32
33 Genetic manipulation, 40
41, 51
52 Genetic modification, 273
275 characterization of, 288 description, 287
288 of plants, transgenic technology for, 273
274 Genetic transformation, 32, 36t of medicinal plants, 217 root cultures, 40
41 technique, 33
34 Genome editing system, 52 application, 59
64 gene regulation, 62
63 high-throughput mutant libraries, 61
62 multiplexing and trait stacking, 59
60 targeted structural changes in crop species, 63
64 in biofortification, 196 high-throughput mutant libraries, 61
62 in iron content. See Iron content, in crop plants in plant pathogens, 57 targeted chimeric sequence-specific nucleases for, 55t techniques, 53
59 CRISPR/Cas9, 58
59 transcription activator-like effector nucleases, 57
58 zinc-finger nucleases, 54
57 transgenic technology, 52 Genome editing tool CRISPR/Cas9, 260
261, 263 gene mining to, 235
238

315

Index

knowledge and competency on, 126 nucleases as, 235
236 technology for crop improvement, 141
144 first-generation, 130
141 second-generation, 141
144 Genome editing with engineered nucleases (GEEN), 52
53 Genome engineering tool, for plant genomes, 220 Genome-wide association studies (GWASs), 146, 196 Genomic information, 69 Geographical indication (GI), 295 Geraniol, 217 Geraniol 10-hydroxylase gene (G10H), 218
219 Germplasm storage, 42
43 G. fasciculatum, 170 Ginsenoside biosynthesis, 216 Glutamine synthetase/glutamate synthase (GS/GOGAT) system, 157
159, 163
164 Glycophytic plants, 3
4 GMO. See Genetically modified organism (GMO) Gossypium arboreum, 218
219 G. pallida, 147 Graminaceous species, 190 Green Revolution, 273, 293
294 GS/GOGAT system. See Glutamine synthetase/glutamate synthase (GS/ GOGAT) system Guide RNA (gRNA), 236
237 GWASs. See Genome-wide association studies (GWASs)

H HaAK gene, 138 Haber
Bosch process, 157
158 Hairy root cultures, 40
41, 217 Halophytic plants, 3
4 Haploid development, in tissue culture, 31 HAST. See High-affinity sulfate transporter (HAST) Heat-resistant Bt toxin, 281

Heat-shock transcription factor (HSF), 98
99 Heat stress tolerance, in plants, 96
101, 97t Heavy metal stress tolerance, transgenic technologies for, 112
114, 112f Helianthus annuus, 173
174 Heliconia bihai, 36
37 Helicoverpa armigera, 138, 144
145 Heliothis virescens, 129
130 High-affinity sulfate transporter (HAST), 175
176, 177f High-throughput mutant libraries loss of function caused by, 61
62 TILLING and T-DNA insertion methods for, 61
62 Histidine-rich proteins, 191
192 HMMTOP, 87 Homology modeling, 88 Hordeum vulgare (barley), 110
111, 160, 160f Horizontal transfer of genetic material, 281
283 Horticulture crops abiotic stress factors, 247
248 biotic stress factors, 247
248 transformation of, 254
257 transgenic, 262
263 HSF. See Heat-shock transcription factor (HSF) Hunch back gene, 137 HvCPK2a, 11
12 HvTOM1, 190 Hybridization program, 32 Hydroxyl radicals, 191
192

I ICMR. See Indian Council for Medical Research (ICMR) IDEF1 gene. See Iron deficiencyresponsive cis-acting elementbinding factor 1 (IDEF1) gene IITA. See International Institute of Tropical Agriculture (IITA) Immobilization scaling, 42 India cereal production in, 278

