Biotechnological Approaches to Enhance Plant Secondary Metabolites: Recent Trends and Future Prospects [1 ed.] 0367473364, 9780367473365

Table of contents :
Cover
Half Title
Title Page
Copyright Page
Dedication
Contents
Preface
Acknowledgements
Editor
Contributors
Chapter 1: Plant Secondary Metabolites—The Key Drivers of Plant’s Defence Mechanisms: A General Introduction
Chapter 2: Generation of Plant Mutant Lines Using Gamma Radiation with Enhanced Secondary Metabolite Contents
Chapter 3: Salinity Stress and Plant Secondary Metabolite Enhancement: An Overview
Chapter 4: Enhancement of Plant Secondary Metabolites Using Fungal Endophytes
Chapter 5: Hydroponic Cultivation Approaches to Enhance the Contents of the Secondary Metabolites in Plants
Chapter 6: Tissue Culture Approaches to Enhance Plant Secondary Metabolites Production
Chapter 7: Hairy Roots and Plant Secondary Metabolites Production: An Update
Chapter 8: Brassinosteroids: The Phytohormones with Potential to Enhance the Secondary Metabolite Production in Plants
Chapter 9: CRISPR-Cas9 Approaches to Enhance Contents of Plant Secondary Metabolites
Chapter 10: RNA Interference for Improvement of Secondary Metabolite Production in Plants
Index

Citation preview

Biotechnological Approaches to Enhance Plant Secondary Metabolites

Biotechnological Approaches to Enhance Plant Secondary Metabolites Recent Trends and Future Prospects

Edited by Mohd. Shahnawaz

First edition published 2022 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742 and by CRC Press 2 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN © 2022 Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, LLC Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging‑in‑Publication Data Names: Shahnawaz, Mohd., editor. Title: Biotechnological approaches to enhance plant secondary metabolites : recent trends and future prospects / edited by Mohd. Shahnawaz. Description: First edition. | Boca Raton : CRC Press, 2022. | Includes bibliographical references and index. Identifiers: LCCN 2021026997 | ISBN 9780367473365 (hardback) | ISBN 9781032122021 (paperback) | ISBN 9781003034957 (ebook) Subjects: LCSH: Phytochemicals–Biotechnology. | Plant metabolites–Biotechnology. | Bioactive compounds–Biotechnology. Classification: LCC QK861 .B563 2022 | DDC 581.6/3–dc23 LC record available at https://lccn.loc.gov/2021026997

ISBN: 978-0-367-47336-5 (hbk) ISBN: 978-1-032-12202-1 (pbk) ISBN: 978-1-003-03495-7 (ebk) DOI: 10.1201/9781003034957 Typeset in Times LTStd by KnowledgeWorks Global Ltd.

Dedication To my parents (Mr. Shamas-ud-Din & Mrs. Muneera Begum) and my better half (Mrs. Mubeena Kousar)

Contents Preface����������������������������������������������������������������������������������������������������������������������������������������������ix Acknowledgements���������������������������������������������������������������������������������������������������������������������������xi Editor��������������������������������������������������������������������������������������������������������������������������������������������� xiii Contributors������������������������������������������������������������������������������������������������������������������������������������� xv Chapter 1 Plant Secondary Metabolites—The Key Drivers of Plant’s Defence Mechanisms: A General Introduction��������������������������������������������������������������������������1 D. Sruthi and C. Jayabaskaran Chapter 2 Generation of Plant Mutant Lines Using Gamma Radiation with Enhanced Secondary Metabolite Contents��������������������������������������������������������������������������������� 27 Lata I. Shukla, P. Vivek Vardhan, T. K. Devika, Sayan Roy and Sourav Bhatacharya Chapter 3 Salinity Stress and Plant Secondary Metabolite Enhancement: An Overview�������������������������������������������������������������������������������������������������������������� 49 Bedabrata Saha, Bhaben Chowardhara, Jay Prakash Awasthi, Sanjib Kumar Panda and Kishore C.S. Panigrahi Chapter 4 Enhancement of Plant Secondary Metabolites Using Fungal Endophytes���������������� 61 Touseef Hussain, Mulla Javed, Samrin Shaikh, Bilquees Tabasum, Kashif Hussain, Moh Sajid Ansari, Amir Khan and Abrar Ahmad Khan Chapter 5 Hydroponic Cultivation Approaches to Enhance the Contents of the Secondary Metabolites in Plants���������������������������������������������������������������������� 71 Yogesh Chandrakant Suryawanshi Chapter 6 Tissue Culture Approaches to Enhance Plant Secondary Metabolites Production���������������������������������������������������������������������������������������������� 89 Vishal N. Patil and Mohd. Shahnawaz Chapter 7 Hairy Roots and Plant Secondary Metabolites Production: An Update��������������������99 Sharada L. Deore, Bhushan A. Baviskar and Anjali A. Kide Chapter 8 Brassinosteroids: The Phytohormones with Potential to Enhance the Secondary Metabolite Production in Plants������������������������������������������������������������� 125 Barket Ali, Zahoor A. Wani and Mudasir Irfan Dar

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Contents

Chapter 9 CRISPR-Cas9 Approaches to Enhance Contents of Plant Secondary Metabolites��������������������������������������������������������������������������������������������� 133 Riddhi Rajyaguru, Nataraja Maheshala, Chandrashekar Mootapally, Neelam Nathani, Rukamsingh Tomar, Hiren Bhalani and Priyanka Sharma Chapter 10 RNA Interference for Improvement of Secondary Metabolite Production in Plants������������������������������������������������������������������������������������������������� 153 Ashutosh Kumar Rai and Pramod Wasudeo Ramteke Index���������������������������������������������������������������������������������������������������������������������������������������������� 165

Preface We have been dependent on plants for food, furniture, construction, and fuel since prehistoric times. Plants in all forms have tendered their services to mankind. We have a long history of plants cultivation and domestication to meet our basic needs. People living in different continents of the world used plants to treat various diseases. Accordingly, various systems of medicines were established viz. Ayurvedic, Unani, Siddha, Homeopathy, Tibetan, folklore and Chinese system of medicines, etc. Due to the advancement of science and technologies, various other non-conventional methods to treat the diseases were also identified. Each system of medicine is having advantages and disadvantages. The system which utilizes plant-based natural products for the discoveries of drugs have attracted the attention globally, due to its least or no side effects. Plant synthesized various secondary metabolites besides its primary metabolites to resist the changing environmental stress and to cross-talk with other biological entities. These secondary metabolites have also been found to have various activities against numerous human diseases. So, to harvest these medicinal principles from the medicinal plants, people have exploited the plant germplasm from the natural habitat at an alarming rate. Most of the medicinal plants are now considered endangered. As per the reports, the contents of these naturally occurring medicinal components were found low in most of the plants. So, it was needed to enhance the contents of such key principal components of the plants. Various people around the globe tried to enhance the contents of the secondary metabolites in the plants using various methods and were reported significant enhancement of the targeted secondary metabolites in the tested plants. The most common methods employed to enhance the contents of the plant secondary metabolites, viz. abiotic and biotic elicitation at in vitro and in situ levels, mutation breeding, plant tissue culture techniques, genetic engineering, RNAi, CRISPR-CAS9 technology etc. Various literature on plant secondary metabolites is available; however, there is a lack of books that provide an overview of all the methods involved in the enhancement of the plant secondary metabolite. This edited volume offers 10 excellent chapters, contributed by various experienced researchers, to fill the lacunae in the literature. Chapter 1 introduces plant secondary metabolites as the key drivers of plant’s defence mechanisms. Chapter 2 describes the gamma radiations usage to enhance the contents of plant secondary metabolites. Chapter 3 and 4 provides an overview of the impact of salinity stress and the role of fungal endophytes to enhance secondary metabolite contents in plants. Chapter 5 reports on the hydroponic cultivation approaches to enhance the contents of the secondary metabolites in plants. Chapters 6 and 7 highlight the tissue culture techniques and hairy root cultures the enhancement of plant secondary metabolites in plants. Chapter 8 documents the impact of brassinosteroids to enhance the secondary metabolite production in plants. Chapters 9 and 10 discuss the application of RNAi technology and CRISPR-CAS9 approaches to enhance contents of plant secondary metabolites.

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Acknowledgements I bow my head before Almighty Allah the most beneficent and most merciful for his endless blessings on me to complete this book. The financial support provided by DST-SERB in the form of the National Post-doctoral Fellowship (DST-SERB (PDF/2017/000178) award is dully acknowledged. I am highly thankful to all the anonymous reviewers, who reviewed the chapters thoroughly and allowed us to make wise decisions about the selection of the best chapters in this current volume. It is of immense pleasure to acknowledge the help and support tendered by the editorial and production team (especially Renu Upadhyay and Jyotsna Jangra) of CRC Press, Taylor & Francis Group. Last but not least, I am thankful to my parents (Mr. Shamas-ud-Din & Mrs. Muneera Begum) and my better half (Mrs. Mubeena Kousar) for their kind love and support throughout this work. Mohd. Shahnawaz ‘Khakii’

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Editor Dr. Mohd. Shahnawaz is currently working as a Lecturer at the Department of Botany, Govt. Degree College Paloura, Mishriwala, Jammu and Kashmir, India, on an academic arrangement basis (2020–2021). In 2020, he was selected as a post-doctoral fellow at Yeungnam University, Gyeongsan, North Gyeongsang, South Korea; however, due to COVID-19 scenario, he was not able to join. He has also completed post-doctoral research (funded by DST-SERB, Govt. of India) from the Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Jammu & Kashmir, India. He worked to enhance the contents of secondary metabolites in Coleus forskohli using in vitro salinity stress. Previously, he has worked as a Lecturer in Botany at the Department of Botany, Govt. Degree College Kishtwar, Jammu and Kashmir, India (2016–2017) on an academic arrangement basis. He has earned his M. Phil. and Ph.D. in Botany from the Department of Botany, Savitribai Phule Pune University, Maharashtra, India under the guidance of Prof. A. B. Nadaf and Prof. A. B. Ade in 2010 and 2016. He is the recipient of various fellowships awarded by the Savitribai Phule Pune University, University Grants Commission (UGC), and Department of Science and Technology (DST)-Science Engineering and Research Board (SERB), India. His research interests are focused on ecology, microbiology, bioremediation of plastic and plant biotechnology. He has served as a referee and editorial board member of various journals of International repute. In 2019, he was awarded the Young Scientist Award by VDGOOD Professional Associations, Vishakhapatnam, India. He has published more than 20 research articles in peer-reviewed international journals and authored 7 books. Previously, he had served as an Academic Editor of the Asian Journal of Biology (Science Domain International).

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Contributors Amir Khan Department of Botany Aligarh Muslim University Aligarh, India Barket Ali Department of Botany Government Degree College Kilhotran Kishtwar, India Moh Sajid Ansari Department of Botany Aligarh Muslim University Aligarh, India Jay Prakash Awasthi Department of Life Science and Bioinformatics Assam University Silchar, India Bhushan A. Baviskar Department of Pharmacognosy and Phytochemistry Government College of Pharmacy Amravati, India Hiren Bhalani Department of Biotechnology and Biochemistry Junagadh Agricultural University Junagadh, India Sourav Bhatacharya Department of Biotechnology Pondicherry University Kalapet, Puducherry, India

Sharada L. Deore Department of Pharmacognosy and Phytochemistry Government College of Pharmacy Amravati, India T. K. Devika Department of Biotechnology Pondicherry University Kalapet, Puducherry, India Kashif Hussain School of Pharmacy Glocal University Saharanpur, India Touseef Hussain Department of Botany Aligarh Muslim University Aligarh, India Mulla Javed Institute of Bioinformatics and Biotechnology Savitribai Phule Pune University Pune, India C. Jayabaskaran Department of Biochemistry Indian Institute of Science Bengaluru, India Abrar Ahmad Khan Department of Botany Aligarh Muslim University Aligarh, India

Bhaben Chowardhara Department of Life Science and Bioinformatics Assam University Silchar, India

Anjali A. Kide Department of Pharmacognosy and Phytochemistry Government College of Pharmacy Amravatima, India

Mudasir Irfan Dar Department of Botany Government Degree College Poonch Poonch, India

Nataraja Maheshala Entomology Section ICAR-Directorate of Groundnut Research Junagadh, India xv

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Contributors

Chandrashekar Mootapally Department of Bioscience Institute of Biotechnology Saurashtra University Rajkot, India

Pramod Wasudeo Ramteke Department of Biological Sciences Sam Higginbottom University of Agriculture Technology and Sciences Prayagraj, India

Neelam Nathani Department of Bioscience Institute of Biotechnology Saurashtra University Rajkot, India

Sayan Roy Department of Biotechnology Pondicherry University Kalapet, Puducherry, India

Sanjib Kumar Panda Department of Biochemistry Central University of Rajasthan Ajmer, India Kishore C.S. Panigrahi School of Biological Sciences National Institute of Science Education and Research Centre for Interdisciplinary Sciences Jatani, India and Life Sciences Homi Bhabha National Institute (HBNI) Mumbai, India Vishal N. Patil Post Graduate Department of Botany Vidya Bharti College Wardha, Seloo, India Ashutosh Kumar Rai Department of Biology Texas A&M University College Station, Texas and Department of Microbiology and Cell Biology Indian Institute of Science Bengaluru, India Riddhi Rajyaguru Department of Biotechnology and Biochemistry Junagadh Agricultural University Junagadh, India

Bedabrata Saha School of Biological Sciences National Institute of Science Education and Research Jatani, India and Weed Research Department Newe Ya’ar Research Centre Agricultural Research Organization (ARO) Ramat Yishai, Israel Mohd. Shahnawaz Department of Botany Govt. Degree College Paloura Mishriwala, India Samrin Shaikh Department of Botany Savitribai Phule Pune University Ganeshkhind, Pune, India Priyanka Sharma Department of Biotechnology Dr. Babasaheb Ambedkar Marathwada University Aurangabad, India Lata I. Shukla Department of Biotechnology Pondicherry University Kalapet, Puducherry, India D. Sruthi Department of Biochemistry Indian Institute of Science Bengaluru, India

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Contributors

Yogesh Chandrakant Suryawanshi Department of Yoga & Naturopathy Vishwakarma University Pune, India

P. Vivek Vardhan Department of Biotechnology Pondicherry University Kalapet, Puducherry, India

Bilquees Tabasum Department of Botany Savitribai Phule Pune University Pune, India

Zahoor A. Wani Department of Botany Government Degree College Kishtwar Kishtwar, India

Rukamsingh Tomar Department of Biotechnology and Biochemistry Junagadh Agricultural University Junagadh, India

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Plant Secondary Metabolites— The Key Drivers of Plant’s Defence Mechanisms A General Introduction D. Sruthi and C. Jayabaskaran

CONTENTS 1.1 Introduction............................................................................................................................... 1 1.2 Classification of Secondary Metabolites...................................................................................2 1.2.1 Terpenes (Terpenoids).................................................................................................... 2 1.2.1.1 Synthesis of Terpenes..................................................................................... 3 1.2.1.2 Classification of Terpenes...............................................................................4 1.2.1.3 Extraction and Identification of Terpenes....................................................... 5 1.2.1.4 Role and Application of Terpenes................................................................... 5 1.2.1.5 Essential Oil.................................................................................................... 6 1.2.2 Alkaloids........................................................................................................................ 8 1.2.2.1 Synthesis of Alkaloids.................................................................................... 8 1.2.2.2 Classification of Alkaloids..............................................................................8 1.2.2.3 Extraction and Identification of Alkaloids......................................................9 1.2.2.4 Role and Application of Alkaloids ................................................................9 1.2.3 Phenolics...................................................................................................................... 10 1.2.3.1 Synthesis of Phenolics.................................................................................. 10 1.2.3.2 Classification of Phenolics............................................................................ 12 1.2.3.3 Extraction and Identification of Phenolics.................................................... 13 1.2.3.4 Role and Application of Phenolics................................................................ 13 1.3 Current Biotechnological Approaches for the Enhancement of Secondary Metabolite Contents in Plants.................................................................................................................... 13 1.4 Conclusion............................................................................................................................... 16 Acknowledgements........................................................................................................................... 17 References......................................................................................................................................... 17

1.1 INTRODUCTION Plants are rich in bioactive phytoconstituents with enormous ethnopharmacological potential and these high-value phytochemicals are broadly categorized into two classes (primary and secondary metabolites) based on their function (Hussein and El-Anssary 2019). The primary metabolites are distributed widely in all plants and carry out metabolic functions which are crucial for normal physiological growth and energy requirements of the plants and are normally evident (Hussain et al. 2012; Wink 2016). In contrast, plants synthesize myriads of organic compounds; most of them do not involve directly in plant growth and development. These phytochemicals, normally known as secondary metabolites, are distributed differentially in restricted taxonomic groups of the plant DOI: 10.1201/9781003034957-1

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Biotechnological Approaches to Enhance Plant Secondary Metabolites

kingdom (Tiwari and Rana 2015). Briefly, secondary metabolites are normally not essential for plant growth and its development, but they are essential for surviving in the ecosystem through chemical defence (Iriti and Faoro 2009). Plant secondary metabolites are the chemical constitutes primarily responsible for the chemical defences to regulate the relationship between the plant and their ecosystem and further take part in protecting plants from abiotic and biotic stress conditions (Mazid et al. 2011). Secondary metabolites are not essentially synthesized under all circumstances but produced undoubtedly for really appreciated reasons, few of such include, toxic materials to protect the plant from predators, as volatile attractants or as an agent to impart colour and thereby warn or attract other species and thus, it is evident that all of them play some crucial function for the safety of their producer (Adeyemi and Mohammed 2014). Plant defence metabolites arise from the isoprenoid, the alkaloid and the phenylpropanoid pathways which form three major secondary metabolite classes viz., terpenes, alkaloids and phenolics, respectively (Iriti and Faoro 2009). Generally, precursors for secondary metabolite synthesis are products of the primary metabolism (Iriti and Faoro 2004). Besides their role in plant protection, secondary metabolites are well studied for various pharmacological and medicinal potentials and many drugs of natural origin against human ailments have been discovered from different plants (Velu et al. 2018). Secondary metabolites provide major pharmacologically active high-value natural products used to treat various diseases since ancient times and it ranges from migraine up to cancer (Hussein and El-Anssary 2019; Jain et al. 2019). Vinca alkaloids (vinblastine and vincristine), taxanes [e.g. paclitaxel (Taxol) and docetaxel (Taxotere)] and combretastatin are some of the secondary metabolites used to treat cancer (Hartwell 1984; Kingston 2012; Pinney et al. 2012; Roussi et al. 2012). Further, secondary metabolite drugs have been obtained from plants with anti-diabetic (e.g. galegine alkaloid from Galega officinalis,), anti-inflammatory (e.g. curcumin and resveratrol) and anti-viral (e.g. betulinic acid and calanolides) efficacies (Min et al. 1999; Dharmaratne et al. 2002; Furst and Zudorf 2014; Rios et al. 2015). So there is a huge demand for medicinal plants to isolate pharmaceutically active molecules (Dar et al. 2017a). These secondary metabolites are produced in low quantity in plants; hence, a large amount of plant material is used to extract the drug molecules (Dar et al. 2017b). To meet the demand of pharmaceutical companies, huge medicinal plants are being exploited at a mass level. This practice leads to threat the germplasm and various plants were extinct from the natural environment (Maxted et al. 2020). Hence, it was needed to enhance the contents of such pharmaceutically active plant secondary metabolites to reduce the exploitation of the medicinal plants from the natural environment (Jimenez-Garcia et al. 2013). With this regard, various biotechnological strategies were used by different workers across the globe (Guerriero et al. 2018). So, in this chapter, an effort was made to overview the plant secondary metabolites, to discuss different classes of plant secondary metabolites, to discuss the biosynthetic pathways that took part in the production of plant secondary metabolites and to highlight various approaches adopted to enhance the secondary metabolite contents in plants.

1.2  CLASSIFICATION OF SECONDARY METABOLITES Secondary metabolites are classified primarily as terpenoids, alkaloids and phenolics depending on their chemical structure (Shamina and Sarma 2001).

1.2.1  Terpenes (Terpenoids) Terpenes are ubiquitous in plants and are the largest class of secondary metabolites with more than 22000 compounds (Freeman and Beattie 2008). The high concentration of compounds in turpentine oil has given the alternate name ‘terpenoid’ to these compounds. All terpenes are said to be the derivative of branched, basic, five-carbon unit isoprene (C5H8) (Bramley 1997; Goodwin and Mercer 2003). Isoprene is a volatile gas emitted by leaves during photosynthesis and that might prevent the damage of plant cell membranes due to high temperature or light (Freeman and Beattie

Plant Secondary Metabolites—The Key Drivers of Plant’s Defence Mechanisms

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2008). Terpenes are the essential metabolite for photosynthesis and also for regulating the plant metabolic processes (Bramley 1997; Goodwin and Mercer 2003). Even though most of the terpenes are categorized as secondary metabolites, extremely few among them (e.g. steroids) are included in the primary metabolite class (Bramley 1997; Goodwin and Mercer 2003). 1.2.1.1  Synthesis of Terpenes The cytosol and the plastids are the sites of biosynthesis of terpenoids (Aharoni et al. 2006). They are also known as isoprenoids and synthesized from acetyl-CoA by mevalonate or isoprenoid pathway (Figure 1.1) (Goodwin and Mercer 2003; Karlic and Varga 2019). Two acetyl-CoA molecules are combined to form acetoacetyl-CoA and, further form HMG-CoA (β-hydroxy-β-methylglutaryl CoA) with an additional acetyl CoA molecule. Both of these reactions are catalyzed by HMG-CoA synthase (Chappell 1995). HMG-CoA reductase further catalyzes the rate-limiting reaction of the isoprenoid pathway, the synthesis of mevalonic acid from HMG-CoA. From the mevalonate, IPP (isopentenyl diphosphate), a five-carbon building block for the synthesis of isoprenoid chains is

FIGURE 1.1  Isoprenoid pathway from acetyl-CoA. (Adapted from Iriti and Faoro 2009.)

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synthesized and is converted into all the different terpenoids found in nature (Bach 1987; Chappell 1995; Goodwin and Mercer 2003). 1.2.1.2  Classification of Terpenes Based on the isoprene unit number, terpenes can be classified as ‘hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpenes and polyterpenes with one, two, three, four, five, six, eight and more than eight isoprene units, respectively’ (Goodwin and Mercer 2003). Table 1.1 represents different classes of terpenoids with their molecular formula. Hemiterpenes are formed from isopentenyl pyrophosphate and mainly found in plants as signalling defence compounds and are represented by parent isoprene, that is a common volatile compound released from woody trees like willows, oaks, spruce and poplar (Dewick 2009; Osbourn and Lanzotti 2009). Other examples for hemiterpenes include berry and fruit metabolite prenol and isovaleric acid, an essential oil constituent (Schnitzler et al. 2012). Monoterpenes represent the simplest terpenoid class with C10 skeleton and are formed from geranyl-PP (Croteau 1987; Goodwin and Mercer 2003). Monoterpenes are generally occurring in secretory glands, as the main constituent of plant essential oil (Goodwin and Mercer 2003). They are primarily non-volatile and can further be categorized as acyclic, cyclopentanoid, cyclohexanoid and irregular monoterpenes based on their structure. Thujene, camphene, pinene, limonene, myrcene, careen, etc. are some of the monoterpenes (Goodwin and Mercer 2003). Monoterpenes are widely seen in higher plants and possess a strong characteristic aroma that gives them huge attention in the perfumery industry (Goodwin and Mercer 2003). They also have applications in food flavourings and pharmacology (Kang and Lee 2016). Because of their repulsiveness or attractiveness, monoterpenes involve in plant-plant, plant-phytophagous and plant-microbe interactions (Croteau 1987). Sesquiterpenes are the largest terpenes and are synthesized from farnesyl-PP (Cane 1990). They have a C15 skeleton and their volatility is less compared to monoterpenes (Cane 1990; Stephane and Jules 2020). However, they co-exist with monoterpenes and found to be the essential part of essential oil (Goodwin and Mercer 2003; Stephane and Jules 2020). Further, sesquiterpenes possess enormous biological activities as pheromones, insect antifeedant, insect juvenile hormone, plant growth regulators, mycotoxins and phytoalexines antibiotics (Bowers et al. 1977; Kalsi et al. 1989; Taylor 1990; Morrissey and Osbourn 1999; Dhadialla et al. 2005; Abdelgaleil et al. 2020). Further, sesquiterpenes can be classified as acyclic, monocyclic, bicyclic and tricyclic sesquiterpenes (Goodwin and Mercer 2003). Sesquiterpenes contain a lactone ring that comes under the class sesquiterpene lactone (Schmidt 2006). Caryophyllene, nerolidol, humulene, elemol, α-cadinene, γ-bisabolene, etc. are the examples for sesquiterpenes (Goodwin and Mercer 2003).

TABLE 1.1 Classification of Terpenoids Name of Terpenes Hemiterpenes Monoterpenes Sesquiterpenes Diterpenes Sesterterpenes Triterpenes Tetraterpenes Polyterpenes

Molecular Formula C5H8 C10H16 C15H24 C20H32 C25H40 C30H48 C40H64 (C5H8)n

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Diterpenes with C20 skeleton are derived from geranylgeranyl-PP and consist of acyclic, monocyclic, bicyclic, tricyclic or tetracyclic diterpenes (Dogbo and Camara 1987; Goodwin and Mercer 2003). More than 500 diterpenes are reported. Agathic acid, phytol, α-camphorene, cassaic acid, steviol, etc. are some of the diterpenes (Goodwin and Mercer 2003). The sesterterpenes have C25 skeleton and are found together with diterpenoids in lichens, seaweeds, fungi, marine organisms and higher flowering plants and originate from geranylfarnesyl pyrophosphate (Bramley 1997; Goodwin and Mercer 2003; Dewick 2009; Osbourn and Lanzotti 2009; Gonzalez 2010; Cimmino et al. 2014). Ophiobolin A and bilosespene A are the examples for sesterterpenes from fungal and marine metabolites, respectively (Rudi et al. 1999; Au et al. 2000). Leucosesterterpenone and leucosterlactone are the tetracyclic sesterterpenes isolated and identified from plant Leucosceptrum canum (Hussain et al. 2008). Sesterterpenoids have been investigated recently for their chemical, structural and biological characterizations and possess a wide spectrum of biological properties against microorganisms and many of them exhibit strong anticancer efficacy (Gonzalez 2010; Wang et al. 2013; Cimmino et al. 2014; Evidente et al. 2015). Triterpenes (C30 skeleton) represent another large set of isoprenoid constituents (Goodwin and Mercer 2003) and are formed from farnesyl-PP (two molecules) through a reductive head-to-head condensation (Iriti and Faoro 2009). It consists of tetracyclic derivatives and pentacyclic compounds (Goodwin and Mercer 2003). Lanosterol, euphol, lupeol, cycloartenol, etc. are some of the cyclic triterpenoids (Goodwin and Mercer 2003). Phytosterols with a cyclopentanoperhydrophenanthrene ring system are the derivatives of tetracyclic triterpenes, whereas saponins are the water-soluble glycosidic triterpenes (Goodwin and Mercer 2003). The tetraterpenes (C40) are synthesized from two geranylgeranyl-PP molecules through head-to-head condensation (Wendt and Schulz 1998) and contain only one group—the carotenoid pigments (α-and β-carotene, xanthophylls, etc.) (Goodwin and Mercer 2003). They are mainly seen in chloroplasts and guard it against photodynamic sensitization and further contribute to the light-harvesting process of photosynthesis (Bramley 1997; Goodwin and Mercer 2003). Polyterpenes (e.g. natural rubber), on the other hand, are the terpenoids widely seen in plants with higher molecular weight (Bramley 1997; Goodwin and Mercer 2003) and composed of more than a thousand isoprene units formed through the polymerization of geranylgeranyl-PP molecules (Wendt and Schulz 1998). The examples for different terpenoid classes are shown in Figure 1.2. 1.2.1.3  Extraction and Identification of Terpenes The non-volatile terpenoids are extracted from the plant samples with organic solvents like hexane or hexane-ethyl acetate mixture and can be separated from other constituents present in the extract using column chromatography with a silica stationary phase (Jiang et al. 2016). On the other hand, the volatile terpenoids from plant tissues can extract directly with traditional extraction techniques (e.g. hydrodistillation and organic solvent extraction) and more novel techniques like microwaveassisted extraction and solid-phase micro extraction (Chemat et al. 2012; Pawliszyn 2012; Jiang et al. 2016). The extracted and purified terpenoids can be identified and quantified using gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GCMS) (Jiang et al. 2016). 1.2.1.4  Role and Application of Terpenes Terpenoids are highly bioactive and play a key function in plant-plant communication and also in plant-environment, plant-insect and plant-pathogen interactions (Dudareva et al. 2004; Cheng et al. 2007; Bouwmeester et al. 2019). Isoprenoids took part in protein prenylation—the synthesis of differentially lengthened isoprenoid chains anchoring proteins like G-proteins, plastoquinone and cytochrome-a in membranes (Wendt and Schulz 1998). Terpenoids have great commercial significance because of their vast applications as insecticides, flavouring agents and anti-microbial agents and further due to their role in pharmaceuticals and perfumes (Martin et al. 2003).

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Biotechnological Approaches to Enhance Plant Secondary Metabolites

FIGURE 1.2  Examples for different classes of terpenoids.

1.2.1.5  Essential Oil The aromatic plants have characteristic fragrance due to certain compounds which collectively form oily volatile liquids called essential oil (Joy et al. 2014; Stephane and Jules 2020). Hence, the essential oil is considered the ‘essence’ of aromatic plants and this volatile liquid is extracted from their different parts (Joy et al. 2014; Morsy 2017). Each plant species has its characteristic blend of volatiles which contain species-specific compounds (Janardhanan and Thoppil 2004). Rosaceae, Lamiaceae, Myrtaceae, Zingiberaceae, Myristicaceae, Acoraceae, Oleaceae, Cupressaceae, Asteraceae, Umbelliferae, etc. are some plant families that have their essential oil constituents (Stephane and Jules 2020). ‘Essential oils are synthesized by glandular trichomes and other secretory structures, specialized secretory tissues mainly diffused onto the surface of plant organs’ (Sharifi-Rad et al. 2017). ‘It is a heterogeneous, complex mixture composed of terpene hydrocarbons (mainly mono and sesquiterpenes), oxygenated compounds, straight-chain non-terpenoid hydrocarbons and their oxygen derivatives (aldehydes, ketones, alcohols, acids, esters and ethers), phenylpropenes and miscellaneous compounds (degradation products formed from lactones, unsaturated fatty acids, glycosides, terpenes, sulphur and nitrogen-containing compounds)’ (Morsy 2017). However, the more volatile fractions of essential oils are contributed by plant terpenes, especially mono and sesquiterpenes (Janardhanan and Thoppil 2004). Considering the physical characteristics of essential oil, most mono and sesquiterpenes have optical activity, whereas phenylpropenes usually do not. However, the oil as a whole has optical activity. Refractive index and specific gravity are the other physical parameters of essential oil,

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which are useful as indicators of oil quality (Waterman 1993). The volatile oils are colourless or may have a wide range of pale colours. The odour, another important physical parameter of essential oil, can be classified into six major types viz., woody (odour of wood like sandal wood), herbaceous (smell of green herbs like dill), fruity (odour of fruits), spicy (smell of spices), green (smell of cut grass, leaves, etc.) and flowery (odour of flowers) (Janardhanan and Thoppil 2004). Hydrodistillation is the traditional and most preferable method for essential oil extraction and can be performed using the Clevenger trap apparatus (AOAC 1975; Rassem et al. 2016; Irshad et al. 2019). Upon boiling, essential oil escapes along with steam and forms a separate layer in the Clevenger trap, which can be collected (AOAC 1975). This method is cheap, easy to conduct, suitable for field operation and the extracted oil will be free from oil glands/ducts/cells in the plant tissues (Janardhanan and Thoppil 2004; Irshad et al. 2019). Steam distillation, solvent extraction, hydrosteam distillation, maceration, enfleurage, expression, microwave-assisted distillation, microwave hydro diffusion and gravity, high-pressure solvent extraction, solvent-free microwave extraction, supercritical carbon dioxide extraction, ultrasonic extraction and the phytonic process are the other essential oil extraction methods (Ranjitha and Vijiyalakshmi 2014; Moghaddam and Mehdizadeh 2017; Mohammed et al. 2019). Supercritical fluid extraction with supercritical CO2 is an advanced technique that is a simple, fast, inexpensive, effective and virtually solvent-free sample pre-treatment method for volatile oil extraction (Pourmortazavi and Hajimirsadeghi 2007). The selection of extraction method is based on the plant material type and the desired end products (Janardhanan and Thoppil 2004). The essential oils are analyzed by various chromatographic techniques like thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC), column chromatography and gas chromatography (GC) (Janardhanan and Thoppil 2004). Nuclear magnetic resonance (NMR) spectroscopy can elucidate the structure of essential oil constituents, and mass spectroscopy (MS) is used for direct analysis of mixtures (Janardhanan and Thoppil 2004). Recently, GC coupled with MS (GC-MS) is extensively used for the separation, identification and quantification of essential oil constituents (Janardhanan and Thoppil 2004). Apart from its role as aroma contributors, the essential oil has many other functions in plants. Essential oils lead to attracting certain insects and are reported to help in pollination (Pichersky and Gershenzon 2002). Certain volatile oils can protect plants from the attack of animals, microbes and plant parasites and are also involved in other non-defensive functions like temperature regulation, decreased water loss and reduced mechanical abrasion (Janardhanan and Thoppil 2004; Sharifi-Rad et al. 2017). Essential oil and its constituents are also reported for allelopathic potential (EI-Sawi et al. 2019). Thus, they are involved in the ecology and physiology of plants (Janardhanan and Thoppil 2004; Sharifi-Rad et al. 2017). Essential oils are well recognized for their application as perfumery contributors, medicaments and flavouring agents and hence, they have immense significance in various industries including perfumery, pharmaceutical and food (Hamid et al. 2011; Chavez-Gonzalez et al. 2016; Sarkic and Stappen 2018). They are used for perfuming soaps, deodorants, cosmetics, oral care products, for flavouring food, beverage products, etc. (Sarkic and Stappen 2018). They are also used as laboratory reagents, as a solvent in the paint industry, as insecticides, as a component of polishes, pastes, ink, etc. (Janardhanan and Thoppil 2004; Sarkic and Stappen 2018; Irshad et al. 2019; Kouznetsov 2019). They have also been reported to be used in pharmaceutical industries due to their antiseptic, stimulative, carminative, expectorant, rubefacient, diuretic, counter-irritant and flavouring properties (Janardhanan and Thoppil 2004). Essential oils are familiar for their anti-spasmodic, antibacterial, anthelmintic, anti-oxidant, disinfectant, anti-cancer, anti-fungal and anti-inflammatory activities and also for irritable bowel syndrome treatment (Perez et al. 2011; Valeriano et al. 2012; Khanna et al. 2013; Cash et al. 2016; Nazzaro et al. 2017; Torres-Martinez et al. 2017; Blowman et al. 2018; Ferreira et al. 2018; Heghes et al. 2019; Man et al. 2019). They are used for the manufacture of ointments, lotions, creams and various syrups (Dosoky and Setzer 2018; Sarkic and Stappen 2018). Essential oil is also used widely in aromatherapy (Ali et al. 2015).

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1.2.2  Alkaloids Alkaloids represent another vast group of heterogeneous, physiologically active and heterocyclic secondary metabolites (Goodwin and Mercer 2003). More than 2000 alkaloids are reported from plants and they are distributed in all plant parts. Alkaloids are majorly basic in nature (Goodwin and Mercer 2003) and accumulate chiefly in four types of tissues viz., vascular sheaths, actively growing tissue, epidermal and hypodermal cells, and latex vessels (Goodwin and Mercer 2003). They are present in vacuoles and thus do not appear in young cells until vacuolation happens (Goodwin and Mercer 2003). Alkaloids are frequently stored in tissues other than their site of synthesis. Likewise, secondary structural modifications of alkaloids often occur at sites other than where primary synthesis took place (Goodwin and Mercer 2003). Dicots are richer in alkaloids than monocots whereas they have limited distributions in lower plants (Goodwin and Mercer 2003). Apocynaceae, Rubiaceae, Solanaceae, Papaveraceae, Leguminosae and Fumariaceae are the families rich in alkaloids, whereas those like Rosaceae and Labiatae contain alkaloids to a lesser extent (Goodwin and Mercer 2003). 1.2.2.1  Synthesis of Alkaloids A common pathway that can explain alkaloid biosynthesis does not exist (Iriti and Faoro 2009). The biosynthetic precursors for alkaloids are almost always amino acids (Goodwin and Mercer 2003). Other multi-carbon units like nicotinic acid, anthranilic acid, isoprenoids, acetate and purine are also incorporated into the final structure of some alkaloids (Iriti and Faoro 2009). Even though the biosynthesis of alkaloids cannot be easily explained, some typical reactions like synthesis of Schiff bases and the Mannich reaction which take part in the synthesis of different classes of the alkaloid can be explained (Plemenkov 2001). The Schiff bases are formed within a molecule, by the reaction of amines with aldehydes or ketones and these reactions are the general method for the production of C=N bonds (e.g. synthesis of piperidine alkaloids) (Plemenkov 2001; Dewick 2002). On the other hand, the Mannich reaction proceeded in both intra and inter molecularly (Plemenkov 2001; Dewick 2002). In addition to a carbonyl compound and an amine, the Mannich reaction contains an integral component, a carbanion, which plays the function of the nucleophile in the nucleophilic addition to the ion formed by the reaction of the carbonyl and the amine (Plemenkov 2001; Dewick 2002). In addition to the biosynthesis of monomeric alkaloids which involved the aforementioned reactions, there are also dimeric, trimeric and tetrameric alkaloids synthesized through condensation of two, three and four monomeric alkaloids, respectively (Hesse 2003). Dimeric alkaloids are normally synthesized from the same type of monomers through the following mechanisms: Michael reaction (e.g. villalstonine), Mannich reaction (e.g. voacamine), condensation of aldehydes with amines (e.g. toxiferine), lactonization (e.g. carpaine) and oxidative addition of phenols (e.g. dauricine) (Hesse 2003). 1.2.2.2  Classification of Alkaloids The alkaloids formed from amino acids and have nitrogen in a heterocyclic ring are commonly known as true alkaloids (e.g. nicotine, morphine) and those without such rings are called protoalkaloids (e.g. ephedrine, cathinone) (Goodwin and Mercer 2003; Wansi et al. 2013). The alkaloids (with or without heterocyclic rings) which are not formed from amino acids are called pseudoalkaloids, in which carbon skeleton is derived from isoprenoids (e.g. actinidine, dendrobine) (Goodwin and Mercer 2003). Besides, steroidal alkaloids are also present and which include, cholestane derivatives, pregnane derivatives and C-nor-D-homosteroidal alkaloids (Goodwin and Mercer 2003). Alkaloids are also grouped based on their biosynthetic precursors and chemical structure (Table 1.2) (Iriti and Faoro 2009). The main alkaloid groups are formed from the amino acid precursors like ornithine (pyrrolidine, tropane and pyrrolizidine alkaloids), leucine (pyrrole alkaloids), lysine (piperidine, indolizidine and quinolizidine alkaloids), tyrosine (catecholamines, tetrahydroisoquinoline, isoquinoline and benzyltetrahydroisoquinoline alkaloids), tryptophan

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TABLE 1.2 Precursors and Different Classes of Alkaloids Precursors

Classes of Alkaloid

Ornithine

Pyrrolidine1, Tropane, Pyrrozidine, Polyamines2 Pyrrole Piperidine, Indolizidine, Quinolizidine Isoquinoline, Tetrahydroisoquinoline, Benzyltetrahydroisoquinoline1, Catecholamines2 Indole1, Carbolines, Quinoline2, Pyrrolindole, Indolamines3

Leucine Lysine Tyrosine

Tryptophan

Histidine Phenylalanine Anthranilic acid Purine Geranylgeranyl-diphosphate Cholesterol Acetate Nicotinic acid

Imidazole Quinazoline, Quinoline, Acridine Terpenoidic Steroidal Piperidine Pyridine

Examples 1. Cocaine, Atropine 2. Putrescine, Spermine, Spermidine 1. Morphine, Curarines, Papaverine 2. Adrenaline, Noradrenaline 1. Vindoline, Catharantine 2. Quinine, Capthotecin 3. Melatonin, Serotonin Capsaicin, Ephedrine Theobromine, Caffeine, Theophylline Solanin Nicotine

Source: Adapted from Iriti and Faoro (2009).

(indolamines, carboline, indole, quinoline, pyrrolindole and ergot alkaloids), histidine (imidazole alkaloids) and phenylalanine (Cordell et al. 2001; Facchini 2001; Hughes and Shanks 2002). 1.2.2.3  Extraction and Identification of Alkaloids The alkaloids can be extracted with different methods. The alkaline alkaloids can be extracted with water or acidic water. The acid extraction method generally uses 0.1% to 1% hydrochloric acid, sulphuric acid, tartaric acid or acetic acid solution as a solvent (Yubin et al. 2014). Both free and salt alkaloids can be extracted using organic solvents like methanol or ethanol (Yubin et al. 2014). Further, lipophilic free alkaloids can be extracted using organic solvents like chloroform, benzene, ether and methylene chloride (Yubin et al. 2014). The extracted alkaloids can be separated using methods like solvent method, precipitation method, fractional distillation method, gradient pH method, dialysis method, crystallization method and chromatographic method (Yubin et al. 2014; Feng et al. 2019). The extracted alkaloids can further go for their purity checking using physical property measurements and chromatographic techniques including TLC, HPLC and GC (Feng et al. 2019). The purified alkaloids can be further identified through structural elucidation using different techniques like ultraviolet-visible spectra, infrared spectra (IR), NMR spectroscopy and mass spectrometry (MS) (Feng et al. 2019). 1.2.2.4  Role and Application of Alkaloids Alkaloids have many ecological, biochemical and physiological roles in plants (Goodwin and Mercer 2003). They protect the plants from insects and herbivores, act as N-excretory products in the same way as uric acid and urea in animals and further as nitrogen reserve and growth regulators (Goodwin and Mercer 2003). They also involve maintaining ionic balance under their chelating power (Goodwin and Mercer 2003). Alkaloids represent massive phytoconstituents with

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nutritional, pharmacological, toxicological and cosmetic potential (Iriti and Faoro 2009). They have great importance in pharmaceutical industries because of their vast pharmacological activities like anti-amoebic, anti-plasmodial, anti-oxidant, anti-cholinergic, anti-malarial, anti-hypertensive, anxiolytic, anti-microbial, anti-inflammatory, anti-depressant, anti-HIV and anti-tumour properties (Wright et al. 1991; Cowan 1999; Kupeli et al. 2002; Hesse 2003; Cos et al. 2008; Kulkarni and Dhir 2009; Liu et al. 2013; Yuan et al. 2019).

