Genetic Engineering of Crop Plants for Food and Health Security: Volume 1 9819950333, 9789819950331

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Siddharth Tiwari Bhupendra Koul   Editors

Genetic Engineering of Crop Plants for Food and Health Security Volume 1

Genetic Engineering of Crop Plants for Food and Health Security

Siddharth Tiwari • Bhupendra Koul Editors

Genetic Engineering of Crop Plants for Food and Health Security Volume 1

Editors Siddharth Tiwari Plant Tissue Culture and Genetic Engineering Lab National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India) Mohali, Punjab, India

Bhupendra Koul Department of Biotechnology, School of Bioengineering and Biosciences Lovely Professional University Phagwara, Punjab, India

ISBN 978-981-99-5033-1 ISBN 978-981-99-5034-8 https://doi.org/10.1007/978-981-99-5034-8

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore Paper in this product is recyclable.

Preface

At the beginning of agriculture, around 8000 BC, there were about 5 million people on the planet. It increased to 200–300 million by the year 1 AD. With the industrial revolution, there was a significant shift in population: from 1 billion in 1800 to 2 billion in the 1930s to 3 billion in the 1960s to 4 billion in 1974 and to 5 billion in 1987. In the twentieth century, the global population increased to 6 billion and, very recently, the world’s population has crossed 8 billion, making it difficult to ensure that everyone has access to food and nourishment. The effects of various biotic (pathogen attack, insect attack, parasitic weed, wounding, anthropogenic activities) and abiotic (temperature: cold/heat, water: drought/flood, radiation: highlight/UV, chemicals: salt/ion/insecticides/herbicides, wind, sound, electrical, magnetic, etc.) factors, as well as projections of future population growth, have also raised serious concerns about sustainable crop production for food and health security. Because of the current situation, agricultural scientists, researchers, academics, and decisionmakers must now develop alternatives to boost crop output in order to feed the thronging millions. The multidisciplinary science of biotechnology has the potential to provide solutions to pressing socioeconomic issues including the lack of wholesome food, fiber, and fuel. In vitro tissue culture and modern genetic modification technologies in crop development have tremendous relevance since the conventional approaches used in crop improvement may not keep up with the growing demands of an expanding population, diminishing land resources, and environmental stressors. Biotechnologies have proven to be beneficial and are able to meet the needs of the average person. By utilizing the novel abilities of Agrobacterium tumefaciens, the natural “Genetic Engineer,” and alternative strategies based on the transfection of plant protoplast or by biolistic devices, are now the possible ways to transfer any gene, regardless of its source, into desired plant species as a routine procedure. With the help of remarkable developments in gene technology, crop plants can now be “tailored” with beneficial genetically engineered traits to fight against various biotic and abiotic stresses and improve a variety of qualitative and quantitative characteristics. These traits include access to an unlimited gene pool and the incorporation and expression of genes from taxonomically unrelated species. Using the methods and tools of genetic engineering, plant genetic transformation is now essential for plant biotechnologists, biochemists, and physiologists to express a v

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foreign gene from a heterologous system into a suitable organism, either for qualitative or quantitative improvement of the organism(s) or their products. However, the amount of expression, purity, affordability, and long-term recovery of the intended products are crucial for biotechnology’s success. We shall constantly be guided by gene cloning, genetic engineering, and biotechnological techniques for additional plant enhancement research. Creating transgenic plants has three main benefits: (1) it can increase a crop plant’s agricultural, horticultural, or nutritional value; (2) it can act as a living bioreactor for the feasible and economical production of industrially or pharmaceutically significant proteins or metabolites; and (3) it can be used as a model for research on the regulation and expression of genes. Contrarily, the environmentally benign crop development method known as cisgenics, which is an alternative to transgenics, entails genetically altering a plant by transferring one or more genes from a sexually compatible plant or any similar source. Cisgenic crops are (1) widely accepted; (2) do not require arduous, expensive, or time-consuming approval processes; (3) do not pose a threat to biodiversity or a potential risk to human health or the ecosystem; and (4) are safer than crops produced through conventional breeding because linkage drag is avoided. Through diverse genetic engineering techniques, significant advancements in agricultural yield and quality traits have been made. Numerous innovative features have been effectively incorporated into different crop plants, such as better nutrition, climatic resistance, and stress management. Notably, genetically modified plants have come to light as a fresh source of hope for the manufacturing of vaccines (edible vaccines) in light of the recent Covid-19 outbreak. The planned book will therefore gather research findings on the most recent developments in agricultural plant transgenesis and genome editing. Through the fusion of molecular biology, genetic engineering, and plant tissue culture regimes, it will largely concentrate on the introduction of critical features like stress tolerance and biofortification. The main goals of this book are to provide sustainable solutions for ending hunger and promoting good health. These goals are in line with the Sustainable Development Goals (SDGs) of the United Nations. In order to generate sustainable crops, this book thoroughly discusses numerous genetic engineering approaches, such as transgenic, cisgenic, and genome editing for the improvement of various traits in crop plants. The book’s chapters also discuss the importance, downsides, difficulties, recent advancements, and potential uses of transgenic and genome-edited crops in the future. The contributors to this edited book are scientists, educators, professors, researchers, experts in genetically modified crops, plant breeders, and policymakers. The book also intends to give interesting and educational content for graduate and undergraduate students specializing in plant sciences, biotechnology, horticulture, environmental sciences, ecology, forestry, and other related topics. We anticipate that this book will achieve the following objectives: 1. Familiarize budding scientists with the current genetic transformation techniques used to introduce value-added traits in crop plants. 2. Provide in-depth information on the importance of modern genetic engineering as a fundamental tool for biotechnologists.

Preface

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3. Elaborate on the utilization of genetically engineered plants as natural and living bioreactors for the production of essential molecules. 4. Present a comprehensive overview of the current landscape and future prospects of genetically engineered crops developed through both transgenic and genome editing approaches. Mohali, Punjab, India Phagwara, Punjab, India

Siddharth Tiwari Bhupendra Koul

Contents

1

Genetic Improvement of Pea (Pisum sativum L.) for Food and Nutritional Security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hardeep Singh, Sejal Asija, Komal Sharma, Bhupendra Koul, and Siddharth Tiwari

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Genetic Improvement of Apple . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chongtham Allaylay Devi, Ashutosh K. Pandey, and Khadija Mika

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3

Genetic Improvement of Carnation . . . . . . . . . . . . . . . . . . . . . . . . . Pooja Sharma, Amarjit K. Nath, Akhil Kumar, and Anshul Shyam

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4

Recent Advances in Genetic Improvement of Cotton . . . . . . . . . . . . Kajal Verma, Pooja Sharma, Kanchan Tripathi, Reena Yadav, and Surendra Pratap Singh

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5

Insights into Genetic Improvement of Cassava . . . . . . . . . . . . . . . . 101 Joel Jose-Santhi and Rajesh Kumar Singh

6

Genetic Improvement of Eggplant: Perspectives and Challenges . . . 123 Pallavi Mishra, Shailesh K. Tiwari, and Kavindra Nath Tiwari

7

Advances in Chilli Pepper (Capsicum spp.) Improvement Using Modern Genetic Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Ratna Kalita, Priyadarshini Bhorali, Manab Bikash Gogoi, and Bornali Gogoi

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Advances in Genetic Engineering for Pathogen Resistance in Capsicum annuum L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Surender Kumar and Anupama Singh

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Genetic Improvement of Poplar . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Deepika Singh and Rajesh Kumar Singh

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Genetic Engineering for Potato Improvement: Current Challenges and Future Opportunities . . . . . . . . . . . . . . . . . . . . . . . 213 Baljeet Singh, Vadthya Lokya, Priyanka Kaundal, and Siddharth Tiwari ix

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Insect Pest Management in Rice Through Genetic Engineering . . . . 233 G. Rajadurai, S. Varanavasiappan, L. Arul, E. Kokiladevi, and K. K. Kumar

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Genetic Engineering of Squash for Food and Health Security . . . . . 263 T. R. Usha Rani, R. N. Yashwanth Gowda, H. Kavya, and R. Pooja

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Genetic Improvement in Peanut: Role of Genetic Engineering . . . . 271 Riddhi Rajyaguru, Nataraja Maheshala, and Gangadhara K

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Transgenic Technologies for Fusarium Wilt Management in Banana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 R. Deepa Sankari, S. Varanavasiappan, L. Arul, K. Eraivan Arutkani Aiyanathan, E. Kokiladevi, and K. K. Kumar

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Genetic Improvement of Banana . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Soni KB, Anuradha T, Pritam Ramesh Jadhav, and Swapna Alex

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Genetic Improvement of Mustard . . . . . . . . . . . . . . . . . . . . . . . . . . 331 Khadija Mika Dawud, Chongtham Allaylay Devi, and Ashutosh K. Pandey

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Genetic Improvement of Mustard for Food and Health Security . . . 355 Gohar Taj, Sandhya Upadhyay, and Anjali Sharma

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Tinkering with Stevia rebaudiana Genome to Improve Its Sweetening Property and Productivity . . . . . . . . . . . . . . . . . . . . 373 Rinku Mondal, Shreyasi Kundu, and Abhijit Bandyopadhyay

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New Advancements in Genetic Improvement of Cash Crop Sugarcane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Anmol Singh Yadav, Shagun Sinha, and Prahlad Masurkar

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Genetic Improvement of Tomato Against Fusarium Wilt Disease Using Biotechnological Interventions . . . . . . . . . . . . . . . . . . . . . . . . 407 Chanchal Kumari, Ishani Shaunak, Parul Sharma, and Rajnish Sharma

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Genetic Engineering Methods for Wheat Improvement . . . . . . . . . . 421 Manisha Godara, Deepak Das, Joy Roy, and Abhishek Bhandawat

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Recent Advances in the Citrus Genetic Engineering for Stress Tolerance/Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 Pankaj Kumar, Komaljeet Gill, Shagun Sharma, Rohit Sharma, and Naresh Thakur

Editors and Contributors

About the Editors Siddharth Tiwari is a Scientist-E at National Agri-Food Biotechnology Institute (NABI), Mohali, Punjab, India. He received his doctoral degree in 2008 from CSIRNational Botanical Research Institute (CSIR-NBRI), Lucknow, U.P., India. His area of specialization includes Plant Tissue Culture, Molecular Biology, and Genetic Engineering. Two major contributions were derived from his doctoral research. The first one was the development of a groundnut-seed-expressed edible vaccine against cholera and rabies, and he received BioAsia Innovation Young Scientist Award in 2010. The second outcome was the development of insect resistance transgenic groundnut against polyphagous foliage insect Spodoptera litura, and he received the 97th Indian Science Congress Association (ISCA) Young Scientist Award in 2010. He joined DBT-NABI, in July 2010 and is presently working as Scientist E. His lab is working on metabolic engineering of staple crops like banana and wheat for nutritional-enrichment by using various biotechnological approaches. He has guided 7 Ph.D. students, >50 graduate/post-graduate trainees, delivered >80 invited lectures and published >50 research articles in the various disciplines of plant biotechnology. He is recently (2023) elected as a fellow of the prestigious Plant Tissue Culture Association of India (PTCA(I)). Bhupendra Koul is an Associate Professor at the Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University (LPU), Punjab, India. During his Ph.D. at the Plant Transgenics Lab, CSIR-National Botanical Research Institute, Lucknow, he worked on the optimization, introduction, and expression of modified full-length and truncated versions of Bt-cry1Ab and 1Ac genes in tomato for developing non-chimeric and stable transgenic lines resistant to two lepidopteran insects (Helicoverpa armigera and Spodoptera litura) and evaluated the stability and efficacy of insecticidal toxin in transgenic plants. He has also optimized the regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni) and has developed herbicide-resistant transgenic Stevia for effective weed management in Stevia cultivation. He was awarded “CSIRSenior Research Fellowship (SRF)” in the year 2013. He also has 9.5 years of teaching experience and received the “Teacher Appreciation Award 2016” from xi

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LPU in the Discipline of Biotechnology, through the MHRD Minister, Government of India. He has designed the full-length synthetic cry1Ac gene (GenBank: KP195020.1) and has published 60 research papers in national and international journals as well as 21 book chapters and 3 authored book with Springer Nature. He has guided 3 Ph.D. students and trained 20 post-graduate students.

Contributors K. Eraivan Arutkani Aiyanathan Department of Plant Pathology, Tamil Nadu Agricultural University, Madurai, Tamil Nadu, India T. Anuradha Department of Molecular Biology and Biotechnology, College of Agriculture, Thiruvananthapuram, Kerala, India L. Arul Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Sejal Asija Plant Tissue Culture and Genetic Engineering Lab, National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India Abhijit Bandyopadhyay Plant Genetics & Biotechnology Section, Department of Botany, University of Burdwan, Burdwan, West Bengal, India Abhishek Bhandawat National Agri-Food Biotechnology Institute, Mohali, India Priyadarshini Bhorali Department of Agricultural Biotechnology, College of Agriculture, Assam Agricultural University, Jorhat, India Chanchal Department of Biotechnology, Dr Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Deepak Das National Agri-Food Biotechnology Institute, Mohali, India Khadija Mika Dawud School of Agricultural Science, Sharda University, Greater Noida, Uttar Pradesh, India Chongtham Allaylay Devi School of Agriculture, Galgotias University, Greater Noida, Uttar Pradesh, India K. Gangadhara ICAR-CTRI Research Station, Kandukur, India Komaljeet Gill Department of Biotechnology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Manisha Godara National Agri-Food Biotechnology Institute, Mohali, India Bornali Gogoi Department of Fruit Science, College of Horticulture & FSR Nalbari, Assam Agricultural University, Jorhat, India

Editors and Contributors

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Manab Bikash Gogoi Department of Agricultural Biotechnology, College of Agriculture, Assam Agricultural University, Jorhat, India Joel Jose-Santhi Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Ratna Kalita Department of Agricultural Biotechnology, College of Agriculture, Assam Agricultural University, Jorhat, India Priyanka Kaundal Plant Tissue Culture and Genetic Engineering Lab, National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India H. Kavya ICAR-IIHR, Division of Basic Sciences, Bengaluru, India E. Kokiladevi Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Bhupendra Koul Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India Akhil Kumar CSIR-IHBT, Palampur, Himachal Pradesh, India K. K. Kumar Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Pankaj Kumar Department of Biotechnology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Surender Kumar Department of Biotechnology, Dr. Y. S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Shreyasi Kundu Plant Genetics & Biotechnology Section, Department of Botany, University of Burdwan, Burdwan, West Bengal, India A. Loganathan Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Vadthya Lokya Plant Tissue Culture and Genetic Engineering Lab, National AgriFood Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India Nataraja Maheshala ICAR-Directorate of Groundnut Research, Junagadh, India

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Editors and Contributors

Prahlad Masurkar Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Department of Plant Pathology, School of Agriculture, Lovely Professional University, Phagwara, India Khadija Mika School of Agricultural Science, Sharda University, Greater Noida, Uttar Pradesh, India Pallavi Mishra Division of Crop Improvement, ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India Department of Botany, Mahila Maha Vidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India Rinku Mondal Plant Genetics & Biotechnology Section, Department of Botany, University of Burdwan, Burdwan, West Bengal, India Amarjit K. Nath Department of Biotechnology, Dr YS Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Ashutosh K. Pandey School of Agricultural Science, Sharda University, Greater Noida, Uttar Pradesh, India R. Pooja ICAR-IIHR, Division of Basic Sciences, Bengaluru, India Jadhav Pritam Ramesh Department of Molecular Biology and Biotechnology, College of Agriculture, Thiruvananthapuram, Kerala, India G. Rajadurai Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Riddhi Rajyaguru Department of Biotechnology, Junagadh Agricultural University, Junagadh, India Joy Roy National Agri-Food Biotechnology Institute, Mohali, India R. Deepa Sankari Department of Plant Pathology, Tamil Nadu Agricultural University, Madurai, Tamil Nadu, India Anjali Sharma Molecular Biology & Genetic Eng., G. B. Pant University, Pantnagar, Uttrakhand, India Komal Sharma Plant Tissue Culture and Genetic Engineering Lab, National AgriFood Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India Parul Sharma Department of Biotechnology, Dr Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India

Editors and Contributors

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Pooja Sharma Department of Biotechnology, Dr. YS Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India NUS Environmental Research Institute, National University of Singapore, Singapore, Singapore Rajnish Sharma Department of Biotechnology, Dr Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Rohit Sharma Department of Forest Product, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Shagun Sharma Department of Biotechnology, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Ishani Shaunak Department of Biotechnology, Dr Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Anshul Shyam Department of Fruit Science, Dr YS Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Anupama Singh Department of Biotechnology, Dr. Y. S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Baljeet Singh Plant Tissue Culture and Genetic Engineering Lab, National AgriFood Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India Deepika Singh Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Hardeep Singh Plant Tissue Culture and Genetic Engineering Lab, National AgriFood Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India Rajesh Kumar Singh Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India Surendra Pratap Singh Plant Molecular Biology Laboratory, Department of Botany, Dayanand Anglo-Vedic (PG) College, Chhatrapati Shahu Ji Maharaj University, Kanpur, India Shagun Sinha Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India K. B. Soni Department of Molecular Biology and Biotechnology, College of Agriculture, Thiruvananthapuram, Kerala, India