316 India (Continued) transgenic hybrids in, 278
279 Indian Council for Medical Research (ICMR), 212
213 Indian Herbal Pharmacopoeia, 212 Indian legislation on PPVFR Act, 301
303 Innovative genome editing tools, 59 Inorganic nitrogen in freshwater, 157
158 Insect pest management in plants, 123
124, 135t control of, 123
124 microinjection-based RNAi technology to, 132t resistance development capability of, 130 transgenic technologies, 124 Bacillus thuringiensis genes. See Bacillus thuringiensis (Bt) genes CRISPR against insects, 144
145 first-generation genome editing technology, 130
141 nematode resistance in crop plants, 145
148 second-generation genome editing technology, 141
144 Insect resistance, non-BT-type strategy of, 125 In silico, structural and functional characterization proteins, 71
84 Integrated pest management (IPM), 124 Intellectual property rights (IPRs), 292
303 copyright and related rights, 296
297 geographical indication, 295 international organization and agreements for, 297
298 patents, 298
301 trademarks, 295
296 trade secrets, 294
295 International Institute of Tropical Agriculture (IITA), 147
148 Introgression line (IL), 16
17 In vitro germplasm storage, 42
43 In vitro haploids, 31 In vitro plant propagation, organ culture for, 29

Index

IPM. See Integrated pest management (IPM) IPRs. See Intellectual property rights (IPRs) Iron, 187 Iron content, in crop plants in Arabidopsis. See Arabidopsis thaliana (Arabidopsis) chelation strategy, 188
191 and long-distance transport of, 191
195 Fenton’s reaction in, 191
192 genetic engineering to improve, 196
201 in maize. See Zea mays (maize) multigene expression, 201 OsYSL9, 193
194 reduction strategy, 188
191 in rice. See Oryza sativa/Oryza sativa japonica (rice) in seeds, 197
200 storage, 197
200 and vacuole sequestration, 195
196 translocation, 200 uptake and transport, 188
196 chelation strategy, 188
191 deoxymugineic acid synthase for, 193 increasing, 200 reduction strategy of, 188
191 Iron deficiency, 188
189 chlorosis, 188
189 stress tolerance, 111f Iron deficiency-responsive cis-acting element-binding factor 1 (IDEF1) gene, 110
111, 111f Iron-deficient phenotype, in plants, 193 Iron ligand, 191
192 IRON-REGULATED TRANSPORTER 1 (AtIRT1), 189
190 Iron
sulfur clusters, 187
188, 195 Irrigation-induced salinization, 2
3 Isolation of protoplast, 37
38

J Japonica rice. See Oryza sativa/Oryza sativa japonica (rice) Jasmonate biosynthetic pathway, 219

Index

K KDEL. See ER retention signal (KDEL) KHP. See Korean Herbal Pharmacopoeia (KHP) Korean Herbal Pharmacopoeia (KHP), 212

L Lacanobia oleracea, 129
130 Lactuca sativa (lettuce), 103
104 Lactuca serriola, 40
41 LdEcR gene. See Ecdysone receptor gene Leaf lupin seeds, 174
175 Lepidopteran, 127
128 Lesquerella fendleri, 37
38 Lipid biosynthesis, 235 Lipids, 233 Loop modeling, 88 Low temperature (LT), 14
16 Lr34, homologous copies of, 18
19 LsGGP2 encodes, 63 LT. See Low temperature (LT) Lupinus angustifolius, 173
174 Lupinus angustifolius L., 174
175

M Macronutrients, 187 MAGE technology. See Multiplex automated genome engineering (MAGE) technology Magnaporthe oryzae, 20 Magnesium-chelatase subunit I (CHLI), 60 Maize. See Zea mays (maize) Malus domestica (apple), 110 Malus zumi, 36 Manihot esculenta (cassava), 113 MAPKs. See Mitogen-activated protein kinases (MAPKs) Marker-assisted selection (MAS), 146 MAS. See Marker-assisted selection (MAS) MdbHLH104 gene, 110 Medicago sativa (alfalfa), 97 Medicinal plants, 207
208 genetic transformation of, 217 history of, 207
209 in pharmacotherapy, 208
209 primary metabolic pathways, 220