1.2.3 Phenolics Phenolics are ubiquitous plant secondary metabolites and these phytoconstituents occur as free phenols or their glycosides (Marinova et al. 2005; Hussein and El-Anssary 2019). This class of compounds consists of more than 8000 biologically active constituents which range from simple phenolic compounds to polymeric structures with a molecular weight of above 30000 Da (Marinova et al. 2005). 1.2.3.1  Synthesis of Phenolics Phenolics are the class of phenylalanine derivatives and they possess a basic C6 ─C3 (phenylpropane) skeleton. They are synthesized through the phenylpropanoid pathway (Figure 1.3)

FIGURE 1.3  Aromatic amino acids biosynthesis from shikimic acid and phenyl alanine derivatives of phenylpropanoid pathway. (Adapted from Iriti and Faoro 2009.)

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and are also called phenylpropanoids (Iriti and Faoro 2009; Hyskova and Ryslava 2019). ‘Phosphoenolpyruvate and erythrose 4-phosphate from the glycolysis and pentose phosphate pathway are the precursors of phenylpropanoid pathway and which leads to the synthesis of two main intermediates, shikimic and chorismic acid’ (Weaver and Hermann 1997). ‘In the succeeding steps, after a branch point, phenylalanine and tyrosine are formed from prephenic and arogenic acid, whereas tryptophan from anthranilic acid’ (Weaver and Hermann 1997). The phenylalanine further subjected to deamination reaction catalyzed by PAL (phenylalanine ammonia-lyase), which leads to the synthesis of cinnamic acid and this forms the key step for the synthesis of phenolic constituents (Iriti and Faoro 2009). After a series of hydroxylation reactions of the benzene ring, hydroxycinnamates (e.g. coumaric, sinapic and ferulic acids) are formed from the cinnamic acid precursor (Iriti and Faoro 2009). Hydroxycinnamates are reduced to their respective alcohols through aldehyde intermediates and collectively termed monolignols which are further subjected to dimerization or polymerization to form lignans and lignin, respectively (Iriti and Faoro 2009). Lignification is a complex reaction in which the polymerization of lignin units is catalyzed by peroxidases, consuming H2O2 (Hahlbrock and Scheel 1989; Iriti and Faoro 2004). Benzoic and hydroxybenzoic acids (C6 ─C1) like salicylic acid are another cinnamic acid derivative group, synthesized by the cleavage of C2 fragment from the phenylpropane structure (Hahlbrock and Scheel 1989; Iriti and Faoro 2004). However, based on the species and the conditions (e.g. pathogen attack), salicylic acid can also synthesize directly from chorismate-isochorismate by isochorismate synthase (Wildermuth et al. 2001). Figure 1.4 explains

FIGURE 1.4  Main steps of the phenylpropanoid pathway leading to benzoic acids, hydroxycinnamates, monolignols and lignin. (Adapted from Iriti and Faoro 2009.)

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Biotechnological Approaches to Enhance Plant Secondary Metabolites

the phenylpropanoid pathway’s major steps which lead to the synthesis of benzoic acids, hydroxycinnamates, monolignols and lignin. 1.2.3.2  Classification of Phenolics The phenolics can be classified as simple phenols, phenolic acids, phenylacetic acids, hydroxycinnamic acids, phenylpropenes, coumarins, quinones, stilbenes, xanthones, lignans, neolignans, melanins, tannins and flavonoids (Goodwin and Mercer 2003). Simple phenols are not widely distributed and the most common simple phenol is hydroquinone. The phenolic acids are extensively present throughout the plant kingdom (Goodwin and Mercer 2003). Protocatechuic acid, p-hydroxybenzoic acid, syringic acid and vanillic acid are some of the phenolic acids (Goodwin and Mercer 2003). Aldehyde or alcohols corresponding to the parent carboxylic acids (e.g. vanillin) are also known (Goodwin and Mercer 2003). Even though the distribution of phenylacetic acids in plants is not known much, 2- and 4-hydroxyphenylacetic acids were reported (Goodwin and Mercer 2003). The hydroxycinnamic acids, like p-coumaric acid and caffeic acid and their methylated derivatives like ferulic acid and synapic acid, are universally present in higher plants (Goodwin and Mercer 2003). Phenylpropenes are not widely distributed, but they occur sporadically in essential oils (e.g. myristicin) (Goodwin and Mercer 2003). Coumarins are lactones, derived from o-hydroxycinnamic acid and occur abundantly in plants (Goodwin and Mercer 2003). The quinones mainly include benzoquinones, naphthoquinones and anthraquinones, whereas lunalaric acid is the most common plant stilbene (Goodwin and Mercer 2003). Over 70 xanthones were reported in plants and their distribution is restricted to Guttiferae and Gentianaceae families (e.g. mangiferin) (Goodwin and Mercer 2003). The lignans are naturally occurring phenylpropanoid dimers in which the two C6 ─C3 units are joined tail-to-tail by the carbon-carbon bond between the middle carbons of their side chains (e.g. conidendrin) (Goodwin and Mercer 2003). If the C6 ─C3 units are joined head-to-tail, the resultant phenolics are called neolignans (e.g. eusiderin) (Goodwin and Mercer 2003). Melanins are brown or black, naturally occurring pigments (Goodwin and Mercer 2003). Melanin, characteristics of plants are called catechol melanins, since they yield catechol on alkali fusion (Goodwin and Mercer 2003). The tannins are class of high molecular weight chemical constituents with tanning properties and mainly include hydrolysable and condensed tannins (Goodwin and Mercer 2003). The flavonoids are the largest group of naturally occurring phenolic compounds and are distributed in different parts of the plant, both in free form and as glycosides (Sulaiman and Balachandran 2012). These compounds function as co-pigments and are responsible for different colours exhibited by different plant parts (Goodwin and Mercer 2003; Iwashina 2015). The flavonoid glycosides are found to be occurring as O-glycosides and C-glycosides, where the latter received less attention (Xiao et al. 2016). The structure of flavonoid consists of 15 carbon atom phenylpropanoid core, together with 2 six-carbon aromatic rings (ring A and B) joined by a heterocyclic ring which contains 3 carbon atoms (ring C), which together can be represented as C6 ─C3─C6 (Cheng et al. 2014). The flavonoids can be classified into the following categories. Flavones (contains benzoγ-pyrone ring; e.g. apigenin), flavanone (have a 2,3-dihydro skeleton in C6 ─C3─C6 structure of flavonoid; e.g. naringenin), flavonol (3-hydroxy derivative of flavone; e.g. kaempferol), dihydroflavonol (3-hydroxy derivative of flavanone; e.g. taxifolin), isoflavones (contain benzo-γ-pyrone ring with phenyl substitution at position 3 of the pyrone ring; e.g. genistein), chalcones (open chain flavonoids; e.g. butein) and dihydrochalcones (dihydro derivatives of chalcones; e.g. phloretin) (Goodwin and Mercer 2003; Alihosseini 2016; Goncalves et al. 2018; Wang et al. 2018; Banoth and Thatikonda 2019; Shao and Bao 2019). They also include the aurones (possess the 2-benzylidene-coumaranone skeleton; e.g. sulphuretin), anthocyanins (glycosides of anthocyanidins; e.g. pelargonidin), leucoanthocyanidins (have flavan-3,4-diol skeleton; e.g. teracacidin) and biflavonoids (dimers of various flavonoids linked by C─C or C─O─C bond; e.g. amentoflavone) (Goodwin and Mercer 2003; Halbwirth 2010; Mercader and Pomilio 2013; Leicach and Chludil 2014; Antal et al. 2016).

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1.2.3.3  Extraction and Identification of Phenolics Various techniques for extraction of phenolic constituents are developed and which include LLE (liquid-liquid extraction), UAE (ultrasound-assisted extraction), MAE (microwave-assisted extraction) and SFE (supercritical fluid extraction) (Jahromi 2019). Maceration, soxhlet extraction and hydro distillation are the main methods for the extraction of phenolic compounds with the LLE technique (Garcia-Salas et al. 2010). The main parameters of these methods are the type and polarity of solvents and their ratio, time and temperature of extraction and also, chemical composition and physical characteristics of the samples (Garcia-Salas et al. 2010). In UAE, using ultrasound waves, cavitation bubbles are created near the sample tissue and then they break the cell wall, and thereby the cell content is released (Altemimi et al. 2016). In the other method, the microwave-assisted extraction, the samples are allowed to interact with the microwave (Sookjitsumran et al. 2016). The microwave generates heat as a result of molecular motion induction and hence the cell wall will be ruptured and which will further result in the release of active substances in the cell (Sookjitsumran et al. 2016). In the supercritical fluid extraction method, the solvent is at a temperature and pressure above its critical point and there is no surface tension in it (Murga et al. 2000). Hence, it concurrently shows the properties of liquid and gas which can be much efficient for phenolic extraction from plants. High diffusivities and low viscosities of supercritical fluids make them able to extract different phenolic constituents in less time with more effectiveness (Murga et al. 2000). Since there are phenolic constituents with various chemical properties and structures, different protocols and techniques like UV-visible spectroscopy, column chromatography, near-infrared reflectance spectroscopy, NMR, HPLC, GC-MS and liquid chromatography-mass spectrometry (LC-MS) are extensively used for their purification, identification and quantification (Kivilompolo et al. 2007; Jahromi 2019). 1.2.3.4  Role and Application of Phenolics The role of phenolic constituents in plant physiology and interactions with biotic and abiotic stress conditions are difficult to overestimate (Bhattacharya et al. 2010). The phenolic constituents are in plant tissues as an adaptive response to unfavourable environmental conditions and they play a pivotal function in the regulation of different environmental stresses, like high light, nutrient deficiency, low temperature and microbial infection (Naikoo et al. 2019). In short, phenolics are synthesized by plants primarily for getting protection from stress and further to get structural integrity and scaffolding support (Bhattacharya et al. 2010). Phenolics secreted by perturbed or wounded plants can kill or repel many microorganisms (Bhattacharya et al. 2010). The phenolic compounds have a broad range of medicinal potentials like anti-oxidant, anti-diabetic, anti-microbial, anti-tumour, anti-viral, analgesic, anti-inflammatory and anti-pyretic efficacies (Chen and Ho 1997; Narasimhan et al. 2004; Sun et al. 2007; Adisakwattana et al. 2008; Cheynier 2012).

1.3 CURRENT BIOTECHNOLOGICAL APPROACHES FOR THE ENHANCEMENT OF SECONDARY METABOLITE CONTENTS IN PLANTS We are dependent on plants for food, furniture, constructions and fuel since pre-historic times. Plants in all forms have tendered their services to mankind (Amjad et al. 2013). Humans have selected plants based on the value and potential for domestication (Ramawat et al. 2009). People living in different continents of the world also used plants to treat various diseases and various systems of medicines were generated viz. Ayurvedic, Unani, Chinese system of medicines, etc. (Nadkarni 1996; Che et al. 2017; Dar et al. 2017a). Due to the advancement of science and technologies, various other non-conventional methods to treat the diseases were also identified. Each system of medicine is having advantages and disadvantages. The system which utilized plant-based natural products for the discoveries of drugs has attracted attention across the globe, due to its least or no side effects (Dubey et al. 2004; Yuan et al. 2016; Roy et al. 2018). Plant synthesized various

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secondary metabolites besides its primary metabolites to resist in the changing environment and to cross-talk between signalling pathways within the plant and with other biological entities (JacoboVelazques et al. 2015; Martin 2017; Rai et al. 2017). These secondary metabolites are reported to serve humans across various domains from food to pharmaceutical industries around the globe (Dar et al. 2017a). To harvest these medicinal principles from the medicinal plants, people have exploited the plant germplasm from the natural habitat at an alarming rate (Maxted et al. 2020). Many of the medicinal plants are now considered endangered (Sharma and Thokchom 2014). As per the reports, the contents of these naturally occurring medicinal components were found low in most of the plants (Verma and Shukla 2015). So, it was needed to enhance the contents of such key principal components of the plants. Various people around the globe tried to enhance the contents of the secondary metabolites in the plants using various methods and were reported significant enhancement of the targeted secondary metabolites in the tested plants (Hussain et al. 2012). The most common methods employed to augment the contents of the plant secondary metabolites are as follows: 1. Tissue culture approaches: Plant cell and tissue culture technologies using explants like leaves, roots, stems and meristems are established for the enhanced in vitro production of secondary plant metabolites as it offers controlled supply of biochemicals independent of plant availability and bioreactors are the key process for their commercial production (Sajc et al. 2000; Hussain et al. 2012). Other advantages of this technique over conventional plant cultivation include the production of a metabolite of interest under controlled environmental conditions, cultured cells could be devoid of contaminations with microorganisms and insects, a cell could be easily multiplied to get their specific metabolite, automated cell growth control and metabolite process regulation can reduce the labour cost and further, the productivity could be improved and the metabolite of interest is extractable from the cultures (Hussain et al. 2012). Taxol, morphine, codeine, L-Dopa, capsaicin and berberine are some of the potentially active natural metabolites produced in large quantities using plant cell and tissue culture techniques (Tam et al. 1980; Yoshikawa et al. 1985; Holden et al. 1988; Morimoto et al. 1988; Wichers et al. 1993; Fett-Neto et al. 1994; Thengane et al. 2003). 2. Abiotic elicitation: The treatment of plant cells with abiotic elicitors is a useful approach to increase the secondary metabolite production in cell cultures (Karuppusamy 2009). Abiotic elicitors represent non-biological origin substances and are grouped into physical, chemical and hormonal factors (Naik and Al-Khayri 2016). Light, osmotic stress, salinity, drought and thermal stress are the physical elicitors (Naik and Al-Khayri 2016). The metabolite production can be affected by light and the effect of light on the enhanced production of two important secondary metabolites—gingerol and zingiberene—in Zingiber officinale callus culture is reported (Anasori and Asghari 2008). Osmotic stress, an important environmental stress, is proven to alter the physiological and biochemical properties of plants and increase the secondary metabolite concentrations in plants (Zobayed et al. 2007). Polyethylene glycol is an osmotic agent which can induce water stress in many plants (Van den Berg and Zeng 2006). Exposure to salinity is also known to stimulate or induce the production of secondary metabolite (Haghighi et al. 2012). Catharanthus roseus grown under salt stress showed increased levels of vincristine—an important alkaloid (Misra and Gupta 2006). Drought stress, which can also greatly reduce plant growth, can increase secondary metabolite content (Naik and Al-Khayri 2016). Heavy metals represent chemical elicitors and agents for abiotic stress in living organisms (Cai et al. 2013). Heavy metal-induced changes in the metabolic activity of plants can alter the production of photosynthetic pigments, nonprotein thiols, proteins and sugars (Naik and Al-Khayri 2016). Plant hormones are also widely used as elicitors and jasmonic acid, salicylic acid and gibberellins are the most studied hormones in this category (Naik and Al-Khayri 2016). Jasmonic acid induces the production of different plant secondary metabolites,

Plant Secondary Metabolites—The Key Drivers of Plant’s Defence Mechanisms

including rosmarinic acid, terpenoids indoles alkaloids and plumbagin in different cell cultures (Krzyzanowska et al. 2012; Almagro et al. 2014; Silja et al. 2014). Salicylic acid is another hormonal elicitor that is reported to enhance the production of various secondary metabolites like tanshinones (Xiaolong et al. 2015), withanolide A, withanone and withaferin A (Sivanandhan et al. 2013), stilbene (Xu et al. 2015), vincristine and vinblastine (Idrees et al. 2010). Another phytohormone gibberellin is also known as an elicitor for secondary metabolite production (Liang et al. 2013). 3. Biotic elicitation: Biotic elicitors represent the substances of biological origin and they contain polysaccharides of plant cell walls (e.g. pectin, chitin, cellulose and chitosan) and microorganisms (Naik and Al-Khayri 2016). The treatment of chitosan in Plumbago rosea cultures enhanced plumbagin content (Komaraiah et al. 2003) whereas chitosan or chitin application induced the synthesis of fluoroquinolone and coumarins in shoot cultures of Ruta graveolens (Orlita et al. 2008). Microorganisms also play a role as biotic elicitors by inducing plant defence response and by triggering the pathways to alter the secondary metabolite production (Naik and Al-Khayri 2016). Researchers use yeast extract for years as one of the important biotic elicitors to stimulate the production of various metabolites like ethylene in tomato and tanshinone in Perovskia abrotanoides root culture (Felix et al. 1991; Arehzoo et al. 2015; Naik and Al-Khayri 2016). The effectiveness of fungal preparations (pathogenic and nonpathogenic) as elicitors is one of the most effective biotic strategies to stimulate a plant’s phenylpropanoid and flavonoid biosynthetic pathways (Dixon et al. 2002; Lattanzio et al. 2006; Naik and Al-Khayri 2016). The production of indole alkaloids serpentine, ajmalicine and catharanthine has increased up to five times in C. roseus cell suspensions and raucaffrincine in Rauwolfia canescens using fungal cell wall fragments (Parchmann et al. 1997; Zhao et al. 2001; Namdeo et al. 2002). Likewise, fungal mycelia increased the diosgenin content in Dioscorea deltoidea by 72% (Rokem et al. 1984). Furthermore, bacterial elicitors induce scopolamine biosynthesis in Scopolia parviflora adventitious hairy root cultures (Jung et al. 2003). Likewise, a significant increase in the glycyrrhizic acid content was observed in Agrobacterium rhizogenes, Bacillus aminovorans and Bacillus cereus challenged cultures (Awad et al. 2014). The secondary metabolite production enhancement in hydroponic cultures using biotic elicitors was also reported (Vu et al. 2006). Datura innoxia Mill., were cultivated in hydroponic conditions in which nutrient solution was inoculated with Agrobacterium rhizogenes and found that there is an increased production of alkaloid content in the plant roots growing in hydroponic culture (Vu et al. 2006). 4. Hairy root culture: The hairy root system based on inoculation with Agrobacterium rhizogenes is another method for the production of secondary metabolites synthesized in plant roots (Palazon et al. 1997; Karuppusamy 2009). 5. Production of transgenic lines: Genetic manipulation became a promising alternative for biotechnological exploitation of plant cells and hence the engineered plants will give an increased enzyme activity and enhanced production of the respective metabolite (Karuppusamy 2009; Hussain et al. 2012). 6. Biosynthetic metabolic pathway engineering: Metabolic pathway engineering is another approach for increased secondary metabolite production through the targeted and purposeful alteration of metabolic pathways (Lessard 1996). Metabolic engineering can offer different strategies to enhance the productivity of the desired metabolite by increasing the number of producing cells, through over expression of genes, by enhancing the ratelimiting enzymes or blocking the feedback inhibition mechanism and competitive pathways (Hussain et al. 2012). 7. Targeted gene editing using CRISPR CAS9: CRISPR-Cas9-based genome editing systems are now flashed to the future efforts in the elucidation and engineering of biosynthetic pathways of novel natural products (Nielsen et al. 2017). The CRISPR/Cas9 system

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exhibits vast applications with high precision and accuracy compared to other genome editing tools. Hence, it can probably be used as a common technique for plant metabolic engineering in the future (Alagoz et al. 2016). The effectiveness of CRISPR-Cas9 genome editing technology in the manipulation of bioactive alkaloid biosynthetic pathways in Papaver somniferum L. has been successfully demonstrated (Alagoz et al. 2016). 8. RNAi technology: RNA interference technology (RNAi) possesses the main impact on the manipulation of secondary metabolites which represents three major groups viz. terpenoids, alkaloids and phenylpropanoids (Wagner and Kroumova 2008). RNAi might prove to be effective for the production of novel natural products from plants, which in turn can give novel and rapid applications (Borgio 2009). Researchers used RNAi technology to yield unnatural plant natural products from lengthy and complex metabolic pathways (Borgio 2009). Runguphan et al. (2009) explained the elimination of monoterpene indole alkaloids (derived from two starting compounds, tryptamine and secologanin) production in C. roseus hairy root culture through RNA mediated suppression of tryptamine biosynthesis. In this study, they have introduced an unnatural tryptamine analogue to the production media and established that the silenced C. roseus culture could produce novel alkaloids derived from this unnatural starting substrate. This provided an advantage that the novel natural product synthesized this way is not contaminated due to the presence of normal natural alkaloids present in C. roseus. 9. Induced mutation (gamma radiation), nuclear radiations and mutation breeding are the other method of enhancement of secondary metabolite production. The effect of gamma radiation on the formation of biomass and the yield of pharmacologically important secondary metabolites in Hypericum triquetrifolium Turra callus cultures induced from different seedling parts has been demonstrated (Azeeza et al. 2017). Photo-elicitation of bioactive secondary metabolites using ultraviolet radiation has also been studied (Matsuura et al. 2013). The role of mutation breeding in the alteration of secondary metabolite production has also been investigated (Kolakar et al. 2018). In periwinkle ‘Dhawal’, a high alkaloid producing variety has been developed by chemical mutagen treatment of seeds followed by rigorous selection in widely cultivated variety ‘Nirmal’ (Kulkarni and Baskaran 2003). An increase in sapogenin (diosgenin) level in Trigonella corniculata following chemical mutagenesis using dimethyl and diethyl sulphate has also been reported (Kolakar et al. 2018). These new approaches will help to explore the plants as a potential source of many more phytochemicals. Continuation and much deeper efforts in this area will hopefully direct to the improved biotechnological production of specific, high-value and yet underexplored plant metabolites (Hussain et al. 2012).

1.4 CONCLUSION This chapter has dealt with plant secondary metabolites, their synthesis, classification, biological function, medicinal applications and an overview of the recent advancements for the enhanced production of novel secondary metabolites in plants. There is a huge range of secondary metabolites present in the plant kingdom, with a very varied distribution. Secondary metabolites are not required directly for the growth and development of plants but they are necessary for the survival of plants in adverse conditions. They protect plants from biotic and abiotic stress conditions through the chemical defence mechanisms. The capability of plants to defend and survive is thus ultimately because of the ecological functions of their secondary metabolites. Such metabolites are studied and clinically proven for their ethnopharmacological potential also and which will aid to human health. However, many of such secondary metabolites are still under-explored and unidentified for their potential and thus the research area of natural products still requires much more attention and thorough study. Further, because of the increased commercial significance of high-value secondary

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metabolites in recent years, there is a great inducement for researchers to focus much on secondary metabolism, especially in the option of their enhanced production by means of different approaches. The common approaches mainly include tissue culture approaches, biotic and abiotic elicitation, metabolic engineering, targeted gene editing, RNAi technology, production of transgenic lines, induced mutation, nuclear radiations and mutation breeding. In future, the aforementioned biotechnological approaches might be approached as an alternative production method to overcome the less accessibility of biologically potential, commercially important and medicinally valuable plant secondary metabolites. Advances in bio techniques, especially methods for culturing plant cell cultures, might give new angles for the commercial processing of even rare medicinal plants and their novel chemicals.

ACKNOWLEDGEMENTS Dr. D. Sruthi acknowledges the Department of Health Research (DHR), Government of India, New Delhi, for her award of Young Scientist-HRD scheme (YSS/2019/000035/PRCYSS). Dr. D. Sruthi is also grateful to Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India, New Delhi, for her award of National post-doctoral Fellowship (PDF/2017/000339). Acknowledgement is further extended to Dr. T. John Zachariah, Former Principal Scientist, and Acting Director, ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, for his valuable guidance on plant secondary metabolites during the doctoral period of Dr. D. Sruthi. Contact Information: Please contact the author at: [email protected]

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Waterman, P. G. 1993. The chemistry of volatile oils. In: Volatile Oil Crops: Their Biology, Biochemistry and Production, eds. R. K. M. Hay and P. G. Waterman, 47–61. Longman Scientific & Technical, Harlow, England. Weaver, L. M. and Hermann, K. M. 1997. Dynamics of the shikimate pathway in plants. Trends in Plant Science 2: 346–351. Wendt, K. U. and Schulz, G. E. 1998. Isoprenoid biosynthesis: Manifold chemistry catalysed by similar enzymes. Current Biology 6: 127–133. Wichers, H. J., Visser, J. F. and Huizing, H. J. 1993. Occurrence of L-DOPA and dopamine in plants and cell cultures of Mucuna pruriens and effects of 2,4-D and NaCl on these compounds. Plant Cell Tissue and Organ Culture 33: 259–264. Wildermuth, M. C., Dewdney, J., Wu, G., et al. 2001. Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414: 562–565. Wink, M. 2016. Secondary metabolites, the role in plant diversification of. In: Encyclopedia of Evolutionary Biology, ed. R. M. Kliman, 1–9. Elsevier Academic Press, UK. https://doi.org/10.1016/ B978-0-12-800049-6.00263-8. Wright, C. W., Bray, D. H., O’Neill, M. J., et al. 1991. Antiamoebic and antiplasmodial activities of alkaloids isolated from Strychnos usambarensis. Planta Medica 57: 337–340. https://doi.org/10.1055/s-2006-960112. Xiao, J., Capanoglu, E., Jassbi, A. R., et al. 2016. Advance on the flavonoid C-glycosides and health benefits. Critical Reviews in Food Science and Nutrition 56: S29–S45. https://doi.org/10.1080/10408398.2015.1 067595. Xiaolong, H., Min, S., Lijie, C., et al. 2015. Effects of methyl jasmonate and salicylic acid on tanshinone production and biosynthetic gene expression in transgenic Salvia miltiorrhiza hairy roots. Biotechnology and Applied Biochemistry 62: 24–31. https://doi.org/10.1002/bab.1236. Xu, A., Zhan, J. C. and Huang, W. D. 2015. Effects of ultraviolet C, methyl jasmonate and salicylic acid, alone or in combination, on stilbene biosynthesis in cell suspension cultures of Vitis vinifera L. cv. Cabernet Sauvignon. Plant Cell Tissue and Organ Culture 122: 197–211. Yoshikawa, T. and Furuya, T. 1985. Morphinan alkaloid production by tissues differentiated from cultured cells of Papaver somniferum. Planta Medica 2: 110–113. Yuan, H., Ma, Q., Ye, L., et al. 2016. The traditional medicine and modern medicine from natural products. Molecules 21: 559. Yuan, Z., Liang, Z., Yi, J., et al. 2019. Protective effect of koumine, an alkaloid from Gelsemium sempervirens, on injury induced by H2O2 in IPEC-J2 cells. International Journal of Molecular Science 20: 754. https://doi.org/10.3390/ijms20030754. Yubin, J., Miao, Y., Bing, W., et al. 2014. The extraction, separation and purification of alkaloids in the natural medicine. Journal of Chemical and Pharmaceutical Research 6: 338–345. Zhao, J., Zhu, W. and Hu, Q. 2001. Selection of fungal elicitors to increase indole alkaloid accumulation in Catharanthus roseus suspension cell culture. Enzyme and Microbial Technology 28: 666–672. Zobayed, S. M. A., Afreen, F. and Kozai, T. 2007. Phytochemical and physiological changes in the leaves of St. John’s wort plants under a water stress condition. Environmental and Experimental Botany 59: 109–116.

2

Generation of Plant Mutant Lines Using Gamma Radiation with Enhanced Secondary Metabolite Contents Lata I. Shukla, P. Vivek Vardhan, T. K. Devika, Sayan Roy and Sourav Bhatacharya

CONTENTS 2.1 Introduction............................................................................................................................. 27 2.1.1 Classification................................................................................................................28 2.1.1.1 Flavonoids.....................................................................................................28 2.1.1.2 Steroids.........................................................................................................28 2.1.1.3 Alkaloids....................................................................................................... 30 2.1.2 Bioactivity of Different Secondary Metabolites.......................................................... 30 2.1.2.1 Anti-Oxidant Activity................................................................................... 30 2.1.2.2 Anti-Microbial Activity................................................................................ 30 2.1.2.3 Anti-Diabetic Activity.................................................................................. 30 2.1.3 Production of Plant Secondary Metabolites................................................................ 32 2.1.3.1 Gamma Irradiation and Effect on Secondary Metabolite Production.......... 33 2.1.4 Units of Measurement of Source for Gamma Rays and the Gamma Irradiation......................................................................................................34 2.2 Mode of Interaction of Gamma Irradiation with Materials Both Living and Non-Living..... 35 2.3 Radiolysis of Water by Gamma Irradiation............................................................................. 36 2.4 Direct and Indirect Effect of Gamma Irradiation on Biological Systems............................... 37 2.5 Effect of Gamma Irradiation on DNA..................................................................................... 38 2.6 Effect of Gamma Irradiation on Proteins................................................................................ 38 2.7 Effect of Gamma Irradiation on the Secondary Metabolite Production................................. 39 2.8 Effect of Gamma Irradiation and the Different Age of the Plantlets...................................... 39 2.9 Effect of Gamma Irradiation on Different Parts of the Plants................................................ 39 2.10 Effect of Gamma Irradiation on the Different Enzymes and Influence on Secondary Metabolite Production.............................................................................................................40 2.11 Summary and Conclusions...................................................................................................... 41 Acknowledgements........................................................................................................................... 43 References......................................................................................................................................... 43

2.1 INTRODUCTION Plants produce a vast and diverse amount of small molecular organic compounds which could act as defences, regulators and primary metabolites, often don’t appear to be directly involved in growth and development (Erb and Kliebenstein 2020). There are alternate enzymatic pathways leading to production of intermediates and products of metabolism which are traditionally referred DOI: 10.1201/9781003034957-2

27

28

Biotechnological Approaches to Enhance Plant Secondary Metabolites

to as secondary metabolites. These are rather complex and specialized metabolites which facilitate plants to withstand the changing environmental conditions and various biotic and abiotic stresses. Secondary metabolites often accumulate in small quantities (1% dry weight) and their quality and quantity varies for a given plant species and depends greatly on the physiological and developmental stages of the plant (Facchini 2001). Production of secondary metabolite in plants occurs in response to a wide range of environmental stress conditions. Nutrient deficiencies, temperature, osmotic shock, salinity, radiation, high intensity light, heavy metal ions and herbicide treatment are some of the factors that often induce the accumulation of secondary metabolites (Isah 2019). They involve in regulating enzymes, defence against microorganisms, insects and animals and in interaction with other plants and with the environment (Demain and Fang 2000; Namdeo 2007). A variety of plant secondary metabolites have been used by humans since their discovery and important for various applications (Bourgaud et al. 2001). Plant secondary metabolites are unique resources for medicines, pharmaceuticals, therapeutics, flavourings, food additives, narcotics and fine chemicals (Singh et al. 2003; Wallace 2004; Patra and Saxena 2010). In this regard, it is essential to address the recent advances in generation of plant mutant lines using gamma radiation with enhanced secondary metabolite contents.

2.1.1 Classification Plant secondary metabolites are usually classified according to their biosynthetic pathways and are of three large molecule families; flavonoids, steroids and alkaloids. 2.1.1.1 Flavonoids Flavonoids are a group of phenolic compounds ubiquitous in photosynthesizing cells and are commonly found in fruits, vegetables, nuts, seeds and flowers. Flavonoids synthesize in particular parts of the plant and distribute to the other parts which are responsible for colour and aroma. Flavonoids protect plants from different stresses (biotic and abiotic) and act as unique UV-filter, signal molecules, phytoalexins, detoxifying agents (Mathesius 2018). Also, flavonoids involve in defence against herbivores and interspecies defence. Several medicinal properties play major role in therapeutic drugs since time immemorial. Important properties include anti-oxidant, anticancer, anti-inflammatory, anti-microbial, anti-allergic and anti-spasmodic activities. Flavonoids inhibit activity of some enzymes such as aldose reductase, xanthine oxidase, phosphodiesterase, ATPase, lipoxygenase, cyclooxygenase etc. (Chaudhry et al. 1983; Nagao et al. 1999; Sadik et al. 2003; Ko et al. 2004; Ribeiro et al. 2015; Ontiveros et al. 2019). Quercetin, luteolin, apigenin, myricitrin and kaempferol are some of the commonly available flavonoids commercially which could regulate the activity of key enzymes across the animal kingdom (Table 2.1). Flavonoids also regulate hormone levels of oestrogens, androgens and thyroid hormones (Lambert and Edwards 2017). 2.1.1.2 Steroids Plant steroids also referred to as phytosterols constitute major groups of steroids of the plant kingdom. They have both structural role as membrane constituents and important role in metabolism as biosynthetic precursors. Phytosterols are the starting materials for some other plant steroids, which humans depend on plant diet since they are essential components and do not biosynthesize in body. Phytosterols are commercially important biochemical sources for steroid compounds, insecticides. Most commonly isolated phytosterols are β-sitosterol, β-stigmasterol, lanosterol and campesterol which have several medicinal and commercial values (Table 2.2). Phytosterols are significantly known for their cholesterol-lowering capacity in animal and human studies. However, recently growing body of evidence suggests their beneficial activities are not only limited to their hypocholesterolaemic capacity but also acts as anti-oxidant and anti-cancer drugs (Jain et al. 2019).

29

Generation of Plant Mutant Lines Using Gamma Radiation

TABLE 2.1 Medicinally Important Flavonoids which Inhibit Enzymes Cross Kingdom in Animals and Humans Flavonoids

Enzymes

Animals

Humans

Quercetin, myricitrin Chrysin, luteolin, kaempferol, quercetin, myricitrin and isorhamnetin Luteolin, genistein, daidzein, eriodictyol, hesperidin, prunetin, diosmetin, biochanin, apigenin and myricetin Quercetin and gossypin Quercetin and luteolin Apigenin, chrysin, luteolin, naringenin, eriodictyol, quercetin, acacetin and taxifolin Apigenin, luteolin, kaempferol, quercetin, rutin and hyperin Apigenin, acacetin, chrysin, isorhamnetin, pinocembrin and tangeretin

Aldose reductase Xanthine oxidase

✓

✓ ✓

Phosphodiesterase

✓

ATPase Lipoxygenase Cyclooxygenase

✓

✓

✓

✓ ✓

Ontiveros et al. (2019) Sadik et al. (2003) Ribeiro et al. (2015)

✓

Cao and Chen (2012)

✓

Šarić Mustapić et al. (2018)

α-glucosidase and α-amylase Cytochrome P450

✓

Reference Chaudhry et al. (1983) Nagao et al. (2014) Ko et al. (2004)

TABLE 2.2 Phytosterols from Plant Sources and Their Therapeutic Properties Phytosterols

Plant Source

Medicinal Property

Phytosterols Sitosterol

Soybean -

Sitosterol Stanols and sterols

Corn oil Wood pulp, vegetables Fruits and vegetables

β-sitosterol and its glycosides

Plant sterols and stanols Plant sterols

-

β-sitosterol, stigmasterol and esters Free plant sterols and esters

-

Fruits and vegetables

Soy bean

Animals

Humans

Reference

Lowers cholesterol Lowers cholesterol

✓ ×

× ✓

Hypocholesterolaemic Lowers cholesterol

× ×

✓ ✓

Immuno-regulatory, anti-inflammatory, anti-neoplastic and anti-pyretic Lowers low-density lipoprotein cholesterol Lowers total cholesterol and low-density lipoprotein cholesterol Lowers serum and liver cholesterol

✓

✓

×

✓

Katan et al. (2003)

×

✓

Hendriks et al. (2003)

✓

×

Tabata et al. (1980)

Lowers cholesterol absorption

×

✓

Mattson et al. (1982)

Peterson (1951) Pollak and Kritchevsky (1981) Ostlund et al. (2002) Plat and Mensink (2000) Bouic and Lamprecht (1999)

30

Biotechnological Approaches to Enhance Plant Secondary Metabolites

2.1.1.3 Alkaloids Alkaloids are a group of complex heterocyclic nitrogen containing compounds which are derived from amino acids tryptophan, tyrosine and lysine. These are widely distributed in the plant kingdom and around 5500 known alkaloids are reported so far (Vonk and Shackelford 2019). Most of the alkaloids that are toxic to other organisms involve in plant defence against herbivores and pathogens. Several alkaloids have been using by the mankind since time immemorial for their beneficial activities; as purgatives, antitussives, sedatives, dyes, spices, drugs, poison etc. (Kurek 2019). Several alkaloids are known to have important medicinal and pharmacological properties (Table 2.3, Figure 2.1). Some commercially used alkaloids include; papaverine has inhibitory effects on several viruses, indoquinoline alkaloids inhibit certain gram negative bacteria and yeast, quinine is known to have anti-malarial activity, codeine and morphine acts as analgesics and anaesthetics (WHO 2014).

2.1.2  Bioactivity of Different Secondary Metabolites 2.1.2.1  Anti-Oxidant Activity Anti-oxidants could be used to alleviate the oxidative stress caused by the free radicals generated during normal metabolism which is harmful for cell organelles and biomolecules. The development of natural anti-oxidants is of great interest for usage in foods, medicines and cosmetics. Therefore, focus on the natural foods which has the ability to scavenge free radicals is gaining importance. Some of the medicinally important plants which have anti-oxidant activity include; i) Limonia acidissima which has kaempferol, quercetin and lutein and other important secondary metabolites which are good anti-oxidants (Pandey et al. 2014), ii) Murraya koenigii has plenty of phenols and flavonoids which act as good anti-oxidants (Zahin et al. 2013) and iii) Duranta erecta has Transcinnamic acid derivatives and other flavonoids which showed anti-oxidant activity (Donkor et al. 2019). Petrea volubilis, Rumex vesicarius, Sisymbrium irio, Digera muricata, Gomphrena celosioides Mart., Trichosanthes cucumerina, Melothria maderaspatana are well known to possess naturally occurring compounds which are efficient anti-oxidants. 2.1.2.2  Anti-Microbial Activity The rapidly growing number of antibiotic resistant bacteria raised the global concern towards developing alternative medicine from naturally occurring compounds. Various plant extracts, phytochemicals and herbal remedies are known to cure microbial infections throughout the history of mankind. Some of them include; i) Rumex vesicarius has anti-bacterial, anti-fungal activities (Panduraju et al. 2009). ii) Sisymbrium irio extracts showed activity against A. niger, F. oxysporum, E. coli, P. aeruginosa (Bibi et al. 2015). iii) Duranta erecta showed activity against A. flavus, P. chrysogenum, Rhizobium spp. (Sharma et al. 2012). iv) Gomphrena celosioides showed activity against E. coli, Pseudomonas aeroginosa and Staphylococcus aureus (Shamra and Vijayvergia 2011). Petrea volubilis, Melothria maderaspatana, Trichosanthes cucumerina, Limonia acidissima, Murraya koenigii are some of the medicinally important plants having anti-microbial activity. 2.1.2.3  Anti-Diabetic Activity India has the largest number of diabetic cases in the world. Type-I diabetes is insulin dependent caused by insulin deficiency which can be treated by administering exogenous insulin. Type-II diabetes is insulin independent caused by insulin insensitivity by the cells. In both Indian and global scenario, non-insulin dependent diabetes mellitus is most prevalent, which is associated with elevated postprandial hyperglycaemia (PPHG). Pancreatic α-amylase is a key enzyme in carbohydrate metabolism, catalyzes the initial step in breaking down starch into simpler sugars. Rapid degradation of dietary starch leads to PPHG. Hence, controlling the activity of α-amylase is an important aspect in treating diabetes by reducing its activity. Hence, α-amylase inhibitors play a

31

Generation of Plant Mutant Lines Using Gamma Radiation

TABLE 2.3 Alkaloids from Plant Sources and Their Therapeutic Properties Alkaloids

Plant Source

Medicinal Property

Animals

Humans

Ajmaline, reserpine, rescinnamine Atropine

Rauwolfia serpentine Atropa belladonna Berberis vulgaris Papaver somniferous Mahonia aquifolium

Antiarrhythmic, antihypertensive Antidote to nerve gas poisoning Bacillary dysentery

×

✓

Mouhssen (2013)

×

✓

Mouhssen (2013)

×

✓

WHO (2014)

Analgesic, anaesthetic

×

✓

WHO (2014)

Muscle relaxant

-

-

Zuo et al. (2006)

Rauvolfia canescens Carapichea ipecacuanha

Antihypertensive, tranquilizer

×

✓

Roy (2017)

Antiprotozoa

✓

✓

Harmine

Peganum harmal

✓

✓

Isoquinoleine

Fumaria capreolata Delphinium linearilobum Trautv. Nicotiana tobacum

Antimicrobial, antifungal, antiplasmodial, antioxidant, antimutagenic, antigenotoxic and hallucinogenic and enhances insulin sensitivity Antioxidant, analgesic, intestinal anti-inflammatory Antioxidant

Fabrican and Farnsworth (2001) Patel et al. (2012)

✓

✓

Bribi (2018)

×

✓

Yang et al. (2006)

Nervous stimulant, insecticide

✓

✓

Anticonvulsant, sedative, muscle relaxant Analgesics, antiseptics, sedatives, stomatics Antiarrhythmic

×

✓

Fabrican and Farnsworth (2001) Pei (1983)

×

✓

×

✓

Bradycardial, hypotensive, sedative Antitumor

×

✓

×

✓

Anticancer, antihypertensive

×

✓

Berberine Codeine, morphine Corstubenne, magnoflorine, isothebaine, isocorydine Deserpidine Ementine

Linearilobin, linearilin, lycotonin, browniine Nicotine

Piperine and derivatives Protoberberine

Piper nigrum

Quinidine

Cinchona officinalis -

Tetrahydropalmatine Vinblastine Vincristine, Vincamine

-

Catharanthus roseus Vinca rosea, Vinca minor

Reference

Da-Cunha et al. (2005) Roy (2017) Da-Cunha et al. (2005) Kaur and Arora (2015) Kaur and Arora (2015)

major role in managing PPHG by reducing starch digestion. Acarbose, miglitol and voglibose are currently in use as inhibitors of α-amylase. But, because of the undesirable side effects, the search for safer, effective compounds which can reduce blood sugar levels has continued to be interesting area. Traditional herbal medicine offers great potential for discovery of new anti-diabetic drugs; (i) Trichosanthes cucumerina extracts show significant anti-amylase activity (Liyanage et al. 2016).

32

Biotechnological Approaches to Enhance Plant Secondary Metabolites

FIGURE 2.1  Some medicinally important plants which produce therapeutic alkaloids; (a) Catharanthus roseus, (b) Berberis vulgaris, (c) Papaver somniferum, (d) Piper nigrum, (e) Atropa belladonna and (f) Vinca minor.

(ii) Melothria maderaspatana extracts show significant inhibition of α-amylase (Balaraman et al. 2010). Aloe vera, Azardirachta indica A Juss., Allium cepa, Allium sativum, Mangifera indica, Andrographis paniculata Nees, Vitex negundo Linn. etc., have potent activity against α-amylase and hence potent anti-diabetic agents.