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Alex Swapna Department of Molecular Biology and Biotechnology, College of Agriculture, Thiruvananthapuram, Kerala, India Gohar Taj Molecular Biology & Genetic Eng., G.B.Pant University, Pantnagar, Uttrakhand, India Naresh Thakur College of Horticulture and Forestry, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Kavindra Nath Tiwari Department of Botany, Mahila Maha Vidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India Shailesh K. Tiwari Division of Crop Improvement, ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India Siddharth Tiwari Plant Tissue Culture and Genetic Engineering Lab, National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India Kanchan Tripathi Plant Molecular Biology Laboratory, Department of Botany, Dayanand Anglo-Vedic (PG) College, Chhatrapati Shahu Ji Maharaj University, Kanpur, India Sandhya Upadhyay Molecular Biology & Genetic Eng., G. B. Pant University, Pantnagar, Uttrakhand, India T. R. Usharani ICAR-IIHR, Division of Basic Sciences, Bengaluru, India S. Varanavasiappan Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Kajal Verma Plant Molecular Biology Laboratory, Department of Botany, Dayanand Anglo-Vedic (PG) College, Chhatrapati Shahu Ji Maharaj University, Kanpur, India Anmol Singh Yadav Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India Reena Yadav Plant Molecular Biology Laboratory, Department of Botany, Dayanand Anglo-Vedic (PG) College, Chhatrapati Shahu Ji Maharaj University, Kanpur, India R. N. Yashwanth Gowda ICAR-IIHR, Division of Basic Sciences, Bengaluru, India

Genetic Improvement of Pea (Pisum sativum L.) for Food and Nutritional Security

1

Hardeep Singh, Sejal Asija, Komal Sharma, Bhupendra Koul, and Siddharth Tiwari

1.1

Introduction

Pisum sativum L. is a highly nutritious diploid (2n = 14) cool-season important legume crop that belongs to the family Fabaceae, with an estimated genome size of about 4.45 Gb (Smýkal et al. 2012). Pea has a significant ecological benefit; it aids in the creation of low-input farming methods, by both fixing atmospheric nitrogen and acting as a break crop, thereby reducing the reliance on external inputs. In the past few decades, pea production has fluctuated due to various biotic and abiotic factors. Fusarium root rot, powdery mildew, common root rot, rust, Ascochyta blight, and Fusarium wilt are some of the biotic stresses which impact its growth severely (Chaturvedi et al. 2022). Alongside abiotic stressors including high temperature, drought, salinity, and cold massively hinder its cultivation. Pea production, like many other pulses, predominantly relies on low-input agriculture globally. As a result, resource-poor farmers producing pea under such conditions are more H. Singh · S. Asija · S. Tiwari (✉) Plant Tissue Culture and Genetic Engineering Lab, National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India e-mail: [email protected] K. Sharma Plant Tissue Culture and Genetic Engineering Lab, National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India B. Koul Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_1

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H. Singh et al.

susceptible to biotic and abiotic stresses. Therefore, researchers are investigating the plant’s genetic makeup to identify genes responsible for stress tolerance, with the goal of developing new, more resilient crop varieties that can better withstand these challenging conditions (Zia et al. 2023). In addition to its ability to tolerate both abiotic and biotic stressors, genetic improvement of pea is being encouraged due to its highly nutritious value (Thavarajah et al. 2022). Pea seeds are a highly nutritious food source containing significant amounts of protein (16–32%), fiber (6–13%), starch (18–54%), oil (0.6–5.5%), and sucrose (1–2%) (Thavarajah et al. 2022). In the case of micronutrients, potassium is the predominant micronutrient, succeeded by phosphorus, magnesium, and calcium (Guindon et al. 2021). Pea also rich in other micronutrients including zinc (41 ppm), molybdenum (12 ppm), iron (97 ppm), and selenium (42 ppm). In addition to minerals, vitamins, and micronutrients, pea seeds also contain polyphenolics, saponins, α-galactosides, and phytic acids in their nutritional profile (Kumari and Deka 2021). Breeding for crop improvement involves selecting and developing varieties that can withstand and perform well under challenging environmental conditions (Chaudhary et al. 2020; Chaturvedi et al. 2021a). This is achieved through a combination of traditional breeding methods and modern biotechnological tools, such as molecular markers and genetic engineering. One of the primary strategies for breeding stress-tolerant pea varieties is to identify and utilize genetic variation within the pea germplasm. This involves screening large numbers of pea accessions for their tolerance to specific stress factors and selecting those with desirable traits for further breeding (Ye et al. 2018). Another approach is to use marker-assisted selection (MAS) to identify and introgress stress-tolerant genes into elite pea varieties. This involves identifying molecular markers linked to genes that confer stress tolerance or are involved in improving quality traits and using them to screen large populations of plants for the presence of these genes (Hasan et al. 2021). In pea breeding program, overall productivity has primarily been enhanced through the targeted breeding of plant types that are resistant to lodging, have optimal plant height, and exhibit resistance to key biotic stressors such as powdery mildew, rust, Ascochyta blight, as well as abiotic stressors including heat, drought, and cold (Huang et al. 2023). In recent years, genetic engineering has also emerged as a promising tool for improving both stress tolerance as well as quality traits in plants (Jangra et al. 2022; Shailani et al. 2021b). The incorporation of genes involved in the improvement of concentration and bioavailability of micronutrients, reduction of the concentration of antinutrients, and the redistribution of micronutrients between tissues has been undertaken in legumes till now (Roriz et al. 2020). For instance, in pea plants, the inactivation of genes encoding less desirable proteins, the manipulation of starch biosynthetic genes and their control, and the development of raffinose synthase mutants to knock out production of these compounds have been performed lately (Robinson et al. 2019). Besides improving quality traits, stress-tolerant crops by overexpressing genes such as the Na+/H+ gene for enhancing salinity tolerance (Ali et al. 2018) and manipulating heat shock factor (HsfA1d) for improving thermal tolerance (Shah et al. 2020). In the current chapter, we summarize the present

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Genetic Improvement of Pea (Pisum sativum L.) for Food and Nutritional Security

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scenario of trait-specific genetic improvement of pea by both traditional breeding techniques and genetic engineering approaches which have aided in quality improvement and enhanced the resilience of this significant legume toward both abiotic and biotic stresses.

1.2

Genetic Diversity, Germplasm Reservoir, and Genomic Resources of Pea

The genetic diversity of specific crop species is a highly valuable resource for enhancing crop development. Pea was originally domesticated around 10,000 years ago from wild and early Pisum species by Neolithic farmers. After domestication, the pea was rapidly distributed throughout Southwest Asia, Europe, and the Mediterranean basin (Zohary et al. 2012). The conservation of pea natural diversity is essential for enhancing genetic gain. In the past two decades, there has been a significant amount of research conducted on pea genetic diversity. Currently, there are around 98,000 pea accessions stored in major gene banks worldwide, with 58,000 of them being distinct. Some accessions also contain wild pea containing only about 1% of the conserved germplasm (Smýkal et al. 2018). To make use of pea germplasm, several core collections of pea have been established. Furthermore, there are a growing number of databases that store vast amounts of genotype and phenotype data including the KnowPulse (https://knowpulse.usask.ca) at the University of Saskatchewan and Food Legume database (https://coolseasonfoodlegume.org) at Washington State University. Together with user-friendly information systems, these germplasm repositories provide a robust foundation for improving the genetic quality of pea varieties through different breeding programs (Byrne et al. 2018). It is also critical in the identification of key trait genes and the development of genomic tools to accelerate crop improvement. Using diverse germplasm can address multiple biotic and abiotic stresses at the same time, resulting in a shorter development time, the ability to handle multiple traits, and an increase in genetic diversity. Over the last decade, genomic tools have advanced significantly, allowing for a more precise transfer of desirable traits while avoiding undesired genomic regions. Furthermore, adopting omics approaches is critical to further facilitating and accelerating these advancements. Omics-based mapping methods have emerged to aid in the discovery of genes/QTLs responsible for desired traits as well as the development of diagnostic markers for breeding purposes (Gali et al. 2018). The combination of high-density genotyping and precise phenotyping in genetic populations has greatly quickened the identification of critical genomic regions and associated markers. There is an increased potential for implementing translational genomics applications in improving pea crops with the advancements of genomic resources and omics-based approaches such as reference genomes, transcriptomic analysis, marker-assisted selection (MAS), and marker-assisted backcrossing (MABC) (Barilli et al. 2018).

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Genomic Libraries

A high-quality, reference genome assembly that is well-annotated is crucial for crop’s genome biology studies. It is also critical for identifying the genes responsible for important traits and developing genomic tools that will accelerate crop improvement (Kreplak et al. 2019). In order to store large and stable genomic clones for crops with extensive genomes, bacterial artificial chromosomes (BACs) were used to create genomic libraries. These libraries played a pivotal role in identifying molecular markers linked to significant agronomic traits. Furthermore, conserved synteny shared by the pea genome and other legumes emphasizes the potential of pea BAC libraries in identifying alleles associated with important agronomic traits and disease resistance (Yu 2012). The first reference genome assembly of the pea cv. “Cameor” was recently completed using complementary methods (BAC library) for physical mapping, positional cloning, genome sequencing, and gene function and structure analysis. This new assembly accounts for approximately 88% (4.45 Gb) of the pea genome size with 82.5% of the sequences attributed to the seven pseudo-molecules and 14,266 unassigned scaffolds. There are 44,756 annotated genes in the genome assembly, with average gene lengths of 2784 bp and exon lengths of 308.5 bp. The annotation also identified 2225,175 repetitive elements, the majority of which are transposable and clustered into 2940 consensus sequences, accounting for 83% of the genome. (Gali et al. 2019). The ready availability of valuable genomic information from various programs has enabled the pea community to rapidly progress in efficient and targeted molecular breeding. For reverse genetics research, genomics approaches such as fast neutron and TILLING were used. The TILLING method uses mutagens such as ethyl methane sulfonate (EMS) to induce point mutations, whereas the fast neutron-induced deletion method causes deletion mutations in genomic DNA targets. The TILLING populations were developed using the pea variety cv. “Cameor” (Dalmais et al. 2008). TILLING population characterization is available in an online database UTILLdb (http://urgv.evry.inra.fr/UTILLdb), containing mutant gene sequences as well as phenotypic information (Moreau et al. 2018). The availability of this reference sequence has greatly improved the understanding of pea genome structure, resulted in the discovery of new genes, and facilitated the development of functional/diagnostic molecular markers. This reference sequence serves as the primary tool for discovering the molecular basis of important agronomic traits, thereby speeding up the progress of pea improvement efforts (Kreplak et al. 2019).

1.2.2

Transcriptome and Proteome Analysis

Microarray or RNA sequencing (RNA-seq) technology can be used to analyze gene expressions and a pioneering transcriptomic analysis identified 346 genes that were differentially regulated using M. truncatula microarray during Peyronellaea pinodes infection in pea. These genes were involved in a variety of processes, including the metabolism of phenylpropanoid, cell wall reinforcement, PR proteins

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(pathogenesis-related), and phytoalexins (Fondevilla et al. 2011a). Furthermore, an EST-based microarray analysis of pea seed aging revealed altered gene expressions linked to programmed cell death and oxidative stress (Chen et al. 2013). Although microarray analysis has the potential to be useful, it is not a reliable method for quantifying transcript splice variants and does not provide data on new genes that are not present on the array. NGS technology has emerged as an effective tool for analyzing transcriptomes. NGS technology has enabled the development of transcriptome repertoires for nonmodel species lacking a sequenced genome, such as pea. This resource facilitates molecular and “omics” approaches for pea. To create this resource, libraries were made from various pea tissues, such as cotyledons, flowers, epicotyl and hypocotyl, and light-treated etiolated seedlings and leaves. These 450 megabase libraries were assembled into 324,428 unigenes and then annotated with M. truncatula, A. thaliana, G. max, and other relevant databases (Fondevilla et al. 2014). The candidate genes for resistance to P. pinodes in peas were identified using a Massive Analysis of cDNA Ends (MACE) (Winter et al. 2016). RNA-seq is accurately measuring gene expression in detail and detecting the variations in nucleotides such as SNPs and SSRs in expressed sequences and identifies alternatively spliced gene products. High-quality transcriptomic data can be used to develop molecular markers, map genomes, and study plant–environment interactions and plant development (Zaman et al. 2019). The first pea gene expression atlas, published by Franssen et al. (2011), involved the sequencing of 20 libraries. Tissue samples from various parts of the pea plant, including the epicotyl, and hypocotyls, flower, cotyledon, and leaf, and from both etiolated and light-treated varieties of the “Little Marvel” pea, were used to create these libraries. However, Zhukov et al. (2015) sequenced RNA from pea nodules and root tips, yielding 37,000 and 58,000 contigs in the “Nodule” and “Root Tips” categories, respectively. Further investigation revealed that approximately 13,000 contigs were identified as protein-coding sequences specific to the nodules, with 581 of them containing complete CDSs and being identified as transcripts specific to pea nodules. Jiao et al. (2017) sequenced 18 samples from three pea accessions at various developmental stages, yielding 9044 high-quality SNPs that were used to build a genetic map. Liu et al. (2015) aimed to identify differentially expressed genes (DEGs) that could explain trait polymorphism. To accomplish this, they sequenced RNA libraries derived from the seeds of two pea cultivars, a vegetable (Zhewan 1) and a grain pea (Zhongwan 6). Through this sequencing analysis, they were able to identify 459 DEGs during early seed maturation and 801 DEGs during late seed maturation. Additionally, Bahrman et al. (2019) identified an expression atlas of DEGs under low-temperature stress in forest-tolerant and frost-sensitive pea varieties, uncovering a total of 4981 DEGs. The study of proteomics is extensively employed in plant research to gain insights into diverse physiological and biological processes. A range of proteins that play a role in biotic and abiotic stress responses have also been detected, such as heat shock proteins, LEA proteins, lipoxygenases, dehydrins, and beta 1,3 glucanase, integrating proteome and metabolome atlases to improve genome annotation (Leonova et al. 2020). The researchers investigated the mechanisms involved in

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various levels of resistance against the parasitic weed Orobanche crenata in pea plants using proteome analysis and 2D DIGE. They discovered 43 distinct protein spots, the vast majority of which were stress-related proteins (Castillejo et al. 2012). A proteomic analysis of the leaves of two pea accessions that responded differently to Acyrthosiphon pisum (pea aphids) revealed 203 differences proteins, and these have been linked to stress responses, signal transduction, amino acid and carbohydrate metabolism, protein folding/degradation, transcription/translation, and photosynthesis. To better understand the mechanism of resistance against Fusarium oxysporum f. sp. pisi, a proteomic analysis was performed. A multivariate statistical analysis was used to identify 53 pea proteins responsible for various functions and used shotgun proteomics and data-independent acquisition analysis to identify potential P. pinodes resistance protein markers in pea. This improved understanding of pea traits from the genome to the proteome will allow for a more precise progression of pea trait improvement (Castillejo et al. 2020).

1.3

Approaches for Biotic Stress Resistance

Pea production is hampered by a variety of biotic stresses such as viral, fungal, and bacterial diseases and many nematodes and insects and pests. Fungal diseases are regarded as the most serious type of biotic stress. Some of the examples are powdery mildew (PM), root rot (RR), Fusarium wilt (FW), Fusarium root rot (FRR), rust (PR), Ascochyta blight (AB), common root rots (CRR), and Aphanomyces root rot, all cause significant yield loss in pea when conditions are favorable for the development of these diseases and have been reported to be severe in pea-growing countries (Bohra et al. 2015).

1.3.1

Traditional Breeding for Biotic Stress Resistance

Attempts have been made to take advantage of the available genetic information of resistance to these important biotic stresses through conventional breeding in order to develop resistant cultivars (Ghafoor and McPhee 2012). High-yielding pea cultivars with PM resistance were developed using traditional breeding techniques by exploiting genes such as er1, er2, and er3 (Fondevilla et al. 2007). In resistant pea accessions, the er1 gene is shown to be more common than the er2 gene. As a result, the majority of pea improvement programs used the er1 gene, which confers resistance via the prepenetration resistance mechanism (Fondevilla et al. 2006). Black spot disease (AB) is the most severe pea disease, which caused yield losses of up to 60%. The rate of transmission from seed to sapling is 40–100%, with the ability to survive on seeds for many years as A. pisi and P. pinodes are seed-borne (Liu et al. 2016). The resistant source for Ascochyta blight has not been discovered, but a significant scale of resistance was discovered in a P. fulvum accession (P651) that is actively used in pea improvement (Sindhu et al. 2014). FW is another severe threat caused by Fusarium oxysporum. f. sp. pisi caused a reduction in yield under

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suitable environmental conditions (Chaturvedi et al. 2021b). The ideal soil temperature for the development of FW disease is between 23 and 27 °C. Fusarium has been identified in 11 different races and discovered in terms of virulence, and of these, races 1 and 2 have become cosmopolitan, whereas races 5 and 6 are dominant (Bani et al. 2018). McPhee (2003) identified resistance sources to races 1 and 2 and develop resistant cultivars, and one CWR accession (PI 344012) has been identified as having resistance to races 1 and 2. Understanding inheritance is essential for incorporating any desired trait into the targeted genotype. As a result, Fop race 1, 5, and 6 inheritance pattern of resistance has been investigated and confirmed to be monogenic with dominance in nature, whereas race 2 resistance is quantitatively regulated. Therefore, many pea cultivars have successfully introgressed the monogenic dominant resistance (Bani et al. 2018). Common root rot reduces grain yield significantly in peas, particularly when there is excess moisture in the soil, and causes root structure damage and wilting of the infected plant (Wu et al. 2018). Traditional disease management approaches, such as seed treatments and crop rotation, are unable to completely control this disease due to the pathogen’s longterm persistence, primarily in the form of oospores, and contaminate the crops. As a result, the development of resistant cultivars has been advocated as the goal of pea breeding programs. The resistant pea accessions to common root rots have been discovered and used in breeding programs to develop new cultivars (Lavaud et al. 2015). Although the use of molecular markers can greatly accelerate the introgression process, as a result, modern genomic tools and procedures have enabled the use and selection of naturally occurring sources of resistance in peas (Smýkal et al. 2012).