317 and secondary metabolites. See Secondary metabolites stabilization methods for, 208
209 in traditional and modern medicine, 212
213 Mediterranean fruit fly, 144
145 Melonic acid pathway, 211
212 MEMBRANE PROTON ATPase 2, 189
190. See also AtAHA2 MEP. See Methylerythritol pathway (MEP) Mesembryanthemum crystallinum, 3 Messenger RNA (mRNA), 133
134 Metabolic engineering, 229
230 approaches, 233 of crop plants, 235
236 crops for ascorbic acid, 258
259 Methionine, 173
174 Methylerythritol pathway (MEP), 209
210 Methyl jasmonate (MJ), 15
16 Mevalonic acid (MVA) pathway, 209
210 Microalgae, 235 Microalgae-derived sugars, for bioethanol production, 232 Microbial biofuels, 227 Micronutrients, 109
110, 187 Micropropagation as multiplication method, 27
29 acclimatization stage, 28
29 initiation stage, 27 multiplication stage, 27 preparation of donor plant stage, 27 rooting stage, 28 Tylophora indica, 27, 28f MicroRNAs (miRNAs), 124
125, 131
133 MIPS. See Myo-inositol-1-phosphate synthase (MIPS) miRNAs role, secondary metabolite production, 217
218 Mitogen-activated protein kinases (MAPKs), 14 Modeller program, 88
89 ModEval, 88
89 ModLoop, 88 Monoterpenes, 209
210 MsHsp23 gene, 97

318 Muconic acid, 234
235 Mugineic acid (MA), 190 Multigene expression, 201 Multiplex automated genome engineering (MAGE) technology, 239 Multiplexing and trait stacking, 59
60 MVA pathway. See Mevalonic acid (MVA) pathway Myo-inositol hexaphosphate, 196 Myo-inositol-1-phosphate synthase (MIPS), 3

N Nanocarrier dsRNA, 136 Nanoparticle chitosan, 136 Natural gene pool, 36
37 Natural products biosynthesis and classification, 209
212 alkaloids, 210
211 phenolics, 211
212 terpenes, 209
210 history of, 207
209 Necrotrophic fungal pathogen, 19
20 NetSurfP, 87 NetTurnP, 87 Neutral/alkaline invertase gene (NINV), 220 Next-generation sequencing (NGS), 51
52, 61 NHEJ. See Nonhomologous end joining (NHEJ) Nicotiana benthamiana genes, 60, 137
138, 235
236, 260
261 Nicotiana glauca, 37
38 Nicotiana langsdorfi, 37
38 NICOTIANAMINE SYNTHASEs, 193 Nicotiana tabacum, 98, 125, 170
171, 235
236 NINV. See Neutral/alkaline invertase gene (NINV) Nitrogen, 157
166 AlaAT-specific activity in, 161
162 atom, 210
211 NUE. See Nitrogen utilization efficiency (NUE) primary assimilatory, 161
162 Nitrogen use efficiency (NUE), 158
159

Index

AlaAT-ox rice, 163 phenotype in crop plant, 159 transgenic crops with, 159
166 in transgenic Hordeum vulgare, 160, 160f Nonbiological delivery systems, 33 Noncell-autonomous RNA interference, 130
131 Nonclimacteric fruits, 249
250 Nonhomologous end joining (NHEJ), 52 Nontarget organism, effect on, 283
284 Notorious insect pests, 123
124 NPK1, 3 NTB-88, 127 NUE. See Nitrogen utilization efficiency (NUE) Nutritional deficiency, 196
197 efficiencies for nitrogen, 274 plant tolerance to, 109
111 Nutritional quality genetic engineering crops with, 259
262 transgenic technology tool for, 252
253

O Oil seed crop, 229
230, 279 Omega fatty acids, 235 Omega-3 long-chain polyunsaturated fatty acids (Ω3 LC-PUFAs), 229
230, 239 Ω3 LC-PUFAs. See Omega-3 long-chain polyunsaturated fatty acids (Ω3 LCPUFAs) Oral delivery method of dsRNA, 133
136 sprayable RNA interference approach, 133
135 Organ culture, 29 Organic crop production, 284
285 Orychophragmus violaceus, 37
38 Oryza sativa/Oryza sativa japonica (rice), 58, 61
62, 103
105, 107, 114, 174
175, 188
191, 193
194 drought stress tolerance in, 107 ferritin, 197
200 FERROPORTIN in, 192 iron content in, 198t mitochondria and chloroplast in, 195 NA synthase genes, 193