2.1.3 Production of Plant Secondary Metabolites Often secondary metabolites can be biosynthesized using primary metabolites or using the raw materials of primary metabolites as prime substrates. In recent years, the commercial importance of the secondary metabolites which is rapidly increasing resulted in great interest in altering their production and quantity using biotechnology. Plant cell and tissue culture technology has been developed as a promising alternative and has been widely used for production and multiplication of various secondary metabolites which are difficult to obtain from plant extraction and chemical synthesis. In vitro production of various types of secondary metabolites in plant cell suspension cultures and tissue cultures has been largely reported. Recent work for improving plant secondary metabolites production was mainly focused on the aspects pertaining to the manipulation of plant cell and tissue cultures to improve productivity by employing elicitors and different stresses. In addition, manipulation of biosynthetic pathways, regulatory mechanisms and cloning and genetic engineering of key regulators was associated with secondary metabolite production. In spite of lot of scientific research in the area, plant secondary metabolite production still faces several biological and biotechnological limitations. One of the major problems is the low yield in plant cell and tissue culture technology. Some strategies have been developed to improve the yield such as treating with various elicitors (biotic and abiotic), signal compounds and different stresses (Akula and Ravishankar 2011; Ahlawat et al. 2014; Srivastava and Srivastava 2014). The stress type and its magnitude are the major factors which determine the effect on secondary metabolite

33

Generation of Plant Mutant Lines Using Gamma Radiation

production (Eilert et al. 1987). Even though most of such treatments effectively promote the yield of secondary metabolites, the productivity requires much enhancement to meet the industrial applications. 2.1.3.1  Gamma Irradiation and Effect on Secondary Metabolite Production Gamma irradiation is one of the promising techniques which could significantly enhance secondary metabolite production without damaging the vigour of the plants. Gamma radiation is sparsely ionizing radiation which typically causes more indirect effect by stimulating stress responses which could lead to accumulation of secondary metabolites. The evidence for increasing the plant secondary metabolite production with γ irradiation has been largely increasing recently. The comparison of enhancement of different secondary metabolites by gamma radiation, biotic and abiotic stresses is shown in Table 2.4 (Vardhan and Shukla 2017). Psoralen content in seeds of Psoralea corylifola increased 32 folds by gamma irradiation of 20 kGy (Jan et al. 2011). Total phenolic content in calli of Rosmarinus officinalis increased five folds (El-Beltagi et al. 2011) and camptothecin content in calli of Nothapodytes foetida increased 20 folds on treating with 20 Gy. The saponinins and ginsenosides in hairy root cultures of Panax ginseng increased 1.5 folds on gamma irradiation of 50 Gy (Zhang et al. 2011).

TABLE 2.4 Comparison of Enhancing the Secondary Metabolite Production Using Gamma Irradiation Treatment with Other Techniques Magnitude of Increase (Fold) Secondary Metabolite

Plant Material

Gamma Irradiation

Biotic Stress

Abiotic Stress

Psoralea corylifola (babchi)

Psoralen

Seeds Cells Root

32 -

9 -

6.6

Capsicum annuum (chili)

Capsaicinoids

Pods Calli cells

0.1 -

2.9 -

2

Rosmarinus officinalis Total phenols (rosemary) Brassica oleraceae “ (broccoli) Arachis hypogea (peanut) “

Calli

5

-

-

Planlets

-

2.2

-

Seeds

-

-

0

Plant Name

Panax ginseng (ginseng)

Saponinins

Hairy root Cells

1.4 -

0.7

0.8

Nothapodytes foetida (ghanera) Ophiorrhiza mungos Camptotheca acuminate Lithospermum erythrorhizan (purple gromwell)

Camptothecin

Calli

20

-

-

“ “ Shikonins

Cells Cells Cells

-

13 -

11 2

References Jan et al. (2011), Ahmed and Baig (2014), Siva et al. (2014) Topuz and Ozdemir (2004), Brooks et al. (1986), Johnson et al. (1991) El-Beltagi et al. (2011), Lola-Luz et al. (2014), Rudolf and Resurreccion (2005) Zhang et al. (2011), Hu et al. (2003), Wu and Lin (2002) Fulzele et al. (2015), Deepthi and Satheeshkumar (2016), Song and Byun (1998), Chung et al. (2006), Fukui et al. (1983)

34

Biotechnological Approaches to Enhance Plant Secondary Metabolites

2.1.4 Units of Measurement of Source for Gamma Rays and the Gamma Irradiation The SI units for the gamma irradiation include gigabecquerel (GBq), gray (Gy) and roentgen (R) is used to measure radiation. The GBq measures the number of gamma rays emitted from a source of radiation and is a unit of radioactivity that is defined as 1.37 × 10 −12 atomic decays per second. Radium of weight unit gram is 37 GBq while 10 −7th of a gram of newly formed radio-sodium is also 37 GBq since both release 3.7 × 10 −10 disintegrations/second (Odum 1971). When dealing with biological systems, generally the small units are put into the application such as the millicurie, microcurie and picocurie which are 10 −3, 10 −6 and 10 −12, respectively. A second measurement of radiation is the gray, Gy. The assimilated portion of 1 Gy implies the retention of 1 joule of radiation vitality per kg of tissue. The unit roentgen is almost equivalent to the Gy, and is utilized as a unit of estimation for introduction to gamma and X-rays. Both are units of the complete portion of radiation got by an organism. The dose rate is the measure of radiation got per unit time. The interaction of gamma irradiation of materials is by ionization. Here the electrons are ejected and the free radicals along with the electrons are the important products of the gamma irradiation. The events that undermine the radiation interactions in terms of different time lines could be seen in Figure 2.2 where, R. and S. are free radicals and M and N are molecular products which are the expected irradiation results of the organic molecules. The essence of chemical characteristics leading to formation of new compounds is ascribed to the amount of energy being transferred, which will create ion, free radicals and excited molecule. Such process of interaction is called the ionization and excitation of the molecules, which can bring chemical changes to irradiated molecule. This happens because all binding energy for organic compound is in the range of 10–15 eV. In those cases where low energy is transferred by photon, the molecule undergoes to the excitation state before it returns to the rest state by emitting X-ray photons or break down to release free radicals in turn undergoing polymerization. Electron which gets knocked out from the irradiated atom (A+) is exposed to solid electric field of framed positive charge. Therefore, the recombination keeps on frequently occurring, either during irradiation or after end of irradiation to create the energetic molecule (A**). Such highly energetic excited molecule will break down into free radicals and new molecule (Denaro and Jayson 1972). Gamma rays square measure fashioned with the self-disintegration of 60Co or 137Cs sources. Among thousands of gamma emitters, solely 137Cs and 60Co square measure indicated for radiation process. The energy of gamma rays, as magnetism quantum waves, is analogous to lightweight, however with higher gauge boson energy and shorter wavelength. The 60Co radionuclide may be made in a very nuclear energy reactor by the irradiation of 59Co (metal),

FIGURE 2.2  The different events associated with interaction of gamma irradiation with matter. (From Kaplan et al. 1955.)

35

Generation of Plant Mutant Lines Using Gamma Radiation

TABLE 2.5 The Half-Life and the Radiations Emitted by Important Radioisotopes Element

Half-Life

Uranium-235 (235U)

7 × 108 years

Radium-226 (226Ra)

1620 years

Potassium-40 (40K)

1.3 × 109 years

The cesium group: Cesium-137 (137Cs) daughter Barium-137 (137Ba) Cesium-134 (134Cs) The Cerium group: Cerium-144 (144Ce) daughter Praseodymium-144 (137Pr)

33 years 2.6 min 2.3 years 285 years 17 min 33 years

Radiations Emitted Alpha Gamma Alpha Gamma Beta Gamma Beta Gamma

Beta Gamma

with quick neutrons. The hot atom is created by nucleon capture as shown in Equation 2.1 (Laughlin 1989).

27

Co59 + 0 n1 → 27 Co60 (2.1)

The unstable nucleus of 60Co will emit photons having energies of 1.17 and 1.33 MeV, having a half-life of 5.2714 years to stable 60Ni (Table 2.5). The radioactive 60Co supply consists of little pellets of metal that are loaded into stainless steel or atomic number 40 alloy sealed tubes (pencil arrays). Radiation is that the distinctive supply of energy which may initiate chemical reactions at any temperature, as well as close, underneath any pressure, in any section (gas, liquid or solid).

2.2 MODE OF INTERACTION OF GAMMA IRRADIATION WITH MATERIALS BOTH LIVING AND NON-LIVING Radiation processing refers to the large-scale application of radiation for industrial-scale alteration of the properties of materials. The word ‘ionizing radiation’ refers to all radiation capable of generating cascades of ionization in matter. The characteristic energy spectrum of ionizing radiation starts at around 1000 eV and reaches its upper limit at about 30 MeV. In order to prevent induced radioactivity, the gamma ray energy higher than 5 MeV or the energy of the fast electrons exceeding 10 MeV is prohibited to use for sterilization. On the other hand, the application of lower energy radiation (below 0.2 MeV) is not rational. Commercial gamma ray irradiation facilities are typically loaded with 60Co of total activity from 0.3 to 3.0 mCi, while commercial e-beam facilities are equipped with one or two electron accelerators generating high power (10–100 kW) beams of 8–10 MeV electrons. When radiation passes through materials, it breaks chemical bonds. Radiation processing has been in commercial use for almost 40 years. X-rays, electron beams, Gamma radiation from 60Co, all are used for sterilization of the medical devices used in operation theatres and in other healthcare treatments. The surgical gloves are sterilized using gamma radiation from 60Co. Blood-bags, gowns implants, artificial joints, syringes, bottle teats for premature baby units and dressings—all are sterilized using radiation. Other industries that benefit from radiation processing include the food, pharmaceutical, cosmetic, horticultural and automotive industries. In the horticultural industry, growing-mats, fleeces and pots may be reused after irradiation-reducing waste and cost and saving the environment from unnecessary waste. Similarly, commercial egg trays may be

36

Biotechnological Approaches to Enhance Plant Secondary Metabolites

recycled after irradiation without risk of proliferating Salmonella. Radiation is the unique source of energy which can initiate chemical reactions at any temperature, including ambient, under any pressure, in any phase (gas, liquid or solid). The radiation process uses extremely penetrating electromagnetic wave from sealed radiation sources traveling at nearly the speed of sunshine, to bombard and kill microorganism in product sealed within their final packaging which means the irradiated product remains sterile until the packaging is removed. The energy carried by the electromagnetic wave is transferred to the object/product being irradiated by collisions between the radiation and therefore the atoms of the object/product. In these collisions, atoms lose their certain electrons in a very method known as ionization. This method ends up in irreparable injury to the life-sustaining chemistry of living organisms and therefore the initiation of cross-linking chemistry or main chair cut in compound materials. There are various factors affecting the resistance of microorganisms to radiation, therefore influencing the form of the survival curve more so with the indirect effects of radiations. 1. Oxygen: The result of oxygen throughout the radiation method will increase the limited result on microorganisms. Below fully anaerobic conditions, the D10 worth of some vegetative microorganism will increase by a factor of 2.5–4.7 as compared with aerobic conditions. 2. Water content: Microorganisms are most resistant once irradiated in dry conditions. This can be the main reason for the low range or absence of free radicals fashioned from water molecules by radiation and therefore the level of indirect result. 3. Temperature: Treatment at elevated temperature, typically within the sub-lethal vary higher than 45°C, synergistically enhances the germicidal effects of radiation on vegetative cells. Vegetative microorganisms are significantly a lot of immune to radiation at the chilling temperatures than at close temperatures; this can be attributed to a decrease in water activity at frozen state. Furthermore, the diffusion of radicals is extremely abundant restricted. 4. Medium: The composition of the medium encompassing the microorganisms plays a vital role within the microbiological effects. D10 values certainly microorganisms will dissent significantly in various media.

2.3  RADIOLYSIS OF WATER BY GAMMA IRRADIATION Radiolytic products of water are mainly formed by action of gamma rays on water molecules yielding radicals OH•, e-aq• and H• (Figure 2.3). The action of the hydroxyl radical (OH•) is responsible for most of the associated indirect effects. Drying or freezing of living organisms can substantially reduce these indirect effects. If we consider pure water (H2O), each 100 eV of energy absorbed will generate: 2.7 radicals OH•, 2.6 e-aq•, 0.6 radicals H•, 0.45 H2 molecules and 0.7 molecules H2O2 as shown in Equation 2.2 (Borrely et al. 1998).

H 2 O → [2.7] OH• + [2.6] e − aq• + [0.6] H• + [2.6] H 3 O+ + [0.7] H 2 O2 + [0.45] H 2 … (2.2)

The lethal effects of ionizing radiation on microorganisms as measured by the loss of cells of colony-forming ability in nutrient medium, has been the subject of detailed study. Much progress has been made towards identification of the mechanism of inactivation, but there still remains considerable doubt as to the nature of the critical lesions involved, although it seems certain that lethality is primarily the consequence of genetic damage. Many hypotheses have been proposed and tested regarding the mechanism of cell damage by radiation. Some scientists proposed the mechanism

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FIGURE 2.3  The effect of gamma radiation on water molecules.

‘radio toxins’, that are toxic substances produced in irradiated cells responsible for lethal effect. Others proposed that radiation was directly damaging the cellular membrane. The effect on the cytoplasmic membrane appears to play an additional role in some circumstances.

2.4 DIRECT AND INDIRECT EFFECT OF GAMMA IRRADIATION ON BIOLOGICAL SYSTEMS Chemical changes are primarily associated with the effects of radiation on living organisms and often depend on physical and physiological factors. Physical parameters include the intensity of dose, the distribution of dose and the quality of the radiation. The temperature, moisture content and oxygen concentration are the most important physiological and environmental parameters. Radiation impacts on living organisms can be divided into two types: direct and indirect. Normally, the indirect effects occur as an important part of the total action of radiation on it. The highest decrease in sensitivity is observed between 0 and –15°C. For example, D10 value of Escherichia coli irradiated in meat increased from 0.41 kGy at +5°C to 0.62 kGy at –15°C. For Staphylococcus aureus, D10 at –76°C was 0.82 kGy instead of 0.48 kGy at +4°C (Sommers et al. 2002). Sub-freezing temperatures provide less protection for spores than for vegetative plants, since they are still poor in humidity. The irradiation of frozen aqueous liquid allowed minimizing the loss of active substance even at 25 kGy dose. The most promising method for terminal sterilization of aqueous solutions by ionizing radiation seems to be this approach. The major radiolysis products formed after interaction of radiation, the more the ejection of the electrons. Indirect effect producing moieties include the hydroxyl free radical. On the other hand, it is very difficult to determine the radiosensitivity of bacteria as a result of the addition of radical scavengers, because many studies have been carried out either on isolated DNA, which does not take into account the effects inside the cell. For experiments conducted on bacteria, the scavenger concentration inside the cell was thought to be equal to that of the extracellular media, which is not necessarily the case. The protection of bacteria against ionizing radiation in the presence of hydroxyl radical scavengers has been shown to be highly dependent on the irradiation conditions (Billen 1984). Scavengers are unable to avoid semi-direct effects from the bound water due to the hydroxyl radicals, as the water lattice around DNA has no solvent capacity (Korystov 1992). Thus, it is almost difficult to scavenge the radicals from the bound water by an exogenous protector. It was observed that thiols can repair DNA damaged sites before a breakage occurs (ABCRI 2001).

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2.5  EFFECT OF GAMMA IRRADIATION ON DNA Effect of low doses of gamma irradiation on DNA: A significant increase in DNA strand breaks and oxidative base damage, determined as formamidopyrimidine-DNA-glycosylase (FPG)sensitive sites, were observed at absorbed doses of 5 and 10 cGy, respectively. HPLC with electrochemical detection (HPLC-ECD) determined that there was no significant increase in the level of 8-oxo-7, 8-dihydro-20-deoxyguanosine (8-oxodG) at doses up to 500 kGy of gamma radiation (Shukla et al. 2004). Chromosomal aberrations as dicentric and deletion frequencies was found to be caused by gamma radiation as low as 5 cGy. This was a significant finding that even at a low dose of 5 cGy, genotoxic effects of gamma radiation can be observed. The mRNA expression of both hOGG1 and XRCC1 repair genes were also decreased by gamma radiation, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and a significant decrease of expression was observed at 20 cGy. hOGG1 and XRCC1 mRNA expression levels were inversely correlated with the levels of FPG-sensitive sites and DNA strand breaks. The finding of decreased expression levels for hOGG1 and XRCC1 in gamma-irradiated lymphocytes has not been reported elsewhere. These observations suggest that a combination of DNA-damaging effects and reduced DNA repair capacity may be the effect of genotoxic effects of gamma radiation, and it may explain the significant increase in health risk from high doses of ionizing radiation (Sudprasert et al. 2006).

2.6  EFFECT OF GAMMA IRRADIATION ON PROTEINS Delincee and Paul (1981) found a cross-linking of the chain influences and physicochemical properties of the tertiary structure of proteins. It has observed decomposition and denaturation in irradiated proteins (Ciesla et al. 2000). The decrease in apparent amylose content may have been due to the breakage or cleavage of long amylopectin chains induced by gamma irradiation (Wu et al. 2002). It also agreed with Descherider and Grant’s observation that the declining apparent amylose content results from the shortening of polysaccharide chains (Descherider 1960, Grant and D’Aponlonia 1991). Ciesla et al. (2000) reported that the indication of protein modifications which occur after gamma irradiation is identical to the transformations that occur under heating. A study by Kanemaru et al. (2005) revealed that the protein content of irradiated semolina derived from irradiated wheat grains was not affected by gamma irradiation and ranged between 10.6% and 10.9%. Similarly, the studies of Marathe et al. (2002), Agundez-Arvizu et al. (2006) and Azzeh and Amr (2009) also agreed with those results. From the research on a Korean garlic cultivar, Kwon et al. (1988) concluded that there is no difference in the amounts of linoleic, palmitic, oleic and linolenic acids, the predominant fatty acids of the bulbs, immediately after gamma irradiation with 100 Gy. The low dose of radiation used may have, in part, generated its long-term effects by inducing lipid degradation, likely mediated by the action of free radicals known to be formed after irradiation (Katsaras et al. 1986; Voisine et al. 1991). The stimulatory response reported on germination due to γ rays can be attributed to the activation of RNA synthesis in coster beans (Kuzin et al. 1975) or protein synthesis (Kuzin et al. 1976) which occurred during the early stage of germination after seeds were irradiated with 4 krad. Thus, the results obtained were in correspondence with the findings of Grover and Dhanju (1980) on Papaver somniferum. Increasing the dose of gamma radiation up to 100 Gy showed a gradual increase in germination percentage and then a gradual decrease with increase in the gamma radiation dose in the second season in Hyoscyamus muticus (Habba 1989). Phaseolus vulgaris seeds on treating with high rates of gamma radiation were reported to exhibit reduced germination (Hell and Silveira 1974). Abo Elsauod and Omran (1976) suggested that snap bean seeds irradiated with 50, 100 and 150 Gy had a higher percentage of germination than the control. The identification of trapped electrons on γ irradiation of barley seeds provided evidence for in vivo release of electrons and its hydration (Israni et al. 1993; Shukla et al. 2001).

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2.7 EFFECT OF GAMMA IRRADIATION ON THE SECONDARY METABOLITE PRODUCTION Interesting information pertaining to the increase in secondary metabolites came from the information from the chromatographic analysis of various herbal extracts which were carried out and it was reported that the changes observed in the total yield and in the components of volatile oil following the irradiation event range from none to slight depending upon the dose-based irradiation of these selected herbal samples (IAEA 1992; Venskutonis et al. 1996; Chatterjee et al. 2000). Therefore, it can be assumed that the dose that can be applied and the extent of the antimicrobial activity may be limited by undesirable changes in volatile oil constituents, their yield and flavour quality. Farag et al. (1995) reported the conversion of terpenes into monoterpe-nesalcohols. An oxygenated monoterpene named β-eudesmol was identified as the major compound in this group; apart from this verbenol, α-eudesmol, verbenone and (E)-r-2-menthen-1-ol were also been detected. The content of α-and β-eudesmol was found to be increased to 9.52% from 6.91%, with no much variation between the different irradiation doses. The levels of remaining oxygenated terpene did not vary significantly during irradiation. There was no variation in the quantity of paeoniflorin in Paeoniae radix as cited by Yu et al. (2004). In addition, Owczarczyk et al. (2000) reported that the content of biologically active substances, including the essential oils, flavonoids, glycosides, anthocyanins and plants mucus did not change significantly after irradiation event. Elevated levels of alkaloids were observed in different organs of plant, mainly in the leaves as a result of irradiation (Abo Elseud 1983, El-Kholy 1987, Habba 1989). By irradiating various doses of γ-radiation (5, 10, 25, 50, 75, 100 and 200 Gy), Zhang et al. (2011) formed mutant cell lines of wild ginseng (Panax ginseng Meyer) hairy root cultures (Table 2.4). They assessed the effect of irradiation on the growth of hairy roots and the development of content of crude saponins and ginsenosides. With growing doses of irradiation, the growth of the roots was inhibited.

2.8 EFFECT OF GAMMA IRRADIATION AND THE DIFFERENT AGE OF THE PLANTLETS In vitro culture of two cultivars of Alpinia purpurata was carried out to produce numerous plantlets, which were then exposed to varying dose of gamma radiation 0, 15, 30 and 45 Gy to study their radio sensitivity (Féréol et al. 1996). Decreased plant survival rate, fresh weight gain and micropropagation rate were observed in plants of C1 and C2 clonal generation with increased doses of γ-radiation. On the basis of survival, LD 50 was reported to be approximately 30 Gy. Gamma radiation dose of 45 Gy was found to drastically affect the different responses studied. It proved lethal or induced dwarf plants. There were morphological abnormalities of leaves or of the whole plants in some plantlets derived from treated plants in vitro. This technique contributed to increased genetic diversity in A. purpurata. The analysis of radio sensitivity of Typhonium flagelliforme resulted in LD50 at 25 Gy (Sianipar et al. 2013). Irradiation at 20 and 25 Gy doses has resulted in production of mutants with significant differences in the percentage of survival, the average plant height and in the average number of shoots as compared to normal mother plantlets. Different responses seen corresponding to different doses have shown that the variation in the irradiation dose caused the variation in the plant height and the number of shoots. The γ irradiation at the 20 and 25 Gy dose can generate T. flagelliforme mutant plantlets with significant differences of plant height and number of shoots from normal mother plantlets.

2.9  EFFECT OF GAMMA IRRADIATION ON DIFFERENT PARTS OF THE PLANTS Gamma irradiation increased the secondary metabolite production in different plant materials including seeds, seedlings, flowers, fruits, roots, hairy roots, callus cultures and suspension cultures (Table 2.4). The psoralen content in seeds of Psoralea corylifolia (babchi) was found to be enhanced

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32 folds by irradiating with 2 kGy (Jan et al. 2011). Total phenolic content of seedlings of Terminalia arjuna (arjuna) was enhanced two folds by irradiating with 25, 150 Gy (Akshatha et al. 2013). Total flavonoids content of flowers of Anethum graveolens (dill) enhanced four folds by irradiation of 640 Gy (Said-Al Ahl et al. 2015). The capsaicinoids content of fruits of Capsicum annuum (chili) was enhanced by 10% with gamma irradiation of 10 kGy (Topuz and Ozdemir 2004). The total phenols and flavonoids of roots of Curcuma alismatifolia (summer tulip) was enhanced 1.5 and 1.8 folds respectively by irradiation with 20 Gy (Taheri et al. 2014). The saponins and ginsenosides of hairy roots of Panax ginseng (ginseng) were enhanced by 1.4 and 1.8 folds respectively by irradiation with 100 Gy (Zhang et al. 2011). The camptothecin content of calli of Nothapodytes foetida (ghanera) increased 20 folds by 20 Gy (Fulzele et al. 2015). Shikonins of suspension cultures of Lithospermum erythrorhizon (purple gromwel) enhanced four folds by 16 Gy (Chung et al. 2006).

2.10 EFFECT OF GAMMA IRRADIATION ON THE DIFFERENT ENZYMES AND INFLUENCE ON SECONDARY METABOLITE PRODUCTION Phenylalanine ammonia-lyase (PAL) is a central enzyme that is responsible for the synthesis of many plant phenolics in the phenylpropanoid pathway (Figure 2.4). It catalyzes the first step of the pathway which is the conversion of phenylalanine into cinnamic acid (Hahlbrock and Scheel 1989; Cheng and Breen 1991; Dixon and Paiva 1995). A number of previous studies have confirmed the role of PAL in the biosynthesis of various phenolics and flavonoids (Cahill and McComb 1992; Lister et al. 1996; Jiang and Joyce 2003). Γ irradiation has been reported to have substantially increased the activity of PAL (Benoit et al. 2000; Hussain et al. 2010). It was also found that there was a positive correlation between the activity of PAL and total phenols, suggesting that an increase in PAL activity is followed by an increase in total phenols. In

FIGURE 2.4  Enzymes influenced by gamma irradiation in flavonoid biosynthetic pathway; Phenylalanine ammonia-lyase (PAL) and Chalcone synthase (CHS). (From Vardhan and Shukla 2017.)

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addition, the rise in phenolics may be attributed to the release of glycosidic components of phenolic compounds and the degradation through γ irradiation of complex phenolics into simple ones (Harrison and Were 2007). By causing oxidative stress in the cells, irradiation exerts its effects on cells. Water radiolysis results in the creation of free radicals such as hydroxyl radicals, radicals of hydroperoxide and hydrated electrons. The glycosidic bonds of procyanidin trimers, tetramers and hexamers present in fruit can be broken by these radicals, leading to the formation of procyanidin monomers that increase the total phenolic content of irradiated fruits (Lee et al. 2009). PAL activity was known to affect the flavonoid biosynthesis which is an essential part of the phenylpropanoid pathway (Oufedjikh et al. 2000). Chalcone synthase (CHS) is the key enzyme in flavonoid biosynthesis, which catalyses the formation of chalcones as basic intermediates in the flavonoid pathway, leading to different flavonoid groups. El-Garhy et al. (2016) reported that a link existed between upregulation of CHS genes and increased content of flavonoids in response to γ irradiation. Ginsenosides are biosynthesized in the roots of ginseng via the mevalonate pathway in the cytosol. The first stages of the pathway leading to different triterpenoids (Han et al. 2010) are catalysed by squalene synthase (SS) and squalene epoxidase (SE). Kim et al. (2013) reported that the SS and SE gene transcription levels increased significantly with ginseng hairy root cultures irradiation. The cyclization of oxidosqualene by oxidosqualene cyclases (OSC) is the first committed step in the biosynthetic pathway of ginsenosides (Choi et al. 2005). There are three types of OSCs, all of which have been found to be upregulated significantly. The overall yields of ghanera callus cultures (N. foetida) of camptothecin and 9-methoxy-camptotheicin were increased by 20 Gy to 20 folds (Fulzele et al. 2015). Camptothecin is a quinoline alkaloid, which belongs to a class of modified monoterpenoid indole alkaloids (TIAs) exhibiting anti-tumour activity by inhibiting DNA topoisomerase-I (Hsiang et al. 1985). Strictosidine synthase (STR) is an important enzyme that catalyses the formation of strictosidine in the TIA biosynthesis (Figure 2.5), a crucial intermediate that is a common precursor for all TIAs (Lu et al. 2009). Though many studies report that γ irradiation significantly influenced the yield of different camptothecin derivatives, none of them reported the correlations. Shikonins and their derivatives are organic hydroxynaphthoquinones with a broad array of properties such as anti-inflammatory, antipyretic, analgesic and anti-oxidant activities that function by inhibiting prostaglandin biosynthesis. The significant step in shikonin biosynthesis is the formation of m-geranyl-p-hydroxylbenzoic acid (GBA), which is catalysed by geranyltransferase PHB (Figure 2.6). Its activity has been substantially increased with Lithospermum erythrorhizon cells suspension cultures irradiation (Chung et al. 2006).

2.11  SUMMARY AND CONCLUSIONS Plants have developed strategies for sustenance in the changing environment by production of small amounts of secondary metabolites which enable them to withstand the various biotic and abiotic stresses. In this regard, the application of gamma irradiation by ionization causes the changes in the materials. The mode of action of sparsely ionizing gamma irradiation is direct where the electrons are ejected, DNA mutation takes place and indirect effects where the damage is caused by radiolytic products like the hydroxyl radicals, electrons which interact with the biological materials. These interactions result in an increase in production of secondary metabolites. The mechanism underlying it is a remarkable source of highly valuable secondary metabolites with wide range of applications in various domains. The gamma irradiation of the plant cell and tissue culture could result in 44 folds increase of biologically active, commercially valuable and medicinally important plant secondary metabolites. Generation of plant mutant lines using gamma irradiation is potential technique sustainable both commercially and ecologically. The enzymes which show an increase in translated products include the phenylalanine amylase in eucalyptus, almonds, strawberry and apples. The

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FIGURE 2.5  Enzymes influenced by gamma irradiation in camptothecin biosynthetic pathway; strictosidine synthase (STR). (From Vardhan and Shukla 2017.)

FIGURE 2.6  Enzymes influenced by gamma irradiation in shikonin biosynthetic pathway; PHB Glucosyl transferase and PHB geranyl transferase. (From Vardhan and Shukla 2017.)

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increase in Chalcole synthase due to gamma irradiation is reported in Silibum marianum. The increase in the levels of secondary metabolites is experimentally validated and role of these enzymes is noted. The increase in enzymatic activity could be due to various point mutations which in turn could play a role at transcriptome levels. The new lines are developed and by the gamma irradiation of seeds, callus and tissue materials, the new plant mutant lines for increased secondary metabolites could be generated.

ACKNOWLEDGEMENTS Dr. Lata I. Shukla thanks Dr. Sushil Kumar, Prof. M. D. Sevilla and Prof. P. C. Kesavan for mentoring and useful discussions. The DBT-GOI and Pondicherry University is gratefully acknowledged. Contact Information: Please contact the author at: [email protected]

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Salinity Stress and Plant Secondary Metabolite Enhancement An Overview Bedabrata Saha, Bhaben Chowardhara, Jay Prakash Awasthi, Sanjib Kumar Panda and Kishore C.S. Panigrahi

CONTENTS 3.1 Introduction............................................................................................................................. 49 3.2 Secondary Metabolite and Salt Stress Amelioration............................................................... 50 3.2.1 Alkaloids...................................................................................................................... 51 3.2.2 Phenolics...................................................................................................................... 51 3.2.3 Lignin ......................................................................................................................... 52 3.2.4 Glucosinolates.............................................................................................................. 53 3.2.5 Tannins........................................................................................................................ 53 3.2.6 Essential Oils............................................................................................................... 54 3.3 Genetic Engineering of Secondary Metabolite to Improve Salt Stress................................... 54 3.4 Conclusions.............................................................................................................................. 55 Acknowledgement............................................................................................................................ 55 References......................................................................................................................................... 55

3.1 INTRODUCTION Salt stress is a major abiotic stressor that drastically affects plant health and yield. Damage to at least 20% of cultivated crops globally is rendered by salt induced stress (Hussain et al., 2018). At extremum situations, salinity affects soil health, ground water and restricts agricultural produce. The detrimentality of salinity is visualized at cellular, tissue and whole plant stratum. High salinity in the growth media induces oxidative, water and ionic stress, and disrupts crucial plant procedures like the osmotic and ionic balance, protein synthesis, photosynthesis, energy and lipid metabolism (Mishra et al., 2014; Saha et al., 2016; Omisun et al., 2018). Salt stress retards plant health; impacts carbon metabolism, ionic, nutritional and redox status; and regulates the status of secondary metabolites which are crucial physiological affiliations in salinity-induced responses. Aggregation of secondary metabolites frequently takes place in plants exposed to stress, either as elicitors or signal molecules. Heightened production of secondary metabolites (flavones, phenolics, anthocyanins and specific phenolic acids) in the cytosol of plants exposed to stress might defend cells from ion-induced oxidative injury by sequestrating the ions and thus depicting enhanced tolerance. Plant secondary metabolites are critical for plant-environment interaction and adaptation. Secondary metabolites developed in plants are biological produce with low molecular weight (Joo et al., 2010). An extensive variety of these products is synthesized in higher plants. Secondary metabolites impart particular scent, colouration and flavour. Plant secondary metabolites are unparalleled DOI: 10.1201/9781003034957-3

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reservoir for food add-ons, flavours, pharmaceuticals and as such commercially important (Bennett and Wallsgrove, 1994). Secondary metabolites reckon several critical roles, like prophylactic character against herbivores, or an appealing character towards pollinators and seed dispersal agents (Selmar, 2008). The tight association among plant secondary metabolism and defence response is extensively realized (Vasconsuelo and Boland, 2007). Alkaloids, steroids, quinones, anthocyanins, lignin, flavonoids and terpenoids are amidst the group of plant secondary metabolites which obtained mercantile applications as pharmaceuticals, colouring agents, taste enhancers, scent, insecticide and antioxidants (Verpoorte et al., 2002). Plant cell culture is an appealing alternate technique for boosting secondary metabolites that are tough to synthesize artificially and are made in plants in restricted amounts. Plant polyphenols constitutes the biggest group among the diverse groups of secondary metabolite antioxidants occurring naturally (Ciesla, 2012). Phenols are presumed to be more powerful antioxidants in comparison to Vitamin C, Vitamin E and carotenoids (Rice-Evans et al., 1996). These compounds are also presumed to make for a prophylactic character against a variety of diseases.

3.2  SECONDARY METABOLITE AND SALT STRESS AMELIORATION Secondary metabolites are organic compounds developed from primary metabolism which neither actively participate in any vital processes of plants nor have any direct character in growth and ontogenesis (Olivoto et al., 2017). On the base of biosynthesis origin, most of the secondary metabolites have been differentiated as lignin, tannins, glycosides, flavonoids, phenols, alkaloids, steroids, essential oils, lectins etc. Salinity, water deficit, temperature and soil pH are key environmental stress factors for discharge of secondary metabolites with enhanced antioxidant functioning (Selvam et al., 2013). Many in vitro experiments proved that compounds like phenolic compounds, flavonoids and tannins are significantly correlated with the antioxidant activity (Khan et al., 2010). Salinity directly influences the induction of the secondary metabolic pathways. At the onset of salt-induced stress, the absorption capacity of water by root is drastically decreased. Higher doses of salt in both plant and soil, lead to loss of water in plant (Munns, 2002; Xu et al., 2016; Adak et al., 2019). As a result, alternations of physiological processes occur that disrupt membrane stability, imbalance redox homeostasis and nutrient imbalance, with overall impact on plant metabolism that sources in secondary metabolites pathways and stomatal function (Chaves et al., 2002; Shabala and Cuin, 2008; Gupta and Huang, 2014; Xu et al., 2016) which may sometimes involve circadian rhythm responses (Chaves et al., 2002). Salt stress tolerance is hastened through systemic changes in physiological processes (Parihar et al., 2015). In plants, physiological adjustment includes extrusion of ions and sequestration, water homeostasis and osmotic adjustment control, tolerance to ion aggregation and reduced loss of K+, along with growth and development adjustments through changes in biochemical and molecular patterns are observed (Munns and Tester, 2008; Sanchez et al., 2008; Shabala and Cuin, 2008; Hamanishi et al., 2015). For extremophiles accustomed to saline growth condition, they pre-adjust by causing increment in the level of secondary metabolites production (Sanchez et al., 2008). Researchers have investigated physiological and molecular effects on growth under salinity stress along with the importance of plant secondary metabolites in crop species. Many reports mentioned that the increment in alkaloids, phenolics, flavonoids, steroids and terpenoids production under salinity stress activated the cellular response to unfavourable condition in plants (Harborne, 1999; Bourgaud et al., 2001; Sytar et al., 2018). For example, concentration of aromatic compounds (alkaloids, phenols and isoprenoids) and phenylpropanoids compounds (flavonoids, tannins and hydroxycinnamate ester) are significantly enhanced due to salt stress in plants and induce the production of free radical scavengers to help sustain themselves (Selmar, 2008; Sytar et al., 2018). Higher activities of antioxidants (enzymatic and non-enzymatic) help to maintain cellular functioning to impart physiological constancy to salt stress, induced by plant secondary metabolites. Sorbitol, mannitol,

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glycinebetaine and fructans impart key role as osmolytes in cell through amelioration of salinity, whose production and generation are significantly enhanced (Shulaev et al., 2008). The halophytes spend major part of their lifecycle under salt stress, as a result the cellular osmotic pressure remains higher and the plant is able to uptake sufficient water when in saline environment meanwhile, synthesizing secondary metabolites to help adapt (Flowers and Colmer, 2008; Xu et al., 2016). Under saline condition, the calli of Solanum nigrum significantly produced proline and solasodine for its tolerance (Å utkoviÄ et al., 2011). Slama et al. (2017) depicted that the synthesis of secondary metabolites and changes in biochemical process with substantial antioxidant capacity is responsible for survival in Sesuvium portulacastrum under extreme saline condition. A number of plants participate in increased synthesis of secondary metabolites based on plant species, genotypes and cultivars to accustom themselves to salt stress (Mahmoudi et al., 2010; Boestfleisch and Papenbrock, 2017; Slama et al., 2017; Hashemi and Shahani, 2019). Let us now look in, on how the production of different classes of secondary metabolites is influenced by salt stress and how the different classes of secondary metabolites modulate salt stress.

3.2.1  Alkaloids Alkaloids are generally nitrogen containing compounds found in higher plants. It has great potential to inhibit various toxic oxidative compounds like singlet oxygen, hydrogen peroxide, etc. For example, the very effective indole alkaloids (strychnine and brucine) are basic nitrogen containing compounds in stiff and cage alike built which are strictly physical quenchers (Larson, 1988). On this basis, the indole alkaloids can immobilize numerous molecules of singlet oxygen per alkaloid molecule. A few of the alkaloids are produced from polyamines. The polyamine (putrescine, spermidine and spermine) are directly involved in various roles of physiology in plant growth and ontogenesis (Kusano et al., 2007). Many reports mentioned that polyamine helps to reduce reactive oxygen species (ROS) production and inhibits lipid peroxidation (Saha et al., 2015). Solanaceae, Papaveraceae, Amaryllidaceae and Ranunculaceae families are rich in alkaloids. Alkaloids are also found in halophytic fodder crops Magnolia sp., Sophora japonica and Pinus ponderosa. Increased alkaloid content in Catharanthus roseus seedlings was observed due to salt stress and different nitrogen sources (Misra and Gupta, 2006). The level of steroidal alkaloids solasodine was directly correlated with the NaCl level in calli of Solanum nigrum (Šutković and Gawwad, 2011). Under stress condition, the alkaloids play crucial role as ROS scavenger. Many reports have mentioned that alkaloids stimulate numerous pathways to overcome ROS production in cells which are undifferentiated (Sachan et al., 2010).

3.2.2 Phenolics Aromatic benzene ring compounds having one or more hydroxyl groups constitute phenolic compounds and are synthesized by plants for protective cover against unfavourable environmental conditions. So, it is also known as anti-stress secondary metabolite. It is very difficult to define the role of phenolic compounds in plant physiology, particularly the crosstalk with biotic and abiotic stressors. The main functions of phenols in plant ontogenesis are the production of lignin and pigments (Bhattacharya et al., 2010). However, the phenolic compounds are considered as antioxidant due to the positive correlation between antioxidant activity and phenols present (Cai et al., 2004; Fu et al., 2011). About 50 medicinal plants were studied and it was observed that phenolics are main component contributing to the functioning of antioxidants. Tripterygium wilfordii, Pyrrosia sheareri, Loranthus parasiticus, Sinomenium acutum, Polygonum aviculare and Geranium wilfordii possess highest antioxidant activity and total phenolics among the 50 medicinal plants (Gan et al., 2010). Under unfavourable condition, glutathione-S-transferase (GST) and phenylalanine ammonium lyase (PAL) get stimulated. PAL corroborates with cinamates-4-hydroxylase to indirectly assist in

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many secondary metabolite’s biogenesis from phenylalanine (Singh et al., 2009). Dehghan et al. (2014) depicted that participation of phenylpropanoid and especially flavonoid pathway in safflower was found under injury and particularly salt stress. Phenolic compound content was found to be in significant increment with increase in the concentrations of NaCl in leaves of buckwheat sprouts. Furthermore, the four compounds (isoorientin, rutin, orientin and vitexin) responsible for enhancing the phenolic compounds accumulation also gradually increased (Lim et al., 2012). Under salinity condition, the antioxidant capacities; antioxidant pool (ascorbic acid, superoxide dismutase and anthocyanins); selected minerals, viz., Na, K, Cl, N, P, and Zn; and lipid peroxidation are significantly enhanced in strawberry fruits (Keutgen and Pawelzik, 2008). Abideen et al. (2015) observed the relationship between the polyphenolic production and salinity in halophyte plant P. karka which showed that salt stress is a powerful instrument to stimulate antioxidant rich biomass for industrial intentions. It has been figured that some concentrations of NaCl can enhance the nutraceutical merit of radish sprouts. Under salt stress, the germination of sprouts improves synthesis of health-promoting compounds. The glucosinolate and total phenolic content were reduced at lower NaCl dose whereas significantly increased at higher concentration (Yuan et al., 2010). Ksouri et al. (2007) observed that the polyphenol capacity and antioxidant functioning increased in Cakile maritima leaves under saline conditions. The total chlorogenic acid concentration was significantly higher in halophytes honeysuckle flower buds under salinity (Yan et al. 2016). Flavonoids, derived from 2-phenyl-benzyl-γ-pyrone, are considered as biggest group of plant phenolics and play a crucial part in plant protection. A total of 9000 compounds of flavonoids are substantially reported (Buer et al., 2010); they aggregate in the plant vacuole as glycosides or exudates on the leaf surface. Flavonoids are grouped into four on the basis of their structure i.e., flavonols, isoflavones, flavones and anthocyanin. These compounds contribute to scent, flavour and colour of the plants and also defend them in inauspicious conditions (Clé et al., 2008; Olsen et al., 2010). Flavonoids have roles to play during environmental constraints (Winkel-Shirley, 2002) in the higher plants and have been suggested to act as plant growth regulators (Stafford, 1991). Isoflavones are intermediate product derived from flavonone, e.g., naringenin plays a vital role in establishment of nitrogen fixing nodules by symbiotic rhizobia in leguminous plants (Sreevidya et al., 2006). Posmyk et al. (2009) mentioned that these flavonoids were synthesized and are also effective against ROS under salinity stress. Azospirillum brasilense helps root branching and also releases nod gene stimulating flavonoid species in saline conditions (Dardanelli et al., 2008). Under salinity condition, increment in anthocyanin content was observed in many plants (Parida and Das, 2005).

3.2.3 Lignin Lignin is the plant secondary metabolite which provide structural rigidity to cell wall. It has been estimated that salinity exercises a synergetic impression in the lignification of both protoxylem and metaxylem vessels, and causes a sooner and stiffer lignification of the phloem fibre cell walls. In plant cells, lignin is the very crucial secondary metabolite which is synthesized from phenylalanine or tyrosine pathways. The biosynthesis of lignin is complex and divided into three steps: (i) lignin monomers biosynthesis, (ii) transport and (iii) polymerization. Lignin and associated metabolism make for a crucial role in the ontogenesis and development. As a polymer of phenol, it increases the rigidity of plant cell wall, hydrophobic characteristics and boosts mineral movement via the vascular bundles in plants (Schuetz et al., 2014). Lignin is also main roadblock against pests and pathogens (Ithal et al., 2007). Many reports have admitted that lignin metabolism actively participated in plant dodging various environmental stress (Tripathi et al., 2003; Van der Rest et al., 2006; Shadle et al., 2007; Derikvand et al., 2008; Moura et al., 2010). The matured casparian strip, in the primary roots of maize, comes near the root tip in saline condition due to reduction in number and length of the cells in the epidermis between the lowest

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position of the strip and the root tip. It was observed that salinity didn’t have any impact on timedependent development of the strip in individual cells (Karahara et al., 2004). Under salt stress, the mean count of lignified cells in the vascular bundle was drastically larger due to enhanced activity of S-adenosyl-L-methionine synthetase, with maximum lignified area in vascular root. Sánchez-Aguayo et al. (2004) mentioned the positive correlation between increment in activity of S-adenosyl-L-methionine synthetase and deposition of lignin in vascular tissue of plants under saline condition. The increased synthesis of lignin hardens cell wall and limits root growth due to saline conditions. Significant increment was observed by Neves et al. (2010) in the total phenols and lignin content; p-hydroxyphenyl and syringyl monomers of lignin in saline condition. Kelij et al. (2015) scored that the functioning of cinnamyl alcohol dehydrogenase, phenylalanine ammonia lyase and lignin capacity was significantly increased.