1.3.2

Genomics-Assisted Improvement for Biotic Stresses

Conventional gene mapping could not be widely used to identify the genes/QTLs (quantitative trait loci) that regulate disease resistance due to their polygenic inheritance and narrow variability pattern. Morphological markers have long been used to select desirable breeding materials in peas based on both qualitative and quantitative traits. However, remarkable advances in molecular marker development have occurred in recent years, greatly facilitating diversity analysis, genetic mapping, and identifying genes/QTLs that regulate quantitatively inherited traits in pea (Gali et al. 2018). Furthermore, environmental factors have a significant influence on quantitatively inherited traits. As a result, DNA-based molecular markers, such as RAPD (random amplification of polymorphic DNA), RFLP (restriction fragment length polymorphism), AFLP (amplified fragment length polymorphism), SNPs (single nucleotide polymorphisms), and SSR (simple sequence repeat), have been developed and successfully used to calculate genetic variations and have been used in genetic studies to improve pea varieties (Gali et al. 2018; Barilli et al. 2020). Many reports focusing on the mining of SSR markers based on expressed sequence tags (EST) were published, and EST-SSR markers developed ranging from 11 to 8899 (Yang et al. 2015). Many gene-based SNP markers for pea genetic improvement

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have been developed using next-generation sequencing technology. The first SNP array (GenoPea 13.2 K SNP) was created from 12,802 transcript-derived SNPs discovered after resequencing 16 different pea accessions (Tayeh et al. 2015). The Pea Marker Database (PMD) was created to store and manage valuable genomic resources, including various types of pea markers. The Pea Marker Database (PMD) was created to store and manage valuable genomic resources, including various types of pea markers (www.peamarker.arriam.ru; Kulaeva et al. 2017). PMD version 1 contained information on 2484 genic markers (Duarte et al. 2014), whereas PMD2 contains information on 15,944 markers with improved features (Tayeh et al. 2015). The initial pea linkage maps were used to map genes/QTLs for biotic stress tolerance using different molecular markers. The genes er 1, er 2, and er 3 and their alleles that confer PM resistance have been identified using different markers. Furthermore, alleles er1-9 have been mapped and validated in pea using co-dominant functional markers, and a new allele er1-6 of gene er1 was identified by sequencing of PsMLO1 cDNA and validated by a closed linked SSR marker (Sun et al. 2019). Next-generation sequencing (NGS) has recently enabled the rapid discovery of SNPs in peas as well as the development of a genotyping array (Duarte et al. 2014). The single dominant gene responsible for FW resistance was identified using dominant and co-dominant markers that were unsuitable for MAS due to their dominant nature and poor linkage to the gene. Thus, a CAPS marker that was co-dominant with 94% accuracy was designed and found to be useful in resistance selection against F. oxysporum race 1 (Jain et al. 2015). In peas, QTL mapping has been used to identify genes that govern partial inherited resistance as well as major or minor QTLs for biotic stress tolerance. Through molecular mapping, scientists have identified major and minor genes/QTLs responsible for resistance against PR (Rai et al. 2011) and further validation of these markers does not provide a complete differentiation between susceptible and resistant genotypes of PR, limiting their use in MAS (Singh et al. 2015). Recent advances in highdensity molecular mapping with SNP markers and the use of heterogeneous inbred family (HIF) and isogenic lines (NILs) populations have provided novel opportunities for fine mapping of genes/QTLs and the identification of closely linked markers for marker-assisted selection. For instance, SNP-based linkage mapping has successfully identified QTLs, i.e., UpDSII, UpDSIV, and UpDSIV.2, responsible for resistance against PR (Barilli et al. 2018). Several QTL mapping studies on AB resistance have identified various genomic regions that regulate resistance. Furthermore, Jha et al. (2015) discovered SNPs (RGA-G3A and PsDof1) within linked genes that have a significant association with resistance to AB. In another study, an interspecific population derived from the crossing of P. sativum (Alfetta) and P. fulvum (P651), nine QTLs were found that were associated with resistance to AB, out of which only QTLs (abIII-1 and abI-IV-2) were stable across locations/ years and further fine mapped in HIF populations (Jha et al. 2017). The genomewide association study (GWAS) validated and refined previously reported QTLs and identified new loci for A. euteiches resistance, revealing 52 QTLs, six of which had previously been identified. On the other hand, the GWAS approach was used in a large set of contrasting pea genotypes (266) using 14,157 SNP markers, and

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11 genomic intervals were identified, with significant association with A. euteiches resistant (Desgroux et al. 2018). SNP marker, linked with disease resistance and root system architecture and mapped to the major QTL Ae-Ps7.6, to reduce the incidence of root rot in peas and used in regular pea breeding programs. Furthermore, genotyping by sequencing (GBS) was used to identify eight novel SNP markers within the abI-IV-2 QTL in RILs, whereas no additional SNPs were found in the abIII-1 QTL. Similarly, using SSR and SNP markers, numerous QTLs have been identified, accounting for up to 53.4% of the phenotypic variation in polygenic inherited FRR resistance (Coyne et al. 2019).

1.4

Genomic Designing and Breeding-Mediated Approaches for Improving Abiotic Stress Resistance

Pea production is severely impacted by abiotic constraints including extremes of temperature (low and high), drought, flood, and salinity conditions. Selecting genotypes that are resistant to abiotic stresses is challenging due to the fluctuation of environmental conditions across different locations over changing time (Chaturvedi et al. 2022). These stressors majorly impact the seed yield and its quality which alters the overall growth of the plant. In order to assess the response of the crop to specific stress and improve breeding efficiency, the evaluation of the crop is done under controlled conditions (Hochmuth 2019). This current section discusses the impact of various abiotic stressors on pea plants along with the progress made in improving stress resistance and quality traits through conventional and molecular breeding along with concurrent advancements in transgenic approaches.

1.4.1

Drought Stress

The water requirement of pea plants is considerably high during the growing season, specifically during the critical stages including germination and flowering, as opposed to the pod-filling stage which is least affected by water deficit conditions (Lahuta et al. 2022). The timing of water stress was reported to affect the total dry matter (DM) production in peas; subsequently, it was reported that a reduction in DM was more pronounced before the flowering stage in pea plants subjected to moisture stress (Gusmao et al. 2012). Alongside this, the protein and starch ratios during the flowering and pod-filling stages are altered during drought, leading to a reduction in yield (Petrović et al. 2016). To sustain moisture-restricted conditions, plants primarily utilize three strategies: escaping unfavorable conditions, avoiding them altogether, or developing a tolerance to them (Jogawat et al. 2021). The escape mechanism in pea is based on earliness in flowering and maturity, which is also the first choice of breeders (Choudhary et al. 2016). However, early flowering and maturing crops are susceptible to moisture stress; hence, dry pea can only perform well if adequate water is available during the flowering and pod-filling stages

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(Nemeskéri et al. 2015). Pea breeding programs in numerous countries are currently prioritizing the selection of stress-resistant cultivars with high yield potential, achieved through both early maturation and elongated flowering periods (Gowda et al. 2015). The drought avoidance strategy utilized by plants to combat stress essentially involves delayed water loss by modulating stomatal conductance, reducing leaf area, and non-transpirational water loss from leaves (Farooq et al. 2019). The semileafless variety extends various advantages under drought conditions and was regarded as drought-tolerant because of its reduced leaf area when compared to the conventional leafy type of pea (Szablińska-Piernik and Lahuta 2021). For, instance, HR1, a semi-leafless breeding line showed promising results under field conditions ascribing to its high yield and stability under moisture stress conditions (Iglesias-García et al. 2017). Moreover, drought stress-related genomic regions have been identified in pea by inducing water stress and evaluating both soil and leaf relative water content (RWC). Ten QTLs associated with drought adaptation were detected in pea using RILs obtained from crossing P665 and Messire, which exhibited a linkage with 9–33% of phenotypic variance. Furthermore, the regenerative markers linked to these QTLs were also identified, providing a useful tool for selecting cultivars for drought tolerance in pea improvements (Iglesias-García et al. 2015). To counteract severe drought conditions, plants suppress stomatal opening by 60% which further reduces the photosynthetic efficiency of the plant (Moisa et al. 2019). Stomatal closure in response to drought stress was recently shown to be modulated by nitric oxide which in turn was influenced by the Gβ subunit of G proteins (Bhardwaj et al. 2020). Furthermore, an E3 ubiquitin ligase, constitutively photomorphogenic 1 (COP1), was observed to regulate stomatal closure under dehydration stress which can be targeted for drought stress tolerance (MoazzamJazi et al. 2018). Another prospective molecular target can be the dehydrationresponsive element binding protein 2A (DREB2A) gene as reported by Jovanovic et al. (2013), wherein drought stress effects were examined in a new variety of pea (NS-Mraz) via expression analysis of DREB2A gene which showed that expression of DREB2A increased two-folds with a concurrent decrease in water content in roots of pea at 10 days post-dehydration stress as compared to its 60% increase in pea leaves 7 days after stress induction suggesting a significant role of DREB2A in dehydration stress response and its promising potential as a molecular target for the development of drought-tolerant pea (Jovanovic et al. 2013). Consequently, these reports indicate that multifaceted approaches toward enhancement of drought tolerance in pea either by selective breeding or by targeting genes such as DREB2A which will culminate in high-yielding drought-tolerant varieties. Furthermore, large-scale high-throughput germplasm screening for the discovery of genes/QTLs and their closely associated markers for different targeted traits will be highly beneficial for pea breeding programs.

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Salinity Stress

Salt stress is a major ecological constraint to worldwide sustainable production and food security (Habib et al. 2016; Shailani et al. 2021a; b). Severe salinity causes a range of physio-biochemical abnormalities that include hyperosmotic effects, nutritional imbalances, ion toxicity, impaired gas exchange, and disrupted water homeostasis. These abnormalities combine to hinder plant growth and reduce yields (Siddiqui et al. 2019). The soils in the northeastern regions of India are predominantly acidic, with a pH range of 5.0–6.0. This acidity results in a high concentration of Fe3+ and Al that leads to a deficiency of K and P (Dambrine 2018). For pea plants, acidic soils pose a challenging situation due to the accumulation of heavy metals including aluminum (Al), iron (Fe), and manganese (Mn) (Bojórquez-Quintal et al. 2017) as pea plants have high requirements of phosphorus for nodule formation and photosynthesis. Recently, a study assessed the salinity-induced physiological responses of eight field pea genotypes. Among these, BD4175 and BD4225 exhibited promising outcomes under a salt stress level of 8 dS m-1, showing the highest levels of germination, shoot length, seedling vigor index, salt tolerance index, and seedling height stress index, suggesting that they can be used for future breeding programs for the development of salt-tolerant pea cultivars (Khan et al. 2022). Furthermore, to develop linkage maps in pea associated with seedling growth stage salinity, SNP markers linked to ESTs were established. In a recombinant inbred line population (Kaspa × Parafield), 768 nucleotide positions out of a total of 36,188 detected positions were designated for genotyping. Single nucleotide polymorphisms (91.7%) were discovered through segregation, with the quantitative trait loci for salinity resistance being identified on linkage groups III and IV to aid in the identification of tolerant cultivars (Leonforte et al. 2013). A mutant population was generated in pea using ethyl methane sulphonates (EMS), in which dehydrins (DHNs) and cinnamyl alcohol dehydrogenase (CAD) were identified as a mutants. These genes play a crucial role in conferring tolerance against various stresses, such as salinity, and can thereby act as potential targets for developing salinity tolerance in pea. (Hameed 2018). Furthermore, in the pea cultivar, meteor, four antioxidant genes, namely alternative oxidase (AOX), peroxiredoxin (PrxIIF), Mn-superoxide dismutase (Mn-SOD), and thioredoxin (Trxo1), were upregulated in response to salt stress and can be manipulated for improving tolerance (Manzoor et al. 2020). Several plant growth regulators like gibberellic acid (GA3) have also been reported to enhance salt tolerance in pea. For instance, two pea varieties Meteor-FSD and Samrina Zard were pretreated with GA3 (10-4 M) for 12 h. The application of GA3 was found to enhance plant growth and decrease Na+ transport in the Samrina Zard pea variety, which is sensitive to salt stress. Additionally, when combined with silicate, GA3 contributed to an increase in salinity tolerance for both varieties Meteor-FSD and Samrina Zard. This combination also resulted in reduced Na+ transport and increased stomatal conductance, K+ transport, and shoot biomass under salt stress. These observations suggest that GA3 plays a regulatory role by affecting Na+ uptake and transport, thereby promoting salt tolerance in peas (Raza Gurmani et al. 2022).

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Heat Stress

Pea plants are highly sensitive to elevated temperature in the field, with reports of flower, fruit, and seed abortion and reduced seed size (Jiang et al. 2015). During the flowering stage in pea, a significant decrease of 600 kg per hectare in pea production was observed in response to every one-degree increase in temperature (Kaushal et al. 2016). This decline is primarily caused by a decrease in the number of pods and seeds, as well as forced maturity. Heat stress majorly impacts the reproductive organs of crops, which results in an acceleration of the crop lifecycle. The development of seeds is particularly affected, with the positioning of the ovules in pea pods playing a crucial role (Hasanuzzaman et al. 2013). Heat stress can cause the production of abortive seeds due to fertilization failure at the basal ovule position (Jiang et al. 2020). Furthermore, temperature plays a fundamental role in seed germination, affecting not only the germination rate but also the growth of the seedlings, including the reduction in the length of the plumule and radical of different legume crops (Lamichaney et al. 2021). Due to the high susceptibility of the flowering stage to heat stress, pea genotypes are being selected based on their ability to produce viable pollen in order to enhance seed setting efficiency (Jiang et al. 2020). As heat tolerance is a quantitative trait, identifying corresponding QTLs and using them effectively are an essential strategy for speeding the breeding strategy for the production of heat-tolerant genotypes. A recent study evaluated 135 accessions of peas across five different environments to identify 10 heat stress-responsive traits using genome-wide association studies (GWAS). The study successfully identified 32 associated markers and 48 candidate genes that may play a role in heat tolerance in peas (Tafesse et al. 2020). Another study on a new recombinant inbred line population of pea (PR-24) successfully identified four QTLs associated with heat stress tolerance across multiple trials, one for each of the following traits: days to flowering (chromosome 7), reproductive node number (chromosome 5), pod number (chromosome 2), and seed protein concentration (chromosome 5). Additionally, the study validated two indices, namely the stress tolerance index and geometric mean yield, which were previously used to assess drought tolerance, as useful criteria for assessing heat tolerance in peas (Chaturvedi et al. 2023; Huang et al. 2023). Besides breeding approaches, transgenic peas obtained by genetic manipulation with Arabidopsis heat shock factor HsfA1d by gateway cloning in plant expression vector pGWB415 conferred resistance to pea plant against heat stress by enhancing the activity of antioxidant enzymes and decreasing hydrogen peroxide compared to wild-type plants (Shah et al. 2020). Pea is a widely used model plant that has been extensively studied at both the phenotypic and molecular levels, with its genome sequence being released in 2019 (Gali et al. 2019). Despite this, progress in identifying the underlying molecular mechanisms of heat stress in pea at the genomic level has been limited when compared to other winter-season legumes, such as chickpeas and lentils (Kreplak et al. 2019), therefore utilizing both breeding and transgenic approaches for improving thermal tolerance can prove to be advantageous in the future.

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Cold Stress

Stress induced by cold temperatures is a significant challenge for global agricultural production, particularly for cool-season food legumes (Bhat et al. 2022). Crops are particularly vulnerable to freezing temperatures during key stages of their growth, such as seedling emergence, flowering, initial pod formation, and seed filling (Bahrman et al. 2019). Cold acclimation refers to the gradual development of a plant’s ability to withstand freezing temperatures, which occurs when the plant is exposed to low temperatures that do not reach a freezing point (Vyse et al. 2019). During the process of cold acclimation, two primary functions take place: Firstly, the metabolic and cellular functions of the plant are adapted to cope with the physical limitations imposed by the low temperatures (known as “chilling responses”), and secondly, the plant undergoes the induction of freezing tolerance (Vyse et al. 2019). The process of adapting to cold temperatures involves the accumulation of several cold stress proteins within the chloroplast which enhances the durability of chloroplast membranes when exposed to freezing conditions, brings about changes in the photochemical properties of photosystem II, and enhances the functioning of ROS scavenging systems, ultimately leading to a reduction in the plant’s sensitivity to photo-inhibition under low-temperature conditions (Nurhasanah Ritonga and Chen 2020). For instance, Champagne and Terese, two pea lines with different acclimation abilities, were evaluated to check chloroplast-related changes occurring during cold acclimatization, wherein freezing tolerance was observed in Champagne ascribing to its high inherent photosynthetic potential, as well as early initiation of metabolic processes designed to sustain photosynthetic capacity suggesting its potential in developing cold stress-tolerant varieties (Grimaud et al. 2013). Furthermore, Baldwin et al. (2014) reported a significant role of esterified pectins in cold stress tolerance of pea plants, wherein cell walls that were extracted from the stipules of plants that were either cold-acclimated or non-acclimated exhibited alterations in the polymers that contain residues of galacturonic acid, xylose, arabinose, and galactose, as a result of cold temperatures. In the tolerant cultivar Champagne, acclimation coincided with an increase in xylogalacturonan, homogalacturonan, and branched rhamnogalacturonan I, which consisted of branched and unbranched (1 → 5)-α-arabinans and (1 → 4)-β-galactans. In contrast, the sensitive cultivar Terese accumulated significant amounts of (1 → 4)-β-xylans and glucuronoxylan, but not pectins, indicating its role in freezing tolerance (Baldwin et al. 2014). Alongside proteomic analysis of Champagne revealed 32 proteins involved in frost tolerance associated with reorienting energy metabolism under stress (Dumont et al. 2011). Moreover, transcription factors known as C-repeat binding factor/dehydration-responsive element binding protein 1 have been reported to play a significant role in cold stress acclimatization in various crops including Solanum tuberosum (Charfeddine et al. 2015), Cucumis sativus (Li et al. 2022), and Vaccinium corymbosum (Polashock et al. 2010) which can be targeted in pea to enhance tolerance to low temperatures.