Index

OsbZIP46 in, 107 OsFRDL1 function in, 192
193 OsMIT1 in, 195
196 OsNAS1 and HvNAATb in, 201 overexpression of, 200 pyruvate decarboxylase in, 109 VIT genes in, 200 OsBAT1. See Rice antigen-B-associated transcript (OsBAT1) OsbZIP46CA1 gene, 107 OsbZIP46 gene, 107 OsENOD93-1 gene, 164
165 OsFRDL1, 192
193 OsMIT1 in rice, 195
196 Osmotin, 291 Osmyb4 gene, 254
257 Os11N3 gene, 58 OsNHX1 gene, 103 OsOPT7 (Osopt7-1), 194
195 OsRab7 gene, 104
105 OsSWEET14. See Os11N3 gene OsTOM1, 190
191 Overexpression of alternative oxidase, 100
101 artemisinin biosynthesis genes, 216 AtCBF1 and AtCBF3, 100 CAT gene, 112
113 of CYP716A52v2, 216 of DaCBF7, 100 MdbHLH104 gene, 110 of OsbZIP46CA1, 107 of OsbZIP46 gene, 107 of OsENOD93-1 gene, 164
165 of OsRab7 gene, 104
105 of P5cs gene, 99, 99f of RAP2.6L gene, 108
109 of rice, 200 Sub1A, 109 of transcription factors, 254
257 Oxalis triangularis, 41
42

P PAE. See Phosphorus acquisition efficiency (PAE) PAM. See Protospacer adjacent motif (PAM) Panax ginseng, 38, 41
42, 215
216

319 Panax ginseng HMGR1 (PgHMGR1), 217 Papaver somniferum, 35, 215
216 Paris Convention, 297 Patent Cooperation Treaty (PCT), 297 Patents, 298
301 Pathogenesis-related (PR) genes, 18
19 Pathogen-free plant propagation, 31
32 pBI121-ZmZIP3 construct, 110 pCAMBIA3301 vector, 97
98, 103 PCT. See Patent Cooperation Treaty (PCT) PDS. See Phytoene desaturase gene (PDS) P450 enzymes, 217 PEPc. See Phosphoenolpyruvate carboxylase (PEPc) Pepsin resistance, 290 Perennial grass plant, 11
12 Perilla frutescens, 214
215 Perovskia abrotanoides, 215
216 Petunia hybrida L., 130 Petunia spp., 99, 99f PGR. See Plant growth regulator (PGR) pgSIT. See Precision-guided sterile insect technique (pgSIT) Phaeodactylum tricornutum, 233 Pharmaceutical terpenoids, 217 Phenolic compounds, 211
212 Phloem vessel, 191 Phosphoenolpyruvate carboxylase (PEPc), 157
158 Phosphorus, 166
172. See also Phosphorus utilization efficiency (PUE) Phosphorus acquisition efficiency (PAE), 167
168 Phosphorus utilization efficiency (PUE), 167, 169f transgenic with, 167
172 Photosynthesis process, 187
188, 230
231 Photosynthetic chassis formation of essential products, 231
233 lipids, 233 sugars, 232 Photosynthetic organisms, 227
230 metabolic genes in, 234
235 sustainable model using, 231
232

320 Phylate biosynthesis in maize, 56
57 PHYRE. See Protein Homology/analogY Recognition Engine (PHYRE) Physical remediation of soils, 112 Phytase genes, 172 Phytic acid (PA), 196 Phytoene desaturase gene (PDS), 260
261 Phytoene synthase gene (PSY), 258
259 Phytosiderophores (PSs), 190 Picrorhiza kurroa, 218 Pioneers of biological sciences, 71 piRNA. See PiwiRNA (piRNA) Pisum sativum, 8
10, 113 PiwiRNA (piRNA), 131
133 Plant acid phosphatase, 172 Plant-based drugs, 212
213 Plant biotechnology, 71, 229 intellectual property rights in, 292
303 Plant breeding program, 32, 36
37 Plant genetic transformations, 33, 36t Plant growth regulator (PGR), 27 Plant lectins, 129
130 Plant-mediated RNAi, 136
141 Plant microRNAs, 217 Plant parasitic nematodes (PPNs), 145
146 Plant Patent Act, 300 Plant phenolics, 70
71, 211
212 Plant resistant (R) genes, 18
19 Plants and endophytic fungi, 41 Plants genomes, 61 Plant species, 4
8, 5t, 147 Plant tissue culture (PTC), 25 based genetic engineering program, 32
33 bioreactor scaling, 41
42 compounds, 25
26 elicitation for enrichment of phytocompounds, 38
39 endophytes and secondary metabolites, 41 hairy root culture and genetic manipulation, 40
41 haploid development via, 31 immobilization scaling, 42 limitations, 43
44 micropropagation as multiplication method, 27
29