3.2.4  Glucosinolates The bigger amalgamation of sulphur and nitrogen-carrying glucosides are glucosinolates. Glucosinolates are majorly detected in Brassicaceae family and their content in tissues is affected by change in environment including saline condition (del Carmen Martínez-Ballesta et al., 2013). Many reports have depicted that glucosinolates content and glucosinolates profile changes under stress to cause plant adaptation. López-Berenguer et al. (2008) depicted that the glucosinolates content significantly increased at 40 mM salt stress but decreased at 80 mM salt stress indicating potential role of glucosinolates in saline conditions. Pang et al. (2012) mentioned that the fluctuation in the content of glucosinolates might be directly correlated to the ontogeny of plants. López-Berenguer et al. (2009) indicated that enhancement of glucosinolates coincides with osmotic adaptation and salt tolerance in broccoli plants. Under saline condition, glucoerucin (a type of glucosinolates) increased significantly in broccoli sprouts whereas the total glucosinolates content drastically reduced. Increased higher biomass along with higher glucosinolates contents in broccoli sprouts under 60 mM saline condition at five days was observed (Guo et al., 2013). Allocation of glucosinolates can be an avoidance scheme to limit stress scenarios at low-energy cost. Bekaert et al. (2012) reported that the allocation of glucosinolates production was directly and positively correlated with the increase in photosynthetic efficiency in Arabidopsis.

3.2.5  Tannins Tannins are large polyphenolic compounds that contain hydroxyls and other appropriate groups (like carboxyl) which frame potent composites with several macromolecules. The tannins are found in diverse plants and play protective character against stresses thus helping in plant growth management (Ferrell and Thorington, 2006). Common plants synthesizing tannins are Arctostaphylos uva-ursi, Potentilla fruticose, Limonium latifolium, Quercus alba, Larix spp, Hemerocallis spp, Quercus rubra etc. Condensed tannins are normally polymeric flavonoids moulded by condensation of monomers like flavan-3-4-diols and flavon-3-ols (Foo et al., 1996). When depolymerized, condensed tannins make cyanidin or delphinidin and they are farther grouped into procyanidin or prodelphinidin (Bruneton, 1999). The most crucial chemical characteristics is their capability to constitute soluble or insoluble composites with macromolecules like fibres and proteins. The roles of condensed tannins are mainly interaction between plant and microorganisms either pathogen or mutualistic, but in response to abiotic stress (Gebrehiwot et al., 2002; Reinoso et al., 2004; Paolocci et al., 2005). Plant height, mass and leaf area are negatively correlated to salinity stress. Significant increment in the heaviness of upper and lower cuticle, upper and lower epidermis and concentrations of tannins with increase in salt dose were observed, while the upper and lower thickness of epidermis,

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and intracellular space of spongy of leaves reduced (Liu et al., 2009). The salt sensitive and tolerant poplar species disclosed the evolutionary adjustment of stress resistance mechanics. Under salt stress condition, two poplar varieties showed increased respiration, tannins and phenolic content (Janz et al., 2010).

3.2.6 Essential Oils Essential oils are secondary metabolites which are volatile, natural, complex compounds, having strong scent and synthesized by aromatic plants. Essential oils are synthesized by all plant organs and stashed in trichome. They participate in plant-animal, plant-microorganisms and plant-plant interactions for endurance and adjustment to environmental scenarios (Castro et al., 2008). The aromatic plants contain diverse volatile molecules like terpenes and terpenoids, phenol-derived aromatic and aliphatic constituents (Bakkali et al., 2008). Normally, terpenoids are main component of plant essential oils but much of these are also formed of various chemicals such as phenylpropenoids. Terpenoids are normally deduced of five carbon isoprene segments and alteration in thousand ways. Maximum of essential oils are complicated in makeup due to existence of variance of extremely functionalized chemical compounds, of various chemical groups (monoterpenoids, phenylpropenoids, sesquiterpenoids, etc.). Essential oils yield is eco-physiological and environmentally well-disposed (Sangwan et al., 2001). Growing and production of essential oils yielding plants from diverse families is essential for a country as it forms integral part of its food and cosmetic industry. Nowadays, the importance of essential oils is in focus due to its relationship with environmental stress. Sometimes, soil salinity promotes synthesis of particular components essential oils. The main factor for accumulation of essential oils is dependent on the development stage of plant and also on storage part or organ, tissue and cells (Sangwan et al., 2001). Ansari et al. (1998) mentioned that the oil yield content was significantly reduced in C. winterianus than C. flexuosus under saline condition. The citral and geraniol content was enhanced in lemongrass and palmarosa under salinity stress condition. Potential of regeneration under higher concentration of salt stress was observed in a salt tolerant line of palmarosa, developed, formulated by an in vitro process (Patnaik et al., 1997). For 4 weeks, 1.8-cineole, β-thujone and camphor content were significantly increased under 100 mM NaCl whereas no impact was observed at lower concentration of NaCl (Tounekti and Khemira, 2015). Essential oil content was either reduced, increased or no changes in pennyroyal (Mentha pulegium L.), peppermint (Mentha x piperita L.) and apple mint (Mentha suaveolens Ehrh.) due to salinity in comparison to control. Under salt stress condition, the essential oil content reduced along with chemical composition that also changed in Carthamus tinctorius L. (Harrathi et al., 2012). Ragagnin et al. (2014) mentioned that salt stress did not have much impact on yield and chemical composition of essential oil in Lippia gracilis at higher concentration of NaCl.

3.3 GENETIC ENGINEERING OF SECONDARY METABOLITE TO IMPROVE SALT STRESS The salinity stress can be affiliated with secondary metabolite synthesis and very specific gene expression for stress amelioration (Falleh et al., 2012; Panich et al., 2010). Numerous secondary metabolites take part in the evolution of salinity-induced responses as antioxidants and hence in the amelioration of salt stress. Various genes are involved in enhancement of secondary metabolite production for conferring salinity tolerance such as OsHsp17.0, OsHsp23.7 genes from rice cultivars (Zou et al., 2012); MYB112 transcription factor isolated from Arabidopsis leads to increase in anthocyanin accumulation (Lotkowska et al., 2015); the sodium potassium antiporter genes NHX1 increases rutin accumulation (Chen et al., 2008); CYP71AV1 from Artemisia annua increases artemisinin production (Sheludko, 2010) etc. The miRNAs biosynthesis from various crops species

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Rauvolfia Serpentine, Arabidopsis thaliana, Diospyros kaki are also needed in the activation of flavonoid production (Gupta et al., 2017). Constitutive silencing of γ-tocopherol methyltransferase (γ-TMT) and homogentisate phytyltransferase (HPT) functioning in tobacco plant lead to 98% decrease in full tocopherol aggregation in transgenic lines and so diminution in capacity of salt stress resistance (Abbasi et al., 2007). F3H, F3′H and LDOX gene expression in Arabidopsis, enhanced anthocyanin aggregation (Van Oosten et al., 2013). Cell wall CWPRX expression from Aeluropus littoralis increased phenolic acids content in cell wall (ferulic acid, p-coumaric acid and sinapic acid) (Haghighi et al., 2014). miRNA 156, 159 and 166 expression patterns in Sacharum sp. led to GA-Myb, SPL5, and Glass III HD-Zip protein 4 overexpression, which raised salinity resistance (Shriram et al., 2016). These genes are also creditworthy for the regulation of the secondary metabolite.

3.4 CONCLUSIONS The secondary metabolites work as antioxidants and are capable to take part in equilibrating redox status of plants. Secondary metabolite such as essential oils, lignin, glucosinolates, tannins and phenols found in halophytes have important role to play in salinity stress resistance. Secondary metabolites are majorly involved in medicine, serve as nutraceuticals for food add-on and beauty products, and adaptation to plant stress physiology. In future, we can explore artificial induction of calculated amount of salinity stress to enhance secondary metabolite synthesis without hampering crop yield.

ACKNOWLEDGEMENT BS would like to acknowledge Science and Engineering Research Board (SERB), Govt. of India for National Post-Doctoral Fellowship (No. PDF/2018/003216 dated 7 March 2019). Contact Information: Please contact the author at: [email protected]

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Enhancement of Plant Secondary Metabolites Using Fungal Endophytes Touseef Hussain, Mulla Javed, Samrin Shaikh, Bilquees Tabasum, Kashif Hussain, Moh Sajid Ansari, Amir Khan and Abrar Ahmad Khan

CONTENTS 4.1 Introduction............................................................................................................................. 61 4.2 Endophyte’s Role in Sustainable Agriculture.......................................................................... 62 4.3 Production of Secondary Metabolites through Fungal Endophytes........................................ 62 4.4 Effects of Fungal Endophyte and its Elicitor on Plant Secondary Metabolite Production..... 63 4.5 Fungal Endophytes and Medicinal Metabolites......................................................................64 4.6 Conclusion............................................................................................................................... 65 References.........................................................................................................................................66

4.1 INTRODUCTION Several plants and their products/by-products are to a great extent utilized for the preparation of medicinal drugs. Plants synthesize both the primary as well as secondary metabolites. Primary metabolites play an important role mainly in the growth and development of plants (Zhi-lin et al. 2007; Guerriero et al. 2018; Yang et al. 2018). In contrast, secondary metabolites assist a variety of cellular functions crucial for physiological processes. A variety of molecules including phenolic, terpenoids and alkaloids are produced so far as secondary metabolites. But their complex structure makes them unable to be synthesized chemically so in this regard, in vitro plant tissue cultures play an important role. Plant material is propagated in vitro for the large-scale production of secondary metabolites. The callus and cell suspension cultures are optimized for the formation of desired bioactive compounds but it does not pacify the current demand. The alternative techniques like optimization of nutrient medium, cell immobilization, metabolic engineering, nutrient manipulations and addition of precursors and elicitors have been used so far by many researchers (Kiran et al. 2011; Archana et al. 2012; Chodisetti et al. 2015). Elicitors are being the bio factors or chemical molecules, which can activate and/or generate the synthesis of secondary metabolites via triggering signal transduction pathway. There are several factors including biotic, abiotic, genetic makeup, morphogenetic, ontogenic, and carbon contents in nutrition which control the production of secondary metabolites (Wink, 2003; Laitinen et al. 2005; Ann Lila, 2006; Yang et al. 2018). Biosynthesis and bioaccumulation of secondary metabolites confer the defence of plants against herbivores, bacteria, viruses, fungi and also other adverse abiotic stress (Wink, 2003; Wink, 2008; Pusztahelyi et al. 2015; Yang et al. 2018). The mutualistic interaction helps in the synthesis of biomolecules and induced resistance in the host plant whereas the antagonistic relationship challenges the yield (Zhi-lin et al. 2007; Millet et al. 2010; Zuccaro et al. 2011; Schulz et al. 2015). Several plant pathogens such as bacteria and fungi cause certain diseases. Thereby, alter the regular function of plants along with low production DOI: 10.1201/9781003034957-4

61

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and deprived quality of plant and their products. To overcome the problem, several studies were done that reported the ability of plant endophytes to yield plant secondary metabolites (Gouda et al. 2016). These endophytes reside inside their host plant without causing any harmful effects (Bacon and White, 2000; Vasundhara et al. 2019). Several recent reviews are explaining the capacity of endophytes in the production of bioactive compounds involving secondary metabolites (LudwigMuller, 2015). Amongst them, the species of Aspergillus, Ramularia, Fusarium, Helianthimum and Neotyphodium have been reported so far that elicit secondary metabolites formation (Bultman and Murphy, 2000; Ding et al. 2018). They reside in intercellular and intracellular spaces of any part of the host plant However, the interaction and complexity of association of endophytic fungi to their host plant is variable with host to host and microbes to microbes depending upon conditions. The outcomes of such association may result in the production of natural active compounds (Kusari and Spiteller, 2011), production of growth promoters, mitigation to abiotic stress, first-line defence against a wide variety of pathogens and/or pests (Aly et al. 2011; Wani et al. 2015), act as enhancers or elicitors of bioactive compounds in callus and cell suspension cultures systems (Dass and Ramawat, 2009; Netala et al. 2016). Some endophytic fungi can survive in seed and spread in the seedling stage while others interact through various processes and make their relationship with the plant (Verma et al. 2009). Overall, endophytic fungi studied in the production of paclitaxel (taxol), emodin, polydatin, chrysophanic, terpenoids, tetralones, xanthones, chrysophanol, physcion and musizin were used as an ailment in several biological activities. For instance, it is used as an antioxidant, antimicrobial, anticancerous, antiasthmatic, immunosuppressive and anti-inflammatory agent (Strobel et al. 1996; Tan and Zou, 2001; Ding et al. 2018; Vasundhara et al. 2019). The practical biotechnological application of these fungal endophytes has been meagrely studied.

4.2  ENDOPHYTE’S ROLE IN SUSTAINABLE AGRICULTURE Fungal endophytes play a crucial role in the survival and adaptation of plants to new conditions through improved plant immunity to cope up with abiotic and biotic stresses (Tan and Zou, 2001). They persuade various methods of promoting plant growth, such as phosphate solvation, production of phytohormone, biological nitrogen fixation and inhibition of ethylene biosynthesis. Also, they promoted plant resistance by secreting several secondary metabolites in the form of enzymes, antibiotics and siderophores to prevent pathogenic attacks. Endophytes contain auxin, cytokinin, gibberellin, adenine ribosides, adenine, acetoin, 2,3-butanediol, indole-3-butyric acid and polyamines that are valuable in the growth and development of the plant (Pirttilä et al. 2004; Spiering et al. 2006; Vadassery et al. 2008; Shi et al. 2009; Khan et al. 2012). Fungal endophytes are demonstrated as a good source of plant growth phytohormones like auxin and gibberellic acid. The effect of the plant defence system is also significantly increased. In the rice, grass and barley, (Redman et al. 2011; Czarnoleski et al. 2012) endophytes also induce yields and an increase in plant biomass (Schafer and Kogel, 2009). Endophytes are used to increase plant resistance to biotic and abiotic stresses, to cover seeds with increased germination, and to induce induced systemic resistance (ISR), which make them important instruments for sustainable agriculture development. The studies demonstrating endophyte’s roles in promoting plant growth and development have been published (Table 4.1). The point-to-point data was explored (Rai et al. 2014).

4.3 PRODUCTION OF SECONDARY METABOLITES THROUGH FUNGAL ENDOPHYTES It might be speculated that horizontal gene transfer and independent secretion by plants and microbes are responsible for secondary metabolite synthesis. But the recent reports envisaged that both plants and endophytic microbes are co-evolved during the process to yield these secondary metabolites. The radio-labelled studies addressed similar but distinct metabolic pathways in both

Enhancement of Plant Secondary Metabolites Using Fungal Endophytes

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TABLE 4.1 The Growth-Promoting Fungal Endophytes in Various Host Plants S. No.

Host Plants

Endophytes

References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Capsicum annuum Chrysanthemum coronarium Cucumis sativus Cucumis sativus Cucumis sativus Cucumis sativus Cucumis sativus Cucumis sativus Dendrobium loddigesii Dendrobium loddigesii Elymus mollis Euphorbia pekinensis Glycine max Glycine max Ipomea batatas Ixeris repenes Monochoria vaginalis Monochoria vaginalis Musa sp. Potentilla fulgens Sesamum indicum Solanum tuberosum Sorghum bicolour

Chaetomium globosum Penicillium sp. Cladosporium sp. Exophiala sp. Penicillium sp. Phoma glomerata Phoma sp. Paecilomyces formosus Fusarium sp. Pyrenochaeta sp. Gliomastix murorum Fusarium sp. Pyrenochaeta sp. Metarhizium anisopliae Fusarium oxysporum Penicillium citrinum Aspergillus sp. Penicillium sp. Fusarium oxysporum Penicillium verruculosum Fusarium oxysporum Aspergillus ustus Helminthosporium velutinum

Khan et al. (2012) Hamayun et al. (2010) Hamayun et al. (2010) Khan et al. (2011) Waqas et al. (2012) Waqas et al. (2012) Khan et al. (2011) Khan et al. (2012) Chen et al. (2010) Chen et al. (2010) Khan et al. (2009) Dai et al. (2008) Khan et al. (2011) Waqas et al. (2012) Hipol (2012) Khan et al. (2008) Nadeem et al. (2010) Nadeem et al. (2010) Machungo et al. (2009) Bhagobaty and Joshi (2009) Hasan (2002) Marina et al. (2011) Diene et al. (2010)

partners (Jennewein et al. 2001). But the question remains the same that whether these metabolites are produced by the plant itself or are an outcome of a mutualistic relationship with advantageous organisms residing inside the host. The inducing elements from both partners increased the build-up of bioactive phytochemicals both in plants and fungi individually. The signalling molecules in the form of elicitors originate from endophytic fungi or bacteria. These molecules help plants to fight against pathogens. Also, they induce the secretion of different phytochemicals such as alkaloids, flavonoids terpenoids, saponins and phenols (Tian et al. 2012). The elicitors are mostly biomolecules involving polysaccharides, polypeptides, unsaturated fatty acids and glycoproteins (Wang et al. 2015). The polysaccharides were released by endophytes upon the action of hydrolytic enzymes of plants (Mohnen and Hahn, 1993). The extracellular secreted polysaccharides were also responsible for the connection among endophytes and host plants (Hiraoka, 1995). Thus, the mutualistic association of such fungal endophytes in the secretion of secondary metabolites is necessary and should be explored. This could provide the initial stage to move towards genetic and metabolic engineering (Engels et al. 2008; Karuppusamy, 2009).

4.4 EFFECTS OF FUNGAL ENDOPHYTE AND ITS ELICITOR ON PLANT SECONDARY METABOLITE PRODUCTION The enormous application and characteristic activities of metabolites produced from endophytic fungi mark them more valuable in recent years. The approaches have been initiated to use fungal endophytes as an elicitor for improvement in the growth and secondary metabolite biosynthesis (Zhang et al. 2009).

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In hairy root cultures of Artem annua, the oligosaccharide components of crude endophytic mycelium of Colletotrichum gloeosporioides have been shown to promote the production of artemisinin. The yield was enhanced from 0.8 mg/g dry weight to 1 mg/g dry weight (Wang et al. 2006; 2001). Recently, plenty of reports were also focused on Fusarium species to promote secondary metabolite production. Thus, it will be fascinating to look at and pinnacle the latest data reported by various investigations on Fusarium species. The red resin of Dracaena cochinchinensis was well known for the treatment of traumatic and visceral haemorrhages in China. The accumulation of red resin was found in the co-culture of root-isolated endophytic Fusarium species of D. cochinchinensis (Jiang et al. 2003). The crude oligosaccharide of Fusarium oxysporum isolate Dzf17 extracted from Discorea zingiberensis was studied and it showed the enrichment of diosgenin within the cell suspension cultures (Zhang et al. 2009). Furthermore, the studies have also been taken by extracting polysaccharides with water and sodium hydroxide along with the only exopolysaccharide (Li et al. 2011a). The water-extracted mycelial polysaccharide (WPS) showed the 3.83-fold increase in diosgenin content in cell culture suspension. However, beauvericin from Fusarium redolens Dzf2 also increases the yield of diosgenin by 3.6-fold in the cultured cells (Yin et al. 2011). Amongst the taxanes, paclitaxel is always the choice of the drug for treating breast cancer (Cremasco et al. 2009). Several efforts are made to elicit paclitaxen production in bioreactors at industrial scale (Li et al. 2009; Li and Tao, 2009). The co-culture technology was applied on suspension cells of T. chinensis with endophytic fungi F. mairei. The 38-fold higher yield of paclitaxel was observed in a co-cultured system within only 15 days. Furthermore, the production of paclitaxel was also dependent on the type of fungal species (Salehi et al. 2019). Remarkably, the defencerelated enzymatic machinery has also up-regulated with the application elicitor of endophytes. Several defence-related enzymes like phenylalanine ammonia-lyase, peroxidase and catalase were found to be increased (Gao et al. 2011). Such enzymatic forms were also increased in volatile oils of Atractylodes lancea when endophytic fungi Gilmaniella sp. were used (Wang et al. 2012; Chen et al. 2016). Some more examples emphasize the diverse role of endophytes and their ability in inducing secondary metabolites. The essential oil content and terpene-producing potential of Mentha piperita get mottled by the inoculation of leaf fungal endophyte (Mucciarelli et al. 2003). The endophytic actinomycetes were able to enhance secondary metabolites (anthocyanin) and nutrient levels (Hasegawa et al. 2006). The up-regulation of withanolide and sterol biosynthetic pathway genes was found in fungal endophyte-treated plants (Kushwaha et al. 2019). The capability of endophytic fungi to desired bioactive compounds also depends on the mutualistic association of other fungi (Ding et al. 2018). For instance, Aspergillus, Fusarium and Ramularia species obtained from Rumex gmelini Turcz (RGT) show similar bioactive compounds as present in the host organism. However, potency gets reduced when the fungus is sub-cultured separately for a long time.

4.5  FUNGAL ENDOPHYTES AND MEDICINAL METABOLITES Endophytic fungi assume a significant function in plant growth and development. Their co-existence favours several enriched bioactive molecules present in the cultures with wider applications in the medical field (Firakovv et al. 2007). It was observed that endophytic fungi fundamentally encourage medicinal plants for the overall growth, struggling against abiotic and biotic factors and finally enhancement in the bioactive metabolites (Firakovv et al. 2007). The various pharmaceutical active components were studied such as podophyllotoxin (Eyberger et al. 2006; Puri et al. 2006), paclitaxel (also known as taxol) (Stierle et al. 1993), deoxy-podophyllotoxin (Kusari et al. 2009a), hypericin and emodin (Kusari et al. 2009b; 2008), camptothecin, and structural analogs (Puri et al. 2005; Kusari et al. 2009c; Shweta et al. 2010; Kusari et al. 2011) and azadirachtin (Kusari et al. 2012). Some pharmaceutically active components have been discussed in Table 4.2.

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TABLE 4.2 Interaction of Endophytic Fungi with Host Plants Produces Secondary Metabolites which Are Used in Different Medicinal Activities Secondary Metabolites

Host Plants

Medicinal Activities

References

Acremonium sp., Shiraia sp. Aspergillus fumigatus Aspergillus nidulans, A. oryzae Alternaria sp. Alternaria sp.

Huperzine A

Huperzia serrate

Anticholinesterase

Li et al. (2007)

Paclitaxel Quercetin

Podocarpus sp. Ginkgo biloba

Antitumor Anti-inflammatory

Sun et al. (2008) Qiu et al. (2010)

Podophyllotoxin Berberine

Antitumor Antibiotic

Lu et al. (2006) Duan et al. (2009)

Blastomyces sp., Botrytis sp. Colletotrichum gloeosporioides

Huperzine A

Sabina vulgaris Phellodendron amurense Phlegmariurus cryptomerianus Piper nigrum

Anticholinesterase

Ju et al. (2009) Chithra et al. (2014)

Cochliobolus nisikadoi

Borneol

Cephalosporium sp., Paecilomyces sp.

Diosgenin

Chaetomium globosum Cladosporium cladosporio Cephalosporium corda

Hypericin Paclitaxel

Hypericum perforatum Taxus media

Antimicrobial, antidepressant, anti-inflammatory, and anticancer Anti-inflammatory, antioxidant Antitumor, antiinflammatory, and cardiovascularprotection Anti-depressant Antitumor

Sipeimine

Fritillaria ussuriensis

Yin and Chen (2008)

Fusarium solani Fusarium solani Fusarium solani

Camptothecin Camptothecin Paclitaxel

Antibiotic and anti-ulcer Apodytes dimidiate Antitumor Camptotheca acuminate Antitumor Taxus celebica Antitumor

Fusarium solani, Metarhizium anisopliae, Mucor rouxianus Fusarium oxysporum Fusarium oxysporum, Neonectria macrodidym, F. solani, F. proliferatum F. oxysporum

Paclitaxel

Taxus chinensis

Antitumor

Puri et al. (2005) Kusari et al. (2009c) Chakravarthi et al. (2008) Deng et al. (2009)

Podophyllotoxin Cajaninstilbene acid

Juniperus recurve Cajanus cajan

Ginkgolide B

Ginkgo biloba

Antitumor Antioxidant, hypotriglycerimic, and hypoglycemic Antishock, antiallergic, and anti-inflammatory

Endophytic Fungi

Piperine

Cinnamomumcamphora chvar. Borneol Parispolyphylla var. yunnanensis

Chen et al. (2011) Chen et al. (2011)

Kusari et al. (2008) Zhang et al. (2006)

Kour et al. (2008) Zhao et al. (2012)

Cui et al. (2012)

4.6 CONCLUSION Desired bioactive compounds production in the form of secondary metabolites in plants with symbiotic association with fungi incites the advancement of alluring approaches that could lead to propagation of medicinal plants into a new era for pharmaceutical use in drug discovery. Recent investigations on the symbiosis of endophytic fungus result in eliciting secondary metabolites

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production. Endophytic fungi might be propagated in vitro along with the host organism under standard conditions to evaluate their role in plant morphogenesis and secondary metabolic pathways. This enables the development of novel approaches that could be used for the commercial production of desired bioactive compounds obtained from plants. However, further exploration is required to understand the exact mechanism of secondary metabolite production induced by fungal endophytes. Contact Information: Please contact author at: [email protected]; [email protected]

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Hydroponic Cultivation Approaches to Enhance the Contents of the Secondary Metabolites in Plants Yogesh Chandrakant Suryawanshi

CONTENTS 5.1 Introduction............................................................................................................................. 71 5.2 Hydroponics............................................................................................................................. 72 5.2.1 What is Hydroponics................................................................................................... 72 5.2.2 History of Hydroponics............................................................................................... 72 5.3 Substrates for Hydroponics...................................................................................................... 72 5.4 Nutrient Solutions.................................................................................................................... 74 5.4.1 Inorganic Hydroponic Solutions.................................................................................. 74 5.4.2 Organic Hydroponics Solutions................................................................................... 74 5.5 Hydroponic Approaches for Secondary Metabolite Production............................................. 74 5.6 Conclusions.............................................................................................................................. 82 References......................................................................................................................................... 83

5.1 INTRODUCTION Plants are an integral part of human life because they are the prominent source of energy. To all human and living things on earth, domestication of plants for food, fodder, fibre, fuel, medicines etc. has started long back in the dawn of civilization. All such environmentally important plants were cultivated using different methods. Soil is usually the universal growing medium for different crops, including medicinal crops. However, soil is responsible to cause many soil-borne diseases to plants. In addition, various toxic metals, salts, pH, soil fertility, water absorption capacity, etc. were affecting the plant growth. Fertile and suitable soil for crop cultivation is a major concern all around the world. Nowadays, new methods and various technologies are used in agriculture to increase the crop production. The secondary metabolites are usually defensive compounds for plants (Torawane et al., 2020). Soil has become the major constrain for cultivation of medicinal plants producing different types of secondary metabolites. This requires a new type of technologies to increase secondary metabolites, where soil quality would not interfere. In this chapter, we will study hydroponic cultivation technique which is a soilless growing of plants in nutrient media. This chapter discusses several key techniques and substrates for enhancing secondary metabolite productions through hydroponic technology.

DOI: 10.1201/9781003034957-5

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5.2 HYDROPONICS 5.2.1  What is Hydroponics The name hydroponics itself says cultivation using water where hydro means water and ponos means labour. Hydroponics simply means growing of plants in mineral nutrient solutions and water excluding the use of soil (Dos Santos et al., 2013). Hydroponics is also known as ‘soilless culture’, ‘soilless agriculture’, ‘nutriculture’, ‘hydroculture’, ‘chemical culture’, ‘tank farming’, ‘aquaculture’ etc. Hydroponics is a form of gardening or growing of plants in which soil is replaced by a mixture of water and liquid nutrients.

5.2.2 History of Hydroponics The first evidence of hydroponics is seen in the Hanging Gardens of Babylon. The Hanging Gardens of Babylon was built 600 years BC (Stanley, 1998). It is believed that a chain pull system was used to water the plants. Some authors suggest that there is no evidence of hydroponics cultivation in Hanging Gardens of Babylon (Hershey, 1994). Evidence of hydroponics cultivation is found near the island city of Tenochtitlan, which was built between the eleventh and twelfth centuries (El-Kazzaz and El-Kazzaz, 2017). The first information about hydroponic cultivation is found in the book A Natural History or Sylva Sylvarum written by Francis Bacon in 1627. After the publication of this book, the method of water cultivation became popular (Pandey et al., 2009). The cultivation experiment of growing plants on water was described by John Woodward in 1699 (Saraswathi et al., 2018). By the eighteenth century, the basic essential elements of plants were discovered by various researchers. Two German botanists Julius von Sachs and Wilhelm Knop developed the technique of growing plants without soil. At that time, the growing of plants without soil in nutrient media was called as solution culture and nowadays, it is a part of hydroponics cultivation techniques. In the nineteenth century, some researchers found that diseases of certain plants were caused by elements in the soil; hence, William Frederick Gericke in 1929 from California began to argue that solution culture could be beneficial to farming and he termed this method as aquaculture. However, at that time aquaculture name was already given to the culture of aquatic organisms. He introduced two names, hydroponics and water culture in 1937, which was suggested to him by W. A. Satchell. Gradually after that, the name hydroponics came into vogue. Hydroponic culture in India was introduced in 1946 by English scientist W. J. Shalto Duglas. He opened his laboratory in West Bengal and wrote a book called Hydroponics: The Bengal System. Later in the 1960s and 1980s, commercial hydroponic farms were developed in many countries (Gericke, 1937; Douglas, 1951; Sardare and Admane, 2013). Over the past few decades, the National Aeronautics and Space Administration (NASA) has conducted extensive studies on hydroponic culture for its Controlled Ecological Life Support System (CLANS) (Roberto, 2003). NASA is also studying whether plants can be grown in space using hydroponics cultivation. Eurofresh Farms in Willcox, Arizona has commercially sold a large number of plants grown with hydroponic cultivation, especially tomatoes. By 2017, hydroponic cultivation in Canada has led to the production of large quantities of tomatoes, pumpkins and peppers. Hydroponics is a very good, reliable and feasible method for obtaining the high-crop productivity, especially secondary metabolites. A lot of experiments on enhancement of secondary metabolites by using hydroponics cultivation have been done and a lot more is yet to be done.

5.3  SUBSTRATES FOR HYDROPONICS The substrate is a surface where plants grow in hydroponic cultivations. It is a very important component of hydroponic cultivation. The substrate acts as growing support materials for plant growth. A variety of substrates are used in hydroponic cultivation. The hydroponic substrates have higher porosity and oxygen penetration capacity compared to soil. Various substrates like expanded clay aggregate, growstones, coconut coir, rice husk etc. are mostly used in hydroponics. Table 5.1 shows the details of different types of substrates used in hydroponic cultivation.

Sr. No.

Substrates Type

Source

Application in Hydroponic Culture

References

1

Expanded clay aggregate

Clay

Boudaghpour and Hashemi (2008)

2

Growstones

Glass waste

3

Coconut coir

Coconut

4

Rice husk

Rice

5

Perlite

Volcanic rock

Also known as expanded clay (exclay) and Lightweight Expanded Clay Aggregates (LECA). It is made by heating the clay at 1200°C in rotary kiln. The gases formed during heating expanded the clay by producing the honeycomb structure. Approximately, ovoid or round shape exclay forms during the heating because of rotary kiln. It can be reused by washing with various chemicals and followed by sterilization process. Sometimes, it is non-reusable because of plant roots which break the clay pebble It contains 0.5–5% calcium carbonate and remaining is soda-lime glass. It has more retention space for air and water than Perlite. It provides best growth conditions for plants It has good water retention, and disease and pest resistance capacity. It requires maturation process before using. Maturation means removing the phenolic compounds, salt and tannin. This process requires six months, lots of water for washing and it is quite dangerous. Hence, use of coconut coir is illegal in Europe and North America. Coir has larger particle size and release more water at lower pressure Parboiled rice husk (PRH) is an agriculture by-product. It holds more water than growstone. Rice husk did not affect plant growth regulator Used in potting soil mixes to decrease soil density. It holds more air and less water. Growth, yield and quality of plants depend on size of perlite

6

Pumice

Volcanic rock

Works like Perlite. Used in cultivation of lettuce, strawberry and green vegetables

7 8 9

Sand Wood fibre Sheep wool

Rock Wood Sheep

10

Rock wool

Molten rock (Basalt)

11 12

Vermiculite Gravel

Volcanic rock Rock

Cheap and easily available. Needs sterilization before use. Water holding capacity is low. Made by steam friction of wood. Very effective organic substrate. Long durability It has great air capacity but it decreases with its use. Gives great product yield. Acts like a biostimulator. Not suitable for tomato cultivation Most widely used in hydroponics cultivation. Inert substrate for run-to-waste. Used typically for seedling stages but can remain at plant base for lifetime. It is non-hazardous, non-carcinogenic material. Naturally, it is in high pH form, to grow the plant it should be neutralized. The rock wool can be modified to hold more water and air. It contains 80% nutrient solutions, 15% air pore space and 5 % fibre Holds more water than perlite. It has natural wicking properties Same like present in aquarium. It is easy to clean, inexpensive, will not become waterlogged and it drains well. However, if continuous water is not supplied to plants, the roots may dry out. Terrestrial plants grow well in gravel medium

Aydogan and Montoya (2011) Choi et al. (2014), Roberto (2003)

Hydroponic Cultivation Approaches

TABLE 5.1 Details of Different Types of Substrates Used in Hydroponics Cultivations

Kumar et al. (2013) Ors and Anapali (2010), Asaduzzaman et al. (2013) Savvas et al. (2006), Marinou et al. (2013) Roberto (2003) Muro et al. (2004) Böhme et al. (2005), Dannehl et al. (2015) Van and George (2004)

Van and George (2004) Jain et al. (2019)

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5.4  NUTRIENT SOLUTIONS The most important thing required in hydroponics is nutrient solutions. The commercial nutrient solutions are easily available in gardening shops. The nutrient solution is the blend of various macro and micro elements or organic materials.

5.4.1 Inorganic Hydroponic Solutions The most important component in hydroponic cultivation is hydroponic solutions which provide important nutrients to plants that are sometimes not present in the soil. Hydroponic solutions do not have the cation-exchange capacity (CEC) so the pH in the hydroponic solution varies. Routine adjustments of pH, use of chelating agents and buffering the solution are often necessary. An important inorganic element used in hydroponics cultivation is explained in Table 5.2 with its roles in plant growth.

5.4.2 Organic Hydroponics Solutions For those who do not want to use inorganic compounds, organic fertilizer is the best supplement available. But there are many problems when using organic fertilizers that are very difficult to remove easily. For example, the nutrients in organic fertilizers are highly variables even they obtained from a similar source of the material. Organic fertilizers are sometimes made from animal waste. The use of animals for organic fertilizers can be dangerous, because of animal diseases that can be transferred to humans and plants. Organic fertilizers can sometimes coagulate; hence, the flow of nutrient solution may be disrupted. Organic fertilizers can degrade easily and gives a foul odour. Nevertheless, if we work carefully, we can use organic fertilizers successfully in hydroponic cultivation (Shinohara et al., 2011). There are various organically sourced materials used in hydroponic cultivation such as blood meal, bone ashes, bone meal, hoof/horn meal, fishmeal, wool waste, wood ashes, cottonseed ashes, grasshopper, leather waste, kelp meal, liquid seaweed, different types of manure (poultry, sheep, goat, horse, cow), guano (bat and birds) etc. These organic materials are rich in essential elements like nitrogen, phosphorus, potassium, calcium, magnesium and sulphur (Brooke, 2000; Snow et al., 2008). Micronutrients can be sourced from organic fertilizers such as composted pine bark which is highly rich in manganese and can be used in hydroponic cultivations (Larsen, 1982). To satisfy the plant nutrients in hydroponic cultivation gypsum, calcite, glauconite etc. can also be added. In addition to increase the nutrient uptakes of plants, humic acid and chelating agents can be added in hydroponic solutions (Lua and Böhme, 2001). Macro elements play an important role in plant nutrition, but a high amount of microelements may not always increase the plant yield. In hydroponics, increasing or decreasing the concentration of nitrogen in nutrient solutions will not affect the plant growth (Nchu et al., 2018).

5.5 HYDROPONIC APPROACHES FOR SECONDARY METABOLITE PRODUCTION The production of secondary metabolites in plants can be stimulated by various agents like precursor molecules, elicitors, environmental factors, genetic transformation etc. By the traditional way, it is difficult to obtain secondary metabolites yield in a large amount. To increase the yield of secondary metabolites, new approaches like hydroponics cultivation can be used. Hydroponics cultivations techniques are best for studying whole plant physiology and nutritional analysis. Static solution culture is a hydroponic method in which plants are artificially grown in water with nutrient solution. In this method, water/nutrient solution is stagnate and does not flow like other hydroponics cultivation methods (Figure 5.1). The plants can be grown in tanks, plastic buckets, mason jars etc. but most preferably plastic tanks are used in static solution culture. There are two

Type

Element

Ionic Forms

Range (ppm)

Role in Plants

References

Macro-nutrient

Nitrogen

NO−3 or NH+4

100–1000

Role in plant metabolism and essential for amino acid production. Combined use of urea and nitrate is effective for adequate plant growth

Potassium

K+

100–400

Phosphorus

PO3−4

30–100

Calcium

Ca2+

200–500

Magnesium

Mg2+

50–100

Sulphur

SO2−4

50–1000

Iron

Fe3+ and Fe2+

2–5

Zinc

Zn2+

0.05–1

Copper

Cu2+

0.01–1

Role in enzyme activation, stomatal activity, photosynthesis, transport of sugar, water and nutrients Energy unit in plants. Stimulate root development. Required for early stages of development, crop maturity and to resist the disease Required for root, fruit and seed formation. Role in cell wall, cell membrane, chromosomes and enzymes activation Important role in chlorophyll pigments and protein synthesis. Secondary metabolite production is affected by Mg2+ Role in production of protein, vitamins, chlorophyll, glucoside oils and in cell walls Essential for chlorophyll synthesis and chloroplast formation. Main role in photosynthesis, respiration, DNA synthesis and hormone synthesis Role in carbohydrate metabolism, protein synthesis, tryptophan synthesis, auxin and membrane action Cofactor for many enzymes and proteins. Role in signalling of transcription and protein trafficking machinery Helps to control plant diseases. Uptake is enhanced by phosphorus Essential for structural integrity of plant membrane Beneficial effect on development of nodule. Important role in nitrate assimilation, sulphite detoxification, synthesis of auxin and purine degradation Essential widely in legumes and grain crops. Used in enzyme urease Essential for plants like pea, maize, sunflowers and cereals. Sometimes, it is toxic to plants Essential in stress conditions. Role in plant metabolism and increase the yield of fibre crops It stimulates chlorophyll content, enzyme activities and uptake of major and minor nutrients

Jones (2016), Ikeda and Tan (1998), UrbanczykWochniak and Fernie (2005) Prajapati and Modi (2012)

Micro-nutrient

Manganese Mn2+ Boron B(OH)–4 Molybdenum MoO–4

0.5–1 0.3–10 0.001–0.05

Nickel Aluminium

Ni2+ Al3+

0.057–1.5 0–10

Silicon

SiO2–3

0–140

Titanium

Ti3+

0–5

Richardson (2001) Jones and Lunt (1967)

Hydroponic Cultivation Approaches

TABLE 5.2 Important Elements Used in Hydroponics Nutrient Solutions with Its Role in Plants

Guo et al. (2016) Scott (1985) Rout and Sahoo (2015) Mousavi (2011) Yruela (2005) Huber and Wilhelm (1988) Pilbeam and Kirkby (1983) Bobko and Savvina (1940), Mendel (2011) Jones (2016) Rout et al. (2001), Douglas (1985) Luyckx et al. (2017) Dumon and Ernst (1988)

75

76

Biotechnological Approaches to Enhance Plant Secondary Metabolites

FIGURE 5.1  Static solution culture.

types of media used in static solution culture; one in which water or nutrient solution is poured/ added into the container as required once in a week or a certain period of time and in second type the solution is added only when it falls below the given specific threshold. To check the concentration of solution, electric conductivity meter can be used. The nutrient solution used in this method should be aerated but sometimes unaerated solutions can be used. The main principle of aeration in the solution is to get enough amount of oxygen to breathe for roots. The aeration can be done by using an aquarium pump, aquarium airline tubing and valves. If the roots are unaerated then the solution in the container must be present in a low amount, which is enough to get oxygen to roots present above the solution. In a single container many plants can grow, the container should be large enough according to the growth of the plant. The downside of this method is that the water/nutrient solution does not flow which causes algae to grow more quickly in the container. Therefore, it is necessary to change the water/nutrient solution every week to avoid algal growth. In the method continuous-flow solution culture, the solution continuously flows through the roots of plants, unlike the solution stagnant in static solution culture (Figure 5.2). This technique is easier than static solution culture as we can automatically adjust pH, temperature and nutrient concentration in the flowing medium. One of the important variations in this method is nutrient film

FIGURE 5.2  Continuous-flow solution culture.

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Hydroponic Cultivation Approaches

techniques (NFT). In this method, very shallow stream water contains all the required nutrients for plants to recirculate through the roots of plants with proper aeration (Wheeler et al., 1990). A well designed NFT provides proper length and slopes for the parallel flow of solution (Spensley et al., 1978). Whichever method in hydroponic cultivation is considered best method when it provides abundant water, oxygen and nutrients for the growth of plants; in this case, NFT is considered as the best method in hydroponic cultivations. The disadvantage of this method is that it has very low buffering against disturbance in the flow, pump failure and disease spread (Burrage, 1992). By using NFT technique, researchers are able to screen tomato genotypes for resistance or tolerance to phosphorus deficiency (Silva et al., 2012). Various vegetable plants are now commercially cultivating all around the world. These vegetables are easy source of various secondary metabolites which can be produced by hydroponics technique all year around (Table 5.3). In aeroponics method, the plant roots get nutrients from surrounding water droplets present in air (Weathers and Zobel, 1992). Although in this method plants get nutrients from air/mist, it is still considered as one of the types of hydroponics because it uses liquid media to transmit the nutrient. In this method, the hanging roots of the plant are sprayed with nutrient-supplemented water in a closed or semi-closed environment (Figure 5.3). The leaves and stalks are preferably on the outside (Hayden, 2006). Aeroponics technology is famous for growing disease and pest-free plants. However, sometimes the atmosphere of aeroponics is not completely closed or there is a risk of disease and pests growing in it. If plants get diseased, it is easy to remove from support structure without infecting or disrupting other plants (Buckseth et al., 2016). The biggest advantage of the aeroponics method is that water as well as air helps to grow plant roots. Oxygen is very essential in the rhizosphere of roots for healthy plant growth. As aeroponics provides oxygen, water and nutrients to roots, it gives optimal growth of the plant (Burgess et al., 1998; Soffer and Burger, 1988). Aeroponics not only provides oxygen to plants but also the carbon dioxide TABLE 5.3 Important Secondary Metabolites from Hydroponically Grown Plants Sr. No.