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Waterlogging

The normal growth and development of plants are adversely affected by factors that limit the availability of oxygen, such as flooding, waterlogging, and partial or complete submergence (Bashar et al. 2019). In order to cope with flooding conditions, plants employ various strategies such as storing energy, elongating their petioles or internodes, regulating stomatal movements to maintain water levels, forming adventitious roots, and developing aerenchyma (Chen et al. 2022). Pea and Lupinus albus (white lupin) plants exhibit severe symptoms, including leaflet abscission and adverse impacts on newly opened flowers post-flooding stress (Malik et al. 2015). These patterns have also been observed in various other crops, such as chickpea, grass pea, lentils, mungbean, pigeon pea, and soybean (Malik et al. 2015). A recent study was performed for evaluating waterlogging tolerance in field pea, wherein an inbred recombinant pea population underwent screening for tolerance to waterlogged and drained soils, as well as evaluation for testa integrity. Plants with dark-colored testa were discovered as waterlogging tolerant, with 90% exhibiting this phenotype. Conversely, plants with a light-colored testa were deemed sensitive to waterlogging, suggesting that testa integrity can be used as a trait for selection (Zaman et al. 2019). Another significant selection criterion is seed size, wherein smaller seeds have been reported to be more tolerant to flooding stress as compared to larger seeds (Sayama et al. 2009). Three genotypes of pea including BM3, NL-2, and Kaspa, which differ in their ability to germinate in waterlogged soil, were subjected to varying durations of waterlogging. These cultivars obtained were concurrently examined for alteration of the gene during growth via analysis of differential gene expression via whole-genome RNA sequencing. Strong activation of tyrosine–protein kinase and inhibition of a gene involved in fat metabolism (linoleate 9S-lipoxygenase 5) were identified in Kaspa. On the contrary, in NL2, stimulation of the fat metabolism gene was observed. BM3 was nontolerant to waterlogging which was correlated to the elicitation of Kuntz-type trypsin/protease inhibitor that results in extreme lipid absorption and membrane outflow related to waterlogging damage. Compared to sensitive genotypes, the tolerant genotypes exhibited upregulated storage protein metabolism which offers a base to generate waterlogging tolerant cultivars (Zaman et al. 2019).

1.5

Marker-Assisted Selection (MAS) for Resistance Traits

Using traditional breeding methods, developing pea cultivars with improved yield, higher nutritional content, and greater disease resistance may take decades or even longer. As a result, marker-assisted selection (MAS) is considered as one of the most important and effective modern breeding techniques for rapid improved cultivars development. The close association of markers with a trait of interest serving as a prerequisite of MAS (Tayeh et al. 2015). To identify gene-linked markers associated with disease resistance in pea, both biparental and association mapping methods were used. These markers are available for marker-assisted breeding and have been

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linked to resistance to FW (Kwon et al. 2013), PM (Lakshmana Reddy et al. 2015), PR (Barilli et al. 2018), pea seed-borne mosaic virus (Swisher Grimm and Porter 2020), FRR (Coyne et al. 2019), CRR (Desgroux et al. 2016), and AB (Jha et al. 2017). The effectiveness of the MABC (marker-assisted backcrossing) technique in transferring QTLs linked to Aphanomyces root rot (ARR) resistance into various recipient genotypes has been reported by Lavaud et al. (2015). One of the main abiotic factors is the frost stress that causes seedling death and a decrease in winter pea yield. A MAS analysis was performed using 267 SSR markers and marker-trait association, 16 out of 672 accessions were discovered to be frost-tolerant. The analysis resulted in the identification of 16 winter-hardy cultivars as well as the detection of seven markers associated with frost tolerance. One of these markers, found on linkage group IV, was found to be functional and responsive to chilling stress in pea plants (Liu et al. 2017). Tafesse et al. (2020) reported marker-trait association (MTA) analyses on pea plants to determine their tolerance to high temperatures. The study utilized a total of 16,877 single nucleotide polymorphisms (SNPs) and identified 32 MTAs that consistently performed consistently well in three different environmental conditions. The study found that four traits, namely reproductive stem length, chlorophyll concentration, internode length, and pod number, were important indicators of heat tolerance in pea plants. These identified MTAs can be used in marker-assisted selection (MAS) to improve the temperature tolerance of pea cultivars. The marker-assisted backcrossing (MABC) strategy was utilized to introduce new traits into pea plants, specifically to increase the frost resistance of pea plants. Lejeune-Hénaut et al. (2008) reported three QTLs (quantitative trait loci) associated with frost resistance were identified. In addition to improving tolerance to a variety of biotic and abiotic stresses, MAS was used to develop folate-profiled pea cultivars, with 85 accessions evaluated using a genomewide association study (GWAS). As a result, SNP markers associated with pea folate profile were discovered (Jha et al. 2020).

1.6

Quality Traits

The development of pea-quality trait breeding has been hampered by the complexity of quality-related traits due to the unavailability of efficient and effective screening tools. Despite these challenges, efforts have been made to enhance the seed quality traits such as starch, seed protein, antinutritional compounds, micronutrients concentration, and seed quality such as seed coat color and seed size, seed protein content and composition, and iron deficiency (Kabir et al. 2012).

1.6.1

Protein

Protein is a crucial food group that plays a vital role in maintaining muscle mass and strength in later life. Recent studies suggest that increasing protein intake during all spans of life can help reduction of muscle decline and prevent health conditions

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(Stevenson et al. 2018). Pea seeds are a good source of protein, containing approximately 15–30% protein, with most cultivars containing 20–28% protein, and globulin and albumin storage proteins are the primary proteins found in pea seeds (Tao et al. 2017). The genetic complexity of pea seed protein composition arises from the involvement of multiple gene families that encode different proteins, which are subsequently subject to extensive posttranslational processing. A study conducted by Bourgeois et al. (2011) reported that the protein composition of pea seeds is largely determined by genetics, with a significant variation (60%) in the abundance of protein spots observed between different genotypes in a two-dimensional electrophoresis analysis. While environmental factors can also impact the composition of pea seed proteins, their effects differ from those of genetic factors. For instance, exposure to heat and drought stress during seed maturation can hinder the accumulation of proteins that typically accumulate in later stages of development, resulting in overall lower protein accumulation. Tar’an et al. (2004) employed a combination of genetic crosses and linkage mapping to locate quantitative trait loci (QTLs) responsible for the total concentration of seed protein in pea. One such QTL was found on linkage group VI, which accounted for 45% of the variation in seed protein concentration between the two parental lines. This was a substantial difference given that the two parental lines only differed by 9 g/kg (0.9%) in seed protein concentration. The QTL was believed to be associated with the synthesis of albumin in pea. Furthermore, SNPs within the O2like gene in pea have previously been discovered to have a significant effect on seed protein concentration. Recently, genome sequencing of the pea cultivar Cam’eor has resulted in the annotation of seed protein information, which includes genes responsible for seed storage proteins such as convicilin (two genes), legumin (12 genes), and vicilin (nine genes), discovered through a search of the assembly on UNIPROT (Kreplak et al. 2019).

1.6.2

Starch

Pea seeds are made up of different starch fractions that are classified based on their structure and digestibility in the gut (Lockyer and Nugent 2017). The starch fraction comprises approximately 45–50% of the dry weight of pea seeds, and resistant starch is a type of dietary fiber with numerous health benefits. Resistant starch is fermented by the gut microflora in the large intestine rather than broken down by amylolytic enzymes in the small intestine. This fermentation process produces short-chain fatty acids such as acetate and butyrate, which interact with pancreatic insulin-producing cells to maintain blood glucose homeostasis (Stephen et al. 2017). The pea varieties used in Gregor Mendel’s inheritance studies most likely had genotypes that produced seeds with varying levels of resistant starch. Although wrinkled seeds have less total starch than round seeds, mutations in genes involved in starch biosynthesis result in higher relative concentrations of resistant starch. In one type of wrinkledseeded pea, a naturally occurring mutation in the SBEI gene results in a higher level of resistant starch (Rayner et al. 2017). Peas also contain long-chain soluble and insoluble polysaccharides, as well as galacto oligosaccharides, including members

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of the raffinose oligosaccharide family (De Almeida Costa et al. 2006). Genetic variation in the levels of raffinose oligosaccharides in pea germplasm has been observed, and mutant lines must be identified in order to develop pea cultivars with significantly lower levels of these sugars, which could improve consumer acceptability of pulse crops. Raffinose synthase is an enzyme that is essential in the biosynthesis of raffinose and could be targeted to reduce raffinose concentrations in pea, potentially increasing consumer interest in pulse crops (Brummer et al. 2015). In recent years, Gali et al. (2018, 2019) have conducted two studies to locate and map quantitative trait loci (QTLs) associated with dietary fiber. To classify fiber fractions, these studies used either acid or neutral detergent for digestion. The first study (Gali et al. 2018) used three recombinant inbred lines (RIL) populations derived from crosses between pea cultivars and breeding lines from Canada and Europe, namely PR-02, PR-07, and PR-15. In the PR-02 RIL population trials, four linkage groups revealed QTLs for acid detergent fiber (ADF), two of which were located on linkage group VII and accounted for 28.0% and 26.2% of the phenotypic variance, respectively. In the PR-07 population, significant ADF QTLs were also discovered on linkage groups IV and VIIa. All PR-02 population trials found QTLs on linkage group Va, while multiple PR-07 RIL population trials discovered QTLs on linkage groups Ia, IV, and VIIa, accounting for up to 44% of the phenotypic variance (Gali et al. 2018).

1.6.3

Micronutrients

Micronutrients found in pea seeds include iron, zinc, and selenium, and iron is especially important for seed embryo development, and pea seeds store a significant amount of iron as ferritin (Khan et al. 2023). Plant ferritin is found in plastids, which are surrounded by multiple membranes, which can impede iron released during digestion (Moore et al. 2018). Purified ferritin and iron from pea and soybean, on the other hand, have been shown in studies to have high bioavailability (Perfecto et al. 2018). During the screening of mutants, two pea mutants known as brz (bronze) and dgl (degenerate leaves) were found to have significantly higher levels of iron uptake, and this increased uptake was observed in both mutants. Despite higher iron intake in brz plants, there was no rise in the iron content of the seeds. When grown under conditions with excessive iron, dgl mutants exhibit an increase in the concentration of seed iron than the wild type. The increase in iron accumulation in dgl mutants results from heightened iron (III) reductase activity and proton efflux in the roots, as well as high levels of nicotinamide, which is an iron (II) chelator associated with both mutations. While the specific genes associated with the dgl and brz mutants remain unidentified, a gene responsible for regulating Fe (III)-chelate reductase (FRO1) expressed consistently in both mutants (García et al. 2013). The average seed iron concentrations in commercial cultivars grown in North Dakota ranged from 45 to 54 mg/kg, which is similar to the values found in Canadian-grown peas (48–58 mg/kg) (Amarakoon et al. 2015). In their study on the marker-trait association. Diapari et al. (2015) discovered that genotype

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accounted for 60.3% of the total variation in seed iron concentration using SNPs in 94 diverse accessions. Another study by Demirbas (2018) revealed that seed iron concentrations varied widely in commercial cultivars and 152 Turkish landraces, ranging from 38 mg/kg to 320 mg/kg. The mean iron concentration in all lines studied was 67 mg/kg, which is comparable to commercial variety average concentrations (Ray et al. 2014). Out of all the accessions, the landrace accession from Tekirda˘g, located in western Turkey, had the highest seed iron concentration, measuring 320.9 mg/kg. According to Demirbas’ 2018 study, the Turkish landrace accession had an unusually high level of iron concentration in its seeds, suggesting that it could be another example of a micronutrient hyperaccumulation mutant. Furthermore, the study discovered seven additional accessions with seed iron concentrations greater than 100 mg/kg, indicating that Turkish germplasm has the potential resource for increasing the iron content of commercial pea cultivars. Several reports have identified genetic markers and QTLs for investigating the seed iron content in germplasm to improve breeding programs. QTLs for seed iron concentration (μg/g dry weight) were discovered by analyzing the population of RILs from a cross between Aragorn and Kiflica cultivars. Linkage groups II (12.3%) and VII (19.4%) had the highest QTLs for seed iron concentration and content, respectively. Diapari et al. (2015) discovered three of the QTLs for iron concentration in a study of 94 different accessions and these QTLs were discovered to be close to markers previously linked to iron concentration, with two QTLs on linkage group V and the third on linkage group VII, which explained the most phenotypic variance (Ma et al. 2017a; b). Research examining the levels of zinc and selenium, which are vital micronutrients, in pea seeds is infrequent. However, a study conducted by Diapari et al. (2015) discovered two single nucleotide polymorphisms (SNPs) on linkage group III that were associated with zinc concentration in seeds explained of the genetic variation of 11.5% and 9.2% respectively. Ma et al. (2017a, b) conducted a study that also discovered a quantitative trait locus (QTL) on linkage group III to be the major determinant of seed zinc concentration. However, the SNPs found in Diapari et al. (2015) research were not closely associated with the QTL on linkage group III. Moreover, Gali et al. (2018) identified four QTLs associated with seed zinc concentration in a study to map QTLs associated with multiple breeding-related traits in pea. One of these QTLs was located in the same region as a previously identified seed zinc QTLs (Ma et al. 2017a, b). This suggests that specific pea genome linkage group III regions have the potential to increase seed zinc concentration, and identifying the underlying genes could help biofortification efforts. The study also examined the concentration of selenium in seeds in three RIL populations and discovered multiple QTLs in PR-02 and PR-7 populations. The environment strongly influenced seed selenium content, and QTLs were only found in PR-02, on two linkage groups, including a locus on linkage group VII. In PR-7, QTLs were discovered on linkage groups IV and Vb, with the locus on the latter explaining the variance of 17.1%.

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Antinutrients

Antinutrients are compounds that can inhibit the absorption of essential nutrients found in food, resulting in a lower nutritional value (Hurrell and Egli 2010). Antinutrients significantly impact the nutrient content of pea crops; one such antinutrient is phytic acid, which is abundant in peas. Therefore, reducing the concentration of phytic acid is considered a potential strategy to enhance nutrient bioavailability in peas. The high concentration of phytate in mature pea seeds is responsible for decreased iron bioavailability, as indicated by Caco-2 cell assays (Moore et al. 2018). Low-phytate (LPA) lines of pea were created from the Bronco cultivar CDC using chemical mutagenesis, which reduced 60–90% of the phytic acid–phosphorus content (Warkentin et al. 2012). Furthermore, Moore et al. (2018) discovered that immature pea seeds have lower phytate levels and were associated with higher iron bioavailability than mature seeds. A linkage map was developed from a RILs population derived from a cross between a wild-type cultivar and low-phytate mutant to map the LPA locus to linkage group V, confirming the single-gene inheritance of phytate concentration. A study that used a larger set of genetic markers confirmed the presence of a significant locus for seed phytate on linkage group V (Gali et al. 2018). Pea seeds contain antinutrients called seed protease inhibitors, which can reduce their nutritional value and have implications for a variety of food and feed industry applications. These inhibitors, such as trypsin and chymotrypsin inhibitors, can hinder the digestion of proteins by gut enzymes, which can require extensive seed processing to neutralize them (Clemente et al. 2015). Pea seeds contain a high concentration of trypsin inhibitors, particularly Bowman–Birk inhibitors, which were discovered to be a type of protease inhibitor over 70 years ago (Birk 1985). Large amounts of these inhibitors can cause the pancreas to overproduce chymotrypsin and trypsin, increasing the demand for the amino acids cysteine and methionine. This, in turn, exacerbates the problem of a lack of sulfur-containing amino acids (Guillamón et al. 2008). Clemente et al. (2015) conducted a study on TILLING mutants and the collection of pea germplasm to examine the effects of mutations that may lead to a reduction in trypsin inhibitor activity in pea seeds. Among the TILLING mutants, three mutations were discovered that were predicted to affect inhibitor activity. C77Y, a TI1 missense mutation that was predicted to affect the formation of intramolecular disulfide bonds, was found to reduce trypsin inhibitor activity by 60%. A genetic marker screening on 2822 accessions from the JIC pea germplasm collection produced a TI1/TI2 double-null mutant in which seed trypsin inhibitor activity was reduced than mutants produced by mutagenesis or transgenesis (Welham and Domoney 2000). This exceptional mutant was a naturally occurring Pisum elatius line from Turkey (John Innes Centre accession JI 262), and analysis of its seeds revealed a significant decrease in trypsin inhibitor activity when compared to the wild-type control. The double-null mutant has successfully been crossed with commercial cultivars to yield double-null progeny lines for comparative studies (Clemente et al. 2015) (Table 1.1).