Index

molecular techniques, 25 organ culture, 29 pathogen-free plant propagation, 31
32 and plant breeding, 32 precursor addition, 40 protoplast culture and somatic hybridization, 37
38 somaclonal variation and plant improvement, 36
37 somatic embryogenesis and synthetic seeds, 29
31 and transgenic plants development, 32
36 in vitro cell and, 26 in vitro germplasm storage, 42
43 Plant Variety Protection Act (PVPA), 301 Plasmid, 112
116 Platycodon grandiflorum, 217 Plumbago rosea, 42 Poa pratensis (Kentucky bluegrass), 98 Podophyllum hexandrum, 40 Polygenic traits, 263 Polymeric nanoparticles, 136 Polyphenols, 211
212 pORFs. See Primary ORFs (pORFs) Porteresia coarctata, 3
4 PPNs. See Plant parasitic nematodes (PPNs) PPVFR Act. See Protection of Plant Variety and Farmers Right Act, 2001 (PPVFR Act) Precision-guided sterile insect technique (pgSIT), 126 Precursor feeding, 40 PR genes. See Pathogenesis-related (PR) genes Primary metabolism, 209, 220 Primary ORFs (pORFs), 63 Primary salinity, 2
3 Primary sequence of protein, 289
290 Programmable nucleases, 54 Prokaryotic proteins, 86 Promoter, CaMV 35S, 99
103, 106
108 Protection of Plant Variety and Farmers Right Act, 2001 (PPVFR Act), 298, 301
302 Indian legislation on, 301
303 Proteinase inhibitor (PIN2), 147
148

321

Index

Protein Homology/analogY Recognition Engine (PHYRE), 88 Proteins amino acid sequence, 84
86 biophysical characterization of, 85
86, 89 function prediction, 89
90 physicochemical properties of, 85 sequence-based approach, 84
89 sharing homology, 89 in silico structural and functional characterization, 71
84 computer-based, 90
91 graphical overview of, 85f using sequence information, 84
85 structure prediction strategy. See Structure prediction strategy subcellular localization of, 86 tools and databases used for, 72t traditional sequence-based characterization, 86 Proteomics, 70
71 Protopanaxatriol (PPT), 217 Protoplast culture, 37
38 Protoplast fusion, 37
38 Protospacer adjacent motif (PAM), 236
237 PROTPARAM, 86 PROTSCALE, 86 P5sc gene, 99, 99f Pseudomonas putida (P. putida), 113
114 PSs. See Phytosiderophores (PSs) PSY. See Phytoene synthase gene (PSY) PTC. See Plant tissue culture (PTC) PUE. See Phosphorus utilization efficiency (PUE) PVPA. See Plant Variety Protection Act (PVPA)

Q QM expression, 15
16 QTLs. See Quantitative trait loci (QTLs) Quantitative resistant (QR) gene, 18
19 Quantitative trait loci (QTLs), 16
17, 146, 196 BnaC.IGMT5.a gene for, 19
20 colocalization and distribution, 17