Name of Plant Important Secondary Metabolites

1

Broccoli

2

Bell Pepper

3

Cabbage

4

Carrots

5 6

Celery Lettuce

7

Parsley

8

Pea

9

Potatoes

10

Spinach

11

Tomatoes

α-Carotene, β-Carotene, Glucoraphanin, Isorhamnetin, Phylloquinone, Quercetin, Rutin, Sinapic acid, Tocopherol Capsorubin, Kaempherol, Luteolin, Myricetin, Rutin, Tocopherol, β-Carotene, β-Cryptoxanthin Caffeic acid, Ferulic acid, p-Coumaric acid, Quercetin, trans-Cinnamic acid, Vanillic acid Caffeic acid, Chlorogenic acid, Cyanidin, Malvidin, Peonidin, Protocatechuic acid, α-Carotene, β-Carotene Apigenin, Apiin, Chrysoeriol, Luteolin, Quercetin Apigenin, Caffeic acid, Caftaric acid, Chicoric acid, Chlorogenic acid, Kaempferol, Myricetin, Phylloquinone, Quercetin, β–Carotene Apigenin, Carotenoids, Chrysoeriol, Isorhamnetin, Luteolin, Neoxanthin, Quercetin, Violaxanthin, β -Carotene, Caffeic acid, Catechin, Coumaric acids, Ferulic, Protocatechuic acid Caffeic acid, Chlorogenic acid, Ferulic acid, Gallic acid, Lectins, p-Coumaric acid, α-Chaconine, α-Solanine Carotenoids, Lutein, Myricetin, Secoisolariciresinol, Zeaxanthin, α-Tocopherol, β-Carotene Chlorogenic acid, Dehydrotomatine, Lycopene, Quercetin, Secoisolariciresinol, α-Carotene, α-Tocopherol, α-Tomatine

References Vasanthi et al. (2009) Sinisgalli et al. (2020) Koss-Mikołajczyk et al. (2019) Fernández et al. (2020) Salehi et al. (2019) Malejane et al. (2018)

Farzaei et al. (2013) Dueñas et al. (2004) Friedman (2006), Fernández et al. (2020) Hedges and Lister (2007), Fernández et al. (2020) Marcolongo et al. (2020)

78

Biotechnological Approaches to Enhance Plant Secondary Metabolites

FIGURE 5.3  Aeroponics.

which is essential for photosynthesis (Nichols et al., 2001). In plants, disease can be transmitted through solid or liquid media but in aeroponics techniques chances of diseases are very rare because nutrient transformation to plant occur through the air. Due to the advantage of disease-free plant cultivation in aeroponics, many plants can grow in high density when compared to other hydroponics cultivation. Therefore, various researchers prefer this technique for preliminary screening of genotypes (Loresto et al., 1983). Du Toit et al. (1997) used the technique aeroponics for screening of maize genotypes for resistance to Fusarium graminearum seedling blight/root rot. The hydroponics technique was also used in selection of recombinant lines of high root growth variety of Oryza sativa L. (Price et al., 1997). Withaferin A is an important secondary metabolite present in many plants but mostly obtained from Acnistus arborescens and Withania somnifera. Von et al. (2014) compared the Withaferin A production in hydroponics and aeroponics. They suggested that plants grown in hydroponics produce (7.8 mg/g) more Withaferin A compare to aeroponics (5.8 mg/g) and demonstrate that hydroponics is efficient for generating Withaferin A. Fogponics is similar to aeroponics, in fogponics the nutrient solutions are transferred to plant by vaporized form (Figure 5.4). In this method, the droplet size is 5–10 µm which is much smaller than aeroponics type. Small drops help to diffuse quickly from the air and deliver nutrients to roots (Nanda, 2018). In the movement of water, there are two phases, i.e. Ebb and Flow. This Ebb and flow principles are used in hydroponics. The Ebb method is also known as the flow (flood and drain) sub-irrigation method. It is one of the simplest forms of hydroponic cultivation method where the pots placed on

FIGURE 5.4  Fogponics.

Hydroponic Cultivation Approaches

79

FIGURE 5.5  Ebb.

a tray are containing nutrient solution above the reservoir (Figure 5.5). In this method, either the tray is filled with clay granules (growing medium) or places the plants over the medium. The timer plays an important role in this method. Timer gets switched on when there is a low medium/nutrient solution on a tray and excess nutrient flow back to the reservoir. This keeps the medium to regularly flow or flush with air and nutrients (Elmer et al., 2012). The run-to-waste method is also sometimes called ‘The Bengal system’ because it was first invented in West Bengal in 1946 (Douglas, 1951). In this method, nutrient solution and water are periodically applied to the surface of the medium (Figure 5.6). There are two subtypes of this method, i.e. simple method and complex method. In the simplest form of a method, everyday nutrient solutions and water are manually applied to the container of inert growing media (rock wool, perlite, vermiculite, chips, coco fibre, sand, etc.). In a complex system, the pump delivers the water and nutrient solution to media. The plant parameters such as plant size, surrounding climate, temperature, pH, water content, etc. were measured by computers or programmable logic controller. Commercially, various vegetables like tomatoes, pepper,

FIGURE 5.6  The Bengal system.

80

Biotechnological Approaches to Enhance Plant Secondary Metabolites

TABLE 5.4 Advantages and Disadvantages of Hydroponics Advantage 1. No need of fertile soil; can grow anywhere 2. Use less water compared to conventional farming. Water can also be reused 3. No environmental damage and no soil pollution 4. Crop grows faster than soil-based crops 5. As the environment conditions are controlled, the crops can produce higher yield compared to traditional farming 6. Artificial stress conditions can be made for the production for high amount of secondary metabolites 7. Harvesting is easier

Disadvantage 1. Initial investment and maintenance cost is high 2. Chances of waterborne diseases 3. Pure water is required 4. Requires continuous power supply for maintaining various instruments 5. Requires a lot of instruments

cucumber etc. are produced by run-to-waste hydroponic technique. The waste that comes out of the tray is nutrient-rich which can be filtered and reused. This makes the system very productive. The hydroponic system costs more than traditional systems because more instruments are being used, i.e. plastic trays, nutrient mediums, stands, pumps, temperature controlling system, more use of electricity, etc. The advantages and disadvantages of hydroponics are shown in Table 5.4. In the deep water culture method, plants are suspended on nutrient-rich oxygenated water. In traditional methods, large containers or plastic buckets containing plants were suspended on the top of the solution. The solution is saturated with oxygen by an airstone. By using this method, the plant gets more oxygen at roots and ultimately the yield of plants gets increased (Saaid et al., 2013). Another method which is similar to deep water culture is Kratky method. The Kratky method uses the non-circulating water reservoir (Kratky, 2008). In deep water culture, the roots of the plants are hanging at the top and for watering they have to grow to reach the solution first, and it takes some time for the plant roots to reach the solution; however, in top-fed deep water culture, a highly oxygenated nutrient solution is pumped straight to the plant root zone (Figure 5.7). The water or nutrient solution is pumped directly to the roots zone and the remaining unabsorbed water returns to the reservoir due to gravitational force. In this method, an airstone is placed in the reservoir which releases air into the water which increases the

FIGURE 5.7  Top-fed deep water culture.

Hydroponic Cultivation Approaches

81

oxygen content of the water (Ernst and Busby 2009; Yoshiga and Umezaki, 2016). The airstone and the pump are running 24 hours a day to ensure that the plants get continuous nutrients and oxygen. The biggest advantage of the top-fed-deep method is that it provides water and nutrient solution to the plants from day one. This makes the growth of plants very fast and quick. In the rotary hydroponic system, plants are grown in a circular frame that rotates continuously for some time until the entire plants grow. Also there is a lot of variation in this method in which the rotation period of the circular frame is different. In particular, the circular frame rotates once an hour, allowing plants to rotate 24 times a day. The high intensity grow light is placed in the centre of the circular frame which mimics the sunlight and helps the plants for photosynthesis. Such circular rotations cause plants to fight with gravitational forces, which accelerate their growth. In this method, the circular frame causes the plants to grow more per square foot of space compared to other hydroponic cultivation methods. The plants get water because circular rotation is not available continuously but at a certain time. Passive hydroponics is also known as passive sub-irrigation, semi-hydroponics or hydroculture and the Kratky method. In passive hydroponics (Figure 5.8) method, plants are grown on inner porous medium that transports water/nutrient to roots of the plant by capillary action (Yıldız, 2008). It is considered as one of the simplest method, the pot containing plant can be placed on the shallow water of fertilizers or water or nutrient solution. The media used in this method have the capability of capillary action, provide adequate space and won’t decompose in the solution. Coconut husk and expanded clay are two important media used in this method. Besides these two rock wool, perlite, vermiculite, chips and diatomite can also be used. These media gives increased oxygen to roots, which is important for plants like orchids and bromeliads. Another importance of this method is additional humidity provided to plants for the reduction of root rot. This method is not productive compared to other method (Sheikh, 2006). Rosmarinic acid can be obtained by hydroponically cultivated basil plants (Kiferle et al., 2011). The study showed the highest rosmarinic acid content found in roots of hydroponically gowned basil plants compared to in vitro grown plants. The authors also suggested the possibility to scale up rosmarinic acid production by hydroponic techniques. Owolabi et al. (2018) reported that hydroponics solutions can be used for spraying biotic and abiotic elicitors on plants for enhancing the secondary metabolite productions. Zhou et al. (2020) established and optimized the hydroponics system for root morphological and nutritional analysis of citrus. They showed the customized hydroponics technique which is useful for low-cost production and to do various scientific experiments on the citrus plant. Apetalic acid and Calanolide B are some important secondary metabolites present in Calophyllum brasiliense and used in the treatment of Mycobacterium tuberculosis. Castillo-Arellano

FIGURE 5.8  Passive hydroponics.

82

Biotechnological Approaches to Enhance Plant Secondary Metabolites

et al. (2019) studied this plant under calcium and potassium-rich nutrient media. Their study showed that in hydroponics cultivations, calcium is essential for the survival of Calophyllum brasiliense and potassium plays a major role in secondary metabolites production. Perilla frutescens are rich in flavonoids most importantly Apigenin (AG) and Luteolin (LT). These two flavonoids are the most potent anticarcinogenic agents. Lu et al. (2018) experimented on Perilla frutescens by using three different types of nutrient solutions (1.0, 2.0 and 3.0 dS m−1) and light intensity (100, 200 and 300 μmol m−2s−1) in hydroponics cultivations. Their results showed different treatments gives different product yield and suggested that electric conductivity and light intensity are important for the production of flavonoids in Perilla frutescens. A similar experiment on Anoectochilus formosanus was performed by Nguyen et al. (2018) using four different nutrient solutions. The results showed that a high level of alkaloid and terpenoids were obtained on Hydro Green media whereas the highest flavonoids were observed in Nitrophoska Foliar media. Another researcher Simeunovic (2002) studied the three different nutrient solutions varying in the macro elements and electric conductivity on Achillea millefolium, Tanacetum parthenium, Leonurus sibiricus and Linum usitatissimum. Giorgi et al. (2009) suggested that Nitrogen starvation in nutrient media significantly increased the content of phenolic acids and phenylalanine ammonia lyase activity in plant leaf and roots. Gontier et al. (2002) demonstrated that secondary metabolites of Datura innoxia and Taxus baccata plants can be ‘milked’ by using hydroponics techniques. Especially hyoscyamine and scopolamine alkaloids present in Datura innoxia were obtained in large amounts from the static solution method (Vu et al., 2006). The use of Agrobacterium rhizogenes in hydroponics nutrient solutions showed the enhanced production of alkaloids in Datura innoxia (Vu et al., 2018). The use of Trichoderma viride in hydroponics showed the enhanced synthesis of plant secondary metabolites (Jurić et al., 2020). Medicinal and aromatic plants have various antibacterial and antifungal activities. These plants are rich in secondary metabolites which can be used to treat various diseases. Various plants (Pelargonium roseum, Cymbopogon citrates, Ocimum gratissimum, Chrysopogon zizanioides, Nepeta transcaucasica, Echinacea angustifolia and Ocimum basilicum, Centella asiatica, Echinacea angustifolia, Valeriana officinalis) have been studied by soilless cultivation for secondary metabolites production (Mairapetyan, 1999; Dayani and Sabzalian, 2006; Maggini et al., 2011). Hydroponic techniques can be used to produce high standard plant material all year round and to enhance secondary metabolites by appropriate manipulation of nutrient solutions. Some new approaches like encapsulated biological agents (calcium and copper ions) can be used to study the secondary metabolite productions not only in plants but also in some fungus. Saline soil can impediments the growth and yield of crops. Hydroponics is considered to be trustworthy and feasible method for screening for salinity tolerance crop like Cotton. Malhotra et al. (2014) did the experiment for the screening of cotton genotype for salinity tolerance and proved that wooden tray and plastic containers are simple, cheap, reusable and practical for hydroponic screening of cotton genotypes.

5.6 CONCLUSIONS Today, most of the cost of agriculture products goes to transportation, which is also consuming high fuel cost and increasing the air pollution. Due to these factors, global warming is on the rise. If the plants can be grown in our own city, it will reduce the amount of greenhouse gases. There have been a lot of efforts taken in the last century to increase agricultural productivity, which is also helping the enormously growing population of the world. However, at the same time, largescale deforestation for agriculture, an enormous amount of water use, soil fertility, pollution in soil, water, air and much of the climate changes are decreasing the agricultural production. Out of all these problems, new technology has emerged which uses less space and less water and gives more production, i.e. hydroponics cultivation which is feasible for commercial production. However, only

Hydroponic Cultivation Approaches

83

a few plants have been studied for the production of secondary metabolites using hydroponics techniques and it shows that the present hydroponics system may require innovative modifications for enhanced secondary metabolite synthesis in plants. This can be achieved by cultivating the plants by various hydroponic techniques. Various environmental factors influence the plant secondary metabolites yield, we can control these conditions in hydroponics and enhance the productivity. This will certainly attract researchers to develop new techniques in hydroponics cultivation for the production of secondary metabolites in a large scale. It will help to capture the global market which is presently captured by East Asian countries. The major concern of hydroponic technique is its cost. The hydroponics takes more than 10 times higher cost than the traditional one. Although the commercial production of hydroponics is in initial phase, but in near time the cost require for hydroponics will definitely get reduced. The secondary metabolites from plants can be produced by conventional, immobilization, in vitro tissue, organ/ cell culture and hydroponics methods. All methods have its pros and cons. From all the methods, hydroponics is emerged as a new, eco-friendly, hygienic and commercial method for the production of secondary metabolites. Currently, various plants have not been tested for production of secondary metabolite by hydroponics. There is an urgent need to set up the standard operating procedures for the commercial cultivation of all the medicinal plants which are rich in secondary metabolites. Contact Information: Please contact the author at: [email protected]

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Tissue Culture Approaches to Enhance Plant Secondary Metabolites Production Vishal N. Patil and Mohd. Shahnawaz

CONTENTS 6.1 Introduction............................................................................................................................. 89 6.2 In Vitro Culture as a Substitute System for Enhancement of Plant Secondary Metabolites ....90 6.3 Approaches to Manipulate the Contents of Plant Secondary Metabolites..............................90 6.3.1 Callus Culture System.................................................................................................92 6.3.2 Cell Suspension Cultures.............................................................................................92 6.3.3 Elicitation: A Biotechnological Tool...........................................................................92 6.3.4 Organ Culture.............................................................................................................. 93 6.4 Scale-up Production................................................................................................................. 93 6.5 Conclusions and Future Prospective........................................................................................ 93 Acknowledgements...........................................................................................................................94 References.........................................................................................................................................94

6.1 INTRODUCTION Presently, plant-based medicines are the preferred choice of patients due to least or no side effect (Dar et al. 2017). Plants synthesize thousands of structurally complex molecules (Narayani and Srivastava 2017) to meet their basic needs viz. primary growth and development (primary metabolites) and to interact with the changing environment to defend themselves against varied stress, predation and diseases (secondary metabolites) (Erb and Kliebenstein 2020). Plant produces such secondary metabolites to enable them to grow and withstand in the challenging and diverse habitat (Taiz et al. 2015). The first conceptual definition of the plant secondary metabolite was given a famous Nobel Laurate, Albrecht Kossel (Jones 1953). Later, another team of scientists lead by Czapek elaborated the concept of secondary metabolites as an end-products obtained from the nitrogen metabolism by secondary modification viz. deamination (Bourgaud et al. 2001). Based on the chemical structure, plant secondary metabolites were grouped into various classes viz. phenolics, alkaloids, saponins, terpenes, lipids and carbohydrates (Hussein and El-Anssary 2019). These secondary metabolites have medicinal value and plants are used in various systems since ancient times. The plants synthesize these drug molecules (plant secondary metabolites) at low concentration in different parts of the plant viz. root, stem and leaves. Due to least or no side effects of plant-based medicines, people preferred plant-based drugs (Dar et al. 2017). Efforts were intended for the extraction, structure elucidation and evaluation of various plant secondary metabolites. About 26–28% of topical medicines are obtained from the plants (Samuelsson 2004) and most of the anticancer drugs are obtained from the plants (Moraes et al. 2017). The plants are the foremost resource for several important bioactive molecules (Dias et al. 2012, Zaheer et al. 2016). So, to meet the increasing demands of pharmaceutical industries, for manufacturing medicines, a huge quantity of the plants is being exploited from the nature, which leads to threaten to the existence of such important medicinal plants. So, it was

DOI: 10.1201/9781003034957-6

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needed to develop such a technique to increase the contents of these key phyto-constituents of the plants to save the plant germplasm. Among the various biotechnological methods reported in literature, the in vitro culture methods are considered as one of the environmentally effective substitute method for the enhancement of plant secondary metabolites (Kolewe et al. 2008, Khani et al. 2012). So, in the present chapter, an attempt was made to discuss the role of plant tissue culture to enhance the contents of the plant secondary metabolites under the following lines: (i) to highlight the in vitro culture as a substitute system for enhancement of secondary metabolites, (ii) to enlist various plant tissue culture system for enhanced production and of the plant secondary metabolites, followed by scale-up production of key drug molecules and (iii) to suggest future prospects of research in tissue culture to enhance the contents of plant secondary metabolites.

6.2  IN VITRO CULTURE AS A SUBSTITUTE SYSTEM FOR ENHANCEMENT OF PLANT SECONDARY METABOLITES An international market has demand about importance plant-based drug molecules for more environmentally suitable as well as economically viable methods for the manufacturing of drugs for steady production of high-quality products. Due to complex constitution and specific stereo chemical requirement of compounds, most of the time, the production of drugs of many plant-derived compounds is very expensive (Rates 2001, Oksman-Caldentey and Inze 2004). The synthetic drug may act as irritants and toxins in the body and it destroys the balance of the whole body (Badisa et al. 2009). Since time immemorial, a diverse plant species are supplying traditional resources for the raw materials for the drugs. As per an estimate, around 28,187 plant species are reported to have medicinal value globally (Allkin 2017). As per report among the 5000 plant species, only 1100 species are widely utilized in Ayurvedic (80%), Unani (46%) and Allopathic medicines (33%) (Gouri 1996). The most of the factors are responsible to decrease the quality of plant material such as infestation, diseases and application of pesticides. In various cases, high-value compounds present in the plants are very difficult to extract and due to over-harvesting, the plant becomes endangered. Greenhouse farming is allied with labour cost, high energy and inadequate availability of area for cultivation. However, by using the plant cell cultures, the developing commercial awareness in innate products resulted in the possible manipulation of plant secondary metabolites to enhance its contents (Jeong and Park 2006). As an alternate and complementary method, instead of the entire plant, only cell and tissue cultures of a plants lead to enhance the contents of plant secondary metabolites in various plants viz. Cruciata glabra, Angelica dahurica (imperatorin), Taxus mairei (taxol), Salvia miltiorrhiza (cryptotanshinone), from Gentiana (gentipicroside and swertiamarin), from Dioscorea doryophora (diosgenin) etc. (Dicosmo and Misawa 1995; Dornenburg and Knorr 1955; Bourgaud et al. 2001; Mulabagal and Hsin-Sheng 2004). As the plant cell is biosynthetically totipotent and hence, they are capable of generating whole range of chemicals compounds which is present in the parent plant (Rao and Ravishankar 2002). The secondary metabolites production provides more advantage by a plant cell cultures such as (i) self-regulating of seasonal disparity, environment, soil circumstances, (ii) continuous and reliable contribution of high quality yield which ensures efficient production system, (iii) through the physical and chemical parameters optimization of manufactured secondary metabolites and (iv) the accumulation of desired compounds through genetic modification. The manufacture of original compounds which is not present in parent plant/parts was observed in case of plant cell cultures by Luczkiewicz and Kokotkiewicz (2005).

6.3 APPROACHES TO MANIPULATE THE CONTENTS OF PLANT SECONDARY METABOLITES For commercial synthesis of high-value potential drug molecules (plant secondary metabolites), the most efficient biotechnological approach must be utilized (Hussain et al. 2012). Murthy et al. (2014) suggested, plants having higher contents of the secondary metabolites are considered as the primary choice to select explant to initiate cell or organ cultures.

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The potential yields of the in vitro cultures are not always sufficient unless the researchers select the good quality plant as a source of ex-plant. It was also reported that after various cycles of cultivation, the yield of the accumulated secondary metabolites decreases. So, various strategies are utilized together viz. conventional mutant lines as a source of explant and enhancement of targeted drug molecules in that efficient mutant line using metabolic pathway engineering technique (Khani et al. 2012; Isah et al. 2018). Cell suspension and callus cultures are reported to enhance various plant secondary metabolites. The production of pigments, flavones, phytoalexins and other defence-related compounds are increased when the higher plant cells triggered with the any other compounds called as elicitors (Savitha et al. 2006). The biotic elicitors mostly of the fungal origin are extensively employed to enhance the plant secondary metabolite contents in plant cell cultures. This approach has been efficient in enhancing the contents of the various plant secondary metabolites viz. coumarin derivatives (Conrath et al. 1989), alkaloids (Godoy-Hernandez et al. 2000), flavonoids (Tamari et al. 1995), phenylethanoid glycosides (Lu and Mei 2003), sesquiterpenes (Ma 2008), terpenoids (Chakraborty and Chattopadhya 2008) and carotenoids (Wang et al. 2006). Barz et al. (1990) reported the accumulation of plant secondary metabolite as a result of active equilibrium in the series of synthesis, transport, storage and degradation in the plants. Hence, the plant cell culture is considered as a flexible structure for the manipulation of yield production. Optimization of the in vitro techniques to enhance the contents of the plant secondary metabolites is accomplished by some of the key strategies (i) to select the elite clones as a source of explant, (ii) to exploit of medium and culture conditions for achieving significant results and (iii) to select the appropriate elicitor or precursor for the enhancement of the desired metabolite (Dornenburg and Knorr 1955). The illustration of in vitro culture strategy utilized for the enhancement of plants secondary metabolites is given in Figure 6.1

FIGURE 6.1  Graphic representation of in vitro culture strategy to generate molecules of interest. (Adapted from Dornenburg and Knorr 1955.)

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6.3.1 Callus Culture System The callus is induced in a plant culture growing on a media due to different stress stimuli of various factors, especially growth regulating hormones viz. auxins and cytokinins (Ikeuchi et al. 2013). Two distinct types of calli are reported in plant culture viz embryogenic or non-embryogenic (Nandhakumar et al. 2018). Embryonic calli have the potential to regenerate a new complete plant through somatic embryogenesis due to the presence of differentiated embryogenically competent cells (Ptak 2013). However, non-embryogenic callus cultures are used for the enhanced production of plant secondary metabolites due to the presence of homogenous clumps of dedifferentiated cells and were reportedly used for flavonoids production (Filova 2014). In literature, several researchers (Ikeuchi et al. 2013; Ali et al. 2016; Murthy et al. 2014; Efferth 2019) got success to report the production of plant secondary metabolites using callus culture system. Besides, callus culture, various other culture system viz. cell suspension cultures and organ culture are also used for the production of plant secondary metabolites at in vitro level (Goncalves and Romano 2018).

6.3.2 Cell Suspension Cultures After callus cultures, cell suspension cultures are reported as a promising tool for the enhancement of potential plant secondary metabolites (Marchev et al. 2014, Yue et al. 2016, Chung et al. 2018). Rajendran et al. (1992) defined about the elicitors as the plants are producing the secondary metabolites only when they are treated by the compound of pathogenic origin and supplementary chemical stress viz. salt, heavy metals etc. These elicitors are efficient in enhancing the assembly of the various plant secondary metabolites (Zhao et al. 2005, Ferrari 2010, Alvarado et al. 2019). In the plant part, the enzymatic oxidization of phenolic compounds consequences in the production of brown substances (Elstner 1987). Aquino-Bolanos et al. (2000) concluded about the cell browning as the phenolic substances and reported increased activity of the phenylalanine ammonia-lyase (PAL) enzymes. Fukuoka and Enomoto (2001) understood the key factors of biosynthetic pathway of phenylpropanoids as PAL and polyphenol oxidase (PPO). Vamos-Vigyazo (1981) studied in the plant tissues as the browning provoked by mechanical damage by the phenolic compounds.

6.3.3 Elicitation: A Biotechnological Tool Chemical substances from varied sources (biotic/abiotic), which enable plant cells to activate the stress response mechanism to increase the accumulation of the molecules in defence, are referred as elicitors (Naik and Al-Khayri 2016). Elicitation is referred to as one of the preferred biotechnological study to increase the contents of the secondary metabolites in plants at in vitro level (Halder et al. 2019). Various researchers have reported the synthesis of secondary metabolites in the plant in response to various induced biotic and abiotic stresses viz. defence response of plants against herbivory (War et al. 2012, Gish et al. 2016, Erb 2018), defence response of plants against pathogens (AbuQamar et al. 2017, Zaynab et al. 2018), defence response of plants against heavy metals (Emamverdian et al. 2015, Ghori et al. 2019), antioxidant defence responses in plants caused due to salinity stress (Stepien and Klobus 2005, Parihar et al. 2015, Polash et al. 2019). Zhao et al. (2005) studied about the control of cellular activities at the molecular and biochemical level by elicitors. The improved yields of secondary metabolites were also reported due to usage of nutrient and precursor feeding (Thimmaraju et al. 2005). Saw et al. (2012) studied and demonstrated that, the combination of various elicitors with physical factors like UV light, temperature regime and pulsed electric field capitulate superior outcome for the production of secondary metabolite. The biotic or abiotic elicitors showed that they encompassed the signalling molecules viz. salicylic acid, methyl jasmonate, microbial cell wall extracts, physical agents (UV radiation) inorganic salts, heavy metals etc. (Vacheron et al. 2013, Farag et al. 2017). A report suggested, by utilizing the

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pre-existing enzyme arrangement, the plant cell cultures are employed to convert precursors into products (Murthy et al. 2014). Cell suspension culture is considered as one of the most useful culture method for the treatment of elicitation and production of secondary metabolites (Zaheer et al. 2016). Pourianezhad et al. (2019) documented the significant production of parthenolide in feverfew using cell suspension technique. Recently, Al-Khayri and Naik (2020) reported enhanced production of various Pharmaceutical molecules viz. catechin, caffeic acid and kaempferol in date palm using pectin, yeast extract (YE), salicylic acid (SA), cadmium chloride (CdCl2) and silver nitrate (AgNO3) as elicitors in its cell suspension culture.

6.3.4 Organ Culture Various potential drug molecules, e.g. plumbagin, forskohlin, tropane alkaloid hyoscyamine and scopolamine etc. were obtained using root culture technique (Li et al. 2002, Pence 2011, Pandey et al. 2014, Roy and Bharadvaja 2018). Due to the slow growth of roots in higher plant, it is significantly difficult to harvest the drug molecules compared to other undifferentiated cells of the plant, so an alternative methods were discovered and highly accepted method is reported as hairy root culture (Pence 2011). Besides, root cultures, various people have also investigated the shoot cultures or induction of somaclonal variation at in vitro level to screen the high yielding plant secondary metabolite producing clones (Suman 2017). Besides, the advantages of various drawbacks of the organ culture are also reported in the literature, e.g. large scale cultures and high cost of the bioreactors used for commercial production of molecules of interest (Filova 2014), so such method is not economically viable. As per reports, only few plant secondary metabolites viz. berberine, ginsenosides, shikonin, scopolamine and rosmarinic acid are produced successfully at commercial level (Goncalves and Romano 2018).

6.4  SCALE-UP PRODUCTION Murthy et al. (2014) stated that in vitro synthesis of secondary metabolites is somewhat difficult method, due to various drawbacks in the plant tissues by having comparatively unsteady efficiency, with high trim sensitivity, slow growth and low oxygen requirement. In early 1940s, research on in vitro cultures was initiated on the assembly of secondary metabolites generations. Afterward, various biotechnological innovations result in in vitro synthesis of secondary metabolites production. For the assembly and extraction of secondary metabolite synthesis, the method of plant part culture is being widely used as a proficient resource. Park and Paek (2014) explained about the number of advantages such as simplicity, predictability and high effectiveness of the secondary metabolites that are repeatedly isolated from cultivation or biomass media. The factors by using bioreactors like optimization of culture environment, measurement of biomass production particularly with organ and tissue culture needs to be considered in scaling up the of plant secondary metabolites (Ruffoni et al. 2010, Steingroewer et al. 2013). Eibl and Eibl (2008) reviewed the developments of bioreactors design for the optimum production of plant secondary metabolites appropriate for plant cell and tissue culture and reported the availability of various types of bioreactors and concluded majority of the in vitro cultures viz. hairy root and cell suspension cultures are cultivated in different types of the reactors.

6.5  CONCLUSIONS AND FUTURE PROSPECTIVE Most of the pharmaceuticals drug molecules are obtained from the plants; however, plant produces these elite drug molecules at low contents. So, huge numbers of plants are utilized to meet the demands of the pharmaceutical industries, results to threaten the plant taxa. The in vitro culture methods are considered as one of the eco-friendly methods for the conservation and increment of plant secondary metabolites.

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There are great signs of progress being made in the last decade. There are several difficulties in scaling up the secondary metabolite synthesis, hence very low yields production was reported. Due to deficiency of precise knowledge about the bioactive molecules of biosynthetic pathways, the production of yields of all the elite drug molecules is inadequate. So, metabolic biosynthetic pathway engineering strategies to enhance the contents of bioactive molecules is considered as one of emerging and an alternate method to increase the synthesis of secondary metabolites in plant cells.

ACKNOWLEDGEMENTS The author is thankful to the Principal, Post Graduate Department of Botany, Vidyabharti College Seloo, Maharashtra, India for providing access to the subscription journals. Mohd. Shahnawaz is highly indebted to his wife Mrs. Mubeena Kousar for her kind support and time to complete this manuscript on time. Contact Information: Please contact the author at: [email protected]

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Hairy Roots and Plant Secondary Metabolites Production An Update Sharada L. Deore, Bhushan A. Baviskar and Anjali A. Kide

CONTENTS 7.1 Introduction����������������������������������������������������������������������������������������������������������������������������99 7.1.1 Historical Aspects of Hairy Root Studies for the Enhancement of Plant Secondary Metabolites��������������������������������������������������������������������������������������������� 100 7.1.2 Mechanism of Hairy Root Induction������������������������������������������������������������������������ 102 7.2 Hairy Root Establishment Protocols by Different Methods������������������������������������������������� 102 7.2.1 In Vitro���������������������������������������������������������������������������������������������������������������������� 102 7.2.2 In Vivo����������������������������������������������������������������������������������������������������������������������� 103 7.3 Analysis and Confirmation of Transformation Techniques�������������������������������������������������� 103 7.3.1 Opine Detection�������������������������������������������������������������������������������������������������������� 104 7.3.2 T-DNA Localization������������������������������������������������������������������������������������������������� 104 7.3.3 Dot Blotting�������������������������������������������������������������������������������������������������������������� 104 7.3.4 PCR Analysis������������������������������������������������������������������������������������������������������������ 104 7.3.5 Colorimetric Assay��������������������������������������������������������������������������������������������������� 105 7.3.6 Most Efficient and Reliable Method of Transgenic Confirmation��������������������������� 105 7.4 Stability��������������������������������������������������������������������������������������������������������������������������������� 105 7.5 Scale Up of Hairy Roots in Bioreactors������������������������������������������������������������������������������� 106 7.6 Diverse and Abundant Uses of Hairy Root Culture������������������������������������������������������������� 107 7.6.1 Phytochemical Production���������������������������������������������������������������������������������������� 107 7.6.2 Molecular Breeding�������������������������������������������������������������������������������������������������� 107 7.6.3 Phytoremediation������������������������������������������������������������������������������������������������������ 107 7.6.4 Biosynthetic Pathway Elucidation���������������������������������������������������������������������������� 107 7.6.5 Recombinant Protein Production������������������������������������������������������������������������������ 107 7.7 Secondary Metabolite Production from Hairy Roots����������������������������������������������������������� 108 7.7.1 Studies on the Enhancement of Alkaloids Using Hairy Root Culture��������������������� 108 7.7.2 Studies for Enhancement of Glycosides Using Hairy Root Culture������������������������� 109 7.8 Conclusion and Future Prospective�������������������������������������������������������������������������������������� 109 Acknowledgement������������������������������������������������������������������������������������������������������������������������� 116 References�������������������������������������������������������������������������������������������������������������������������������������� 116

7.1 INTRODUCTION Secondary metabolites are compounds synthesized by plants as excretory product which are released by plants in specific environmental conditions (Isah 2019; Tiwari and Rana 2015). These secondary metabolites possess immense therapeutic potential and are helpful for mankind to cure dreadful DOI: 10.1201/9781003034957-7

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diseases (Khadabadi and Deore 2019). Many plant-derived drugs like atropine, morphine, quinine, taxol, vincristine, vinblastine, digoxin etc. have been approved by FDA; hence, secondary metabolites obtained from plants got immense importance since last century (Seca and Pinto 2018; Atanasov and Waltenberger 2015). According to https://www.bccresearch.com/market-research/, it is estimated that, global market for plant derived drug products will grow to 36.6 billion dollars by 2022 while it was 29.4 billion dollars in 2017. This highlights increasing demand for herbal products. Before plant tissue culture technology, traditional methods were utilized for obtaining secondary metabolites. However, due to season-based collection of desired secondary metabolite containing plant parts and periodic gap between seasons, it was difficult to expect sufficient, uniform and timely supply of desired raw material (Rao and Ravishankar 2000; Sarfaraj and Fareed 2012). This emerging need gave birth to the exploration of plant tissue culture techniques to fulfil the demand and supply gap. In addition, to meet increasing demand of plant drugs, enhancement in yield of secondary metabolites were found essential (Sheludko 2010). Different methods like organ tissue culture, cell suspension culture and various genetic transformation techniques investigated for yield enhancement of secondary metabolite/s. (Sheludko 2010). However, hairy root culture has emerged as a most attractive alternative to intact plants as well as cell cultures for the production of secondary metabolites (Giri and Narasu 2000). Hairy root is a disease caused in plants by infection of gram-negative bacteria called Agrobacterium rhizhogenes i.e. Rhizhobium rhizhogenes belonging to family Rhizhobiaceae (Ricker and Banfeild 1930). This microorganism combines its Ri plasmid with genetic material of host plant in natural environment which results in hormonal imbalance in host plant and overgrowth of hair like structures at infected region. These hairs like structures are characterized by stability and high productivity of secondary metabolites in host plants (Srivastava and Srivastava 2007). This characteristic behaviour of hairy roots attracted attention of researchers as a key opportunity to enhance secondary metabolites in short period of time in order to meet industrial demand (Rao and Ravishankar 2000). Today, a number of medicinal plants have been genetically transformed by using different virulent strains of Rhizhobium rhizhogenes strains to obtain high yield of pharmaceutically important secondary metabolites (Guillon and Guiller 2008). However, high cost to set up of plant tissue culture laboratory as well as variations in outcomes when placed in alternate type of bioreactors for large-scale production are limiting factors for hairy root establishment in industries (Mishra and Ranjan 2008). Hence, more efforts need to be taken to improve this technology in order to mitigate over exploitation of valuable medicinal plant species. The main objective of the present chapter is to provide extensive coverage (Figure 7.1) of developments that took place in hairy root technology since past three decades including hairy root culture establishment protocols, advancements in molecular analysis, methodologies for confirmation of genetic transformation, recent applications of hairy root culture other than secondary metabolite production and insight of successfully established hairy root culture protocols for phytochemicals like alkaloids, flavonoids, glycosides and other commercially valuable plant secondary metabolites.

7.1.1 Historical Aspects of Hairy Root Studies for the Enhancement of Plant Secondary Metabolites The term ‘Hairy root’ was first time introduced by (Stewart and Hall 1900) while conducting fruit disease survey in New York in 1900. During survey, fruit plants observed suffering from weird disease consisting of knot like structures with fine hair like outgrowths. In 1930, Ricker AJ et al. studied hairy root diseased apple trees obtained from different nurseries and found causative organism named Phytomonas rhizhogenes which was renamed as Agrobacterium rhizhogenes. Ricker noted that pathogenesis case due to Agrobacterium rhizhogenes strain is found different than Agrobacterium tumefaciens but exact mechanism was unknown (Ricker and Banfeild 1930).

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FIGURE 7.1  Aspects of hairy root culture covered in the present chapter.

Agrobacterium tumefaciens was brought in focus by researchers Erwin Smith and Charles Townsend in 1907 as tumour-forming agent in crown gall disease in dicotyledonous plants (Smith and Townsend 1907). After this, extensive research was conducted to understand mechanism behind tumour formations and role of Agrobacterium tumefaciens. In 1947, a study conducted by Braun A, a research scholar at Rockefeller Institute revealed DNA of pathogen may act as a ‘Tumour Inducing Principle’ (Braun 1947). Braun A also reported that, Agrobacterium tumefaciens can infect permanently plant cells and also grows in vitro on phytohormone free culture medium (Braun 1947). In 1969, first report of genetic transformation through plasmid from virulent strain to non-virulent strain was produced by researcher Allen Kerr (Kerr 1969). Later in 1974, Schell J and Montagu VM published remarkable study based on 11 Agro bacterial strains and reported that pathogenic Agro bacterial strains contained large plasmid while it was absent in non-virulent strains which highlighted strongly role of plasmid in pathogenesis (Zaenen and Larebeke 1974). Heating experiment on Agrobacterial strain C58 at 37°C proved that bacteria loose pathogenicity at higher temperature due to loss of large plasmid (Zaenen and Larebeke 1974). Further investigations revealed that Agrobacterium rhizogenes contains larger Ri plasmid than Ti plasmid present in Agrobacterium tumefaciens. These plasmids contain genes which encode synthesis of opines in transformed hosts which serves as food for bacteria (Zaenen and Larebeke 1974). In 1977, Ackermann C induced hairy roots in tobacco plants and established protocol for regeneration of plants from hairy roots which was a beginning of application of this marvellous technology for enhancement of secondary metabolites (Ackermann 1977). Since mid-1980, biosynthetic potential of hairy roots was identified by researchers and hairy roots were successfully established in plants belonging to Solanaceae family for Alkaloid enhancement (Chilton and Tepfer 1982). Today, more than 400 medicinal plants are successfully genetically transformed with hairy roots inducing Rhizobium rhizhogenes for enhancement of secondary metabolites in plants belonging to families Solanaceae, Polygonaceae, Asterceae, Fabaceae, Liliaceae, Caesalpinaceae, Brassicaseae etc. (Tian and Ono 2011).

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7.1.2  Mechanism of Hairy Root Induction The causative agent of hairy root disease in plants is gram negative soil bacterium, Agrobacterium rhizhogenes. They cause invasion at wounded site of most of the dicotyledonous plants because dicotyledons releases phenolic compounds at their wounded site which creates chemotaxic effect and attracts A. rhizhogenes towards wounded site. After infection, a number of small roots protrudes as fine hairs at the sight of infection and proliferates rapidly (Srivastava and Srivastava 2007). A DNA segment (T-DNA) from large root inducing (Ri) plasmid of this gram-negative soil bacterium transfers in to the genome of infected plant (Chilton and Tepfer 1982). Auxin and cytokinin biosynthesis controlling enzymes encoding genes are present in this T-DNA. (Guillon and Guiller 2006). The integrated segment T-DNA also contains genes for opine biosynthesis, sole source of carbon and nitrogen for A. rhizhogenes (Srivastava and Srivastava 2007). These opine serve as marker at the time of confirmation of transformation (Aleksey and Kuluev 2017). Polyphenolic compounds play an important role in host–pathogen relationship of Agrobacterium induced neoplasm (De Cleene 1988). Extensive literature survey and previous studies have shown that dicotyledonous plant families are extensive accumulators of polyphenolic compounds thus they are highly sensitive to Agrobacterial infection. Monocotyledonous plant family lacks this ability since they are not accumulators of polyphenolic compounds; however, there are some exceptions like Liliaceae family (Agastian 2013). It has been reported that this family contains Arbutin, a monoglucoside of hydroquinone which can help for A. rhizogenes transformation in Liliaceae family (De Cleene 1988). In literature, researchers have reported transformation in monocotyledonous family by external employment of polyphenolic compounds like acetosyringone, δ–hydroxyacetosyringone (De Cleene 1988). Hairy root induction in Gloriosa superba belonging to Liliaceae family was successfully achieved to enhance colchicin level (Agastian 2013). Studies show that acetosyringone increased rate of transformation in both monocotyledonous and dicotyledonous plant families (Winans 1992).

7.2  HAIRY ROOT ESTABLISHMENT PROTOCOLS BY DIFFERENT METHODS Mainly, there are two establishment protocols (Figure 7.2) for genetically transformed hairy root development (Christey and Braun 2005) as follows.

7.2.1  In Vitro For in vitro development of hairy root cultures, either explant from previously cultured plants or excised from fresh plant and later developed in to callus can be utilized (Karami 2008). While using freshly incised explants, it should be sterilized carefully before employing for further process to avoid contamination. Selected explants are incised slightly by sharp blade on surface or near the meristematic region and are inoculated by dipping in the liquid culture medium containing selected activated Agrobacterium rhizogenens for a period of 1 s to 15 min depending upon requirement of the experiment. This process is known as inoculation. After inoculation, explants are kept on hormone-free culture medium for time period of 24–48 hours in dark condition at room temperature. Absence of light is highly suitable condition for Agrobacteria to incorporate their gene in to the host cell. Different types of culture media like Murashige and Skoog (MS 1962), White (1963), Gamborg B5 (1968), Anderson (1978, 1980), Kao & Michayulk (K & M 1975), Nitsch & Nitsch (N & N 1969), Coke (Westvaco WV3 1996) etc. are used without incorporating growth hormones for induction of hairy roots (Carlin 2015). Out of these, MS media is still highly preferable for hairy root culture development in different strengths at laboratory as well as industrial level due to simplicity, cost effectiveness and modifications (Murashige and Skoog 1962). After induction period, explants are washed with antibiotics like cefataxime to kill excess of agrobacteria. After washing in aseptic condition, explants are transferred to suitable half strength or full-strength culture medium.