Ascochyta blight (Peyronellaea pinodes)

Common root rot (Aphanomyces euteiches)

Powdery mildew

Rust (Uromyces fabae)

AA446/SSR, AA505/SSR, AD146/SSR, AA416/ SSR AD146/SSR, AA416/SSR KASPar-er1 PSMPSAD51/SSR, OPX04-880/SSR, KASPar-er1-8 and KASPar-er1-9 AD60/SSR, c5DNAmet AA446-486, PA8, AB23-376, AA430942 AA122, AA387, AB101 Ps115429/SNP OPAI14_1353/AA175, OPAI14_1273/ OPAI14_1353 Sc33468_44352/SNP, Sc33287_25420/SNP, Sc12023_67096/SNP, Sc34405_60551/SNP

Traits Markers For biotic stress improvement Ps900299/SNP Fusarium root rot CAPS/dCAPS (Fusarium solani f. sp. pisi) Fusarium wilt (race1) THO/CAPS, AnMtL6, Mt5_56, PR X1TRAP13, (Fusarium Subt_SNP2, Sus3_SNP8, Cwi1.SNP1, PPT2.SNP1, oxysporum. f. sp. Hlhrep_SNP1, Cwi1_SNP3, pisi), FVE.SNP6, Trans_SNP1 Rust (U. pisi) AD280/SSR, 3567800/DArT, 3563695/DArT, 3569323/DArT, OPV171078/RAPD

Ae-Ps4.5, Ae-Ps7.6 Ae-Ps7.6, Ae-Ps4.4-4.5 MpIII.3_DS_06, MpIII.3_DRl_06 abIII, abI-IV-2, abI-IV-2.1, abI-IV-2.2

er-1 er1-8, er1-9

II, IV III

UpDSII, UpDSIV, UpDSIV.2 Up1 Qruf, Qruf1, Qruf2

I-IV, III, VII

III

II, III, IV, V, IV, VII

VI VI

I VII

II, IIa III, VI, VII II, III, V, VI, VII

Linkage Group

Fsp-Ps3.2, Fsp-Ps6.1, Fsp-Ps7.1 Fw

Genes/QTLs

Table 1.1 Markers and genes/QTLs regions associated with different traits for genetic improvements in pea (Pisum sativum L.)

Fondevilla et al. (2011b) Jha et al. (2017)

Lavaud et al. (2015); Desgroux et al. (2018)

Ma et al. (2017a, b) Sun et al. (2019)

Rai et al. (2016)

Barilli et al. (2018)

Cheng et al. (2015)

Coyne et al. (2019)

Reference(s)

20 H. Singh et al.

Dumont et al. (2011)

III, V and VI

(continued)

Beji et al. (2020)

V

WFD3.1, WFD 5.1, WFD 6.1 WFDcle.a, WFDmon.b WFDcle.b, WFDmon.c, WFDcle.c, FD164.a, FD164.c

Barilli et al. (2020)

I, II, III, IV and V

Jain et al. (2013)

Carrillo et al. (2014)

Jha et al. (2019) Fondevilla et al. (2012) Ashtari et al. (2020)

Gali et al. (2018)

ApI, ApII, ApIII, ApIV.1, ApIV.2, ApV

III

II, III, V

MpII.1, MpIII.5, MpV.3, MpV.2

OPZ10_576/Sugtrans_SNP3, agpl1_SNP2/MSU515_SNP3, OPM4_490/ OPK6_887, sut1_SNP1/OPRS4_699 CNGC, tRNAMet2 En

III

III III, VI

QTLs/abIII-1 Psy1 and Psy2 13 QTLs

IIIb

QTLs

Chr5LG3_562563492, Chr5LG3_568430003, Chr5LG3_568430003, Chr5LG3_569648908

3,535795/DArT, 3537104/3568590/3569349/ 3536355/DArT DArT, 3535012/ DArT,3536533, DArT/3568629 For abiotic stress improvement Frost AD59/SSR, AD141/SSR, AA200/SSR, AD159/SSR AA67/SSR, AGL20a/SSR, AD141/SSR, SucSyn/SSR, AA475/SSR, I01.600/SSR, AB64/SSR, AGL20a/SSR

Pea enation mosaic virus (PEMV) Pea aphid (Acyrthosiphon Pisum)

White mold (Sclerotinia sclerotiorum) Ascochyta blight (Didymellapinodes)

Pseudomonas Syringae pv. Syringae

Sc8865_149928 PsC1846p336—Sc5317_256613/ SNP, Sc7388_112888/SNP Sc3030_71736—PsC7000p195/SNP sC8780p118/SNP OPW5387/RAPD, OPJ121504/OPO61121

1 Genetic Improvement of Pea (Pisum sativum L.) for Food and Nutritional Security 21

Vicilin Legumin Lectin Pea albumin 1 Pea albumin 2

Seed zinc concentration Total seed protein concentration

Zinc Protein

Iron

Raffinose Stachyose Seed iron concentration

II, III, V II, V VII VI VI

QTLs VicB, Vc-2-5 Leg A-D, J-M, S LecA PA1 PA2

V

rfs sts QTLs QTLs QTLs SNP in O2like gene/QTLs

QTLs

Seed starch concentration

IIIa, IIIb VIb Ps III and VII

Linkage Group I, III, and VI

IVa, Ia, IIIb, IIIc, VIIb III V II, VII III III IIIb and VIIb

QTLs 10 QTLs QTLs

Genes/QTLs audpc_rwcs-1, rwcsF-1, audpc_rwcl rwclF3, audpc_rwcs-2, rwcsF-2, rwclF-2

Markers SSR, DipeptIV-SP1/SSR, SSR, Psblox2/SSR, PsAAP2-SNP4 AC74/SSR, AD57/SSR, AB 141/SSR, AB64, 248 SSRs and SNPs 6 SNPs SNPs SNPs

RFO

Lodging resistance Heat resistance Salinity stress For quality traits Starch

Traits Drought

Table 1.1 (continued)

Ellis et al. (2018) Ellis et al. (2018) Ma et al. (2017a, b) Gali et al. (2018) Gali et al. (2018) Jha et al. (2015); Gali et al. (2018) Krajewski et al. (2012) Le Signor et al. (2017) Kreplak et al. (2019) Domoney et al. (2013) Eyraud et al. (2013) Vigeolas et al. (2008)

Gali et al. (2018)

Gali et al. (2018) Huang et al. (2017). Leonforte et al. (2013)

Reference(s) Iglesias-García et al. ( 2015)

22 H. Singh et al.

1684 markers (SSR and SNP) SNP

1684 markers (SSR and SNP) 1866 SNP

1684 markers (SSR and SNP) 1684 markers (SSR and SNP) 1684 markers (SSR and SNP) 1684 markers (SSR and SNP) 1684 markers (SSR and SNP) 1684 markers (SSR and SNP) 1866 SNP 1684 markers (SSR and SNP) 1866 SNP 3355 SNP

3408 SNP

Ca Fe

Fe Fe

Mg Mn

Phytic acid

Mo P K S Se Zn Zn Zn

Seed selenium concentration Seed phytate concentration

Selenium Trypsin inhibitors Phytate

9 QTLs

5 QTLs 5 QTLs 6 QTLs 5 QTLs 4 QTLs 5 QTLs 4 QTLs 15 QTLs

4 QTLs 5 QTLs

5 QTLs 5 QTLs

5 QTLs 15 QTLs

QTLs TI1 and TI2, QTL &Tri locus 4 QTLs

I III, V, VI III, IV, V, VII III, V, VI, VII IVa, Va, VII II, III, V, VII Ia, IIIb, VI Ia,Ib, IIb, IIIb, IV, VIIa IIIa, V, VIa

4, 5, 7 I, III, IV, V, VII, II, V, VII IIb, IIIb, IVb, VI III, IV, V I, II, IV, V, VII

IV, V, VII V V

Klein et al. (2014)

Ma et al. (2017a, b) Ma et al. (2017a, b) Ma et al. (2017a, b) Ma et al. (2017a, b) Klein et al. (2014) Ma et al. (2017a, b) Klein et al. (2014) Klein et al. (2014)

Ma et al. (2017a, b) Ma et al. (2017a, b)

Ma et al. (2017a, b) Klein et al. (2014)

Gali et al. (2018) Clemente et al. (2015) Shunmugam et al. (2015) Ma et al. (2017a, b) Diapari et al. (2015)

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Transgenic Technology and Genome Editing

Conventional breeding methods may not always suffice in addressing both biotic and abiotic stresses in crops, and genetic transformation can be a useful supplement to improve crop resilience. However, due to its large genome and difficulty with genetic transformation, the pea crop faces challenges in developing new cultivars with improved traits such as resistance to detrimental diseases, interaction with beneficial species, and adaptation to environmental stress (Warkentin et al. 2015). Nonetheless, recent biotechnological advances have allowed the successful development of transgenic pea resistant to various insect and pests and diseases through the expression of different genes. Other desirable traits to focus on include enhanced pea grain yield and resistance to various biotic and abiotic stresses and other desirable traits to improved pea interactions with beneficial species like mycorrhiza and rhizobia and adapting to environmental stress, as well as adjusting plant phenology and morphology to better fit new crop systems, all of which would benefit farmers (Foyer et al. 2016). For several detrimental diseases and insect and pests, limited resistance sources exist among cross-compatible germplasm in pea. As a result, resistance genes transferring from other non-cross-compatible species is one method for developing resistant cultivars, possibly through the development of transgenic plants (Warkentin et al. 2015). However, advances in biotechnology have enabled the development of transgenic peas for disease and insect–pest resistance in recent years. Although genetic transformation in peas is challenging, these studies demonstrate its potential for developing resistant cultivars with improved traits and have been particularly effective in expressing cry1Ac protein from soil bacterium Bacillus thuringiensis for the development of insect-resistant cultivars (Teressa Negawo et al. 2016). Weevils are the most destructive insect to food legumes like peas. There is currently no genetic resistance to this insect in crosscompatible germplasm. However, in the common bean, an alpha-amylase inhibitor-1 (aAI) gene has been identified that completely protects against weevil destruction and this gene was transferred to pea through genetic transformation, and developed transgenic lines demonstrated pest resistance (Lee et al. 2013). A recent study sought to improve disease tolerance in European pea cultivars by introducing four antifungal genes: polygalacturonase-inhibiting proteins (PGIPs), 1–3 b glucanase (G), endochitinase (C), and stilbene synthase (S). As a result, transgenic lines with a single antifungal gene or all four genes stacked through hybridization were developed (Kahlon et al. 2018). Additionally, Shivakumara et al. (2017) have demonstrated the potential of pea DNA-Helicase 45 in mitigating various abiotic stresses through agrobacterium-mediated transformation. Moreover, transgenic pea has been reported to show improved salt stress tolerance. Genetically stable transgenic pea plants with salt-tolerant properties have successfully developed by overexpression of the AtNHX1 allele from Arabidopsis thaliana and its combination with PGPR resulted in the promotion of salt tolerance in pea (Ali et al. 2018).

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Furthermore, the HsfA1d heat shock factor, from Arabidopsis thaliana, has been introduced using the expression vector pGWB415 to develop a heat-tolerant transgenic pea line. The transgenic pea plants showed a fivefold increase in HsfA1d expression under heat stress conditions of 42 °C. In comparison to wild-type plants, the transformed pea plants displayed reduced levels of hydrogen peroxide and increased activity of antioxidant enzymes (Shah et al. (2020). In recent years, functional gene analysis and the introduction of new alleles for the trait of interest into commercial crop plants have been revolutionized through genome editing or modification techniques. Various genome editing approaches have been developed for this purpose, including CRISPR/Cas, Meganucleases, TALENs, and ZNFs. Among these, CRISPR/Cas is the most widely used method for genetic improvement of different crops (Kaur et al. 2020; Awasthi et al. 2022; Singh et al. 2023). The development of CRISPR/Cas-based genome editing systems has led to significant advancements in fundamental plant research and crop breeding. By harnessing this powerful gene editing tool, there is a considerable prospect to improve global food security and promote sustainable agricultural development. Susceptibility (S) and resistance (R) genes in crop plants have been identified as potential targets for escalating crop protection. These genes were chosen as the best candidates for gene editing in order to confer disease or pest resistance in a crops (Das et al. 2019). This approach has the potential to drastically reduce the time required to produce transgenic seeds with homozygous genotypes (Ahmar et al. 2020). Transcriptomic analysis in pea elucidates the genes and pathways involved in disease or pest resistance. Furthermore, research into protein expression, modification, and interaction at the plant–pathogen interface revealed key proteins involved in pathogenesis. This data is a valuable resource for editing or modifying the genome of a crop or pathogen in order to develop resistant cultivars (Barakate and Stephens 2016). Recent advancements in genetic engineering have enabled the precise and stable editing of aphid lineages in pea plants using the CRISPR-Cas9 system (Le Trionnaire et al. (2019). A transient transformation system for hairy roots, mediated by Agrobacterium rhizogenes, was developed and used to test the efficacy of a CRISPR/Cas9 system. The engineering reagents for CRISPR/Cas9 constructs were optimized and this improved CRISPR/Cas9 system by Agrobacteriummediated genetic transformation was used to edit the pea phytoene desaturase (PsPDS) gene. This novel approach resulted in the production of albino-phenotype mutants in the T0 generation, which makes a significant contribution to functional genomics research and has the potential to improve gene editing technology to improve agronomic traits in pea. Furthermore, it connects this fundamental genetic model to the current era of gene functionality (Li et al. 2023).

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Conclusion and Future Prospective

Pea is a significant and productive crop that grows best in cool seasons all over the world. However, its full production potential is limited by biotic as well as abiotic stresses, which can infect the crop at various stages of growth and have disastrous consequences, affecting the sustainable production of pea (Rubiales et al. 2019). A number of significant and minor genes/QTLs that regulate significant biotic and abiotic stresses in pea have been discovered and mapped using different genomic tools. Accurate molecular markers flanking the genes/QTLs of interest could hasten the introduction of resistance alleles from various sources and accelerate pea breeding program more efficiently and precisely. It is recommended to focus on largescale, high-throughput germplasm screening to be used to precisely identify genes/ QTLs and their closely linked markers for specific traits using advanced mapping populations. The omics-based data and advanced breeding tools available for pea provide valuable resources for creative approaches in both fundamental research and practical breeding. A schematic diagram depicts different genetic and genomic resources, and advanced biotechnological tools including transgenic and genome editing technology can be used in tandem to develop improved cultivars in the pea improvement program (Fig.1.1). In the future, more focused efforts are required to conduct transcriptomic and proteomic analyses to unravel the molecular mechanism of pea disease and pest resistance. Despite the fact that few commercial pea cultivars have been released as a direct result of omics tools due to their recent application history, many breeding programs are adopting user-friendly MAS and MABC markers, and new cultivars are expected to emerge soon. Researchers and breeders can use biotechnological and breeding interventions to continuously improve pea production and quality in order to meet rising demand. Numerous studies have been conducted to identify the genetic mechanisms underlying variations in these nutritional traits. Several QTLs and SNPs that correspond to specific nutritional traits have been identified in recent studies, and further exploration of these markers using genome sequences can support research into the underlying genes responsible for these effects and aid in breeding programs targeting various end uses. The emerging genome editing technologies utilizing programmable nucleases and CRISPR/Cas provide novel opportunities for genetic improvement in crops by targeted editing of genes regulating traits such as stress tolerance and quality trait improvement and can be utilized to exploit the variability of the primary and secondary gene pool of crops and overcome crossability constraints. There is still significant potential for achieving new heights in productivity enhancement for stress-resistant pea cultivars.

Fig. 1.1 Major genomic-assisted crop enhancement strategies. Smaller collections of large-scale germplasm, such as a core/mini-core germplasm, can be characterized that can be evaluated for desired traits. Following that, specialized genetic stocks such as a natural population, mutant population, and mapping population can be developed and subjected to genotyping by sequencing (GBS), whole-genome sequencing (WGS), or array-based genotyping to identify QTLs, SNPs, superior alleles, and haplotypes. By combining these superior alleles using modern breeding tools such as marker-assisted selection (MAS), marker-assisted backcrossing (MABC), and genomic selection, cultivars with improved stress tolerance, nutritional quality, and yields can be developed. Furthermore, genetic engineering can be used to improve desirable traits by focusing on candidate genes identified through “omics” approaches

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Acknowledgements The authors express their gratitude to the National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology (DBT) and Biotechnology Industry Research Assistance Council (BIRAC), Government of India for research support and facilities. The present work is also supported through the India-Poland Joint Research Project (DST/INT/POL/P-45/ 2020) funded by Department of Science & Technology (DST), Government of India and Polish National Agency for Academic Exchange – NAWA (Poland) to Siddharth Tiwari. Authors acknowledge to DBT-eLibrary Consortium (Del-CON) for providing access to online journals.