pyramiding, 16 for stem resistance, 19
20

R RaMPK1 transcript, 14
15 RAP2.6L gene, 108
109 Rauwolfia canescens, 215
216 Rauwolfia serpentina, 38 Recombinant-DNA plant, 286
288 Recombinant DNA technology, 36
37, 252
253, 257 Reduction strategy, of iron uptake, 188
191 Repeat variable diresidues’ (RVDs), 57 Resistant allele Lr34res, 18
19 Reverse genetic approaches, 196, 251
252 Reverse transcription PCR (RT-PCR), 4
8 RGENs. See RNA-guided engineered nucleases (RGENs) Rheum australe genes, 14
15 Rice. See Oryza sativa/Oryza sativa japonica (rice) Rice antigen-B-associated transcript (OsBAT1), 8
10 Rice-breeding programs, 16
17 Rice genome, 61
62 RISC. See RNA-induced silencing complex (RISC) RNA-guided engineered nucleases (RGENs), 58
59 RNAi. See RNA interference (RNAi) RNA-induced silencing complex (RISC), 133 RNA interference (RNAi), 124
125, 130
131, 218 based sprayable, 133
135 Bemisia tabaci (whitefly) by, 138 cell-autonomous, 130
131 knockdown technology, 138
139 mechanism, 140f nanoparticles-coated, 136 noncell-autonomous, 130
131 oral delivery method of dsRNA, 133
136 pathways and mechanism, 131
133 plant-mediated, 136
141

322 RNA interference (RNAi) (Continued) several experiments of, 144
145 Root development stage, 28 Root-knot nematodes, 145
146 RT-PCR. See Reverse transcription PCR (RT-PCR) Rubia akane cell culture, 38 Ruta graveolens, 215
216

S Saccharomyces cerevisiae, 238
239 Safety assessment of genetically modified technology Codex Alimentarius and Codex Alimentarius Commission, 285
286 framework for, 286
288 SaINO1 gene, 3 Salicylic acid (SA), 15
16 Salinity, 2
10 stress tolerance in plants, 101
104, 102t, 103f various plant species, 5t Salt concentration, 2
3 Salt stress, 2
4 Salt-tolerant grasses (STGs), 3
8 drought and, 11
12 gene, 3
8, 9f Saturating electron transfer, 231 SAVES. See Structure Analysis and Verification Server (SAVES) Scenedesmus obliquus, 233
235 Sclerotinia sclerotiorum (Lib.) de Bary, 19
20 Scopolia parviflora, 215
216 SE. See Somatic embryogenesis (SE) Secondary metabolites, 41, 215
216 artificial miRNAs for, 218 biosynthesis key genes, homologous overexpression, 216 ectopic expression of genes, 217 elicitors, 213
216 abiotic, 214
215 biotic, 215
216 engineering plant metabolic pathways to, 220
221 in plant cells, 213
214 in plants, 209, 210f

Index

production, miRNAs role in, 217
218 technologies for enhancement, 213
220 terpenoid biosynthesis, phytohormones levels in, 219 in traditional and modern medicine, 212
213 transcription factors, expression of, 218
219 Secondary salinization, 2
3 Secondary structure prediction strategy, 87 Secreted insecticidal proteins (Sip), 127 Seeds iron content in, 200 by overexpressing YSL2, 200 protein, 174
175 Seeds Act of 1966, 25
26 Sequence-based approach, proteins, 84
89 Sequence similarity search methods, 89 Sequence-specific nucleases (SSNs), 52, 54, 259
260 Serine proteases, 129
130 Serum screening, 290 sfa8 gene, 174 sgRNA. See Single-guided RNA (sgRNA) Shelf life, 248
249 and fruit ripening, 249
250 improvements, 252
253 metabolic alterations incorporating, 250
252 Shikimic acid pathway, 211
212 Sieve element plasma membrane (SEPM), 164
165 Silybum marianum, 40 Single-guided RNA (sgRNA), 141
144 Single-stranded amiRNA, 218 siRNA. See Small interference RNA (siRNA) Sitobion avenae (grain aphid), 137, 139
141 Small interference RNA (siRNA), 131, 133 Soils, physical remediation of, 112 Solanum lycopersicum, 177f Solanum tuberosum, 37
38, 100, 102
103, 170
171 Somaclonal variation, and plant improvement, 36
37 Somatic embryogenesis (SE), 25, 29
31