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Hairy roots can also be initiated from stems of in vitro cultured plants by giving small incision on stem by using needle or any other sharp tool. Another method in which explants are excised from in vitro seedlings by cutting hypocotyls and placed in inverted position on culture medium where excised portion of hypocotyls will be in contact with Agrobacterum rhizogenes strains also showed good results (Christey and Braun 2005).

7.2.2  In Vivo For in vivo establishment of Hairy roots, green house plants or pot plants grown in green house are used (Collier and Fuchs 2005). These plants are initially grown in aseptic area by tissue culture then transferred to green house after hardening (Christey and Braun 2005). In this case, stems, juvenile leaves or petioles are pricked with needle which is infected with virulent Agro bacterial strain (Mandal and Sinharoy 2019). In vivo hairy root establishment requires highly humid condition for genetic transformation. Hence, in this process, infected sites are needed to be covered with gauze to maintain humidity at site. But success rate is lower as compared to in vitro method hence rarely used. (Christey and Braun 2005). This method is widely used for study of composite plants. This method is gaining popularity due to its simplicity, cost effectiveness and less tediousness than in vitro culture (Collier and Fuchs 2005). After successful genetic transformation and sufficient growth of hairy roots, roots are excised and transferred to half strength liquid culture medium either on lab scale or in large bioreactors.

7.3  ANALYSIS AND CONFIRMATION OF TRANSFORMATION TECHNIQUES Plant cell transformed with A. rhizhogenes can be confirmed by distinctive morphology of transformed roots growing at the site of infection and their transformed regenerants (White and Sinkar 1987). Generally, abundance of lateral roots with lack of geotropism in transformed roots is common altered phenotype (Tepfer 1984) while wrinkled leaves and shortened internodes can also be observed in transformed regenerants compared to normally grown counterparts (Chilton and Tepfer 1982; Ooms and Karp 1985; Guerche and Jouanin 1987). Following confirmatory methods (Figure  7.2) are commonly used in Hairy root analysis:

FIGURE 7.2  Analysis and confirmation of transformation of hairy root disease and its applications.

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Biotechnological Approaches to Enhance Plant Secondary Metabolites

7.3.1 Opine Detection After transformation, T-DNA of Ri plasmid encodes opine synthesis in A. rhizhogenes infected plant cell. The most effective biochemical marker to confirm transformation in cultured root tissue is these opine. Type of opine present helps in classifying wild type A. rhizhogenes strains used for induction of hairy roots as follows (Petit and David 1983). • Agropine strains—induce agropine, mannopine and agropinic acid production. • Mannopine strains—induce the production of single opine. • Cucumopine strains—induce the production of single opine. Two regions, called TL and TR-DNA of T-DNA of Ri plasmid of A. rhizhogenes, are found to be integrated and stably retain in the plant genome. • Agropine strain—transfers independently both TL and TR-DNA to the plant genome. • Mannopine strains—transfers the TL-DNA only. Auxin and cytokinin (Estrutch and Chriqui 1991) susceptibility of plant cells is enhanced by four rol genes A, B, C and D (Schmulling and Schell 1988; Peterson and Stummann 1989) present in TL-DNA. All four rol A, B, C, D genes but more specifically rol B gene (Nilsson and Olsson 1997) are responsible for hairy root formation. However, rol c gene alone can also induce hairy roots in Atropa belladonna (Bonhomme and Laurain 2000; Bensaddek and Gillet 2001). Paper electrophoresis is used by many researchers for detection of opines (Srivastava and Srivastava 2007).

7.3.2  T-DNA Localization T-DNA localization in host plant genome is also a reliable genetic marker to confirm transformation (Mukundan 1997). Southern hybridization also called Southern blotting is a widely used technique for T-DNA localization in genetic material of transformed root line (Southern 1975). In this technique, DNA fragments on the basis of size and charge are separated by Gel electrophoresis and then labelled probe hybridization is used for identification (Gitschier 2013; Bernatzky 1992; White and Barker 1983). This technique emerged as an efficient method to direct a DNA sequence from complex mixture of DNAs (Gitschier 2013; Yang and Platt 1987). Southern hybridization is used for confirmation of transformation of hairy roots in Daccus carrota (David and Petit 1988), Cinchona ledgeriana (Hamill and Robins 1989), Nicotiana rustica (Rhodes 1994), Artemisia annua (Chen and Liu 1999; Srivastava and Srivastava 2007). This method is comparatively simple and cost effective but laborious and time consuming. Still, it is preferred at first place with conventional PCR in many hairy root establishment studies (Gitschier 2013).

7.3.3 Dot Blotting This method screens presence of a foreign gene sequence and thus verifies transformed nature of a tissue (Draper and Scott 1988). This technique involves direct application of DNA fragments on nylon membrane instead of electrophoresis like in southern hybridization. Later, it goes under probe hybridization in order to detect desired DNA molecule sequence (Brown 1993). This method is simple, robust, inexpensive and highly sensitive as compared to other blotting techniques (Falconar 2013); however, it doesn’t provide information about size of DNA fragment. It can detect only identical proteins present in transformed root line (Brown 1993).

7.3.4 PCR Analysis This technique is commonly used by many researchers as a reliable source of confirmation (Erlich and Arnheim 1992; Trijatmiko and Arines 2016). This technique was first invented by Nobel Prize

Hairy Roots and Plant Secondary Metabolites Production

105

winner Dr. Kary B Mullis in 1983 (Garibyan and Avashia 2013; Valones and Guimarães 2009; Mullis 1990). PCR technique is based on synthesis of billion copies of DNA fragments by using DNA polymerase enzyme which is profoundly involved in synthesis of complimentary DNA primers. These DNA primers attach themselves at selected site on small DNA strand and start synthesis process which results in amplification of small fragment of DNA (Mullis 1990). It involves verification by polymerase chain reaction for tissue culture-mediated transgenic as well as alteration in a specific gene sequence. (Jazari and Yoshimatsue 1994; Dong and Sun 1992). This technology became popular as a molecular analysis tool within short period of time due to advantages like high sensitivity, accuracy, precision, reproducibility, qualitative and quantitative output in short period of time (Smith and Osborn 2009); however, there are few limitations too. Due to high sensitivity, there are many chances of obtaining misleading output if contaminants are present in sample. Primer designing can be a tedious step if DNA sequence to be studied is unknown (Smith and Osborn 2009).

7.3.5 Colorimetric Assay Presence of arginine derived opines like nopaline and octopine in plant tissue extract can be confirmed by Colorimetric assay. Paper electrophoresis is most general technique used for detection of nopalines and octopines by observing UV–phenanthroquinone reacted fluorescent products. Heat treatment compatible with paper electrophoresis results in quick production of red–purple pigment. Sensitivity of this method is up to 1.25 µg quantities of opines. This method eradicates that common background fluorescence problem occurs while analysing crude plant extract in the typical assay. (Yang and Platt 1987)

7.3.6  Most Efficient and Reliable Method of Transgenic Confirmation Transgenic confirmation involves verification of presence of transgene in host plant cells to claim successful genetic transformation (Passaricha and Saifi 2016). As above discussed, various efficient tools and techniques like southern blotting, blot dot and PCR analysis have been developed for confirmation of presence of transgene in genetic material of host plant. Before invention of PCR technique of molecular analysis, Southern hybridization or Southern blotting was highly preferable technique for confirmation (Brown 1993). Although this technique is simple or less complicated to access, it has limitations like laborious technique, time consuming and high amount of DNA sample required for study (Podzorski and Loeffelholz 2006). Thus, PCR analysis is considered as sensitive and most reliable method for both quantitative and qualitative analysis of genetic transformation in medicinal plants (Valones and Guimarães 2009). Modified PCR techniques like real-time PCR (RT-PCR) are prime choice by many researchers for Agrobacterial genetic transformation (Safaei and Aghaee, 2019). In RT-PCR, both amplification of DNA and detection of amplified DNA fragments occur simultaneously which prevents extra time required for Agarose gel step in conventional PCR technique (Nain and Jaiswal 2005). Quantification of DNA fragment amplification is possible in RT-PCR which is absent in conventional PCR technique Hence, RT-PCR is also known as quantitative PCR (qPCR) (Ingham and Beer 2001). However, due to expensiveness, Southern hybridization is an alternative choice by researchers for genetic transformation confirmation along with conventional PCR analysis (Podzorski and Loeffelholz 2006; Tuteja and Verma 2012).

7.4 STABILITY Stability of hairy root culture medium depends on culture medium, pH of culture medium, temperature, method of sterilization of explants and high aseptic conditions are important factors on which success of hairy root culture depends (Gutierrez and Hakkinen 2020; Sivakumar and Yu 2005). The growth medium has significant effect on hairy root induction (Panda and Meheta 2017; Pakdin and Farsi 2014). High salt media like B5 and MS favour hairy root formation in some plants (Carlin and Tafoya 2015; Verma and Singh 2015). Previous studies have revealed that MS medium serves as an

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Biotechnological Approaches to Enhance Plant Secondary Metabolites

ideal medium for hairy root culture (Das and Raut 2000). There is significant effect of pH on hairy root development (Verma and Singh 2015). Hairy root culture for glycosides like Diosgenin (Merkil and Christen 1997), irridoid glycoside (Verma and Singh 2015) containing herbs have shown highest yield between pH ranges 5 and 6, while hairy roots cultures of flavanolignan containing plants have shown highest yield at acidic pH (Rahimi and Tahereh 2016; Thimmaraju and Bhagyalakshmi 2003). The ideal temperature for hairy root growth in most of the hairy root studies is reported as 25 ± 5°C (Srivatanakul and Park 2001; Wu and Sparks 2003). Sterilization of explants for hairy root cultures must be proper because if it is not sterilized well, fungal or bacterial contamination arises again and again which destroys the culture (Srivastava and Srivastava 2007). Mercuric chloride solution (0.1–1%) is effective in killing fungal spores and bacteria on explants surfaces (Rebecca 2012). Sodium hypochlorite (1.5%), 70% ethanol treatment also helps to irradiate contamination (Jan and Bhat 2013). All the transformation work must be performed inside the laminar air hood to avoid contamination. Incubator should be fumigated time to time to irradiate fungal spores or bacterial growth inside it (Evans and Dingle 1973). Generally, fumigation with formalin + potassium permanganate is very effective (Connolly 1983); however, these fumes are hazardous for human health so tremendous care must be taken. Alternative techniques are swabbing incubator with 1% sodium hypochlorite then with water (Rebecca 2012). After swabbing, it is cleaned with 70% ethanol. In this way, sterilization of incubator is also an important factor during hairy root culture development.

7.5  SCALE UP OF HAIRY ROOTS IN BIOREACTORS Heterotrophic and respiratory nature of hairy roots depends on energy generating oxygen and other metabolic functions (Walker and Harsh 2003). Dissolved oxygen concentration in medium reduces due to solid phase nature of roots and growth of oxygen gradients within root tissues (Mehrotra and Srivastava 2018). It badly affects to growth rate and synthesis of secondary metabolites due to formation of a pocket of senescent tissues as a result of restricted nutrient oxygen delivery to the central mass of tissue. Growing hairy roots are unable to get sufficient oxygen due to mass transfer resistances near liquid and solid boundary. (Khan and Siddiqui 2019). Thus, scale up of hairy root culture requires carefully designed bioreactor system controlling a number of physical and chemical parameters like nutrient availability and uptake, oxygen and hydrogen levels, mixer and shear sensitivity, support matrix requirement and possibility of flow restriction etc. (Khan and Siddiqui 2019). Such modified bioreactors (Figure 7.3) can only provide most excellent environment for most

FIGURE 7.3  Classification of bioreactors used for hairy root culture to produce plant secondary metabolites. (Modified from Srivastava and Srivastava 2007.)

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107

favourable growth and secondary metabolite production in hairy roots compared to normal roots (Mehrotra and Srivastava 2018).

7.6  DIVERSE AND ABUNDANT USES OF HAIRY ROOT CULTURE 7.6.1 Phytochemical Production Biotic and or abiotic stimulations can be used for production of phytochemicals (Guillon and Guiller 2006). Synergistic effect of use of high sorbitol concentration and yeast elicitor leads to enhanced production of diterpenoid tanshinone in hairy root culture of salvia miltorhiza (Wu and Shi 2008). A new phenomenon of electro elicitation has been introduced by researchers to enhance phytochemical production. Electric current is found as clean elicitor because the culture system or compound of interest remains unaltered. This is also the most attractive method for industrial purposes due to better and strong penetration ability to bulky root mass inside bioreactors as compared to other elicitors (Tian and Ono 2011). Electro elicitation in hairy root culture of Pisum sativum showed enhanced (+)-pisatin accumulation to same levels to CuCl2 elicitation (Kaimoyo and Farag 2008).

7.6.2  Molecular Breeding Regeneration of whole plants from A. rhizhogenes-infected hairy root cultures of same plants is called as molecular breeding. This technique yields plants with desirable phenotypes such as compact size for horticulture purposes (Giri and Narasu 2000).

7.6.3 Phytoremediation Phytoremediation is a utilization technique of plant’s ability to absorb and accumulate heavy metals or to convert toxic organic molecules to nontoxic form enzymatically (Guillon and Guiller 2006). Phytoremediation of toxic substances and reactive dyes can be studied by hairy root culture considering as model system (Eapen and Suseelan 2003). Cadmium, nickel or uranium is found to be absorbed by the hairy roots from hyper accumulator plant (Boominathan and Doran 2003; Eapen and Suseelan 2003). Pesticides and/or few antibiotics from industrial waste are successfully detoxified by Hairy roots of Brassica species, Chicorium intibus and Helianthus annuu (Agostini and Coniglio 2003; Gujarathi and Haney 2005; Suresh and Sherkhane 2005).

7.6.4  Biosynthetic Pathway Elucidation Biosynthetic pathway elucidation for phytochemicals is accelerated due to simplified molecular, biochemical and genetic studies and thus metabolic engineering in hairy root cultures (Kadabadi and Deore 2019). Hairy root culture of Medicago trancatula helped to reveal distinct role of MtDXS2 in isoprenoid biosynthesis that is separate from DXS1 (Floss and Hause 2008). Hairy root culture also helped to reveal biosynthetic pathways of lignin which have important role in plant defence and human health. To elucidate biosynthetic pathways, stable isotope feeding studies through use of hairy root culture is possibly same as cell culture systems (Tian and Ono 2011). Biosynthesis of camptothecin from the MEP and shikimate pathway elucidated by using stable isotope tracers ([1–13 C] Glc) and in silico prediction of labelling patterns methods (Yamazaki and Kitajima 2004).

7.6.5 Recombinant Protein Production The use of plants for synthesis of human therapeutic proteins in plant cells is called molecular farming (Kamenarova and Abumhadi 2005). Plant-mediated production of therapeutic proteins like vaccines, antibodies and mammalian enzymes is an attractive technique because of the cost effectiveness, uncomplicated control system and availability of more human like post translational

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modifications machinery compared to mammalian and or microbial cells (Tian and Ono 2011). Hairy root cultures are beginning to be established as a method for recombinant therapeutic proteins production. Human acetyl cholinesterase, a bio scavenger of organophosphate toxin, was synthesized by hairy root culture that can be used in post exposure treatment (Woods and Geyer 2008). Hairy root culture in tobacco plants was successfully established for secretion of full-size homogeneous monoclonal antibody M12 and 30-fold increase was observed after elicitation (Hakkinen and Raven 2014). Thus, hairy roots offer an excellent platform for development of homogeneous, mammalian toxin free and cost-effective therapeutic proteins with higher yield (Mehrotra and Mishra 2015).

7.7  SECONDARY METABOLITE PRODUCTION FROM HAIRY ROOTS Production of highly complicated phytochemical compounds of commercial value from simple building blocks makes plants attractive ‘chemical factories’ (Tian and Ono 2011). But yield of these phytochemicals always depends on environmental factors (Ramakrishna 2011). Thus, hairy root culture is an attractive option due to genetic stability and very high yield of desired phytochemical/s under optimised conditions (Abraham and Thomas 2017) compared to cell or callus cultures (Chandra and Chandra 2011). More than 400 plant species are studied and hairy root cultures developed successfully (Tian and Ono 2011). Many scientists are working on more and more plants for establishment of hairy root culture for various commercial applications and leading to small scale laboratory to large scale industrial production. High commercial value having camptothecin and podophyllotoxin producing German company ROOTec is one of the best examples (http://www. rootec.com) (Guillon and Guiller 2006). Nutritional and environmental factors like composition of culture medium, quantity of sucrose and exogenous growth hormone while both quantity and quality of nitrogen source, light, temperature and other chemicals are responsible for total yield of secondary metabolites, biosynthesis rate and overall biomass yield of hairy roots (Giri and Narasu 2000; Rhodes and Parr 1994). Elicitation, precursor feeding, improving cell permeability and released molecules trapping in the liquid medium are most effective ways for enhancement of secondary metabolite production in hairy root culture (Pistelli and Giovannini 2010). Many studies confirmed very promising role of methyl jasmonate in secondary metabolite enhancement without altering the growth rate of cultures (Guttierez and Hakkinen 2020). Sequence tags (ESTs) determining specific limiting enzyme/s in the genesenoside biosynthesis is verified from methyl jasmonate treatment in ginseng hairy root culture (Choi and Jung 2005). It is also found that methyl jasmonateis used to enhance secretion of metabolites into medium. Use of Phytophthora cinnamoni in hairy root culture of Oxalis tuberose leads to liberation of more amounts of harmine and harmaline (Bais and Vepachedu 2003). Similarly, stimulation of secondary metabolite secretion found due to use of various elicitors and permeability enhancing chemicals like surfactants, calcium chealators, pH modifiers and physical elicitors—sonication, temperature and oxygen stresses (Thimmaraju and Bhagyalaxmi 2003). Adsorbent/s can also enhance the secretion rate of secondary metabolites. Mixture of alumina and silica adsorbents (1:1) provided 97.2% of betalain from red beet hairy roots (Thimmaraju and Bhagyalakshmi 2004).

7.7.1 Studies on the Enhancement of Alkaloids Using Hairy Root Culture Alkaloid have emerged as an important plant secondary metabolite after evolution of pharmaceutically important drug candidates like vincristine, vinblastine, atropine, reserpine, nicotine, morphine etc. Alkaloids are nitrogen containing compounds produced by plants as excretory compound and eliminated during unfavourable conditions of plants (Wink 1998). These are highly beneficial to mankind due to medicinal properties. Thus, its demand increased tremendously since last two decades for formulation development in pharmaceutical industries. They are present in

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plants in very minute quantity. Hence to obtain desired alkaloidal compound in bulk, tons of crude plant material was required. Thus, transgenic approach by using various strains of Agrobacterium rhizhogenes/Rhizhobium rhizhogenes was essential to meet industry requirements for alkaloid compound. Hairy root culture was successfully established for enhancement of alkaloids with significant yield in medicinal plants having anticancer activity like vincristine, vinblastine, catharanthine from Cantharanthus roseus (Hanafy and Matter 2015), camptothecin from Ophiorrhiza pumila (Yamazaki and Mochida 2013), anticholinergic agents like Hyoscyamine, scopolamine from Hyoscyamus reticulatus L (Moharrami and Hosseini 2017), antitussive and sedative agents like codeine, morphine, sanguinarine from Papaver somniferum L (Bonhomme and Laurain 2004), anti-malarial agents like quinine, qunidine from Cinchona officinalis (Geerlings and Hallard 1999) etc. (summarized in Table 7.1 along with further details). Elicitors like Methyl jasmonate, quercetin, salicylic acid, chitosan, aluminium chloride etc. have shown remarkable increase in yield of alkaloidal compounds (Zayed 2014; Mawla 2010; Qaderi and Akbari 2016) which have been summarized in Table 7.1.

7.7.2 Studies for Enhancement of Glycosides Using Hairy Root Culture Glycosides are the secondary metabolites derived from plants which upon hydrolysis divide into glycone and aglycone moiety to exert therapeutic effects. Therapeutic effect is based on aglycone moiety present in particular glycoside (Kren and Martinkova 2001; Cheeke 2001). Like alkaloids, glycosides also gained immense importance as therapeutic agent after discovery of famous cardiac glycosides, digoxin derived from Digitalis purpuria (Toro and Jimenez 2019). Researchers started to focus on glycosides for discovery of lead molecule for dreadful diseases like cancer, cardiovascular disorders, diabetes etc. Many miraculous glycosides found by researchers had potent anticancer, cardiovascular, antidiabetic, neuroprotective, antiviral and antibacterial activities. Hence, it was necessary to enhance production of these metabolites inside plants by genetic transformation methodologies. Researchers have successfully established hairy root cultures by using different Agrobacterial strains in many glycoside yielding medicinal plants, for example, immunomodulators like Panax ginseng and Withania somnifera genetically transformed by Agrobacterial strains KCTC2703, LBA9402 enhance yield of ginsenosides and withanolides, respectively (Sivakumar and Yu 2005; Ray and Ghosh 1996), anti-diabetic activity exerting medicinal plants like Swertia chirata and Momordica charantia were successfully genetically transformed by Agrobacterial strains like ATCC158, LBA9402, TR105, MTCC 532 for enhancement of amarogentin, charantin, respectively (Kiel and Hartle 2000; Swarna and Ravindhran 2012). In this way, a number of medicinal plants have been Agrobacterial strains in order to enhance level of glycosides. Few important genetically transformed glycosides containing medicinal plants have been summarized in Table 7.2.

7.8  CONCLUSION AND FUTURE PROSPECTIVE Secondary metabolites are commercially important molecules for food, agro, cosmetic and/or pharmaceutical applications. Acute as well as chronic diseases/disorders can be treated using regulated use of phytochemicals. Since in this century global awareness regarding herbal medicines is increased tremendously, demand for plants as well as purified secondary metabolites are greater than before which is nearly impossible to meet with tradition techniques. Not only dicot but many monocot plant species are also successfully genetically transformed with Rhizhobium rhizhogenes. Cost-effective protocols for hairy root establishment have been developed and reported by many researchers. Hence, industrial scale up hairy root culture technology with its wide application will be a promising alternative in near future to meet this global demand for herbal-based food, agro, cosmetic formulations and/or drugs. Hairy root culture is found to protect environment from harmful heavy metals released from agriculture or chemical industry pollution through phytoremediation.

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TABLE 7.1 Hairy Root Culture Development for Production of Alkaloids Sr. No.

Name of Plant

1

Agro-Bacterial Strain Used

Elicitors Used

% Yield Obtained

Catharanthus roseus

Ajmalicine

DMSO: 12 folds, Triton-X 100:16-fold

2

Hyoscyamus reticulatus L A7

3

Datura metel

A4

Hyoscyamine Scopolamine Scopolamine

DMSO, Triton-X 100, n-hexadecane, Tween 80 Iron oxide nanoparticles

4

Ophiorrhiza pumila

15834

Camptothecin

Bacillus cereus Staphylococcus aurous --

5

Hyocyamus muticus

Scopolamine

--

6

Rauwolfia serpentina

LBA9402 pLAL21 (carrying 35S-h6h) A4

Ajmaline and Ajmalicine

7

Solanum khasianum

A4

Solasodine, α-Solacine

8

Ophiorrhiza pumila

15834

Camptothecine

9

Nicotiana tobacum

15834, TR105, LBA

Nicotine

10

Cinchona officinalis

LBA9402

11

Arnebia hispidissima

A4

Tryptophan, Strictosidine, Quinine, quinidine Shikonin

NaCl as abiotic elicitor, Cellulase from Aspergillus Niger as biotic elicitor NaCl as abiotic elicitor, Cellulase from Aspergillus niger as biotic elicitor Polystyrene resin (Diaion HP 20) Methyl Jasmonate, Quercetin, Salicylic acid cDNA integration with TDC and STR genes from C. rosus

12

Tribulus terrestris L

AR15834, GMI9534

13

Duboisia myoporoides

AR4

β-carboline alkaloids – Harmine Scopolamine

Reference

Thakore and Srivastava (2013) 2.4 folds than normal plant Moharrami and Hosseini (2017) 0.03 g and 0.017 g of scopolamine after Shakeran and Keyhanfar 12 hours and 24 hours, respectively (2017) 0.1% about 2.5 folds higher than Udomsom and Rai normal leaves (2016) 65–67 folds higher than normal plant Häkkinen and Movano after 16 years continuous subculture (2016) 14.8 folds increase with abiotic elicitor, Srivastava and Sharma 2.9 folds increase with biotic elicitor (2016) 4.0 folds, 3.6 folds increase with Srivastava and Sharma abiotic elicitor, 1.6 folds increase with (2016) biotic elicitor 16 folds increase in 1 week Yamazaki and Mochida (2013) 7 folds increase Zayed (2014) 1200 µg g−1, 1950 µg g−1, 500 µg g−1, 1000 µg g−1

Geerlings and Hallard (1999)

--

0.85 mg g−1 compared to callus

--

1.7 µg g−1

--

2.5 folds increase

Chaudhary and Pal (2010) Sharifi and Sattari (2014) Yukihito and Yasuhero (1994)

Biotechnological Approaches to Enhance Plant Secondary Metabolites

Name of Alkaloid

LBA9402

Pyrolizidine Alkaloids

15 16

Leucojum aestivum L Solanum aviculare

Galanthamine Solasodine

17

Lobelia inflata

LBA9402 A4, ATCC 11325, ATCC15834, ATCC43057 R1601

18

Trypterygium wilfordii

A4, ATCC 15834

Wilforine

19

Papaver somniferum L

15834, LBA9402

20

Lithospermum canescens (Michx). Lehm

21

Peganum harmala

22

Brugmansia candida

ATCC15834, LBA9402, NCIB 8196 15834, TR105, LBA9402 LBA9402

Codeine, Morphine, Sanguinarine Acetylshikonin Isobutyrylshikonin

23

Scopolia japonica

24

Coleus forskohlii

25 26

Gloriosa superba Razhya stricta

ATCC 15834, A4, NCPPB 1855, NCPPB 2659 15834, k599, LBA9402, A4 MTCC2364 LBA 9402,

27

Vinca minor L

DC-AR2

28

Chonemorpha fragrans

A4, NICM 5140, ATCC15834

Lobeline

Methyl Jasmonate, Quercetin, Salicylic acid -Variation of Salt and Sucrose composition in culture medium 3% sucrose in MS medium Methyl Jasmonate, Salicylic acid

19 folds

Mawla (2010)

1.14–6.79 × 10−3 dry wt 6.2 mg g−1

Diop and Hehn (2007) Kittipongpatana and Hock (1998)

1.7 mg g−1 in hairy roots, 14.3 mg in culture medium 10% Less than naturally grown roots

Banyai and Balvanyos (2003) Zhu and Miao (2014)

3 folds more than normal plant

Flem-Bonhomme and Laurain (2004) Pietrosiuk and Syklowska (2006)

--

10 folds than normal

β carboline alkaloids, Harmine Scopolamine Hyoscyamine Scopolamine Hyoscyamine

Hydrogen Peroxide, Tryptophane Aluminium Chloride Methyl Jasmonate --

6 folds than normal

Zayed (2011)

43–83% increase

Giuiletti and Pitta (2000)

0.5% and 1.3% on dry wt

Yoshihiro and Shigeyasu (1986)

Forskolin

Salicylic acid, Copper sulphate, Methyl jasmonate -Agrobacterial strain carrying pK2WG7-gusA binary vector, Light

2–12-fold increase

Reddy and Pravina (2012) Agastian and Bai (2013) Akhgari and Yrjonen (2015)

--

--

--

0.024–0.030% dry wt

Colchicine Vincanine, Lipacine I isomer, Lipacine isomer II, Strictosidinelactum, serpentine Vincamine Camptothecin

Higher than normal

Tanaka and Takao (1994) Kedari and Malpathak (2014) (Continued)

111

Echium rauwolfii

Hairy Roots and Plant Secondary Metabolites Production

14

112

TABLE 7.1  (Continued) Hairy Root Culture Development for Production of Alkaloids Name of Plant

Agro-Bacterial Strain Used

26

Razhya stricta

LBA 9402,

27

Vinca minor L

DC-AR2

28

Chonemorpha fragrans

29

Catahranthus roseus

30

Tinospora cordifolia

31

Name of Alkaloid

Elicitors Used

% Yield Obtained

Reference

Vincanine, Lipacine I isomer, Lipacine isomer II, Strictosidinelactum, serpentine Vincamine

Agrobacterial strain carrying pK2WG7-gusA binary vector, Light

Higher than normal

Akhgari and Yrjonen (2015)

--

--

A4, NICM 5140, ATCC15834 K599 Harbouring p35SGFPGUS+ Plasmid LBA2402

Camptothecin

--

0.024–0.030% dry wt

Tanaka and Takao (1994) Kedari and Malpathak (2014) Hanafy and Matter (2015)

Berberine

L-Tyrosin, Methyl jasmonate

0.034 mg, 0.047 mg

ATCC15834

Trigonelline

Methyl jasmonate, Chitosan

32

Trigonella foenum–graecum Rauvolfia micrantha

ATCC15834

Ajmaline, Ajmalicine

IBA, NAA

33 34

Taxas brevifolia Taxus cuspidate Sieb

A4 (ATCC31798) R1000, A4, 15384

Taxol Taxol

-Methyl Jasmonate

36.7 mM/g in MJ and 37.33 mM/g chitosan 0.001 mg/g Ajmaline and 0.006 mg/g Ajmalicine greater than normal 1.3–8 times higher than callus culture 52.2 mg/l higher than normal

Vincristine, Catharanthine

Vincristine 1434.3-fold than control

Verma and Juvekar (2006) Qaderi and Akbari (2016) Sudha and Reddy (2003) Huang and Mu (1997) Kim and Baek (2009)

Biotechnological Approaches to Enhance Plant Secondary Metabolites

Sr. No.

Sr. No.

Name of Plant

Agro Bacterial Strain Used

Name of Glycoside

Elicitors Used

% Yield Obtained

Reference

1

Calendula officinalis

ATCC 15834

Oleanolic acid

--

4.59 mg g−1

2

Sylibum marianum

AR15834

Sylimarin

3.33 folds, 1.52 folds, 1.42 folds

3. 4

Withania somnifera Catalapa ovata

Withanolide D Cis-trans verbacosides Martynoside

5

Calendula officinalis

LBA 9402 A4, LBA 9402, ATCC 15834, TR105 LBA 9402pRi 1855

A. niger, F. Proliferatum, R. solani 3% sucrose --

Dlugosz and Wiktorowska (2013) Hasanloo and Ahmadi (2013) Ray and Ghosh (1996) Wysokinska and Lisowska (2014)

Half strength MS medium with 3% sucrose

2 folds higher than normal

Malarz and Stojakowska (2013)

6 7

Sylibum marianum Withania somnifera

ATCC 15834 MTCC 532

Chitosan, GA3, 4% sucrose --

0.7% DW, 0.65% DW --

8 9

Catalapa ovata Gentiana scabra

ATCC 15834 ATCC 15834

10

Polygonum multiflorum T Abrus precatorius L Panax ginseng

KCTC 2703

Gypenoside Iridoids, Secoiridoids like Loganic acid, gentiopicroside, swertiamarin Emodin, Physcion

Hwang (2009) Swarna and Ravindhran (2012) Chang and Chang (2005) Huang and Vishwakarma (2014)

MTCC 532 KCTC 2703

Glycyrrhizin Ginsenoside

11 12

Hydroxycinnamate, 8-Deoxylactucin (Crepidiaside A) Catapol Charantin

Ziatin, NAA

30 g/l sucrose

38 mg g–1 Loganic acid 6.6 folds in presence of Zeatin, gentiopicroside 1.8 folds, swertiamarin 2.5 folds

3.7 folds higher emodin, 3.5 higher Thiruvengadam and Pravin Physcion (2013) 71.35 mg/g higher than normal 35 mg/g Dixit and Vaidya (2010) 16.11 mg/g Sivakumar and Yu (2005) 8.93 mg/g 15.77 mg/g 13.27 mg/g 14.44 mg/g 12.58 mg/g 19.84 mg/g (Continued)

113

Phenylalanine Caffeic acid Catechin Chitin Gum karaya Fucoidan Pepton

0.30 mg g−1 3 folds verbacoside, 10 folds martynoside than normal

Hairy Roots and Plant Secondary Metabolites Production

TABLE 7.2 Updates on Secondary Metabolite—Glycoside Enhancement by in Hairy Root Culture by Using Different Agrobacterial Strains and Biotic, Abiotic Elicitors

Agro Bacterial Strain Used

Name of Plant

13

Gentiana cruciata

A4, 15834, 8196, R1000

14

Rhaponticum carthamoides

A4, ATCC 15834

15 16

Fogopyrum esculentum 15834 with binary vector pBI121 Rhamnus fallax A4M70GUS

17

Centella asiatica L

18

Scutellaria lateriflora

R1000 harbouring pCAMBIA1302 A4

19

Picrorhiza kurroa

LBA 9402

20

Calotropis gigantea

Cardenolides

21

Rhodiola kirilowii

LBA 9402 (NCPPB 1855) LBA 9402

22

Harpagophytum procumbens Physalis minima

A4, ATCC 15834

Verbacoside, Isoverbacoside Solasodine

23

ATCC 15834

Name of Glycoside

Elicitors Used

% Yield Obtained

Reference

Gentiopicroside, -Gentiopicroside lower in transformed Hyta and Gruel (2011) Loganic acid, roots than wild roots Swertiamarin, sweroside Glycosidal flavanoides Varying Media composition and Total flavanoid content found 2.93 mg/g Sakala and Kicel (2015) like Quercetagenin, light–dark condition higher than normal Quercetin, luteolin, patuletin hexosides Rutin -2.6 folds higher than normal Kyoung and Xu (2010) Anthraquinone glycosides Asiaticoside

--

16.43 mg/g, 14.21 mg/g

Methyl jasmonate

7.12 mg/g higher than normal

Acteoside, baicalin, Wogonoside Iridoid glycoside like picroliv

Yeast extract, Pectobacterium carotovorum lysate Various nutrient medium B5, MS, WP, NN and Sucrose concentrations (1–8%) Methyl jasmonate, Yeast extract, Chitosan Cinnamyl alcohol and 1% sucrose --

1.4 folds higher than normal

Rosavin

8% sucrose, NAA, Benzyladanine

27 folds increase in picroliv in bioreactor than normal 2.7 folds higher than normal with chitosan 505 mg/L with CA 8.12 mg/g, 9.97 mg/g higher than in normal tubers 900 g dry wt, 20 times higher as compared to normal

Rosic and Momcilovic (2006) Kim and Bang (2007) Wilczanska and Krolicka (2012) Verma and Singh (2015)

Sun and Xiao (2011) Grech and Syklowska (2014) Grabkowska and Krolicka (2009) Putalun and Prasarnsiwamai (2004)

Biotechnological Approaches to Enhance Plant Secondary Metabolites

Sr. No.

114

TABLE 7.2  (Continued) Updates on Secondary Metabolite—Glycoside Enhancement by in Hairy Root Culture by Using Different Agrobacterial Strains and Biotic, Abiotic Elicitors

25

Centaurium maritimum L Saussurea involucrata

26 27

Helicteres isora L Fogopyrum tataricum

28

Swrtia chirata

29

30 31 32

Bacopa monnieri

33

Calotropis gigantea

34

Senna alata

35

Polyginium multiflorum T Chlorophytum laxum (Figure 7.4) Rubia aken

37

Secoiridoid glycosides Used bioreactor

8% higher than normal

Misic and Siler (2013)

A4, LBA9402, R1000, R1601 ATCC15834 ATCC15834

Syringin

--

Fu and Zhao (2005)

Diosgenin Rutin

-Endophytic fungal isolates (Fusarium oxysporum) Fat9, (Alternaria Sp.) Fat 15 Salicylic acid, Chitosan, Tween 20 in bioreactor Methyl jasmonate, Salicylic acid Fungal cell wall elicitors --

R1601 gave 50 folds higher Syringin conc. than normal 8 folds higher than normal 3.1–3.2 folds than normal

1.5 folds increase than normal in bioreactor 10.5–18.3 mg/g (1.5–3 folds) increase than normal

Kiel and Hartle (2000)

Significant increase in diosgenin observed 7.12 mg/g dry wt

Yong (2009)

ATCC15834, LBA9402, TR105 Scutellaria baicalensis ATCC15834

Dioscorea zingiberensis Centella asiatica

36

A40M70GUS

Amarogentin Baicalin

R1601, A4, LBA9402 Diosgenin R1000 harbouring pCAMBIA1302 A4, R1000, SA79, MTCC532, MTCC2364 LBA9402 (NCPPB1855) ATCC15834 LBA9402 R1000

Kumar and Desai (2014) Zhao and Dabing (2014)

Hwang (2006)

Asiaticoside

Methyl Jasmonate

Bacoposide A

--

10.02 mg/g DW higher than untransformed roots

Bansal and Kumar (2014)

Cardenolides

Methyl Jasmonate, Yeast extract, chitosan 5% sucrose

Sun and Xiao (2012)

--

1.05 g/l which is 2.7-fold higher than control 169 µg/g DW, 34 µg/g higher than control 2.8 folds higher than control

--

1.5 folds higher than control

Deore and Kide (2015)

--

R1601 Shown 4.3 mg/g Alizarin and 4.9 mg/g purpurin higher than normal

Lee and Kim (2010)

Sennosides A, Sennosides B Anthraquinine glycoside Rhein Saponin glycosides

13333,15834, R1000, Alizarin, Purpurin R1200, R1601

Kim and Bang (2007)

Hairy Roots and Plant Secondary Metabolites Production

24

Putalun and Pimmeuangkao (2006) Wang and Yu (2002)

115

116

Biotechnological Approaches to Enhance Plant Secondary Metabolites

FIGURE 7.4  Hairy root development in Chlorophytum laxum. (From Deore 2015.)

From present review, it can be concluded that, profound research is undergoing to understand and develop various protocols and applications of hairy root culture. This method has wide range of applications which will play an important role in reduction of exploitation of commercially useful plants as well as environmental protection. This method is a savvier for endangered or rare plant species. However, still this method is far away from industrial set ups due to skill and variations in bioreactor outputs. Hence, more research is needed in this field to encourage industries to accept this technology for uniform and continuous raw material supply compared to indirectly killing thousands of plants by traditional raw plant material collection techniques. Huge commercialization of this technique is expected in near future. Contact Information: Please contact the author at: [email protected]

ACKNOWLEDGEMENT The authors are thankful to AICTE, Delhi for the RPS grant sanctioned to author Sharada L. Deore to explore applications of hairy root culture in herbal drug industry.

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8 The Phytohormones with Potential Brassinosteroids

to Enhance the Secondary Metabolite Production in Plants Barket Ali, Zahoor A. Wani and Mudasir Irfan Dar CONTENTS 8.1 Introduction........................................................................................................................... 125 8.2 Occurrence and Structure...................................................................................................... 126 8.3 Secondary Metabolites.......................................................................................................... 127 8.3.1 Terpenes or Terpenoids.............................................................................................. 127 8.3.2 Plant Phenolics........................................................................................................... 127 8.3.3 Nitrogen Containing Compounds.............................................................................. 128 8.3.4 Effect of Brassinosteroids on Secondary Metabolites............................................... 128 8.4 Conclusion............................................................................................................................. 130 References....................................................................................................................................... 130

8.1 INTRODUCTION Plants are bestowed with a large number of biochemical molecules, which serve as endogenous messengers. Some of these biochemical substances are produced at one site and are translocated to another site of the plant where in micro molar concentration they exert a profound influence on a physiological response (Hopkins 1995). All such compounds are designated as ‘phytohormones’. They are represented by five classical phytohormones namely, auxins, gibberellins, cytokinins, abscisic acid and ethylene. However, Mitchell (1970) discovered another group of plant hormones, which were initially named as ‘brassins’ after their source, Brassica napus pollen grains. However, subsequent investigations later revealed their polyhydroxylated steroidal nature and they were named as ‘Brassinosteroids’ (BS) (Fariduddin et al 2014). Initially, they were implicated in pollen tube growth or reproductive development. However, their role expanded to larger spectrum of responses related to physiology, growth and development of the plant. These responses included cell division, cell elongation, seed germination, reproductive development, vascular differentiation, senescence and ethylene production (Clouse and Sasse 1998). One of the most important roles that has been attributed to BS is their ability to enhance resistance against different stress situations that arise due to abiotic factors such as moisture, water, temperature, heavy metals, pesticide, salinity or biotic factors such as pathogen attacks (Gomes 2011, Sirhindi 2013). In addition, the exogenous application of BS also enhances the yield of different cereal crops, leguminous crops and oil seed crops (Hayat et al 2007) and thereby helps in improving the quality and quantity of fruits in various horticultural plant (Ali 2017, 2019).

DOI: 10.1201/9781003034957-8

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8.2  OCCURRENCE AND STRUCTURE The concentration of BS varies from one plant part to the other and one species to another. However, the maximum amount of BS has been observed in pollen grains (1–100 micro grams per kg fresh weight) (Clouse and Sasse 1998). Moreover, the younger tissue possesses BS quantity more when compared to the older ones. Furthermore, the occurrence of BS is spread throughout the plant kingdom which includes monocots, dicots, gymnosperms, pteridophytes, bryophytes and algae. The number of plants possessing BS is 69 (Figure 8.1). The structures of the most stable analogues of BS are given in Figure 8.2.

FIGURE 8.1  Different groups and number of plant belonging to them wherein BS occurrence has been demonstrated. (Adapted from Bajguz and Tretyn 2003.)

FIGURE 8.2  The most stable analogues of BS: Brassinolide (BL) (A) and 24-Epibrassinolide (EBL) and 28-Homobrassinolide (HBL) (B).

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8.3  SECONDARY METABOLITES Plant metabolites may be broadly categorized into two groups, viz., (a) primary metabolites such as amino acids, proteins, lipids and carbohydrates, which are essential and exists in almost every plant and (b) secondary metabolites, which are restricted to specific plants or related species. Earlier, secondary metabolites were considered as functionless or waste product. However, further investigations by chemists have revealed their importance in drugs, flavours, poisons and related industries (Rupasinghe et al 2014). From ecological point of view, they serve the following important functions in plant. a. As a repellent to herbivorous animals, thus ensuring the survival and existence of specific plant. b. As preventive agent against the microbial infections. c. As an attractant to pollinators for successful pollination. d. As an attractant to animals for wider seed dispersal. e. As a facilitator in plant-microbe interaction. f. Plant-plant competition. Secondary metabolites are classified into three broad categories (Taiz and Zeiger 2010).