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Lavaud C, Lesné A, Piriou C, Le Roy G, Boutet G, Moussart A, Poncet C, Delourme R, Baranger A, Pilet-Nayel M-L (2015) Validation of QTL for resistance to Aphanomyces euteiches in different pea genetic backgrounds using near-isogenic lines. Theor Appl Genet 128:2273–2288 Le Trionnaire G, Tanguy S, Hudaverdian S, Gléonnec F, Richard G, Cayrol B, Monsion B, Pichon E, Deshoux M, Webster C (2019) An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid. Insect Biochem Mol Biol 110:34–44 Lee R-Y, Reiner D, Dekan G, Moore AE, Higgins TJV, Epstein MM (2013) Genetically modified α-amylase inhibitor peas are not specifically allergenic in mice. PLoS One 8(1):e52972 Lejeune-Hénaut I, Hanocq E, Bethencourt L, Fontaine V, Delbreil B, Morin J, Petit A, Devaux R, Boilleau M, Stempniak JJ, Thomas M (2008) The flowering locus Hr colocalizes with a major QTL affecting winter frost tolerance in Pisum sativum L. Theor Appl Genet 116:1105–1116 Leonforte A, Sudheesh S, Cogan NOI, Salisbury PA, Nicolas ME, Materne M, Forster JW, Kaur S (2013) SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.). BMC Plant Biol 13(1):1–14 Leonova T, Popova V, Tsarev A, Henning C, Antonova K, Rogovskaya N, Vikhnina M, Baldensperger T, Soboleva A, Dinastia E (2020) Does protein glycation impact on the drought-related changes in metabolism and nutritional properties of mature pea (Pisum sativum L.) seeds? Int J Mol Sci 21(2):567 Li J, Li H, Quan X, Shan Q, Wang W, Yin N, Wang S, Wang Z, He W (2022) Comprehensive analysis of cucumber C-repeat/dehydration-responsive element binding factor family genes and their potential roles in cold tolerance of cucumber. BMC Plant Biol 22(1):1–13 Li G, Liu R, Xu R, Varshney RK, Ding H, Li M, Yan X, Huang S, Li J, Wang D (2023) Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea (Pisum sativum L.). Crop J 11(1):132–139 Liu N, Zhang G, Xu S, Mao W, Hu Q, Gong Y (2015) Comparative transcriptomic analyses of vegetable and grain pea (Pisum sativum L.) seed development. Front Plant Sci 6:1039 Liu N, Xu S, Yao X, Zhang G, Mao W, Hu Q, Feng Z, Gong Y (2016) Studies on the control of Ascochyta blight in field peas (Pisum sativum L.) caused by Ascochyta pinodes in Zhejiang Province. China. Front Microbiol 7:481 Liu R, Fang L, Yang T, Zhang X, Hu J, Zhang H, Han W, Hua Z, Hao J, Zong X (2017) Marker-trait association analysis of frost tolerance of 672 worldwide pea (Pisum sativum L.) collections. Sci Rep 7(1):1–10 Lockyer S, Nugent AP (2017) Health effects of resistant starch. Nutr Bull 42(1):10–41 Ma Y, Coyne CJ, Grusak MA, Mazourek M, Cheng P, Main D, McGee RJ (2017a) Genome-wide SNP identification, linkage map construction and QTL mapping for seed mineral concentrations and contents in pea (Pisum sativum L.). BMC Plant Biol 17(1):1–17 Ma Y, Coyne CJ, Main D, Pavan S, Sun S, Zhu Z, Zong X, Leitão J, McGee RJ (2017b) Development and validation of breeder-friendly KASPar markers for er1, a powdery mildew resistance gene in pea (Pisum sativum L.). Mol Breed 37:1–7 Malik AI, Ailewe TI, Erskine W (2015) Tolerance of three grain legume species to transient waterlogging. AoB Plants 7. https://doi.org/10.1093/aobpla/plv040 Manzoor S, Ahmad R, Shahzad M, Sajjad M, Khan N, Rashid U, Afroz A, Khan SA (2020) Salt stress reduces the pea growth and induces the expression of selected antioxidant genes. Pak J Agric Sci 57(2):393–399 McPhee K (2003) Dry pea production and breeding: a minireview. J Food Agric Environ 1:64–69 Moazzam-Jazi M, Ghasemi S, Seyedi SM, Niknam V (2018) COP1 plays a prominent role in drought stress tolerance in Arabidopsis and Pea. Plant Physiol Biochem 130:678–691 Moisa C, Copolovici D, Lupitu A, Lazar L, Copolovici L (2019) Drought stress influence on pea plants (Pisum sativum L.). Sci Tech Bull Ser Chem Food Sci Eng 16:20–24 Moore KL, Rodríguez-Ramiro I, Jones ER, Jones EJ, Rodríguez-Celma J, Halsey K, Domoney C, Shewry PR, Fairweather-Tait S, Balk J (2018) The stage of seed development influences iron bioavailability in pea (Pisum sativum L.). Sci Rep 8(1):1–11

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Moreau C, Hofer JMI, Eléouët M, Sinjushin A, Ambrose M, Skøt K, Blackmore T, Swain M, Hegarty M, Balanzà V (2018) Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum). New Phytol 220(1):288–299 Nemeskéri E, Molnár K, Vígh R, Nagy J, Dobos A (2015) Relationships between stomatal behaviour, spectral traits and water use and productivity of green peas (Pisum sativum L.) in dry seasons. Acta Physiol Plant 37:1–16 Perfecto A, Rodriguez-Ramiro I, Rodriguez-Celma J, Sharp P, Balk J, Fairweather-Tait S (2018) Pea ferritin stability under gastric pH conditions determines the mechanism of iron uptake in Caco-2 cells. J Nutr 148(8):1229–1235 Petrović G, Jovičić D, Nikolić Z, Tamindžić G, Ignjatov M, Milošević D, Milošević B (2016) Comparative study of drought and salt stress effects on germination and seedling growth of pea. Genetika-Belgrade 48(1):373–381 Polashock JJ, Arora R, Peng Y, Naik D, Rowland LJ (2010) Functional identification of a C-repeat binding factor transcriptional activator from blueberry associated with cold acclimation and freezing tolerance. J Am Soc Hortic Sci 135(1):40–48 Rai R, Singh AK, Singh BD, Joshi AK, Chand R, Srivastava CP (2011) Molecular mapping for resistance to pea rust caused by Uromyces fabae (Pers.) de-Bary. Theor Appl Genet 123:803– 813 Rai R, Singh AK, Chand R, Srivastava CP, Joshi AK, Singh BD (2016) Genomic regions controlling components of resistance for pea rust caused by Uromyces fabae (Pers.) de-Bary. J Plant Biochem Biotechnol 25:133–141 Ray H, Bett K, Tar’an B, Vandenberg A, Thavarajah D, Warkentin T (2014) Mineral micronutrient content of cultivars of field pea, chickpea, common bean, and lentil grown in Saskatchewan, Canada. Crop Sci 54(4):1698–1708 Rayner T, Moreau C, Ambrose M, Isaac PG, Ellis N, Domoney C (2017) Genetic variation controlling wrinkled seed phenotypes in Pisum: how lucky was Mendel? Int J Mol Sci 18(6): 1205 Ritonga FN, Chen S (2020) Physiological and molecular mechanism involved in cold stress tolerance in plants. Plan Theory 9(5):560 Robinson GHJ, Balk J, Domoney C (2019) Improving pulse crops as a source of protein, starch and micronutrients. Nutr Bull 44(3):202–215 Roriz M, Carvalho SMP, Castro PML, Vasconcelos MW (2020) Legume biofortification and the role of plant growth-promoting bacteria in a sustainable agricultural era. Agronomy 10(3):435 Rubiales D, González-Bernal MJ, Warkentin T, Bueckert R, Patto MCV, McPhee K, McGee R, Smykal P (2019) Advances in pea breeding. In: Achieving sustainable cultivation of vegetables. Burleigh Dodds Science Publishing, Cambridge, pp 575–606 Sayama T, Nakazaki T, Ishikawa G, Yagasaki K, Yamada N, Hirota N, Hirata K, Yoshikawa T, Saito H, Teraishi M (2009) QTL analysis of seed-flooding tolerance in soybean (Glycine max [L.] Merr.). Plant Sci 176(4):514–521 Shah Z, Iqbal A, Khan FU, Khan HU, Durrani F, Ahmad MZ (2020) Genetic manipulation of pea (Pisum sativum L.) with Arabidopsis’s heat shock factor HsfA1d improves ROS scavenging system to confront thermal stress. Genet Resour Crop Evol 67:2119–2127 Shailani A, Joshi R, Singla Pareek SL, Pareek A (2021a) Stacking for future: pyramiding genes to improve drought and salinity tolerance in rice. Physiol Plant 172(2):1352–1362 Shailani A, Wungrampha S, Dkhar J, Singla-Pareek SL, Pareek A (2021b) Genetic improvement of rice for food and nutritional security. In: Genetically modified crops: current status, prospects and challenges, vol 1. Springer, Cham, pp 13–32 Shivakumara TN, Sreevathsa R, Dash PK, Sheshshayee MS, Papolu PK, Rao U, Tuteja N, UdayaKumar M (2017) Overexpression of Pea DNA Helicase 45 (PDH45) imparts tolerance to multiple abiotic stresses in chili (Capsicum annuum L.). Sci Rep 7(1):2760 Shunmugam AS, Liu X, Stonehouse R, Tar’An B, Bett KE, Sharpe AG, Warkentin TD (2015) Mapping seed phytic acid concentration and iron bioavailability in a pea recombinant inbred line population. Crop Sci 55:828–836. https://doi.org/10.2135/cropsci2014.08.0544

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Siddiqui MH, Alamri S, Al-Khaishany MY, Khan MN, Al-Amri A, Ali HM, Alaraidh IA, Alsahli AA (2019) Exogenous melatonin counteracts NaCl-induced damage by regulating the antioxidant system, proline and carbohydrates metabolism in tomato seedlings. Int J Mol Sci 20(2):353 Sindhu A, Ramsay L, Sanderson L-A, Stonehouse R, Li R, Condie J, Shunmugam ASK, Liu Y, Jha AB, Diapari M (2014) Gene-based SNP discovery and genetic mapping in pea. Theor Appl Genet 127:2225–2241 Singh AK, Rai R, Singh BD, Chand R, Srivastava CP (2015) Validation of SSR markers associated with rust (Uromyces fabae) resistance in pea (Pisum sativum L.). Physiol Mol Biol Plants 21: 243–247 Singh S, Chaudhary R, Deshmukh R, Tiwari S (2023) Opportunities and challenges with CRISPRCas mediated homologous recombination based precise editing in plants and animals. Plant Mol Biol 111(1–2):1–20 Smýkal P, Aubert G, Burstin J, Coyne CJ, Ellis NTH, Flavell AJ, Ford R, Hýbl M, Macas J, Neumann P (2012) Pea (Pisum sativum L.) in the genomic era. Agronomy 2(2):74–115 Smýkal P, Trněný O, Brus J, Hanáček P, Rathore A, Das RR, Pechanec V, Duchoslav M, Bhattacharyya D, Bariotakis M (2018) Genetic structure of wild pea (Pisum sativum subsp. elatius) populations in the northern part of the Fertile Crescent reflects moderate crosspollination and strong effect of geographic but not environmental distance. PLoS One 13(3): e0194056 Stephen AM, Champ MM-J, Cloran SJ, Fleith M, Van Lieshout L, Mejborn H, Burley VJ (2017) Dietary fibre in Europe: current state of knowledge on definitions, sources, recommendations, intakes and relationships to health. Nutr Res Rev 30(2):149–190 Stevenson EJ, Watson AW, Brunstrom JM, Corfe BM, Green MA, Johnstone AM, Williams EA (2018) Protein for life: towards a focussed dietary framework for healthy ageing. Nutr Bull 97: 102 Sun S, Deng D, Duan C, Zong X, Xu D, He Y, Zhu Z (2019) Two novel er1 alleles conferring powdery mildew (Erysiphe pisi) resistance identified in a worldwide collection of pea (Pisum sativum L.) germplasms. Int J Mol Sci 20(20):5071 Swisher Grimm KD, Porter LD (2020) Development and validation of KASP markers for the identification of pea seedborne mosaic virus pathotype P1 resistance in Pisum sativum. Plant Dis 104(6):1824–1830 Szablińska-Piernik J, Lahuta LB (2021) Metabolite profiling of semi-leafless pea (Pisum sativum L.) under progressive soil drought and subsequent re-watering. J Plant Physiol 256:153314 Tafesse EG, Gali KK, Lachagari VBR, Bueckert R, Warkentin TD (2020) Genome-wide association mapping for heat stress responsive traits in field pea. Int J Mol Sci 21(6):2043 Tao A, Afshar RK, Huang J, Mohammed YA, Espe M, Chen C (2017) Variation in yield, starch, and protein of dry pea grown across Montana. Agron J 109(4):1491–1501 Tar’an B, Warkentin T, Somers DJ, Miranda D, Vandenberg A, Blade S, Bing D (2004) Identification of quantitative trait loci for grain yield, seed protein concentration and maturity in field pea (Pisum sativum L.). Euphytica 136:297–306 Tayeh N, Aluome C, Falque M, Jacquin F, Klein A, Chauveau A, Bérard A, Houtin H, Rond C, Kreplak J (2015) Development of two major resources for pea genomics: the GenoPea 13.2 K SNP Array and a high-density, high-resolution consensus genetic map. Plant J 84(6):1257–1273 Teressa Negawo A, Baranek L, Jacobsen H-J, Hassan F (2016) Molecular and functional characterization of cry1Ac transgenic pea lines. GM Crops Food 7(3–4):159–174 Thavarajah D, Lawrence TJ, Powers SE, Kay J, Thavarajah P, Shipe E, McGee R, Kumar S, Boyles R (2022) Organic dry pea (Pisum sativum L.) biofortification for better human health. PLoS One 17(1):e0261109 Vigeolas H, Chinoy C, Zuther E, Blessington B, Geigenberger P, Domoney C (2008) Combined metabolomic and genetic approaches reveal a link between the polyamine pathway and albumin 2 in developing pea seeds. Plant Physiol 146(1):74–82 Vyse K, Pagter M, Zuther E, Hincha DK (2019) Deacclimation after cold acclimation—a crucial, but widely neglected part of plant winter survival. J Exp Bot 70(18):4595–4604

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Warkentin TD, Delgerjav O, Arganosa G, Rehman AU, Bett KE, Anbessa Y, Rossnagel B, Raboy V (2012) Development and characterization of low-phytate pea. Crop Sci 52(1):74–78 Warkentin TD, Smýkal P, Coyne CJ, Weeden N, Domoney C, Bing D-J, Leonforte A, Xuxiao Z, Dixit GP, Boros L (2015) Pea. Grain Legum, pp 37–83 Welham T, Domoney C (2000) Temporal and spatial activity of a promoter from a pea enzyme inhibitor gene and its exploitation for seed quality improvement. Plant Sci 159(2):289–299 Winter P, Rubiales D, Fondevilla S (2016) Use of MACE technology to identify positional and expressional candidate genes for resistance to Didymella pinodes in pea. Second International Legume Society Conference Wu L, Chang K-F, Conner RL, Strelkov S, Fredua-Agyeman R, Hwang S-F, Feindel D (2018) Aphanomyces euteiches: a threat to Canadian field pea production. Engineering 4(4):542–551 Yang T, Fang L, Zhang X, Hu J, Bao S, Hao J, Li L, He Y, Jiang J, Wang F (2015) High-throughput development of SSR markers from pea (Pisum sativum L.) based on next generation sequencing of a purified Chinese commercial variety. PLoS One 10(10):e0139775 Ye H, Roorkiwal M, Valliyodan B, Zhou L, Chen P, Varshney RK, Nguyen HT (2018) Genetic diversity of root system architecture in response to drought stress in grain legumes. J Exp Bot 69(13):3267–3277 Yu K (2012) Bacterial artificial chromosome libraries of pulse crops: characteristics and applications. J Biomed Biotechnol 2012:493186 Zaman MSU, Malik AI, Erskine W, Kaur P (2019) Changes in gene expression during germination reveal pea genotypes with either “quiescence” or “escape” mechanisms of waterlogging tolerance. Plant Cell Environ 42(1):245–258 Zhukov VA, Zhernakov AI, Kulaeva OA, Ershov NI, Borisov AY, Tikhonovich IA (2015) De novo assembly of the pea (Pisum sativum L.) nodule transcriptome. Int J Genomics 2015 Zia MAB, Ul-Allah S, Sher A, Ijaz M, Sattar A, Yousaf MF, Chaudhry UK, Qayyum A (2023) Genomics for abiotic stress resistance in legumes. In: Sustainable agriculture in the era of the OMICs revolution. Springer, Cham, pp 327–342 Zohary D, Hopf M, Weiss E (2012) Domestication of plants in the old world: the origin and spread of domesticated plants in Southwest Asia, Europe, and the Mediterranean basin. Oxford University Press, Oxford

2

Genetic Improvement of Apple Chongtham Allaylay Devi, Ashutosh K. Pandey, and Khadija Mika

2.1

Introduction

The apple (Malus domestica Borkh.), which has been cultivated in Asia and Europe since antiquity, is the most common temperate fruit. Today, orchards may be found everywhere from high altitudes in Indonesia and Colombia that are crossing the equator to northern China and Siberia, where winters can reach -40 °C. Apples have been grown in temperate, subtropical, and tropical regions all over the world. Globally their there are more than 6000 regionally significant landraces and cultivars, although only a few of these are globally dominant. Since fruit plants were domesticated thousands of years ago, their inherent variety has been harnessed through genetic manipulation of physiological properties and developmental aspects relevant to climate adaptation. Domestication of apple provides a clear opportunity for the production of cultivars that contain all of the desirable commercial, horticultural, qualities in a single genotype. The two main goals of apple breeders around the world are to increase fruit quality and create varieties that are disease-resistant. Another key goal that breeders are working toward is the development of cultivars that mature quickly and are winter-hardy. So, both breeding and the selective apple cultivars have taken into account the study of local germplasm in terms of early maturing and fruit quality. Native cultivars are a crucial component of breeding efforts. The trans-Himalayan Ladakh’s indigenous apple cultivars are very diverse and early blossoming compared to the newly introduced variety. The Ladakh region produces petite, delicate apples with a mean weight of 60.1 ± 29.1 g. The C. A. Devi (✉) School of Agriculture, Galgotias University, Greater Noida, Uttar Pradesh, India e-mail: [email protected] A. K. Pandey (✉) · K. Mika School of Agricultural Science, Sharda University, Greater Noida, Uttar Pradesh, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_2

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distinctive characteristics of apples from the Ladakh region, such as early blossoming and early fruit maturity, offer a an oppurtunity to research local varieties for prospective breeding initiatives to produce early maturing cultivars. The native cultivars’ flowering times, according to these researchers, ranged from 9 to 17.7 days, depending on the cultivar and the year (Rasool et al. 2022).