323

Index

asynchronous development of, 29
31 Somatic hybridization, 37
38 Soybean ferritin, 197
200 Soybean, transgenic, 276 Spartina alterniflora (smooth cordgrass), 3
8 SSA. See Sunflower seed albumin (SSA) SSNs. See Sequence-specific nucleases (SSNs) SSNs gene editing tools, 62 Stabilization methods for medicinal plants, 208
209 StALS1 function, 261 Staphylococcus aureus (SaCas9), 60 Starch degradation genes, 162 StarLink, 281 StDREB1 gene, 102
103 STGs. See Salt-tolerant grasses (STGs) STIF. See Stress Gene Transcription Factor (STIF) Strain EHA105, 35, 97 Strains LBA4404, 35
36 Streptococcus thermophilus, 60 Stress Gene Transcription Factor (STIF), 12
13 Stress-responsive genes, 12
13 Structural bioinformaticians, 87
88 Structure Analysis and Verification Server (SAVES), 88
89 Structure prediction strategy, 86
89 computer-based, 88
89 function prediction, 90 model validation and evaluation, 88
89 secondary, 87 tertiary, 87
88 Sugar-based polymers, 232 Sugars, 232 Sulfate transporters (SULTRs), 172
173 Sulfate transport system, 175
176 Sulfur, 172
178 incorporation of, 173 nutritional deficiency, 174
175 utilization efficiency, 173
178 Sulfur-rich sink protein, 174
175 Sulfur utilization efficiency (SUE), transgenic with, 173
178 SULTRs. See Sulfate transporters (SULTRs)

Sunflower seed albumin (SSA), 173
174 accumulation, 175 sulfur-rich, 174
175 SuperLooper, 88 Susceptible allele Lr34sus, 18
19 Sweet potato peroxidase anionic 2 (SWPA2), 113 SWISS-MODEL, 88 SWPA2. See Sweet potato peroxidase anionic 2 (SWPA2) Synthetic biology, 229
230 Synthetic chemodisruptive peptides, 147
148 Synthetic seed methodology, 29
31 technology, 29
31 Systemic RNA interference, 131

T TAGs. See Tandemly arrayed genes (TAGs) TALENs. See Transcription activator-like effector nucleases (TALENs) Tandemly arrayed genes (TAGs), 63 Tanshinone production, 215
216 Targeted structural changes in crop species, 63
64 Target genome sequence nucleases, 59 Target-independent tracrRNA, 58
59 Target-induced local lesions in genomes (TILLING), 61
62 TAS. See Taxadiene synthase gene (TAS) Taxadiene synthase gene (TAS), 220 Taxus chinensis, 215
216 Taxus media, 41 Terpenes, 209
210, 234 Terpenoid biosynthesis pathway gene expression, 218
219 phytohormones levels in, 219 Terpenoid indole alkaloid (TIA), 218
219 Terpenoid synthases/cyclases (TPSs), 217 Tertiary structure prediction, 87
88 TFs. See Transcription factors (TFs) Therapeutic efficacy of medicinal plants. See Medicinal plants 3D structure of proteins, 87
88 TILLING. See Target-induced local lesions in genomes (TILLING)

324 Ti (tumor-inducing) plasmid transfers, 32 TMHMM, 87 Tobacco plants, 128
129 Tobacco streak virus (TSV), 138
139 Tolerance mechanism, 3
4 Tomato, 249
250 Toxic protein accumulation, 125 Toxins, 288 TPSs. See Terpenoid synthases/cyclases (TPSs) tracrRNA. See Transactivating RNA genes (tracrRNA) Trademarks, 295
296 Trade-Related Aspects of Intellectual Property Rights (TRIPS) agreement, 295
298 Trade secrets, 294
295 Traditional and modern medicine, 212
213 Traditional sequence-based characterization of protein, 86 Transactivating RNA genes (tracrRNA), 236
237 Transcription activator-like effector nucleases (TALENs), 52
54, 57
58, 220, 259
260 zinc-finger nucleases, 60 Transcription factors (TFs), 12
13, 235
236, 238 expression of, 218
219 gene families, 12
13 overexpression of, 254
257 Transfer DNA (T-DNA) technology, 32, 40
41, 259
260 insertion methods, 61
62 of pBIHSP construct, 98
99 Transformation methods, 33
34 Transformed root culture, 40 Transgenes, 125 rice plant, 197
201 sequence of protein, 289
290 Transgenic approach, 12
13 Transgenic broccoli, 33
34 Transgenic cotton, 276 Transgenic crops (TC), 144
145 with nitrogen use efficiency, 159
166