8.3.1  Terpenes or Terpenoids They constitute the largest group of secondary metabolites which are synthesized from acetyl Co A or the intermediates of glycolysis. Structurally, they are composed of branched 5-C units called isoprene. Depending upon the number of isoprene units, they are named monoterpenes (10-C), sesquiterpenes (15-C), diterpenes (20-C), triterpenes (30-C), tetraterpenes (40-C) and polyterpenes. Terpenes are also known to play some primary roles in the plants. For example, they are the important constituents of phytohormones (gibberellins, abscisic acid), phytol tail of chlorophylls, carotenoids and lipid constituents of plasma membrane (Taiz and Zeiger 2010). Many plants possess the mixture of monoterpenes and sequiterpenes called essential oils. They give characteristic smell to the plant and act as insect repellent. Some of the commonly occurring essential oils include Limonene (lemon), Menthol (Mentha sp.), Azadirachtin (Azadirachta indica) and Phytoecdysone (Polypodium vulgare) (Figure 8.3).

8.3.2 Plant Phenolics Plant phenolics comprise a class that includes thousands of secondary metabolites. These are of ubiquitous occurrence in higher plants as well as in bryophytes. Some of them are soluble in organic solvents as well as in water (Nardini and Ghiselli 2004). They are synthesized both by Shikimic acid

FIGURE 8.3  Chemical structure of Limonene (A) Menthol (B) Azadirachtin (C) Ecdyosone (D).

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FIGURE 8.4  Chemical structure of caffeic acid (A), ferulic acid (B), vanilin (C) and salicylic acid (D).

pathway and/or Malonic acid pathway (Taiz and Zeiger 2010, Lattanzio 2013). Chemically, phenolic compounds are phenyl with one or two or more phenyl rings bearing one or more hydroxyl groups (Lattanzio 2013). Phenolics are classified into two classes; (a) simple phenolics such as caffeic acid, ferulic acid, vanillin and salicylic acid (Figure 8.4) and (b) complex phenolic macromolecules such as lignins, tennins and flavonoids (anthocyanin, anthocyanidin, flavones, flavanones, flavonols, flavanonols, flavan-3-ols, chalcones, dihydrochalcones, anthocyanidins, isoflavones, aurones and biflavonoids (Julkunen-Tiitto et al 2015).

8.3.3 Nitrogen Containing Compounds Some secondary metabolites possess nitrogen in their structure and are synthesized from one or more amino acids such as lysine, tyrosine and tryptophan. These compounds constitute a group of hundreds of molecules called alkaloids such as cocaine, nicotine, morphine and caffeine, and highly poisonous groups of cyanogenic glycocides and glucosinolate (Figure 8.5).

8.3.4 Effect of Brassinosteroids on Secondary Metabolites BS play an important role in the regulation of diverse primary and secondary metabolic responses in plant. However, the predominant part of research has focused on their involvement in primary metabolite synthesis and accumulation in plants. The primary metabolites on the other hand include amino acids, proteins, enzymes, nucleic acids, carbohydrates and other cellular components. Secondary metabolites include thousands of the molecules which are not essential for plants, although few of them are known to play some important roles in plants. BS have a great similarity with the secondary metabolites in the sense that they follow a common biosynthesis pathway. Both are known to be synthesized from isopentenyl diphosphate precursor via mevalonate pathway (Verma and Shukla 2015). The biosynthesis of secondary metabolites, specifically the plant phenolics involve some important enzymes like phenylalanine ammonia lyase (PAL) and γ-glutamyl-2-oxoglutrate amino transferase (GOGAT). Li et al (2016) observed an increase in the activity of these enzymes in tea plant when treated with 24-epibrassinolide (EBL). The increased activity of these enzymes led to an increase in the accumulation of total phenolics. The elevation in the concentration of phenolics was dependent on (a) concentration of EBL and

FIGURE 8.5  Chemical structure of cocaine (A), nicotine (B), morphene (C) and caffeine (D).

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(b) the collection time of the sample. The enhancement took place at 3 hours after treatment until 24 hours of the treatment. The stability was accomplished afterwards, at 0.5 ppm concentration, which proved to be the most effective in this study. Here, EBL also enhanced the level of catechein and theanine. However, the concentration of caffeine remained unaffected by the EBL treatment. Likewise, BS also increased the activity of PAL in Vitis vinifera (Xi et al 2013). The promontory effect of BS on PAL and possibly other biosynthesis-related enzymes led to the accumulation of total phenolics in the above mentioned study as well as in Ocimum basilicum (Koca and Karaman 2015) and Camellia sinensis (Li et al 2017). 28-Homobrassinolide (HBL) at 10 −6 M concentration improved the level of carvocrol and paracymene essential oil components in Satureja khuzzestanicas (Eskandari and Eskandari 2013). Similarly, mint (Mentha arvensis L.) plants treated with 10 −8, 10 −7 and 10 −6 M of HBL regularly at 10 days interval, starting from 2–3 true leaf stage, also exhibited an increase in the yield of the herbage as well as the content of most of the active constituents (menthol, L-methone, isomenthone and menthyl acetate). 10 −7 M concentration of the BS was the most effective, which was closely followed by the next higher concentration (10 −7 M) (Naeem et al 2012). The application of EBL to the foliage of peppermint (Mentha piperita L.) favourably affected its growth, and the total essential oil and phenolic contents, both under normal and salt stress conditions. Intermediate concentration (0.5 mg l−1) of BS significantly increased the essential oil content in non-stressed plants, which was 27.01% higher, compared to the control plants. However, the salt stress increased the concentration of phenolics and decreased that of the essential oil. BS treatment also improved the phenolics and essential oil contents in the plants exposed to the salt stress, thus minimizing the damage caused by salt stress (Çoban and Baydar 2016). In lavandin (Lavandula intermedia), the application of EBL (0, 0.75, 1.5 and 2.25 mg/L) to the foliage twice (at the beginning of the budding stage and 10 days after the first treatment) affected the plants, both quantitatively and qualitatively. EBL enhanced the total phenolics and essential oil contents significantly and also modified essential oil composition where 1.5 mg/L was the most effective dose (Asci et al 2019). Likewise, the foliar application of black mustard (Brassica nigra L.) with 0.1 μmol EBL at two stages (before flowering and at pod filling) significantly enhanced the phenolic compounds, flavonoids and anthocyanin (Ghassemi-Golezani et al 2020). Moreover, in Catharanthus roseus, the exogenous application of EBL enhanced the level of total alkaloids in the leaves and the roots. However, the alkaloid content remained unaffected in the stem. 10 −7 M concentration was the most effective that increased the alkaloid yield up to 9.8% in leaves and up to 60.9% in the roots (Alam et al 2016). Exploring the possible mechanism of flavonoid accumulation in tea (Camellia sinensis L.), in response to BS application, Li et al (2017) established that the BS regulate the synthesis of flavonoids via the synthesis of nitric oxide (NO). To verify the hypothesis, these scientists used a BS biosynthesis inhibitor, brassinazol (BRz) and a NO scavenger, 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Exogenous application of BS increased the concentration of both NO and flavonoids. However, BRz application decreased the concentrations of both NO and flavonoids. Similarly, NO scavenger cPTIO also decreased the level of flavonoids. BS follow-up treatment to the BRz-affected plants reversed the impact of BRz and also restored the levels of NO and flavonoids. Likewise, the follow-up treatment of cPTIO-affected plants with NO also improved the concentration of flavonoids. These observations clearly elucidated the mechanism of BS-induced flavonoid biosynthesis, which takes place via the intermediate accumulation of NO. The involvement of BS in secondary metabolism was further proved by the studies wherein microRNA (miRNA) analysis was done. MicroRNA (cs-mir414) regulates the endogenous BS level in tea/plants (Jeyaraj et al 2014). The synergism among the BS accumulation, miRNA and total phenolics in tea leaves clearly indicates that the synthesis and the accumulation of phenolics is facilitated by the BS, specifically in tea (Li et al 2016). BS also regulates the synthesis of some other metabolites like catechins and theanine in tea by regulating the expression of the genes involved in their biosynthesis (Li et al 2016). The expressions of the genes like GST1 (representing glutathione-s-transferase), SKDH (representing shikimate dehydrogenase), PAL (representing phenylalanine ammonia lyase) and CAD

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(representing cinnamyl alcohol dehydrogenase) were also enhanced by the EBL treatment. This upregulated expression of the genes was associated with the levels of phenols and flavonoids content. This synergistic effect of BS treatment with that of the expression of genes and subsequently the proteins (corresponding to them) clearly elaborates the enzyme-mediated role of BS in secondary metabolite synthesis and accumulation in plants (Ahammed et al 2013).

8.4 CONCLUSION Following conclusions can be drawn from the above. a. BS is a group of steroidal plant hormones implicated in diverse physiological responses including conferring resistance to abiotic and biotic stress conditions. b. BS also stimulate the synthesis of primary and secondary metabolites, although the number of studies related to their involvement in secondary metabolite synthesis is quite less. c. BS and secondary metabolites share the common precursors and pathway of biosynthesis. Both are synthesized from isopentenyl diphosphate precursor via mevalonate pathway. d. There are numerous plants species which exhibit an elevated level of different secondary metabolites including phenolics. Some of these secondary metabolites have medicinal significance. e. BS maintain the accumulation of secondary metabolites by involving the expression of the genes corresponding to the enzymes associated with their biosynthesis. f. Some of the plants which show promising response to BS treatment, in respect of secondary metabolite biosynthesis include Vitis vinifera, Ocimum basilicum, Satureja khuzzestanica, Brassica nigra, Camellia sinensis, Mentha piprata, Catharanthus roseus and Lavandula intermedia. g. Although BS treatment has promising influence on secondary metabolite synthesis, yet the studies are quite scarce. Therefore, more such studies need to be undertaken to elucidate further with respect to the role played by BS in secondary metabolite production in plants. Contact Information: Please contact the author at: [email protected]

REFERENCES Ahammed, G.J., Zhou, Y.H., Xia, X.J., Mao, W.H., Shi, K., and Yu, J.Q. 2013. Brassinosteroid regulates secondary metabolism in tomato towards enhanced tolerance to phenanthrene. Biologia Plantarum 57: 154–158. https://doi.org/10.1007/s10535-012-0128-9 Alam, M.M., Naeem, M., and Khan, M.M.A. 2016. Exploiting the epibrassinolide as a plant growth promoter for augmenting the growth, physiological activities and alkaloids production in Catharanthus roseus L. Journal of Medicinal Plants Studies 4: 88–93. Ali, B. 2017. Practical applications of brassinosteroids in horticulture – Some field perspectives. Scientia Horticultrae 225: 15–21. http://dx.doi.org/10.1016/j.scienta.2017.06.051 Ali, B. 2019. Brassinosteroids – The Promising plant growth regulators in horticulture. In Brassinosteroids: Plant Growth and Development, 349–365, eds. S. Hayat et al., Springer. https://doi. org/10.1007/978-981-13-6058-9_12 Asci, Ö.A., Deveci, H., Erdeger, A., Özdemir, K.N., Demirci, T., and Baydar, N.G. 2019. Brassinosteroids promote growth and secondary metabolite production in Lavandin (Lavandula intermedia Emeric ex Loisel.). Journal of Essential Oil Bearing Plants 22: 254–263. https://doi.org/10.1080/0972060X. 2019.1585298 Bajguz, A., and Tretyn, A. 2003. The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry 62: 1027–1046. Clouse, S.D., and Sasse, J.M. 1998. Brassinosteroids: essential regulators of plant growth and development. Annual Review of Plant Physiology and Plant Molecular Biology 49: 427–451. Çoban, Ö., and Baydar, N.G. 2016. Brassinosteroid effects on some physical and biochemical properties and secondary metabolite accumulation in peppermint (Mentha piperita L.) under salt stress. Industrial Crops and Products 86: 251–258. https://doi.org/10.1016/j.indcrop.2016.03.049

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Eskandari, M., and Eskandari, A. 2013. Effects of 28-homobrassinolide on growth, photosynthesis and essential oil content of Satureja khuzestanica. International Journal of Plant Physiology and Biochemistry 5: 36–41. Fariduddin, Q., Yusuf, M., Ahmad, I., and Ahmad, A. 2014. Brassinosteroids and their role in response of plants to abiotic stresses. Biologia Plantarum 58: 9–17. Ghassemi-Golezani, K., Hassanzadeh, N., Shakiba, M-R., and Esmaeilpour, B. 2020. Exogenous salicylic acid and 24-epi-brassinolide improve antioxidant capacity and secondary metabolites of Brassica nigra. Biocatalysis and Agricultural Biotechnology 26: 101636. https://doi.org/10.1016/j.bcab.2020.101636 Gomes, M.M.A. 2011. Physiological effects related to brassinosteroids applications in plants. In Brassinosteroids: A Class of Plant Hormone, 193–242. eds. S. Hayat and A. Ahmad, Springer, Dordrecht Heidelberg, London. Hayat, S., Ali, B., Hasan, S.A., and Ahmad, A. 2007. Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea. Environmental and Experimental Botany 60: 33–41. Hopkins, W.J. 1995. Introduction to Plant Physiology. John Wiley and Sons, Inc., New York. Jeyaraj, A., Chandran, V., and Gajjeraman, P. 2014. Differential expression of microRNAs in dormant bud of tea [Camellia sinensis (L.) O. Kuntze]. Plant Cell Report 33: 1053–1069. https://doi.org/10.1007/ s00299-014-1589-4 Julkunen-Tiitto, R., Nenadis, N., Neugart, S., Robson, M., Agati, G., Vepsäläinen, J., Zipoli, G., Nybakken, L., Winkler, B., and Jansen, M.A.K. 2015. Assessing the response of plantflavonoids to UV radiation: an overview of appropriate techniques. Phytochemistry Reviews 14: 273–297. Koca, N., and Karaman, S. 2015. The effects of plant growth regulators and L-phenylalanine on phenolic compounds of sweet basil. Food Chemistry 166: 515–521. https://doi.org/10.1016/j.foodchem.2014.06.065 Lattanzio, V. 2013. Phenolic compounds: Introduction. In Natural Products, 1543–1580, eds. K.G. Ramawat and J.M. Mérillon, Springer-Verlag, Berlin Heidelberg. https://doi.org/10.1007/978-3-642-22144-6_57 Li, X., Ahammed, G.J., Li, Z-X., Zhang, L., Wei, J-P., Shen, C., Yan, P., Zhang, L-P., and Han, W-Y. 2016. Brassinosteroids improve quality of summer tea (Camellia sinensis L.) by balancing biosynthesis of polyphenols and amino acids. Frontiers in Plant Science 7: 1304. https://doi.org/10.3389/fpls.2016.01304 Li, X., Zhang, L., Ahammed, G.J., Li, Z-X., Wei, J-P., Shen, C., Yan, P., Zhang, L-P., and Han, W-Y. 2017. Nitric oxide mediates brassinosteroid-induced flavonoid biosynthesis in Camellia sinensis L. Journal of Plant Physiology 214: 145–151. https://doi.org/10.1016/j.jplph.2017.04.005 Mitchell, J.W., Mandava, N., Worley, J.F., Plimmer, J.R., and Smith, M.V. 1970. Brassins: a new family of plant hormones from rape pollen. Nature 225: 1065–1066. Naeem, M., Idrees, M., Alam, M.M., Aftab, T., Khan, M.M., Moinuddin. 2012. Brassinosteroid-mediated enrichment in yield attributes, active constituents and essential oil production in Mentha arvensis L. Russian Agricultural Sciences 38: 106–113. Nardini, M., and Ghiselli, A. 2004. Determination of free and bound phenolic acids in beer. Food Chemistry 84: 137–143. Rupasinghe, H.P.V., Nair, S.V.G. and Robinson, R.A. 2014. Chemopreventive properties of fruit phenolic compounds and their possible mode of actions. In Studies in Natural Products Chemistry, 42: 229–266, ed. F.R.S. Atta-ur-Rahman, Elsevier, Amsterdam, The Netherlands. http://dx.doi.org/10.1016/ B978-0-444-63281-4.00008-2 Sirhindi, G. 2013. Brassinosteroids: Biosynthesis and role in growth, development, and thermotolerance responses. In Molecular Stress Physiology of Plants, 309–329, eds. G.R. Rout and A.B. Das, Springer, India. Taiz, L. Z., and Zeiger, E. 2010. Plant Physiology. Sinauer Associates, Sunderland, pp. 607–611. Verma, N., and Shukla, S. 2015. Impact of various factors responsible for fluctuation in plant secondary metabolites. Journal of Applied Research on Medicinal and Aromatic Plants 2, 105–113. http://dx.doi. org/10.1016/j.jarmap.2015.09.002 Xi, Z. M., Zhang, Z. W., Huo, S. S., Luan, L. Y., Gao, X., and Ma, L. N. 2013. Regulating the secondary metabolism in grape berry using exogenous 24-epibrassinolide for enhanced phenolics content and antioxidant capacity. Food Chemistry 141: 3056–3065. http://dx.doi.org/10.1016/j.foodchem.2013.05.137

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CRISPR-Cas9 Approaches to Enhance Contents of Plant Secondary Metabolites Riddhi Rajyaguru, Nataraja Maheshala, Chandrashekar Mootapally, Neelam Nathani, Rukamsingh Tomar, Hiren Bhalani and Priyanka Sharma

CONTENTS 9.1 Introduction........................................................................................................................... 133 9.2 Secondary Metabolites (SMs) in Plants................................................................................. 134 9.3 Classification of SMs............................................................................................................. 134 9.4 Role and Significance of SMs................................................................................................ 134 9.5 Genetic Engineering for Plant SMs....................................................................................... 134 9.6 Transgenic Approaches.......................................................................................................... 134 9.7 RNA Interference.................................................................................................................. 136 9.8 Advanced Genome Editing Tools.......................................................................................... 136 9.9 Clustered Regularly Interspaced Short Palindromic Repeats............................................... 136 9.10 Brief History of CRISPR Technology................................................................................... 137 9.11 Classification of CRISPR/Cas............................................................................................... 138 9.12 Workflow CRISPR/Cas9 System for Genome Editing of Plants........................................... 139 9.13 Advantages of CRISPR Technology...................................................................................... 140 9.14 CRISPR Applications for Production or Disruption of Plant SMs........................................ 140 9.14.1 Terpenes..................................................................................................................... 140 9.14.2 Phenolics.................................................................................................................... 141 9.14.3 Nitrogen-Containing Compounds............................................................................. 141 9.15 Limitations of CRISPR/Cas Systems.................................................................................... 146 9.16 Future Prospectus of CRISPR/Cas Systems in Plant Genetic Engineering for SMs............ 146 Acknowledgements......................................................................................................................... 146 References....................................................................................................................................... 146

9.1 INTRODUCTION CRISPR/Cas system is the most recent and advanced genetic engineering tool available for researchers today. Since its discovery and successful demonstrations, it is widely used for genome editing of various organisms, including plants. In plants, genome editing for secondary metabolism has attained greater significance due to increased demand for nutritional and medicinal compounds. In this chapter, authors have made efforts to describe secondary metabolites and application of CRISPR/Cas systems with illustrations and examples, wherever applicable. This chapter begins with a description on secondary metabolites in plants, their classification along with their role and significance. Followed by genetic engineering for secondary metabolites in plants with special emphasis on CRISPR/Cas systems.

DOI: 10.1201/9781003034957-9

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9.2  SECONDARY METABOLITES (SMs) IN PLANTS SMs are chemical compounds produced by plant cells and are byproducts of primary metabolite biosynthesis. Their production can be demonstrated genetically, physiologically and biochemically. They are low molecular weight organic compounds and have no role in growth, photosynthesis, reproduction or other ‘primary’ functions that are necessary for survival of plants. They are actively involved in plant-insect, plant-microbe and plant-plant interactions. More than 200,000 SMs have been identified from the kingdom Plantae, and they tend to be species specific.

9.3  CLASSIFICATION OF SMs SMs are broadly classified into terpenes, phenolics, nitrogen (N)- and sulphur (S)-containing compounds (Mazid et al. 2011). A comprehensive list of economically important SMs is furnished in Table 9.1.

9.4  ROLE AND SIGNIFICANCE OF SMs SMs have very diverse roles in plants: terpenoid carotenes, phenolics and flavonoids impart colour; terpenes and phenols add odour and flavour to flowers and edible parts. Presence of SMs negatively affect herbivory by insects (Rosenthal and Berenbaum 1991) and interferes in growth and survival of pathogens (Ramírez-Gómez et al. 2019). Adaptation of plants to edaphoclimatic stresses like, drought, cold, salinity etc. depends on SMs (Ramakrishna and Ravishankar 2011). Plants experiencing either biotic or abiotic stress produce excessive SMs, as a defence response (Naik and Al-Khayri 2016; War et al. 2012). Moreover, several plant SMs are used to produce medicines, dyes, insecticides, flavours and fragrances. Alkaloids, flavonoids and phytosterols are pharmaceutically active with antioxidant, anti-inflammatory, antispasmodic, anti-allergic, anti-cancer and antimicrobial properties (Jain et al. 2019). Primary health care largely depends on herbal/medicinal plant extracts and their derivatives (Ekor 2013).

9.5  GENETIC ENGINEERING FOR PLANT SMs An unceasing demand exists for exploration and exploitation of plant SMs on commercial scale. There has been enormous interest in genetic manipulation through either conventional breeding or genetic engineering approaches. Limitations of time and resources favour greater use of plant genetic engineering tools. Plants with altered biochemical pathways cause enhanced (sometimes reduced) production of SMs which can bring desired changes in the plant traits such as, better flavour, aroma, nutrition, resistance to insects and pathogens, resistance to abiotic stresses and production of drugs and other valuable biochemicals (Gómez-Galera et al. 2007; Nishihara and Nakatsuka 2010; Pichersky and Dudareva 2007; Sugiyama et al. 2011). The only prerequisite of genetic engineering for SMs is the availability of information on key genes involved in the biosynthesis of SMs. Once such genes have been identified, genetic engineering tools can be effectively employed for achieving targeted traits.

9.6  TRANSGENIC APPROACHES Enhanced production of existing metabolites or the production of new metabolites may result after a gene of interest is identified in a foreign organism and transferred into a target host plant. Indican (3-hydroxyindole-β-D-glucoside), a new metabolite which is not present in wild-type tobacco was produced in transgenic tobacco when genes associated with BX1, an enzyme in the biosynthesis of hydroxamic acids and human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) were introduced (Warzecha et al. 2007). In the absence of catalytically efficient native genes, engineered genes can

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TABLE 9.1 Economically Important SMs of Plant Origin Class of SMs

Description

Terpene

Largest and most Hemiterpenes (n = 1) diverse group of Monoterpenes (n = 2) Plant SMs; resin-like; polymers of isoprene Sesterterpenes (n = 2.5) Sesquiterpenes (n = 3) Diterpenes (n = 4) Triterpenes (n = 6) Phenol group present Simple phenolics in the structure

Phenolics

Nitrogen (N) containing compounds

Extremely diverse group

Sulphur (S) containing compounds

Rarely found in plants

Types

SMs of Economic Importance Angelic acid, Isovaleric acid Geraniol, Limonene, Linalool, Linalyl Acetate, Citronellal, Carvone Geranyl farnesol Farnesol, Bisabolol, Caryophyllene Vitamin K1, Vitamin A

Quassin, α-Boswellic acids, β-Boswellic acids Gallic acid, Eugenol, Vanillin, Salicylic acid, Ferulic acid, Caffeic acid, Hydroquinone, Capsaicinoids, Catechol, Phloroglucinol, Thymol Tannins Gallotannins, Ellagitannins, Proanthocyanidins, Geranilin, Tellimagrandin Coumarins Esculetin, Umbelliferone, Scopoletin Flavonoids Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Proanthocyanidins, Theaflavins, Thearubigins, Kaempferol, Myricetin, Quercetin, Isorhamnetin, Apigenin, Luteolin, Baicalein, Chrysin, Eriodictyol, Hesperetin, Naringenin, Daidzein, Genistein, Glycitein, Biochanin A, Formononetin Chromones and xanthones Eugenin, Khellin, Euxanthone Stilbenes Resveratrol Lignans Lariciresinol, Pinoresinol Secoisolariciresinol, Matairesinol Alkaloids Coniine, Nicotine, Berberine, Papapverine, Morphine, Codeine, Thebaine, Quinine, Atropine, Colchicine, Strychnine, Brucine, Cocaine, Cannabidiol, Lupinine, Reserpine, Ergatomine, Tomatin, Solanine Cyanogenic Glycosides Amygdalin, Linamarin, Lotaustralin, Dhurrin, and Glucosinolates Benzylglucosinolate Non-Protein Amino Acids Canavanine, Albizziine, Ornithine, Mimosine S-2-Propenyl L-Cysteine Sulfoxide (Alliin), Trans S-1-Propenyl L-Cysteine Sulfoxide, S-Propyl L-Cysteine Sulfoxide, S-Methyl L-Cysteine Sulfoxide, Camalexin (3-Thiazol-2′-Yl-Indole), 5-(3-Buten-1-Ynyl)-2,2′-Bithiophene, Trithiophene Α-Tertienyl

Source: Hussein and EL-Anssary (2018).

also be used to achieve higher production of metabolites. For example, overexpression of the HMGCoA reductase (HMGR) gene led to the dramatic increase of phytosterols in transgenic tobacco seeds (Hey et al. 2006). Similarly, a 78-fold increase in flavonoid production was achieved in tomato peel by inserting the Petunia CHI gene which codes for Chalcone Isomerase, an enzyme in the flavonoid biosynthesis pathway (Muir et al. 2001). Agrobacterium-mediated gene transfer is a well understood technique in genetic engineering of plants. It was used to successfully transfer the geraniol 10-hydroxylase gene to Catharanthus

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roseus (Pan et al. 2012), the squalene synthesis (PgSS1) gene to Panax ginseng (Seo et al. 2005). Similarly, Deng et al. (2017) transferred 3-hydroxy-3-methylglutaryl CoA reductase (PnHMGR) and squalene synthase (PnSS) in Panax notoginseng to regulate triterpenoid saponin biosynthesis. Multiple enzymes are often involved in secondary metabolism. Wu et al. (2006) achieved higher levels of sesquiterpenes, patchoulol and amorpha-4, 11-diene by over expressing patchoulol synthase and avian farnesyl diphosphate synthase genes in transgenic tobacco. Fujisawa et al. (2009) introduced the complete biosynthesis pathway of in ketocarotenoid by transferring a set of seven genes into Brassica napus. Similarly, ‘Golden Rice’ is another example of pathway engineering (Ye et al. 2000) where, genes from various sources have been transferred to create a functional β-carotene biosynthetic pathway in rice. Farhi et al. (2011) have used a mega-vector harbouring five genes of the artemisinin biosynthetic pathway in transgenic tobacco plants. Full understanding of the complexity of secondary metabolism and their temporal and spatial regulations is necessary for a successful transgenic approach (Dudareva and Pichersky 2008; Lange and Ahkami 2013). Apart from structural genes, regulation of transcription factors (TFs) controlling expression of structural genes can be beneficial (Ding et al. 2017). For instance, Butelli et al. (2008) co-expressed two TFs bHLHDelila and MYBRosea1 from Antirrhinum majus in transgenic tomato resulted in dramatic accumulation of anthocyanin pigments.

9.7  RNA INTERFERENCE Some secondary metabolites are antinutritional or detrimental and their elimination can be achieved by down regulation of the genes responsible. RNA interference technology (RNAi, dsRNA-mediated gene silencing) is where small molecules known as interfering RNA are used to suppress a target gene. Down regulation of the HCT gene coding for hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase, reduced accumulation of lignin (Hoffmann et al. 2004). Similar results were observed in Arabidopsis, Nicotiana bentamiana and Pinus radiate (Wagner and Kroumova 2008). In contrast, Van der Rest et al. (2006) suppressed the CCR (cinnamoyl-CoA reductase) gene in tomato to cause accumulation of soluble, health-related phenolics. However, knockdown of any gene of interest through RNAi has some disadvantages such as, unpredictable off-target effects and temporary inhibition of gene function (Gaj et al. 2013).

9.8  ADVANCED GENOME EDITING TOOLS Tools like, Zinc-Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) permanently and precisely knock out any targeted gene from complex genome of plants. Both ZFNs and TALENs are protein-DNA based while, CRISPR is RNA-DNA based. They cause double-strand break at specific sites in the plant genome, which get repaired by either nonhomologous end-joining break (NHEJ) or homology-directed repair (HDR) resulting in gene mutation at target site (Upadhyay et al. 2013; Xiong et al. 2015). Zhang et al. (2010) employed ZFNs to induce a high frequency mutation in tt4 to down regulate anthocyanins production in the seed coats of Arabidopsis. TALENs have tremendous advantages over ZFNs, because of their one-to-one recognition of nucleotides, which makes them easier to design and construct (Boch et al. 2009). TALENs for plant genome editing have been successfully demonstrated in Arabidopsis, tobacco, rice, barley, maize and Brachypodium (Holme et al. 2017; Liang et al. 2014; Shan et al. 2013). However, there is no literature available with respect to the application of TALENs for altering secondary metabolism in plants.

9.9  CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS CRISPR is an adaptive immunity acquired by prokaryotes like bacteria and archaea over evolutionary history (Barrangou 2015). They incorporate the DNA segments from invading phages or plasmids into their own genome and transcribe them; this is known as a CRISPR array. These series

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FIGURE 9.1  CRISPR/Cas9-based gene modification. Cas9 protein complex with guide RNA. After the gRNA binds to the target site of genomic DNA, the Cas9 protein creates a double strand break around the PAM site. Random indels or precise modifications introduced into the genomic DNA by the NHEJ or HDR pathway. Illustration is created on BioRender.com.

work as memory against the same phage or plasmids if reencountered by generating RNA fragments that target the viral genome. CRISPR-associated protein 9 (Cas9) is the most used enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA, complementary to the CRISPR sequence (Figure 9.1). CRISPR and Cas9 together form very effective CRISPR technology, with a wide range of applications including but not limited to functional genomics, genetic engineering of organisms, development of biotechnology products, and treatment of diseases (Hsu et al. 2014). Guide RNAs (gRNA) are also coded by CRISPR arrays which guide Cas endonucleases to cleave target DNA sequences.

9.10  BRIEF HISTORY OF CRISPR TECHNOLOGY Ishino et al. (1987) first observed an interval of a non-repetitive sequence of 32 nucleotides and tandem repeats downstream from the iap gene in the gut microbe Escherichia coli. These tandem repeats were again observed in archaea, Haloferax mediterranei by Mojica et al. (1993). With advances in genomic studies, sequence similarities between spacer regions in unique sequences of bacteriophages, archaeal viruses, and plasmids finally shed light on the function of these sequences in the immune system. In 2002, these tandem repeats were named as Clustered Regularly Interspaced Short Palindromic Repeats. Three independent research groups observed CRISPR protospacer sequences that shared high homology with exogenous sequences with bacterial plasmid and phages (Bolotin et al. 2005; Mojica et al. 2005; Pourcel et al. 2005). Bolotin and co-workers (2005) identified a novel Cas gene coding for a large protein with nuclease activity, which was later named Cas9. They also observed presence of a common sequence, protospacer adjacent motif (PAM) which is crucial for target recognition.

138

Biotechnological Approaches to Enhance Plant Secondary Metabolites

Barrangou et al. (2007) experimentally demonstrated adaptive immunity imparted by the CRISPR/Cas9 system in Streptococcus thermophilus. In 2008, phage derived spacer sequences in E. coli were transcribed into small RNAs (crRNA) that guided Cas proteins to the target DNA (Brouns et al. 2008). The target molecule for the CRISPR/Cas9 system is only DNA as demonstrated by Marraffini and Sontheimer (2008). However, different types of CRISPRs are available to target RNA molecules (Hale et al. 2009). Later, Garneau et al. (2010) used the CRISPR/Cas9 system to induce double-stranded breaks in DNA, 3 nucleotides upstream of the PAM. The final critical information came from Deltcheva et al. (2011) where they showed that tracrRNA formed a duplex with crRNA, and this duplex guides Cas9 to its targets in S. pyogenes. Research groups of Emmanuelle Charpentier and Jennifer Doudna simplified the CRISPR/Cas9 system by synthesizing a single gRNA, fusion of crRNA and tracrRNA (Jinek et al. 2012). This revolutionized CRISPR technology for wider usage in genome editing. Immediately, Zhang and co-workers successfully harnessed CRISPR/Cas9 for genome editing in eukaryotic cells of humans and mouse (Cong et al. 2013). More recently, CRISPR/Cas9 technology has found greater application in agriculture, gene therapy and medicine, drug discovery, and producing knockout genes, etc., (Liu et al. 2017).

9.11  CLASSIFICATION OF CRISPR/Cas There are two different classes of CRISPR-Cas systems namely, Class I and Class II systems, divided based on architectural principles of the effector modules with unique signature proteins (Figure 9.2). For degrading foreign nucleic acids, Class I systems use a complex of multiple subunits of Cas proteins, while Class II systems use a single large Cas protein. The Class I system is further divided into Types I (Cas3 cascade), III (Csf1) and IV (Cas10). Likewise, the Class II system is further divided into Types II (orthologs and variants of Cas9), V (orthologs and variants of

FIGURE 9.2  Broad classification of Cas proteins, their types and sub-types.

CRISPR-Cas9 Approaches to Enhance Contents of Plant Secondary Metabolites

139

Cas12), and VI (Cas13). These 6 types further comprise a total of 19 sub-types and are exclusively characterized by presence of a ‘signature gene’ (Westra et al. 2016). Type II (Cas9) is the most used CRISPR/Cas system in the genetic engineering of plants for secondary metabolite production or disruption.

9.12  WORKFLOW CRISPR/Cas9 SYSTEM FOR GENOME EDITING OF PLANTS A typical CRISPR/Cas9 system for genome editing in plants usually comprises the following four steps, as detailed in Figure 9.3 (Schiml et al. 2016; Yin et al. 2017). First, one must design a gRNA sequence for a selected genome region. It includes a target sequence of 20 bp followed by a PAM. Note that the choice of PAM sequences is determined by species source of the applied Cas9 protein (Srivastava, 2019). The second step is to assemble a codon optimized Cas9/sgRNA construct with a plant RNA polymerase III promoter (U6 or U3). The efficiency of sgRNA is confirmed in protoplasts prior to its use in genome editing. The third step is to transform the CRISPR/Cas9 into plant tissues, via particle bombardment, Agrobacterium-mediated transformation, or polyethylene glycol (PEG)-mediated transformation for stable integration of Cas9 and sgRNA cassette into the genome. The fourth step is to verify the transformation. Mutant or transformed (regenerated) plants with the anticipated changes are identified by polymerase chain reaction (PCR) genotyping (restriction enzyme loss) or a surveyor assay (such as Mismatch cleavage, Heteroduplex mobility, High resolution melting etc.) and confirmed by next generation sequencing (NGS).

FIGURE 9.3  Typical CRISPR/Cas9 system workflow for genome editing in plants. Cas9/gRNA construct’s legend as follows: LB = left border, P = promoter, Tor = target of rapamycin, gRNA = guide RNA, Cas9 = CRISPR Associated protein9, SM = selection marker, and RB = right border.

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Biotechnological Approaches to Enhance Plant Secondary Metabolites

9.13  ADVANTAGES OF CRISPR TECHNOLOGY CRISPR technology enjoys the following advantages over the RNAi, ZFNs and TALENs. It is (i) highly specific and relatively simple due to its plasmid design and construction, (ii) easily programmable by changing the guide sequence of the sgRNA to any DNA sequence of interest with its capacity to recognize multiple targeted genes, and (iii) has very low off-target effects due to reduced CpG methylation. According to Khan et al. (2019) it can modify chromosomal targets with the Cas9 protein for any number of different sequence specific gRNAs.

9.14 CRISPR APPLICATIONS FOR PRODUCTION OR DISRUPTION OF PLANT SMs As described in the earlier sections, SMs of plant origin are very vast and in high demand. Apart from traditional breeding, transgenic approaches, and advanced techniques (RNAi, ZFNs and TALENs), CRISPR systems can be very effectively employed to either enhance or disrupt production of SMs in plants. For ease of understanding, applications of CRISPR systems in plants are grouped into terpenes, phenolics, and nitrogen (N)-containing compounds (Table 9.2). No research efforts have been reported in the application of CRISPR systems for altering production of Sulphur (S)-containing compounds.

9.14.1  Terpenes A huge chunk of work on the application of CRISPR/Cas9 has been conducted on gene coding for phytoene desaturase (PDS), which converts 15-cis-phytoene into zeta-carotene (Stanley and Yao-Wu 2019). The carotenoid biosynthesis pathway is responsible for production of coloured compounds like, lycopene, carotenes (α, β, γ, and δ), zeinoxanthin, β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, lutein, etc. PDS silenced plants display photobleaching phenotype of leaves (albino) and impaired pigment formation in fruits. Due to this, PDS was extensively used in demonstrating CRISPR systems applications in plants (Table 9.2). Tobacco (Nicotiana benthamiana) was the first plant where, CRISPR/Cas9 was used for knocking out the gene, NbPDS to obtain an albino phenotype with a mutation rate of 2.1 (Nekrasov et al. 2013) to 14.2% (Li et al. 2013). Over expression and inversion/deletions of NbPDS were respectively achieved by Ali et al. (2015) and Gao et al. (2015). In cultivated tobacco (N. tobacum), NtPDS was knocked out to achieve 8.2% mutation with CRISPR/Cas9 by Chen et al. (2018) and Ren et al. (2020). In common wheat, Upadhyay et al. (2013) produced an albino phenotype by knocking out CtPDS1 with a mutation rate of 12.7%. Similarly, in rice disruptions to the gene OsPDS effected through CRISPR/Cpf1 and CRISPR/Cas12, respectively with mutation rates of 13.6 to 21.4 and 32.3% (Banakar et al. 2020; Xu et al. 2017). Dong et al. (2020) knocked out two genes involved in the carotenoid cassette (GR1 and GR2) to enhance carotene content in rice using CRISPR/Cas9. Apart from SlPDS (Parkhi et al. 2018) in tomato, CRISPR/Cas9 was also successfully utilized for knocking out five genes (SGR1, Blc, LCY-B1, LCY-B2 and LCY-E) involved in enhancing lycopene content with mutation rates ranging from 0 to 95.8% (Li et al. 2018). Strigolactone content in tomato was reduced by CRISPR/Cas9 mediated knocking out of CCD8, carotenoid cleavage dioxygenase 8 (Bari et al. 2019). CRISPR/Cas9 successfully realized albino phenotypes in vegetables like Chinese kale, cabbage and carrots by knocking out PDS with mutation rates of 76.5, 37.5 and 35.3%, respectively (Ma et al. 2019; Sun et al. 2018; Xu et al. 2019). Also, in commercial crops like cassava a high mutation rate of 90–100% was achieved in albino phenotypes by knocking out PDS by employing CRISPR/Cas9 (Odipio et al. 2017) while, it was only 4.5% in chicory (Bernard et al. 2019). In fruit crops CRISPR/Cas9 was highly successful with very high mutation rates ranging from 42 to 100%. Knocking out of the PDS gene resulted in an albino phenotype in the case of sweet

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orange (Jia and Wang 2014), grape (Nakajima et al. 2017), banana or plantain (Kaur et al. 2018; Ntui et al. 2019), strawberry (Wilson et al. 2019; Zhou et al. 2018), kiwifruit (Wang et al. 2018), watermelon (Tian et al. 2017), and melons (Hooghvorst et al. 2019). Similarly, PDS was knocked out using CRISPR/Cas9 to develop albino phenotypes in flower or ornamental plants like, garden petunia with 55.6 to 87.5 per cent mutation rate (Zhang et al. 2016); Chinese white poplar with 51.7 per cent mutation (Fan et al. 2015); and lilies with 63.4 to 69.6 per cent mutation rate (Yan et al. 2019). In contrast, CRISPR/Cas9 was used to regulate the CCD gene to elevate carotene content in petals of the Japanese morning glory (Watanabe et al. 2018). Production of triterpenoids like, soyasapogenols and soyasaponin were halted when, CRISPR/Cas9 knocked out the cytochrome P-450 family of genes (CYP716A51 and LjCYP93E1) in lotus (Suzuki et al. 2019). Plants with pharmaceutical importance like, red sage and Chinese yam were the first to be exploited for CRISPR/Cas9 applications (Table 9.2). Li et al. (2017) successfully reduced production of tanishones and achieved white roots in red sage by knocking out diterpene synthase gene (SmCPS1) with a 42.3% mutation rate. Similarly, reduced transcription of the farnesyl pyrophosphate synthase gene (DzFPS) was observed with a 60% mutation rate in Chinese yam (Feng et al. 2018).

9.14.2 Phenolics Phenolic compounds like, flavonoids, coumarins, lignins and phenolic acids have also been targeted in plants with the CRISPR/Cas9 system (Table 9.2). Many researchers have successfully altered the anthocyanin biosynthesis pathway. Cermak et al. (2015) achieved over expression of anthocyanin mutant 1 gene (ANT1) with 29.0% mutation rate in tomato. In contrast, CRISPR/Cas9 knocked out genes coding for enzymes like, favanone-3-hydroxylase (F3H) in carrot with a 90% mutation rate (Klimek-Chodacka et al. 2018) and in wishbone flower with an 80% mutation rate (Nishihara et al. 2018); anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3′-O-glycosyltransferase (Gt3′GT), anthocyanin 5/3′-aromatic acyltransferase (Gt5/3′AT) in Gentian with mutation rates ranging from 4.9 to 8.6% (Tasaki et al. 2019) and in wishbone flower (Nishihara et al. 2018). Xu et al. (2020) produced purple pigmented carrots by knocking out R2R3-MYB transcription factor gene (DcMYB113) using CRISPR/Cas9 with a 36.4% mutation rate while, Yang et al. (2017) reduced production of phenolics by over expressing R2R3-MYB transcription factor gene (PtoMYB156) involved in phenylpropanoid biosynthesis pathway of poplar, with a mutation rate of 48.0%. CRISPR/Cas9 was also successfully used to disrupt genes encoding, 4-coumarate: CoA ligase (4CL) with 100% mutation rate in cottonwood and squalene synthase (SQS) with an 84.6% mutation rate in sweet wormwood for elevated artemisinin content, a malarial drug (Koerniati and Simanjuntak 2020; Zhou et al. 2015). Similarly, function of rosmarinic acid synthase gene (SmRAS) was disrupted in red sage by Zhou et al. (2018). A fibre-specific knockout of HCT gene, responsible for lignin production in Arabidopsis was achieved with a 9 to 14% mutation rate by Liang et al. (2019).

9.14.3  Nitrogen-Containing Compounds Very meagre research has happened in the application of CRISPR/Cas systems for altering production of nitrogen-containing compounds (Table 9.2). Gene knockout was achieved with CRISPR/ Cas9 specifically in alkaloid biosynthesis pathways. Alagoz et al. (2016) accomplished reduced production of benzylisoquinoline alkaloid (BIAs) like morphine and thebaine in Opium poppy by targeting R, S-reticuline 7-O-methyltransferase gene (7OMT) and 3′-hydroxyl-N-methylcoclaurine 4′-O-methyltransferase gene (4′OMT2) with an 80 to 85% mutation rate. Likewise, Schachtsiek and Stehle (2019) realized reduced production of nicotine in tobacco by knocking out the berberine bridge enzyme-like family gene (BBL) while, Nakayasu et al. (2018) completely abolished accumulation of steroidal glycoalkaloids (SGAs) in potato by a 2-Oxoglutarate-dependent dioxygenase (2OGD or St16DOX) gene knock out.