2.1.1

Genetic Resources

Vavilov proposed that Malus sieversii and Malus domestica may have come from Turkistan in 1930. According to Eldin and Jo (2014), several Malus species with enormous fruits have evolved from Central Europe to Middle Asia. They believe that Malus sieversii played a major role in the Middle Asian gene pool that eventually gave rise to Malus X domestica. It is possible that the “Silk Road” was responsible for the arrival of Malus orientalis and Malus sylvestris var. praecox from Caucasia and southeast Russia. Previous studies contend that the native species Malus sylvestris var. sylvestris, found in Central Europe, were not involved in the domestication of the apple. Malus asiatica, Malus orientalis, Malus niedzwetzkyana, and Malus prunifolia can be grouped with the domesticated apple and the wild apple of Central Asia (Harris et al. 2002). Apple cultivars with desired traits may have been produced later through hybridizations after apple selections from wild species were introduced to Western Europe. Hybridization may played an important role to diversify the local apple varieties (Bramel et al. 2019).

2.1.2

Breeding Objectives

Despite a decline in the number of commercially grown apple varieties, polymorphism causes an unusually high level of variation in apples (Un et al. 2020). This decline in cultivars used in breeding initiatives can be related to the germplasm banks’ lack of information, which restricts their potential utility. (Caligari 2014). Future generations’ inbreeding is the key issue when employing a small number of cultivars. Modern technique is feasible to increase genetic variability in commercial releases using dated cultivars, such as by collecting seedling from the alleged original species or sieversii (Lamichhane and Thapa 2022). Owing to the high heterozygosity in Malus genus, inbreeding issues are not yet apparent in seedling populations. Thomas Andrew Knight is credited with creating the first cultivar by crosspollination (1759–1838). The selection of mutations and chimeras has been another strategy for creating novel cultivars (Yu and Li 2022). The benefit of genetic modification is that it preserves cultivar uniqueness (Nasri et al. 2022). Using certain cultivars can benefit from having some unique qualities, such as the following: 1. Sensitivity to russeting, which gives some cultivars, like “Reineta Gris de Canada” and “Boskoop,” a distinctively brown look.

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Genetic Improvement of Apple

2. 3. 4. 5. 6.

Weeping, spur kinds, and growth habits. Flowering later. Strong resistance to cold. Pest- and disease-resistant. Local cultivars that are desired in their area and require less care than commercial varieties.

2.1.3

41

Polyploidy in Crop Plants

Angiosperms frequently exhibit polyploidy or entire genome multiplication. Due to interspecific hybridization and polyploidy, many crop species are allopolyploids. In order to understand crop plant domestication, crop improvement, and the evolution of angiosperms as a whole, it is crucial that we comprehend the evolutionary consequences of (allo)polyploidy. Moreover, polyploidy crops, such as cotton, wheat, tobacco, sugarcane, and apples, have provided numerous contemporary discoveries in plant biology. Polyploidy genomes are impacted by a wide range of evolutionary events, including quick and significant genome reorganization, gene conversion, downsizing of genome, transgressive gene expression changes, gene fractionation, and sub- and neo-functionalization of duplicate genes. In comparison to closely related diploids, these genetic modifications frequently come with heterosis, resilience, and an increase in crop load. In the past, polyploidy crops’ genomewide assessments lagged behind those of diploid crops and other model species. This delay is partly due to the difficulties in genome assembly induced by the complexity of the genomes that result from combining two or more evolutionarily different genomes into a single nucleus, as well as the sizeable size of polyploidy genomes. In this study, Akagi et al. (2022) explored the role of polyploidy in the evolution of angiosperms, domestication, and agricultural improvement. We emphasize the capability of modern technology, in particular next-generation sequencing, to offer details on the patterns and mechanisms governing polyploidy crop improvement and phenotypic change following domestication.

2.1.4

Use of Rootstock in Plant Breeding

The most typical type of apple tree is a rootstock and scion graft chimera. In addition to absorbing nutrients and secreting, rootstocks also serve as a function for support and fixation. Additionally, the quality and production of the fruit, as well as the ability to withstand biotic and abiotic stressors, are all significantly influenced by rootstocks (Santhi et al. 2020). The three main categories of apple rootstocks are cloning-propagated rootstocks, apomictic seedling rootstocks and seedling rootstocks. They include rootstocks from different genera as well as rootstocks from apples that are domesticated, wild, or semi-wild. Horticultural traits should come first when carrying out apple rootstock breeding, because they are quite important (reproductive capacity, grafting compatibility, impacts on scion growth,

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APPLE EMLA111/MM1 11

Suit locations that are prone to drought

EMLA.7/M-7

Fit for muddy,undev eloped and moderately avtive topography

EMLA.106/MM. 106

EMLA9/M 9

Suitable for moderately,muddy and also lower clay soil

Very small plants are best for growing at higher densities having reliable irragation and rich soils.

M779

For Uttarakhand's and H.P, mountaino us regions.

Fig. 2.1 Promising rootstocks suitable for multiplication, Caligari (2014)

dwarf, etc.). The biological factors of the planting region (woolly apple aphids, bacteria, viruses, nematodes, and other venomous critters) and the environmental parameters (soil pH, temperature, irrigation and fertility, etc.) must also be considered (Wang et al. 2019a). For instance, in Poland, rootstock breeding is primarily focused on conquering the cold climate as well as improving propagation, dwarfism, and increasing yields (Basheer-salimia et al. 2020). The apple rootstock breeding program in Russia places a strong emphasis on traits such as high crop load, better rooting capacity, and cold resistance. The vast terrain of Russia necessitates the development of cloning-propagated rootstocks to adapt to the various terroir conditions (Demian and Jaksa-czotter 2020). Canada’s main research areas in North America include freezing tolerance and dwarfism. The USA is carrying out lengthy breeding programs with the objectives of dwarfing, replanting tolerance, cold tolerance, antilatent viral traits, early fruiting, and high yield. In the USA, apple rootstocks that are resistant to collar rot (Phytophthora cactorum) and fire blight have been successfully screened (Vahdati et al. 2021). The few of promising rootstocks with different salient features needs have been described in Fig. 2.1.

2.2

Apple Rootstock Breeding Research Priorities and Key Areas

Seedling selection, hybrid breeding, bud mutation selection, and molecular breeding are the main breeding techniques used for apple rootstocks. The fundamental idea is that the target trait must be controlled by a gene that is both persistently expressed and inherited allowing for efficient, exact, and quick selection throughout the early

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developmental stage. Therefore, the following are the areas of research and breeding for rootstocks.

2.2.1

Selection of Parent and Cross-Setting

Under plant breeding strategies breeders consider wild resources in order to breed for traits like biotic and abiotic resistance and for enhanced productivity and quality of horticultural crops. The parent choice during setting crosses will be aided by identifying the natural resources like rot disease (Malus komarovii), salt-tolerant (Malus zumi), Fe deficiency (Malus xiaojinensis), and wild (Malus baccata) with traits for early fruiting and flowering (Zha et al. 2016; Jain and Priyadarshan 2009). To obtain rootstocks that exhibit the dwarf and salt stress resistance qualities, Malus micromalus and M9 can be used as parents because of the ability of ideal comprehensive traits like good fixing ability, dwarf, and salt-tolerant. Malus micromalus, for instance, has demonstrated great stress resistance and effective root function.

2.2.2

Target Gene Acquisition and its Selection Using Molecular Marker

Apple is a woody, perennial plant with a lengthy juvenility and significant heterozygosity. Additionally, a lot of significant apple features are quantitative variables governed by several genes. Due to these traits, conventional breeding is inefficient and has a lengthy cycle (Haroon et al. 2020). The guiding principles of molecular marker-assisted selection (MAS) call for the use of molecular markers that are closely connected to or co-segregate with the desired trait to identify potential candidates. In genetics, molecular markers can precisely reflect the genetic diversity of phenotypes and can demonstrate the individual genome variations. Breeders can successfully differentiate between heterozygous and homozygous genotypes by using MAS. Additionally, MAS does not depend on the growth of the plant or other environmental conditions; its focus is the genome. Because of this, MAS may significantly increase breeding accuracy and efficiency. In future, MAS or molecular breeding will be the trends for the crop improvement program of apple rootstock (Kaiser et al. 2020).

2.2.3

Linkage and Selection

Similar to molecular marker-assisted selection, screening rootstocks early on based on a model of the root configuration also offers a significant potential to reduce the breeding cycle and enhance breeding efficiency which have been crucial study areas in recent years. Hasan et al. (2021) discovered that three of the apple grafts employing the semi-vigorous rootstock MM106, semi-dwarf rootstock M26, and dwarf rootstock Mark had increased root length densities. According to Lo Bianco

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et al. (2003), the dwarfing rootstock M9 had a much lower total root length density than the robust rootstock MM106. Through dynamic observations of apple grafts that were 1 or 2 years old and had varied rootstock-scion combinations, a preliminary root configuration was developed. A “shallow root” is seen in Fuji/M9 (a dwarf rootstock), and a “deep-wide root” is found in Fuji/M. micromalus (a vigorous rootstock). Luo et al. (2014) used minirhizotron tubes to further monitor the dynamic changes of fine root initiation in apple trees that were 4 or 5 years old and had various rootstock-scion combinations. In contrast to Fuji/M9 and Fuji/SH40, they discovered that the roots of Fuji/M. micromalus had the maximum root length density. Five genes (RGF, LOB, SPL, SAU, and LAX) were found to be connected to root configuration by whole-genome sequencing of pools of individuals with enormous phenotypes. The discovery of these genes will aid in elucidating the regulatory processes governing root configuration and help the methodology for quickly identifying the best root configuration in the early stages.

2.2.4

Utilization of Apomictic Apple Resources

The characteristics of apomictic seedling rootstocks are superior to those of sexually generated seedling rootstocks in that they can be securely inherited. Additionally, seedlings can be used to directly grow apomictic seedling rootstocks rather than cuttings or layering propagation. Malus sieboldii, Malus hupehensis, Malus sikkimensis, Malus platycarpa, Malus rockii, Malus sargentii, Malus toringoides, Malus xiaojinensis, Malus coronaria, and Malus lancifolia are the 10 species of Malus that have been identified as having apomixes features (Haroon et al. 2020). Malus hupehensis var. pinyitiancha seeds that had been exposed to gamma rays (γ rays) were used by Dwivedi et al. 2021 to screen the apomictic variety Qingzhen 2. From M. xiaojinensis seedlings, Pan et al. (2015) selected the Chistock 1 apple rootstock variety. Few of the positive traits are strong root system, semi-dwarfism, good fixation, cold tolerance, iron-deficiency tolerance, and early flowering after grafting (Pan et al. 2015). In China, 300,000 hectares of orchards have been planted using Chistock apple rootstock. Wang et al. (2019a; b) looked into the genetic stock of apples used in breeding facilities. Apple breeding uses the valuable parental varieties McIntosh, Jonathan, Wealthy, Golden Delicious, Melba, Antonovka Obyknovennaya, Ranetka Purpurovaya, and Papirovka in Russia and around the world. Although though a sizable genetic collection was available, only 48 cultivars were used as the initial parental forms. The McIntosh, scab-immune Seyanetz, Antonovka Obyknovennaya, Papirovka Tetraploid, etc., are some of the top ten sources of new types. As a result, suggest sources and donors for breeding apples for traits that are commercially valuable (winter hardiness, columnar tree type, precocity and high productivity, long fruit storability, high resistance and immunity to scab), donors of diploid gametes for breeding triploid cultivars, sources and donors for high sugar, titrated acid, and biologically active substance (ascorbic acid and P-active substance) contents in fruits, among others. It describes how it is essential to choose and breed complicated sources and donors that have two or more desirable qualities

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(such as immunity to scab and tetraploidy, immunity to scab and the columnar tree type, etc.).

2.2.5

Utilization of Genetic Engineering in Apple Rootstocks

Apple rootstocks can be made better in terms of tree structure, organogenesis, and stress resistance using transgenic technology, which has emerged as a key development for effective and precise rootstock breeding. Transgenic technology is now being used by apple rootstock breeders to enhance important traits. By preventing the synthesis of endogenous brassinosteroid, overexpression of the MdWRKY9 gene caused the transgenic apple rootstock M26 to become even more dwarfed (Zhu et al. 2001); overexpression of the MdNHX1 gene significantly increased the transgenic rootstock’s tolerance to salt (Vahdati et al. 2021); and overexpression of the MsDREB6.2 gene enhanced drought tolerance by lowering endogenous cytokinin concentration (Liao et al. 2017). M26 and Jork 9 rootstock have the better rooting and this was greatly enhanced by rolB gene overexpression (Malling and Malling 1998).

2.2.6

Graft Incompatibility

It was found that the Maruba-kaido (Malus prunifolia) rootstock could not be grafted onto the dwarf and compact kinds of cv. Fuji mutant apples. The majority of the mutants displayed weak growth in the diameter and height of the trees when grafted onto the rootstock. The Institute of Radiation Breeding used either chronic or acute gamma-ray exposure to create the cv. Fuji mutant. Modifications in vegetative organs served as the selection criteria in their selection procedures. We discuss their graft compatibility with the Maruba-kaido (Malus pyunifolia Borkhausen var., riiago asami) rootstock, which is the most popular rootstock in Japan and exhibits typical congeniality and good nature of bud union with most cultivars, particularly with Fuji when grown in practical cultivation. Three mutants of spur compact growth and eight mutants of dwarf growth were studied against their originals. When compared to their originals, the majority of the mutants exhibited weak diameter development and slow height growth. Few mutants, particularly IRB 500-10, 500-14, and 500–20, however, displayed a very noticeable overgrowth of the top. For instance, IRB 500r-l4 had a stock-top ratio of 0.89, whereas the original was 1.29. The combination of these scions has not yet reached fruiting and blooming. These weaker growth responses to the grafted top might be due to selection of early mutants with weak growth habits. As a result of their apparent graft incompatibility, mutants showing weak growth habits when grafted onto Maruba-kaido. In the future, experiments should be conducted to find a new rootstock that is suited for induced mutants with limited growth habits and the rate of successful for the multiplication of apple crop under asexual propagation under Indian conditions has been illustrated in Fig. 2.2.

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100 90 80 70 60 50 40 30 20 10 0

RECOVERY RATE OF MULTIPLICATION

Wedge grafting

Tongue grafting

T-budding

APPLE

Fig. 2.2 Propagation method and efficiency of multiplication in budding and grafting of temperate fruit and nut crops under Indian conditions (Verma et al. 2014)

2.3

Compact-Type Mutants in Apple

According to physiological research, the leaves of spontaneous apple mutants of the compact type are more efficient at photosynthesis than the leaves of the corresponding standards. Despite this, branch ringing methods revealed that there is no difference in productivity between the leaves of compacts and those of standards. The tissue culture approach to mutant breeding appears to offer a variety of advantages.

2.4

Research on Apple Mutants with Respect to Physiological and Biochemical Parameters

Previous investigations made use of Starkrimson Delicious and Golden Delicious spur. Six-year-old seedling rootstocks were used to grow each tree and in comparison, to their respective compacts, the rate of photosynthesis in the leaves of standards was much lower. However, there were no variations in the rate of respiration. The data seem to indicate that, despite the faster rate of photosynthesis on compact plants’ leaves, this does not affect the productivity of such plants. Parts of apple roots were cultivated on agar media, and the results thus far obtained suggest that adventitious buds formed on removed apple root sections may be capable of developing into shoots. Important promising scion varieties of apple crops cultivated in different agro-climatic zone of India were listed in Table 2.1.