Index

with phosphorus utilization efficiency, 167
172 with sulfur utilization efficiency, 173
178 Transgenic cucumber, 33
34 Transgenic hybrids, in India, 278
279 Transgenic maize, 138
139 Transgenic plants, 8
10 development, 32
36 Transgenic plants, with nutrient utilization efficiency nitrogen, 157
166 use efficiency, 158
166 phosphorus, 166
172 utilization efficiency, 167, 169f sulfur, 172
178 utilization efficiency, 173
178 Transgenic rice, 8
10 Transgenic soybean, 276 Transgenic technologies, 52 for abiotic stress tolerance in plants, 114
116 drought, 104
107, 105f, 106t flooding, 107
109, 108t heat and cold, 96
101, 97t heavy metal stress, 112
114, 112f nutrient deficiency through genetic engineering, 109
111 salinity, 101
104, 102t, 103f agriculture biotechnology, intellectual property rights in, 292
303 Arabidopsis plant, 138 in biofuel production. See Biofuel production for genetic modification of plants, 273
274 for heavy metal stress tolerance, 112
114, 112f improve therapeutic efficacy of medicinal plants. See Medicinal plants natural products. See Natural products for insect pest management in plants, 124 Bacillus thuringiensis genes. See Bacillus thuringiensis (Bt) genes CRISPR against insects, 144
145

325

Index

first-generation genome editing technology, 130
141 nematode resistance in crop plants, 145
148 second-generation genome editing technology, 141
144 potato plant, 129
130, 147 potential accumulation of, 292 progress in, 303
304 shelf life. See Shelf life tobacco plants, 98, 217 tool for nutritional quality, 252
253 Transgenic tomatoes (Del/Ros1), 252 Transplastomic potato plants, 125
126 TRANSPORTERS OF MUGINEIC ACID 1 (HvTOM1), 190 Triacylglycerol production, 233 Triacylglycerols, 235 Trichoderma harzianum T34 hsp70 gene, 98
99 Trifolium repens L., 171 Trifolium subterraneum L., 170
171 Tri-iron(III), 191
192 Triticale wheat, 285 TSV. See Tobacco streak virus (TSV) Tylophora indica, micropropagation of, 27, 28f

VirulentPred, 86 Virulent protein, 86

W Wet-rice farming, 190
191 Wheat cultivar Jinghual, 137 White clover, 171 White eye gene, 144
145 WHO. See World Health Organization (WHO) Wild Asian rice, 3
4 Wild-type plants, 100 WIPO. See World Intellectual Property Organization (WIPO) World Health Organization (WHO), 285
286, 289
290 World Intellectual Property Organization (WIPO), 297
299

X Xanthomonas oryzae pv. oryzae (Xoo), 58, 61
62 Xylem vasculature, 192
193 Xylem vessels, 191

Y Yeast, 215
216

U

Z

Union Internationale pour la protection des obtentions végétales (UPOV), 298 uORFs. See Upstream open reading frames (uORFs) UPOV. See Union Internationale pour la protection des obtentions végétales (UPOV) Upstream open reading frames (uORFs), 63 Utility patents, 299
300

Zea mays (maize), 103, 188
190, 200
201, 276
278 Arabidopsis, 110 FERROPORTIN in, 192 iron content in, 198t mitochondria and chloroplast in, 195 reproductive system, 190 VIT genes in, 200 ZFNs. See Zinc-finger nucleases (ZFNs) Zinc-finger nucleases (ZFNs), 52
57 DNA-binding domain of, 56 and TALENS, 60 Zingiber officinale, 214
215 ZmNAS genes, 193 ZmZIP3 transgenic lines, 110

V Vacuole sequestration, iron, 195
196 Valeriana officinalis, 217 Vegetative insecticidal proteins (Vip), 127 VICMPred, 86