142

TABLE 9.2 Application of CRISPR Systems for Plant Secondary Metabolites Production/Disruption Plant Species

Target Gene(s)

SM Pathway Targeted

Screening of Edited Plants

Mode of Action

Nuclease Used

Mutation Rate (%)

NbPDS

-do-

-do-

Gene knockout

Cas9

14.2 2.1

NbPDS NtPDS

-do-do-

-do-do-

Cas9 Cas9

81.8

-do-

Gene over expression Gene inversions/ deletion Gene knockout

NtPDS

-do-

Cas9

8.2

CtPDS1

-do-

-do-

-do-

Cas9

12.7

OsPDS OsPDS Carotenoid cassette (GR1 and GR2) PDS

-do-do-do-

Gene disruption Gene disruption Gene knockout

Cpf1 Cas12 Cas9

13.6–21.4 32.3 –

Gene knockout

Cas9

–

Stay-green 1 (SGR1), lycopene ε-cyclase (LCY-E), beta-lycopene cyclase (Blc), lycopene β-cyclase 1 (LCY-B1), lycopene β-cyclase 2 (LCY-B2) Carotenoid Cleavage Dioxygenase 8 (CCD8)

Lycopene biosynthesis

-do-doHigh carotene content Albino phenotype Enhanced lycopene content

-do-

Cas9

0.0 to 95.8

Gene knockout

Cas9

–

Bari et al. (2019)

BoPDS

Carotenoid biosynthesis

Reduced strigolactone production Albino phenotype

Gene knockout

Cas9

76.5

Sun et al. (2018)

Reference(s)

A. Terpenes Tobacco (Nicotiana benthamiana)

Wheat (Triticum aestivum) Rice (Oryza sativa)

Tomato (Solanum lycopersicum)

Chinese kale (Brassica oleracea var. sabellica)

-do-

Strigolactone biosynthesis

Chen et al. (2018) and Ren et al. (2020) Upadhyay et al. (2013) Xu et al. (2017) Banakar et al. (2020) Dong et al. (2020) Parkhi et al. (2018) Li et al. (2018)

Biotechnological Approaches to Enhance Plant Secondary Metabolites

Tobacco (Nicotiana tobacum)

Li et al. (2013) Nekrasov et al. (2013) Ali et al. (2015) Gao et al. (2015)

BoPDS

-do-

-do-

-do-

Cas9

37.5

Ma et al. (2019)

DcPDS

-do-

-do-

-do-

Cas9

35.3

Xu et al. (2019)

MePDS

-do-

-do-

-do-

Cas9

Odipio et al. (2017)

CiPDS

-do-

-do-

-do-

Cas9

90.0 to 100.0 4.5

Phytoene desaturase (CsPDS)

Carotenoid biosynthesis -do-

Albino phenotype -do-

Gene regulation

Cas9

3.5

Jia and Wang (2014)

-do-

Cas9

61.5

Nakajima et al. (2017)

AdPDS

-do-do-do-do-do-

-do-do-do-do-do-

-do-do-do-do-do-

Cas9 Cas9 Cas9 Cas9 Cas9

59.0 100.0 49.0 to 75.0 – 0.0 to 91.7

ClPDS

-do-

-do-

-do-

Cas9

100.0

CmPDS

-do-

-do-

-do-

Cas9

PhPDS

-do-

-do-

-do-

Cas9

PtoPDS

-do-

-do-

-do-

Cas9

LpPDS

-do-

-do-

-do-

Cas9

Carotenoid Cleavage Dioxygenase (CCD)

Carotenoid biosynthesis

Lotus (Lotus japonicus)

Cytochrome P-450 family genes (CYP716A51 and LjCYP93E1)

Triterpenoid biosynthesis

Elevated Gene regulation accumulation in petals Absence of Gene knockout soyasapogenols and soyasaponin

VvPDS MaPDS PDS

Bernard et al. (2019)

Kaur et al. (2018) Ntui et al. (2020) Zhou et al. (2018) Wilson et al. (2019) Wang et al. (2018) Tian et al. (2017)

42.0 to 45.0 Hooghvorst et al. (2019) 55.6 to 87.5 Zhang et al. (2016) 51.7

Fan et al. (2015)

63.4 to 69.6 Yan et al. (2019)

Cas9

–

Watanabe et al. (2018)

Cas9

–

Suzuki et al (2019)

CRISPR-Cas9 Approaches to Enhance Contents of Plant Secondary Metabolites

Cabbage (B. oleracea var. capitata) Carrot (Daucus carota subsp. sativus) Cassava (Manihot esculenta) Chicory (Cichorium intybus) Sweet oranges (Citrus sinensis) Grape (Vitis vinifera) Banana (Musa sp.) Strawberry (Fragaria × ananassa) Kiwifruit (Actinidia deliciosa) Watermelon (Citrullus lanatus) Melon (Cucumis melo) Garden petunia (Petunia hybrid) Chinese white poplar (Populus tomentosa) Lilly (Lilium longiflorum) Japanese morning glory (Ipomoea nil)

143 (Continued)

144

TABLE 9.2  (Continued) Application of CRISPR Systems for Plant Secondary Metabolites Production/Disruption SM Pathway Targeted

Target Gene(s)

Red sage (Salvia miltiorrhiza)

Diterpene synthase gene (SmCPS1) Tanshinone biosynthesis

Chinese yam (Dioscorea zingiberensis)

Farnesyl pyrophosphate synthase gene (DzFPS)

Diosgenin biosynthesis

Tomato (S. lycopersicum)

Anthocyanin mutant 1 (ANT1)

Anthocyanin biosynthesis

Carrot (D. carota subsp. sativus) Wishbone flower (Torenia fournieri)

Favanone-3-hydroxylase gene -do(F3H) F3H, -doanthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3′-O-glycosyltransferase (Gt3′GT), and anthocyanin 5/3′-aromatic acyltransferase (Gt5/3′AT) -doGt5GT, Gt3′GT, and Gt5/3′AT

Screening of Edited Plants

Nuclease Used

Mutation Rate (%)

Reference(s)

White roots and -doreduced tanshinones Reduced -dotranscription of DzFPS

Cas9

42.3

Li et al. (2017)

Cas9

60.0

Feng et al. (2018)

Purple pigmented tomatoes Discolouring of callus Discolouring of callus

Over expression

Cas9

29.0

Cermak et al. (2015)

Gene knockout

Cas9

90.0

-do-

Cas9

80.0

Klimek-Chodacka et al. (2018) Nishihara et al. (2018)

Elevated anthocyanin content Purple pigmented carrots Reduced production of phenolics

-do-

Cas9

4.9 to 8.6

-do-

Cas9

36.4

Xu et al. (2019)

Over expression

Cas9

48.0

Yang et al. (2017)

Mode of Action

B. Phenolics

Gentian (Gentiana sp.) Carrot (D. carota subsp. sativus) Poplar (Populus sp.)

R2R3-MYB transcription factor gene (DcMYB113) R2R3-MYB transcription factor gene (PtoMYB156)

-do-

Phenylpropanoid biosynthesis

Tasaki et al. (2019)

Biotechnological Approaches to Enhance Plant Secondary Metabolites

Plant Species

4-coumarate: CoA ligase gene (4CL)

Sweet wormwood (Artemisia annua)

Squalene synthase gene (SQS)

Red sage (Salvia miltiorrhiza)

Rosmarinic acid synthase gene (SmRAS)

Rockcress (Arabidopsis thaliana)

Hydroxycinnamoyl‑CoA shikimate/quinate hydroxycinnamoyltransferase gene (HCT)

-do-

Elevated Artemisinin content Sterol biosynthesis Elevated Artemisinin content Phenolic acid Elevated biosynthesis rosmarinic acid content Lignin biosynthesis Reduced production of lignin

Gene disruption

Cas9

100.0

Zhou et al. (2015)

-do-

Cas9

84.6

Koerniati and Simanjuntak (2020)

-do-

Cas9

–

Zhou et al. (2018)

Fibre-specific knockout

Cas9

9.0 to 14.0

Liang et al. (2019)

R, S-reticuline 7-O-methyltransferase gene (7OMT) and 3′-hydroxylNmethylcoclaurine 4′-O-methyltransferase gene (4′OMT2) Berberine bridge enzyme-like family gene (BBL)

Benzylisoquinoline Reduced alkaloid (BIAs) production of biosynthesis BIAs like, morphine and thebaine

Gene knockout

Cas9

80.0 to 85.0 Alagoz et al. (2016)

Alkaloid biosynthesis

-do-

Cas9

–

Schachtsiek and Stehle (2019)

2-Oxoglutarate-dependent dioxygenase (2OGD or St16DOX)

Steroidal glycoalkaloid (SGAs) biosynthesis

-do-

Cas9

–

Nakayasu et al. (2018)

C. Nitrogen-Containing Compounds Opium poppy (Papaver somniferum)

Tobacco (N. benthamiana) Potato (S. tuberosum)

Reduced nicotine production Complete abolition of SGAs accumulation

CRISPR-Cas9 Approaches to Enhance Contents of Plant Secondary Metabolites

Cottonwood (P. tremula × P alba)

145

146

Biotechnological Approaches to Enhance Plant Secondary Metabolites

9.15  LIMITATIONS OF CRISPR/Cas SYSTEMS Although CRISPR/Cas system is the most advanced tool for genetic engineering, it is still pestered with some limitations as listed by Manghwar et al. (2019): (i) off-target mutations at very low frequency, (ii) low homology-directed repair efficiency, (iii) limited availability of PAM sites, (iv) bigger protein size affecting mutation efficiency, (v) high commercialization costs and (vi) policy regulations governing varietal release. Apart from these, lack of knowledge regarding the functional genes involved in secondary metabolism is also a huge stumbling block.

9.16 FUTURE PROSPECTUS OF CRISPR/Cas SYSTEMS IN PLANT GENETIC ENGINEERING FOR SMs Since, 2012 several researchers have successfully demonstrated the application of CRISPR/Cas systems for SMs in plants (Table 9.2). However, many of them were only to check efficiency and suitability of the system. This was evident, as most of them (ca. 50%) worked on highly visible traits like, albino phenotype controlled by the PDS gene. This may be due to lots of uncertainties still surrounding the CRISPR/Cas system. Elucidation of enzymes and genes involved in secondary metabolism is foundational for successful CRISPR/Cas applications in plants. Functional genomics of currently unknown genes can be studied by knocking them out followed by phenotyping. A tremendous scope exists for CRISPR/Cas system in the genetic engineering of plants (Manghwar et al. 2019) with, (i) identification of smaller sized CRISPR/Cas9 systems that can utilize virus as vectors, (ii) multiple PAM sites which can enhance the targeting mechanism, (iii) development of tissue-culture free genetic engineering with CRISPR/Cas system will reduce off-target mutations, and (iv) research efforts required to improve HDR and viral vector efficiencies.

ACKNOWLEDGEMENTS The authors are thankful to Dr. Jennifer Baltzegar from North Carolina State University, Raleigh for conducting a critical review of this manuscript. Contact Information: Please contact the author at: [email protected]

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RNA Interference for Improvement of Secondary Metabolite Production in Plants Ashutosh Kumar Rai and Pramod Wasudeo Ramteke

CONTENTS 10.1 Introduction........................................................................................................................... 153 10.2 The History and Fundamental Mechanism of RNAi............................................................ 154 10.3 Secondary Metabolites in Plant............................................................................................. 155 10.3.1 Phenolics.................................................................................................................... 156 10.3.2 Flavonoids.................................................................................................................. 156 10.3.3 Stilbenes..................................................................................................................... 156 10.3.4 Terpenes..................................................................................................................... 156 10.3.5 Steroids...................................................................................................................... 156 10.3.6 Alkaloids.................................................................................................................... 156 10.4 Enhancement of Secondary Metabolites............................................................................... 157 10.5 Application of RNAi in Crop Improvement.......................................................................... 157 10.5.1 Plant Architecture and Flowering Time Alteration................................................... 157 10.5.2 Development of Seedless Fruits................................................................................ 157 10.5.3 Enhancement of Resistance to Biotic Stresses.......................................................... 157 10.5.4 Altered Flowers Colour/Scent................................................................................... 157 10.5.5 Weeds Resistance...................................................................................................... 158 10.5.6 Development of Male Sterility.................................................................................. 158 10.5.7 Elimination of Allergen and Toxin ........................................................................... 158 10.5.8 Nutritional Improvement........................................................................................... 158 10.6 Conclusion............................................................................................................................. 158 References....................................................................................................................................... 159

10.1 INTRODUCTION The basic needs of mankind heavily rely on plants and plant-derived products such as fossil fuels, gums, medicines, wood, fibre, paint and oils. Given the global degradation of agricultural land, the main restraining factors contributing to poor plant yield are water supplies, environmental stress factors and global warming. As the global population is growing rapidly, demand for crops and crop based products have increased, leading to future food insecurity, undernourishment and starvation (Brown and Funk, 2008; Lobell et al., 2008; Godfray et al., 2010). To tackle these challenges, molecular breeding and genetic engineering will need to increase crop output by creating resilient and high yield crop varieties (Mittler and Blumwald, 2010; Tester and Langridge, 2010). So far, the traditional plant breeding methods have increased the quality and volume of plants effectively. However, these approaches are time consuming and arduous and constitutes many other environmental barriers. DOI: 10.1201/9781003034957-10

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The accuracy of bioengineering techniques has led to crucial crop biotechnology advancements by providing a wide range of genetic variants and phenotypes that would be reliably incorporated into stellar crops to increase yield, nutrient enrichment and resistance against abiotic stress factors (Sharma et al., 2002). However, currently there are biosafety concerns about the impact of genetically modified crops on human health and environment, especially when genes found in microorganisms are used (Wolfenbarger and Phifer, 2000; Herdt, 2006). Transgene escape to noxious wild relatives, loss of genetic diversity and environmental degradation are some of the major concerns. Therefore, transgenic plants should be tested precisely to detect any potential hazard risks before release for public use. Therefore, new tactics and safer ways to improve crop yield is needed that can public can accept without hesitation. In this respect, RNA interference (RNAi) has attracted the attention of researchers worldwide. The RNAi technique is a combined activity of small interfering RNAs (siRNA) and microRNAs (miRNA) causing protein degradation. The discovery of RNAi has also improved our understanding of gene expression and function, opening new avenues for advanced crop biotechnology. The ability of RNAi has been developed to influence the growth and development, circadian rhythm and other crucial physiological processes of plants. Further, the RNAi has been successfully implemented in modifying the hormone signal transduction and pathogen attack (Liu et al., 2009). On the basis of these results, a framework of synthetic RNAi development was conceptualized that can suppress the activity of targeted genes. Many articles are published that are focused on the role of RNAi in current plant biotechnology landscape (Shukla et al., 2008; Tang et al., 2008; Belostotsky and Sieburth, 2009). Besides facilitating a wide variety of developmental activities, RNAi has been effectively used for diseases control and protection against pest invasion (Eamens et al., 2008). There is three natural RNA silencing mechanism in plants has been shown, i.e., 1) cytoplasmic siRNA silencing, 2) silencing of endogenous mRNA by miRNA and 3) DNA methylation. These pathways involve the cleavage of dsRNA by Dicer into small RNA fragments (21–26-nucleotide) (Baulcombe, 2004). The Food and Drug Administration (FDA) department of the United States of America has authorized the use of recombinant crop maize (Zea mays) SmartStax Pro, designed to express dsRNA against the corn rootworm (Diabrotica virgifera virgifera). Nevertheless, despite considerable success, RNAi-based transgenic plants are not produced at a high commercial scale. In general, transgenic plants and GMOs face massive protest by public and non-government organizations that their prospects for widespread acceptance appear relatively low in near future. According to some predictions, transgenic crops may cost ~$140 million to be introduced in current market. However, even if it does successfully, it is likely to face criticism from many anti-GMO organizations (Rosa et al., 2018). Therefore, taking these concerns into account, a GMO-free RNAi methods is currently needed to allow RNAi that is not activated by transgenic viruses or gene products but by direct delivery of inhibitory dsRNA or sRNAs fragments in crops. This book chapter aims to summarize some of the current knowledge on RNAi and presents an outlook for their successful implementation in managing future food security.

10.2  THE HISTORY AND FUNDAMENTAL MECHANISM OF RNAi Before the establishment of RNAi, researchers employed multiple techniques such as the incorporation of T-DNA (Transfer DNA of tumour inducing plasmid) components and transposable elements, irradiation treatment and inhibition by antisense RNA to produce loss-of-function mutations. However, the successful implementation of these methods requires meticulous skills as they are prone to error. For example, transposable and T-DNA elements target the random sites in genome and may lead to an irregular variation in gene expression. Further, in most of these cases the observed phenotype could not be linked rationally to the expected function of the target gene. Therefore, to rectify these unpredictable effects, a method was needed that could change the expression of genes without physically altering the genetic architecture of target organism. The further research in this direction leads to the discovery and implementation of RNAi mechanism in crop biotechnology. The

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FIGURE 10.1  The simple illustration of RNA interference (RNAi) mechanism.

basic mechanism of RNAi technology involves the stable hybridization between the complementary antisense RNA and mRNA, leading to the hindrance for ribosome binding sites and subsequent inhibition of translational activity (Knee and Murphy, 1997; Brantl, 2002; Arenz and Schepers, 2003). The RNAi mechanism is naturally found in almost all domains of life including fungal mycelium, plants and animals (Romano and Macino, 1992). The first successful implementation of RNAi was demonstrated in genetically transformed Petunia hybrida (Napoli et al., 1990). It was observed that this genetically modified plant had white flowers unlike the purple flowers of wild-type. This accomplishment showed that RNAi has potential to manipulate the plant traits in a desired manner. In plants, the mRNA suppression occurs in three distinct ways viz. the suppression by dsRNA leading to mRNA depletion in cytoplasm, the regulation of by miRNA leading to mRNA depletion in nucleus and post-transcriptional gene silencing (PTGS) (Romano and Macino, 1992). Additionally, RNAi is a homology based processes that regulates the gene expression by binding the dsRNA to target which leads to translation inhibition (Mansoor et al., 2006). The pathway of RNAi in plants involves the generation of staggered cut small interfering RNAs (21–24 nt long) created by the action of Dicer enzyme (ribonuclease III type enzyme) on a lengthy natural or alien dsRNA molecules (Hamilton and Baulcombe, 1999; Zamore et al., 2000). The generated siRNAs (21–24 nt) fragments are then integrated into the RISC (RNA-induced suppression complex) containing multiple proteins such as AGO (Argonaute) protein in addition to siRNAs (Baumberger and Baulcombe, 2005; Vaucheret, 2008). The RISC (RNA-induced silencing complex; see later) induced by adenosine triphosphate (ATP) then unravels the double stranded siRNA. Martinez et al. (2002) reported a RNA helicase action that integrates the siRNA antisense strand into the target gene comprising the RISC complex. The RISC-antisense RNA complex attacks the corresponding mRNA through base-pairing and blocks the mRNA eventually leading to the termination of transcription and translation (Figure 10.1) (Bartel, 2004). The different kinds of RNAs such as dsRNA, siRNA, plasmid or virus-based short hairpin RNA (shRNA) and miRNA are responsible for activation of RNAi in the host. The most important enzyme involved in this process is Dicer, which processes the long dsRNA, shRNA and pre-miRNA into siRNA duplexes of smaller sized fragments (21–23 nt) with the generation of symmetric overhangs, 2-nt 3′overhangs and 5′-phosphate groups. After this processing, the siRNAs with cellular proteins form RISC complex. During the process of RISC assembly, one of the strand is degraded, while the other strand forms an active RISC. This active RISC finally triggers the mRNA degradation (complete complementary) or translation repression (partial complementary miRNA).

10.3  SECONDARY METABOLITES IN PLANT The plant secondary metabolites are mainly generated at various stages of tricarboxylic acid (TCA) cycle such as pyrrolic enzymes, cinnamic acid, oritinite phospholipids, mevalonate, triacylglycerol, glycosides and phenols. The mevalonate derives terpenoids, stigmasterol and carotenoids also form a larger pool of plant metabolites.

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Secondary metabolites are varied products that are extensively divided into three categories: alkaloids, steroids and terpenes and phenolics (Harborne, 1999; Bourgaud et al., 2001). These substances perform useful functions in sustaining the growth, development and survival of plants under optimal and changing environmental conditions.

10.3.1 Phenolics The phenolics have primarily structure-based roles in plants (e.g., lignin strengthening specialist cell walls) but some of the members are also involved in plant protection (e.g., stilbenes, coumarins, proanthocyanidins, isoflavonoids, condensed tannins), photosynthesis, photosensing and embryo development. Chemically, they are distinguished by one or more acidic hydroxyl groups of aromatic compounds. While exposed to the environment they decompose rapidly and mix with proteins.

10.3.2 Flavonoids Flavonoids comprise a diverse family of flavonols, anthocyanidins, isoflavones, flavan-3-ols, flavanones and flavones. The other flavonoids are dihydroflavonols, aurons, coumarins, dihydrochalcones, flavan-3, 4-diols and chalcones. Flavonids are mostly found in the cell membrane and control the colour of many fruits and flowers, serving as pollinator inducers (delphinidins, cyanidins, pelargonidins), defending crops (kaempferol against Ultraviolet-B radiation) inducers for insect grazing (isoquercetine) and antifeedants (proanthocyanidins). They also exhibit antioxidant and anti-inflammatory properties (Fang et al., 2005; Comalada et al., 2006).

10.3.3 Stilbenes Stillbenes are phenolic compounds that are involved in the plant protection, resveratrol being the most prevalent stilbene. Resveratrol are mostly present in plant cells and are known for their antioxidant properties against infection during myocardial infarction in humans (Bradamante et al., 2004). The trans-resveratrol have applications in beauty products (Burns et al., 2002; Counet et al., 2006).

10.3.4  Terpenes Terpenes can be categorized as polyterpenes (>C80), hemiterpenes (C5), tetraterpenes (C40), monoterpenes (C10), triterpenes (C30) and diterpenes (C20). Terpenoids can be found in glandular trichomes, glandular epidermis and secretory cavities. Monoterpenes are popular compound in perfumery and flavouring industries due to their aromatic nature.

10.3.5 Steroids Steroids are beneficial for the synthesis of animal growth factors, stigmasterol, campesterol and β-sitosterol (95–98% of all plant sterols) being the most common phytosterols. Sterols can lower the cholesterols in human diet and perform a cardiopulmonary therapeutic function (García-Llatas and Rodríguez-Estrada, 2011; Sabater-Jara and Pedreño, 2013). They also play important role in preventing inflammation, atherosclerosis and oxidation (Delgado-Zamarreño et al., 2009). Brassinosteroids stimulate cellular proliferation and elasticity, vascular segregation and microtubular reconfiguration. The steroids such as Carotenoids are strong antioxidants and precursor of vitamin A.

10.3.6  Alkaloids In plants, the function of alkaloids is mainly in the protection against invaders such as insects, herbivores and microbial pathogens. Majority of alkaloids are derived from the catalyst of amino acids,

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nicotinic acid or anthranilic acid. Alkaloid are typically synthesized in specialized tissues where they aggregate and released from the transcription site (De Luca and St-Pierre, 2000; De Luca and Laflamme, 2001).

10.4  ENHANCEMENT OF SECONDARY METABOLITES The RNAi technology has been effectively used in plant metabolic engineering programs. The RNAi-mediated suppression of the tomato regulatory gene DET1 resulted in a ‘high pigment’ fruit phenotype, ~3.5 times increase in flavonoid volume and an increased carotenoid content (Schijlen et al., 2006). The second successful implementation of RNAi has been demonstrated in delaying of tomato fruit ripening by targeting aminocyclopropane oxidase (ACC) enzyme. This approach resulted in a significantly increased shelf life of tomato (Xiong et al., 2005).

10.5  APPLICATION OF RNAi IN CROP IMPROVEMENT 10.5.1 Plant Architecture and Flowering Time Alteration The gene encoding carotenoid cleavage dioxygenase (CCD) enzyme has an important role in regulating branch growth in model plants (Drummond et al., 2009). The downregulation of AcCCD8 transcripts in genetically modified kiwifruit (Actinidia chinensis L.) via hpRNA-induced suppression is found to increase the total amount of root sections and delay leaf senescence up to two seasons of crop growth (Ledger et al., 2010). Such an enhancement in vegetation structure can boost the number of the flowers grown on kiwifruit vines.

10.5.2 Development of Seedless Fruits Farmers and consumers prefer the exclusion of seedlings from fruit for regular consumption, preservation and refined fruit items. In tomato plants, fruits without seeds were obtained by suppressing the auxin response factor 7 (ARF7) (De Jong et al., 2009). Transgenic tomato expressing the Aucsia genes encoding a small receptor in the ovary were suppressed by hpRNA and developed fruits without seeds after flower denigration (Molesini et al., 2009).

10.5.3 Enhancement of Resistance to Biotic Stresses Biotic stress cause serious loss of crop yield. Particularly, viruses are difficult to control as they harbour wide variety of dispersal tactics. The potato plant was transformed with a vectors simultaneously encoding the antisense and sense transcripts of the viral helper-component proteinase (HCPro) gene (Waterhouse et al., 1998). This approach lead to the increased resistance of potato plant against potato virus Y (PVY). Further, the papaya plants expressing the truncated virus coat protein (cp) gene demonstrated a significant decrease in virus coat protein mRNA causing a significant resistance against them (Kertbundit et al., 2007). Transgenic potato expressing hpRNA against plasma membrane-localized Syntaxin related 1 (SYR1) gene exhibited a higher tolerance to the oomycete pathogen, Phytophthora infestans (Eschen-Lippold et al., 2012). A cytochrome P450 gene CYP6AE14 is confers immunity to insects against a poisonous cotton alkaloid gossypol. Transgenic tobacco expressing hpRNA targeted against CYP6AE14 has been reported to successfully inhibit the expression of genes (CYP6AE14 and GSTI) (Mao et al., 2007).

10.5.4  Altered Flowers Colour/Scent Flowers have been a valuable commodity in decor market and fragrance industry. Researchers have shown that RNA suppression of polyacylated anthocyanins can induce flower colour regulation

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(Seitz et al., 2007; Nakatsuka et al., 2010). Further, Nishihara et al. (2005) had suppressed the chalcone isomerase (CHI) gene using RNAi in tobacco. This suppression resulted into decreased discolouration and alteration of flavonoid compounds in flower petals.

10.5.5  Weeds Resistance The weeds (plant parasite) are found extensively in crop fields and cause a huge loss of farm yields. There are several traditional methods for controlling them. Since the traditional methods have many constraints, an advanced tool for controlling parasitic weeds is required. RNAi has been used to develop transgenic tomato carrying cartridge expression of M6PR dsRNA (Aly et al., 2009). It has been reported that the amount of mannose-6-phosphate receptor (M6PR) mRNA in Orobanche aegyptiaca tuber and buried shoots was lowered by 60–80% with a substantial drop in mannitol and rise in the percentage of dead Orobanche tubercle.

10.5.6 Development of Male Sterility The generation of male sterility is among the most significant feature to assure the purity to eventually create a hybrid plant. Additionally, RNAi has been used to recover the vitality of male sterile plant (Nizampatnam and Kumar, 2011). Sandhu et al. (2007) used RNAi technology to inhibit the expression of MutS HOMOLOG 1 (Msh1) in tobacco and tomato plants, resulting in alterations of mitochondrial DNA linked to normal intracellular male sterility.

10.5.7 Elimination of Allergen and Toxin Eliminating toxins and hazardous chemicals can be accomplished by using RNAi by modifying the gene expression of allergens. RNAi has been used to suppress the transmission of Mal d1 allergens of Apple (Malus domestica) which corresponds to a pathogenesis-related protein group PR10. Gilissen et al. (2005) succeeded in reducing the Mal d1 transcription using RNAi.

10.5.8 Nutritional Improvement RNAi provides tomatoes with additional nutritional values such as antioxidative properties and enrichment of basic elements such as phosphorous, zinc, sulphur, iron, calcium, magnesium, selenium, coppers etc. (Niggeweg et al., 2004). The leaf starch deterioration route is less defined in maize (Weise et al., 2011). Kusaba et al. (2003) were able to minimize glutenin levels using GluB hpRNA and developed a rice version called LGC-1 (Low Glutenin Content-1). This trait is beneficial for patients with inability for glutenin ingestion and processing. Reducing the content of α-linolenic acid is useful for improving the flavour and health factor of soybean oil. In presence of ω-3 fatty acid phosphatase, the linoleic acid is transformed into α-linolenic acid. Flores et al. (2008) developed hpRNA for seed suppression of ω-3 fatty acid desaturase with a glycinin inhibitor. Genetically modified soybean seed is documented to have 1–3% of α-linoleic acid compared to 7–10% in nongenetically modified soybean seed (Table 10.1).

10.6 CONCLUSION Malnourishment is a challenging issue in the developing world (World Health Organization, 2000). More than 750 million people are affected by food shortage worldwide including 20% of the developing world. To provide a healthy diet for a healthy world, biofortified vegetables, fruits and cereals need to be developed by enriching them with nutritionally important ingredients, crucial antioxidants, amino acids, minerals, fatty acids and vitamins. Stress (biotic and abiotic) resistance is among the most significant characteristics that should be imparted in crops. Plant species need to be evolved

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TABLE 10.1 List of Genes Targeted by RNAi for Plant Improvement Gene

Source

Traits Improved

References

Cor

Morphine

Allen et al. (2004)

OsPCS1 CaMxMt 1 Apx sbeIIa and sbeIIb sbeIIa and sbeIIb ZLKR and SDH AtGWD and AtSEX4 ZmGWD bch DET1

Papaver somniferum (Opium poppy) Rice Coffea canephora Tomato Barley Wheat Maize Arabidopsis thaliana Maize Potato Brassica napus

Li et al. (2007) Ogita et al. (2003) Zhang et al. (2011) Regina et al. (2010) Regina et al. (2006) Houmard et al. (2007) Weise et al. (2012) Weise et al. (2012) Van et al. (2007) Wei et al. (2009)

DET1 ε-cyc NCED1

Tomato Brassica napus Tomato

Cadmium Caffeine Vitamin C Amylose Amylose Lysine Starch Starch β-Carotene and lutein Carotenoid with decreased sinapate esters Carotenoid and flavonoid Carotenoid β-Carotene and lycopene

Davuluri et al. (2005) Yu et al. (2008) Sun et al. (2012)

to resist phytopathogenic fungi and parasites, along with ecological concerns such as extreme temperatures, droughts and flooding, effects on the soil content (hard rock concentration, alkalinity, declining fertility etc.). To feed the growing population, these variants of plants will be needed for future food security. The development of engineered crops, using RNAi technology is required to fulfil the global demand for fuel, fibre and food (Chapotin and Wolt, 2007). There are many possibilities for RNAi’s use in the science of crops to enhance it, such as stress resistance and higher nutrient level. Thus, RNAi has enormous potential to increase crop output dramatically. RNAi will, therefore, be a mandatory requirement in the future and not a choice. Contact Information: Please contact the author at: [email protected]; rak5838@ rediffmail.com

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Index Note: Locators in italics represent figures and bold indicate tables in the text.

A Abiotic elicitation, 14–15 Acetyl CoA molecule, 3 Acid extraction method, 9 Adenosine triphosphate (ATP), 155 Aeroponics method, 77, 78 Agrobacterium rhizogenes, 15, 100 Agrobacterium tumefaciens, 101 Alkaloids classification, 8–9 extraction, 9 hairy root culture, 108–109, 110–112 identification, 9 plant sources and therapeutic properties, 30, 31 RNAi, 156–157 role and application, 9–10 salt stress, 51 synthesis, 8 Allopathic medicines, 90 Alpinia purpurata, 39 Altered flowers colour/scent, 157–158 Anti-diabetic activity, 30–32 Anti-microbial activity, 30 Anti-oxidants, 30 Aquaculture, 72 Aromatic plants, 6 ATP, see Adenosine triphosphate Atropa belladonna, 32 Ayurvedic, 90 Azospirillum brasilense, 52

B The Bengal system, 79 Benzoic acids, 11 Berberis vulgaris, 32 Bioreactors, 106–107 Biosynthetic metabolic pathway engineering, 15 Biosynthetic pathway elucidation, 107 Biotic elicitation, 15 Biotic stresses, 157 Brassinosteroids (BS) nitrogen containing compounds, 128 occurrence, 126 plant phenolics, 127–128 secondary metabolites, 128–130 structure, 126 terpenes/terpenoids, 127 BS, see Brassinosteroids

C Calophyllum brasiliense, 81, 82 Callus culture system, 92

Carotenoid cleavage dioxygenase (CCD), 157 Catharanthus roseus, 14, 32 CCD, see Carotenoid cleavage dioxygenase Cell suspension cultures, 92 Chalcone synthase (CHS), 40, 41 Chemical culture, 72 Chlorophytum laxum, 116 CHS, see Chalcone synthase CLANS, see Controlled Ecological Life Support System Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, 15–16, 136–137 advantages, 140 application, 142–145 classification, 138–139 genome editing of plants, 139 history, 137–138 limitations, 146 nitrogen-containing compounds, 141 phenolic compounds, 141 plant genetic engineering, 146 terpenes, 140–141 Co-culture technology, 64 Colorimetric assay, 105 Continuous-flow solution culture, 76 Controlled Ecological Life Support System (CLANS), 72 Coumarins, 12 Crop improvement, RNA interference (RNAi) allergen and toxin, 158 altered flowers colour/scent, 157–158 biotic stress, 157 flowering time alteration, 157 male sterility, 158 nutritional improvement, 158, 159 plant architecture, 157 seedless fruits, 157 weeds resistance, 158

D Datura innoxia, 82 Deep water culture method, 80 Dioscorea deltoidea, 15 Diterpenes, 5 Docetaxel, 2 Dot blotting, 104

E Ebb method, 78, 79 EBL, see 24-Epibrassinolide Elicitation, 92–93 24-Epibrassinolide (EBL), 126, 128–129 Escherichia coli, 38, 137 Essential oils, 6–7, 54 β-Eudesmol, 39

165

166 F Flavonoids, 12, 28, 29, 156 Flowering time alteration, 157 Fogponics, 78 Formamidopyrimidine-DNA-glycosylase (FPG), 38 Fungal endophytes elicitors, 63–64 and medicinal metabolites, 64–65 plant growth and development, 63 polysaccharides, 63 sustainable agriculture, 62

G Gamma irradiation age of plantlets, 39 biological systems, 37 DNA, 38 enzymes, 40–41 materials, 35–36 medium, 36 oxygen, 36 parts of plants, 39–40 proteins, 38 radiolysis of water, 36–37 secondary metabolite production, 33, 39 temperature, 36 units of measurement, 34–35 water content, 36 Gamma radiation, 16 Gamma rays, 34–35 Gas chromatography-flame ionization detector (GC-FID), 5 Gas chromatography-mass spectrometry (GCMS), 5 GC-FID, see Gas chromatography-flame ionization detector GCMS, see Gas chromatography-mass spectrometry Genetic engineering, 54–55, 134 Glucosinolates, 53 γ-Glutamyl-2-oxoglutrate amino transferase (GOGAT), 128 Glutathione-S-transferase (GST), 51 Glycosides, 109, 113–115 GOGAT, see γ-Glutamyl-2-oxoglutrate amino transferase gRNA, see Guide RNAs GST, see Glutathione-S-transferase Guide RNAs (gRNA), 137

H Hairy roots, 15 alkaloids, 108–109, 110–112 in bioreactors, 106–107 Chlorophytum laxum, 116 diverse and abundant, 107–108 glycosides, 109, 113–115 history, 100–101 mechanism, 102 stability, 105–106 transformation techniques, 103–105 in vitro development, 102–103 in vivo development, 103 HBL, see 28-Homobrassinolide

Index HDR, see Homology-directed repair Hemiterpenes, 4 Histidine, 9 28-Homobrassinolide (HBL), 126, 129 Homogentisate phytyltransferase (HPT), 55 Homology-directed repair (HDR), 136 HPLC-ECD, see HPLC with electrochemical detection HPLC with electrochemical detection (HPLC-ECD), 38 HPT, see Homogentisate phytyltransferase Hydroculture, 72 Hydroponics advantages, 80 definition, 72 disadvantages, 80 history, 72 nutrient solutions, 74 production of secondary metabolites, 74–83 substrates, 72–73 Hydroxycinnamates, 11 Hydroxycinnamic acids, 12 Hyoscyamus muticus, 38 Hypericum triquetrifolium Turra callus cultures, 16

I Inorganic hydroponic solutions, 74 In vitro culture methods callus culture system, 92 cell suspension cultures, 92 elicitation, 92–93 organ culture, 93 plant secondary metabolites, 90, 90 scale-up production, 93 Ionizing radiation, 35 Isoprenoids, 5

K Kratky method, 80, 81

L Leucine, 8 LGC-1, see Low Glutenin Content-1 Lignans, 12 Lignin, 11, 52–53 Liquid-liquid extraction (LLE), 13 Low Glutenin Content-1 (LGC-1), 158 Lysine, 8

M MAE, see Microwave-assisted extraction Male sterility, 158 Medicinal metabolites, 64–65 Melanins, 12 Micronutrients, 74 MicroRNA (miRNA), 129, 154 Microwave-assisted extraction (MAE), 13 miRNA, see MicroRNA Molecular breeding, 107 Monolignols, 11 Monoterpenes, 4

167

Index N

R

Neolignans, 12 Nuclear magnetic resonance (NMR) spectroscopy, 7 Nutriculture, 72 Nutrient solution, 76

Rauwolfia canescens, 15 Reactive oxygen species (ROS), 51 Real-time PCR (RT-PCR), 105 Recombinant protein production, 107–108 Reverse transcriptase-polymerase chain reaction (RT-PCR), 38 RGT, see Rumex gmelini Turcz Rhizhobium rhizhogenes, 100 RISC, see RNA-induced suppression complex RNAi, see RNA interference RNA-induced suppression complex (RISC), 155 RNA interference (RNAi), 16, 136 crop improvement allergen and toxin, 158 altered flowers colour/scent, 157–158 biotic stress, 157 flowering time alteration, 157 male sterility, 158 nutritional improvement, 158, 159 plant architecture, 157 seedless fruits, 157 weeds resistance, 158 history, 154–155 mechanism, 154–155 secondary metabolites in plant, 155–157 ROS, see Reactive oxygen species Rosmarinic acid, 81 Rotary hydroponic system, 81 Rumex gmelini Turcz (RGT), 64 Run-to-waste method, 79; see also The Bengal system Ruta graveolens, 15

O Opine detection, 104 Organ culture, 93 Organic fertilizers, 74 Organic hydroponics solutions, 74 Ornithine, 8

P Paclitaxel, 64 Paeoniae radix, 39 PAL, see Phenylalanine ammonia-lyase Pancreatic α-amylase, 30 Papaver somniferum, 32, 38 Paper electrophoresis, 105 Passive hydroponics, 81 PCR, see Polymerase chain reaction Perilla frutescens, 82 Perovskia abrotanoides, 15 Phaseolus vulgaris, 38 Phenolic acids, 12 Phenols, 12 classification, 12 CRISPR/Cas9 system, 140–141 extraction, 13 identification, 13 RNAi, 156 role and application, 13 salt stress, 51–52 synthesis, 10–12 Phenylacetic acids, 12 Phenylalanine, 9 Phenylalanine ammonia-lyase (PAL), 40, 51, 128 Phenylpropanoids, 10 Phenylpropenes, 12 Phytochemicals production, 107 Phytoremediation, 107 Phytosterols, 28, 29 Piper nigrum, 32 Plant architecture, 157 Plant cell, 32 Plant phenolics, 127–128 Plumbago rosea, 15 Polyamines, 51 Polymerase chain reaction (PCR), 104–105 Polysaccharides, 63 Polyterpenes, 5 Postprandial hyperglycaemia (PPHG), 30

Q Quantitative PCR (qPCR), 105 Quinones, 12

S S-adenosyl-L-methionine synthetase, 53 Salt stress alkaloids, 51 essential oils, 54 genetic engineering, 54–55 glucosinolates, 53 lignin, 52–53 phenolics, 51–52 salinity, 50 soil pH, 50 tannins, 53–54 temperature, 50 water deficit, 50 Scopolia parviflora, 15 Secondary metabolites (SMs) in plants alkaloids, 8–10 bioactivity, 30–32 biotechnological approaches, 13–16 BS, 127–130 classification, 134, 135 endophytes, see Fungal endophytes gamma irradiation, see Gamma irradiation genetic engineering, 134 hairy roots, 108–109 hydroponic, 74–82 palkaloids, 10–13 production, 32–33

168

Index

RNAi, 136, 155–157 role and significance, 134 salt stress amelioration, see Salt stress terpenes, 2–7 transgenic approaches, 134–136 in vitro culture methods, 90, 90 Seedless fruits, 157 Sesquiterpenes, 4 Sesterterpenes, 5 Sesuvium portulacastrum, 51 SFE, see Supercritical fluid extraction Short hairpin RNA (shRNA), 154 Small interfering RNAs (siRNA), 154 Soilless agriculture, 72 Soilless culture, 72 Solanum nigrum, 51 Static solution culture, 74, 76 Steroids, 28, 156 Stilbenes, 12, 156 STR, see Strictosidine synthase Strictosidine synthase, (STR), 41 Supercritical fluid extraction (SFE), 13 Sustainable agriculture, 62

Tetraterpenes, 5 Tissue culture approaches, 14, 32 γ-Tocopherol methyltransferase (γ-TMT), 55 Top-fed deep water culture, 80 Toxin elimination, 158 Transcription Activator-Like Effector Nucleases (TALENs), 136 Transfer DNA (T-DNA), 104 Transgenic confirmation, 105 Transgenic lines, production, 15 Transgenic tobacco, 134–136 Triterpenes, 5 Tryptophan, 8 Type-I diabetes, 30 Typhonium flagelliforme, 39 Tyrosine, 8

T

Vinca alkaloids, 2 Vinca minor, 32 Volatile oil, see Essential oil

TALENs, see Transcription Activator-Like Effector Nucleases Tank farming, 72 Tannins, 12, 53–54 Taxanes, 2, 64 Taxus baccata, 82 T-DNA, see Transfer DNA Terpenes BS, 127 classification, 4–5 CRISPR/Cas9 system, 140–141 essential oil, 6–7 extraction, 5 identification, 5 RNAi, 156 role and application, 5–6 synthesis, 3–4 Terpenoids, see Terpenes

U Ultrasound-assisted extraction (UAE), 13 Unani, 90

V

W Water-extracted mycelial polysaccharide (WPS), 64 Water radiolysis, 36–37 Weeds resistance, 158 WPS, see Water-extracted mycelial polysaccharide

X Xanthones, 12

Z Zinc-Finger Nucleases (ZFNs), 136 Zingiber officinale, 14