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Table 2.1 Promising scion varieties of apple fruit crops for multiplication (Verma et al. 2014) Crop Apple

2.5

Regions Himachal Pradesh Top Red, Royal Delicious, Red Chief, Starking Delicious, Granny-Smith, Starkrimson, Tydeman’s Early, Gold Spur, Mollies Delicious, Silver Spur, Red Chief, Rich-a-red, Oregon Spur, Vance Delicious

Uttar Pradesh Red Chief, Starking Delicious, Oregon Spur, Rich-a-Red, Well Spur, Red Delicious, Early Shanburry, Fanny Benoni, Chaubattia Princess, Starkrimson

Jammu & Kashmir Gold Spur, Top Red, Golden Delicious, Silver Spur, Red Fuji, Gala Spartan, Scarlet Spur, Vista Bella, Scarlet Gala, Gala Mast, Vance Delicious, Ambri, Oregon Spur, Red-a-Red

Signaling Mechanisms Initiating Apple Fruitlet Abscission

The process of fruit development is incredibly plant-specific and is governed by a complex interplay of endogenous and external variables. Numerous molecular studies have mostly examined the final stages of fruit development and the ripening process due to its significant ecological effects (Handa et al. 2012). The hormone ethylene plays a dominant role in controlling the ripening of climacteric fruits, according to a significant body of experimental evidence gathered using the tomato (Solanum lycopersicum) as a model system. The process of fruit set, which is defined as the commitment of the ovary tissues to undergo metamorphosis into a fruit (Din et al. 2019), is gaining interest due to its potential application to control parthenocarpic fruit development in the absence of pollination or fertilization. In the inductive stage of fruit set and the parthenocarpic growth of fruits, auxins and GAs are crucial. The hypothesis that auxins may be the primary signal initiating cell division and that their interaction with GAs may be necessary for maintaining cell growth is supported by a number of studies (Sarma et al. 2020). Auxin/indole acetic acid (AUX/IAA) and auxin response factor (ARF) proteins appear to exert a negative control over the ovary’s ability to turn into fruit, according to research from the tomato and Arabidopsis (Arabidopsis thaliana) plants. Following fertilization, pollination, or auxin treatment, the elimination of this negative regulation causes fruit set and cell growth. For the purpose of researching fruit abscission, the apple (Malus X domestica) is an intriguing model tree crop (Bertiter and Droz 1991). By employing chemicals for thinning, like benzyladenine (BA), to produce fruits with higher quality and marketability, the physiological fruitlet drops occurring in this species can be easily amplified. By combining the available transcriptomic and metabolomic data, a speculative model for apple fruitlet abscission was created. This model predicts that BA treatment will cause the tree to experience a nutritional stress that is largely felt by the fruitlet cortex, whose growth is inhibited in a manner similar to the ovary growth suppression seen in other species. A block in embryogenesis and the subsequent activation of the abscission zone accompany this stress, which is quickly noticeable also at the seed level in weaker fruits. This stress is most likely transmitted by reactive oxygen species/sugar and hormone signaling

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cross-talk. While most of the transcriptionally active components implicated in hormone signaling appear to be downstream of the induction of abscission, hormones appear to have a relatively significant role during the early stages of abscission in the cortex (Depuydt and Hardtke 2011). In Russia, a number of apple cultivars with the Vf gene have been created that are scab-resistant. Yet, it is possible to create cultivars with longer resistance by combining the genes Vf and Vr, Vf and Vm, etc., on a digenic basis (Gao and Van De Weg 2006). It is essential to develop cultivars that are extremely resistant to pests like the apple moth, the European red mite, and powdery mildew. We require apple varieties with a range of disease and pest resistance. From several chromosome crossings, including 2x X 4x and 4x X 2x, triploid apple cultivars were produced for the first time in Russia. They stand out for having more regular fruit producing, fruit that is more marketable, and increased autogamy. In the future, this breeding strategy has to be developed. For the purpose of enhancing fruit production, columnar apples produced by Russian research institutions are of interest. To begin with, they are useful in novice gardens. Apple cultivars with high-quality fruit must be triploid, columnar, and scab-resistant, according to breeders. The development of autogamous cultivars using apetalous plants and true-rooted apple cultivars should be the future goals of breeding. It might be required to change the range of apple varieties and breeding practices with change to global warming.

2.6

Genes and Effects

The national field gene bank CITH and the NBPGR have ex-situ preserved a significant number of collections in temperate fruits that were made through introductions and indigenous collections. They were produced using different types of fruit trees, including apple (743 Nos). These accessions have been used in an effort to develop further varieties adapted to the climatic conditions of India. Several temperate fruit crops grown in the nation have a relatively limited genetic base. The genetic base must be expanded with high-yielding varieties with regular bearing, precocious, resistance to biotic and abiotic stresses, and export quality and processing attributes in order to increase product variety, lower biotic and abiotic risks, and ensure fruit availability for a longer period of time. Here are some of the notable introductions that have been made (Sedov 2014). Apple The specific achievements mentioned about varietal development are listed below in Table 2.2 and Fig. 2.3 under different headings. Jain and Priyadarshan (2009) have analyzed breeding initiatives that began in the USA in 1953. Many of the selections from the Cornell Geneva series (CG) are fire blight-resistant and are studied widely. “Michigan Apple Clone 9,” “MAC 9,” is a well-known American rootstock which is a poor performer under hot and dry soil (Agdex 2014). Al-Hinai and Roper (2004) studied that different rootstocks influence the growth and quality of “Gala” fruits and concluded that rootstocks had no effect on fruit growth, final size, or yield, whereas apple fruit size is effected by the by the crop load. A study conducted by Marini et al. (2002) found that the effect of apple

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Table 2.2 List of mutants discovered from cv. Red Delicious (Verma et al. 2014; Li et al. 2023)

Royalred Starkspur Redspur Super Starking Stark Earlibrite Starkrimson Adams Apple, Burchinal Red Delicious Red Chief Starkspur or Ultrastripe

1954 1960 1959 1964 1971 1957 2004

Vallee Spur Eve’s Delight

1989 1985

Super Clone

1982

Mutated form Shotwell Starking Red King Spokane Beauty Richard Starking Starking Starking Ryan Red Starking Oregon Spur II Starkrimson Oregon Spur Red Chief Spokane Beauty Starking

Early Red One Silverspur Super Chief Top Spur Rose Red

1974 1977 1988 1984 1974

Brauns Hi Early Red Chief Starkrimson Starking

Trade name Top Red Sturdyspur Oregon Spur Eve’s Delight

Year of release 1960 1964 1968 1985

1974 1985

Habit Standard Spur Spur Stripe

Color Darker Dark Darker Light

Standard Spur Spur Standard Standard Spur Spur Spur Spur

Lighter Deeper Brighter Fuller Bright Similar More uniform, deeper, purple, bloom Deeper, brighter More consistent

Spur Stripe

Dark red with bloom Light

Spur, dwarfing Standard Spur Spur Spur Spur

Light Darker, blackish purple Bright Red stem Deeper, brighter Dark

rootstocks on the weight of “Gala” fruits for crop load and the differences between rootstocks but agreed that longer-term study would be necessary to conclude. Rootstock can influence gene expression patterns and, therefore, over-grafted scions. Jensen et al. (2003) reported that the difference between the two, “M.7” rootstocks (with reduced susceptibility to fire blight) and “M.9 NAKB T337” (“M.9 T337”) rootstocks (highly susceptible to fire blight). New challenges with apple dwarfing rootstocks are being taken into consideration (Friedt 2010) from Russia (“B.146” and “B.491”), Sweden (“BM 427”), USA (“G.65” and other CG- and G-rootstocks), Japan (“JM.1,” “JM.5,” and “J.M.8”), Czech Republic (“J-TE-G”), UK (“M.20”), Poland (“P.22,” “P.59,” “P.61,” “P.66”), Canada (“V.3”), Germany (“Supporter” 1 to 4), and Romania (“Voinesti 2”). With recent advancements in molecular marker and its availability (Jensen et al. 2010), for example, isoenzymes, restriction fragment length polymorphisms (RFLP), RAPD, microsatellites, amplified fragment length polymorphism (AFLP), SCAR, or ISSR that allows differentiating varieties. Lawson et al. (1995) investigated that blooming is correlated with Prx-c. Kovic 1999

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Apple (M. x domestica Borkh) Fruit Crops

Traits

Research Work

Reduced Ethylene suppression -Juvenile stage reduced -Scab resistance -Polyphenol Oxidase Levels -Altered sorbitol levels -Resistance to fire blight

nptII PPO suppression transgenes (AP14, APO5, PGAS, PGAS2) -ACC synthase S6PDH sorbitol 6 phosphate dehydrogenase -nptII BpMADS4 -nptII, gusA ech42 -PPO suppression transgene -nptII ACC oxidase -GUS

Fig. 2.3 Features of qualitative trait loci in apple cultivar

found that linkage to woolly aphid resistance was studied using stylar ribonucleases and Got-1. Din et al. 2019 studied the use of isozymes for tracing the transfer of resistance trait to mildew (P. leucotricha (Ell. et Ev.) Salm.) between M. hupehensis and cultivated apples. James and Evans (2004) used a set of microsatellites, AFLP and RAPD primers, to identify markers linked to mildew resistance. Bretting and Widrlechner 2010 found that screening of seedlings for peroxidase allozyme variation reliable method to preselect apple dwarf types. The availability of molecular markers and genetic linkage maps can help in identifying and analysis of major resistance genes and of QTL contributing to the resistance of a genotype (Kumar Meena et al. 2017). In scab-resistant breeding, the cultivar “Antonovka kamienna” was used at first as a polygenic scab-resistant source and later the M. floribunda and other wild species with different resistant sources (Vf, VA, Vm) were also investigated (Bus et al. 2012). Breeding program for resistance were extended in Pillnitz for mildew, fire blight, bacterial canker, red spider mite, and different abiotic stresses, for example, winter frost and spring frosts. The major finding of the Pillnitz apple-resistant breeding program was that the selection of a number of cultivars with resistance to economically important diseases was identified using conventional recombinant breeding methods.

2.7

Biotechnological Tools and Transgenic Approaches

It is one of the top requirements for biotechnological methods needed to boost temperate fruit crops. It can be used using a variety of methods, including as micropropagation, meristem culture, micrografting, somaclonal variants, embryo culture, and other cultures. Meristem cultivation is the practice of cultivating

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meristem tips. The tissue in plants that are located in areas of the plant where development is possible and that contains rapidly proliferating, undifferentiated cells (meristematic cells) is known as a meristem. A classic illustration is the creation of apples that are devoid of the apple mosaic virus. In fruit crops like the apple, it has been successfully standardized. Instead of using natural or artificial pollination, transgenic technology inserts a gene. The gene sequence that has been introduced may be from unrelated or related plant species. The transgenic techniques give researchers the ability to locate and isolate the genes responsible for particular traits in a particular plant species and then transfer copies of those genes into an entirely new organism. It has been determined that the technology is appropriate for the creation of varieties with pest and disease resistance, tolerance to salinity and drought, production of high-yielding hybrids, improvement of protein and oil quality, postharvest traits, metabolic manipulation, therapeutics, edible vaccines, and phytoremediation. The researchers already several examples of transgenic technology being successfully used in temperate fruits (Verma et al. 2018). Fruit Trees Being Trans-Grafted: Trans-grafting is the grafting of a standard wild-type scion with a transgenic rootstock (Lobato-Gómez et al. 2021). This is apple improvement biotechnology of the newest generation. Genetic Engineering: The understanding of precision breeding techniques has recently undergone a revolution thanks to the development of genomics and molecular-based methodologies, opening up new possibilities for more effective and manageable plant breeding. To help with the breeding pace, a number of markers have been developed in this area. The creation of comparable linkage maps has been substantially assisted by the introduction of novel markers, particularly microsatellites, and the amount of data on the location of key genes and QTLs for the most significant agronomic traits on maps is fast expanding. The technique of QTL mapping involves using molecular markers to find genes that have an impact on quantitative traits. The era of genome sequencing is a new one for molecular breeding. Apple, pear, peach, and strawberry genomes have been sequenced in temperate fruits for gene prediction and functional annotation.

2.8

Breeding for Processing Traits

The decision a breeder must make is whether cultivars should be multifunctional or specialized to fulfill certain market needs. Growers will often push for multipurpose cultivars that allow them marketing flexibility because a cultivar that the processing industry views perfect is frequently too specialized for broad use. All temperate fruit plants must have color for canning. It should be a deep enough red to prevent the need for artificial coloring, and processing must leave it this color rather than turning it brown. There are less and fewer apple cultivars that are only used for processing, while in Europe, particular high-tannin apple cultivars are utilized to manufacture cider (an alcoholic beverage). Apple cultivars that are appropriate for the processing

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market must be highly productive, large in size, and have the desired qualities for various processed products (usually high soluble solids, a particular sugar-to-acid ratio, yellow flesh color, nonbrowning flesh, and a “apple” taste). Most apple cultivars can be used to make applesauce, but only a select number are thought to be the best. Hussin et al. (2010) describe the qualities of raw apples that result in a high-quality product. High sugar contents, high acid, aromatic flavor, bright golden or white flesh, variable grain or texture, and adequate water-holding capacity are all desirable qualities in apples for applesauce. Cultivars which possess different processing traits are as follows: 1. Making Slices: York, Stayman, Golden Delicious, Northern Spy, Rhode Island Greening, Yellow Newtown, and Jonathan. 2. Beverages Varieties: Golden Delicious, Jonathan, gala, Fuji, Granny and Gala. 3. Cider Varieties: Baldwin, McIntosh, Northern Spy, Russet, James Grieves and Bramley’s Seedling. 4. Sauce Varieties: Granny Smith, Jonathan, McIntosh, Golden Delicious, Lodi, Northern Spy, Cortland, Gala, Liberty, Rome Beauty, and Mutsu. 5. Pies Making Varieties: Granny Smith, Jonathan, McIntosh, Golden Delicious, Lodi, Northern Spy, Cortland, Gala, Liberty, Rome Beauty, and Mutsu. 6. Baking-Type Varieties: Granny Smith, Jonathan, McIntosh, Golden Delicious, Lodi, Northern Spy, Cortland, Gala, Liberty, Rome Beauty, and Mutsu. 7. Varieties Suitable for Freezing: Golden Delicious, Jonathan, Mutsu, and Liberty. 8. Salad Varieties: Granny Smith, Cortland, Golden Delicious, red Delicious, Mutsu, and Baldwin.

2.9

Conclusion

Diverse researchers have made sincere efforts to comprehend the apple tree, including (1) cultivar and rootstock selection and breeding; (2) botanical considerations; (3) knowledge of pests and diseases; (4) genetic resources and variability; (5) breeding for resistance; (6) development of potent tools like molecular markers to support breeding programs; and (7) technical and genetic aspects in the improvement of the crop. Thoughts should be given to the following questions in the future: dwarf or semi-dwarf rootstocks with strong enough vigor to avoid trellis, great compatibility, and exceptional resilience to cold winters (columnar habit in new cultivars should help it). A phylogeny review using modern tools like molecular markers will help us better understand this complex genus and make it easier to create a wider variety of cultivars that combine high quality, excellent postharvest preservation, and resistance to the main pests and diseases, as well as dwarf or semi-dwarf rootstocks with good resistance to cold winters, excellent compatibility, and enough vigor to avoid trellis (columnar habit in new cultivars should help to do it).

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Rasool A, Bhat KM, Mir MA, Jan A, Dar NA, Mansoor S (2022) Elucidating genetic variability pertaining to flowering, maturity and morphological characters among various apple (Malus ✕ domestica Borkh.) cultivars. S Afr J Bot 145:386–396. https://doi.org/10.1016/j.sajb.2021. 06.010 Santhi VP, Nireshkumar N, Vasugi C, Parthiban S, Masilamani P (2020) Role of rootstocks to mitigate biotic and abiotic stresses in tropical and subtropical fruit crops: a review. September. https://doi.org/10.22271/chemi.2020.v8.i5g.10348 Sarma B, Das K, Bora SS (2020) Physiology of fruit development. Int J Curr Microbiol Appl Sci. 9(6):504–521. https://doi.org/10.20546/ijcmas.2020.906.066 Sedov EN (2014) Apple breeding programs and methods, their development and improvement. Russ J Genet Appl Res 4(1):43–51. https://doi.org/10.1134/S2079059714010092 Un S, Virendra N, Manoj KB, Govind KY (2020) Association of Apple necrotic mosaic virus ( ApNMV ) with mosaic disease in commercially grown cultivars of apple (Malus domestica Borkh ) in India. Biotech 3:1–9. https://doi.org/10.1007/s13205-020-2117-6 Vahdati K, Sarikhani S, Arab MM, Leslie CA, Dandekar AM, Alet N, Bielsa B, Gradziel TM, Montesinos Á, Sideli GM, Serdar Ü, Akyüz B, Beccaro GL, Donno D, Rovira M, Ferguson L, Akbari M, Sheikhi A (2021) Advances in rootstock breeding of nut trees: objectives and strategies, pp 1–34 Verma MK, Mir JI, Lal S, Sahoo T (2014) Recent advances in temperate fruit crop improvement. Int J Sci Technol Soc 4(1–2). https://doi.org/10.18091/ijsts.4121 Verma MK, Mir JI, Lal S, Sahoo T (2018) Recent advances in temperate fruit crop improvement. Int J Sci Technol Soc. 4(1–2). https://doi.org/10.18091/ijsts.4121 Wang Y, Li W, Xu X, Qiu C, Wu T, Wei Q, Ma F (2019a) Progress of apple rootstock breeding and its use. Hortic Plant J 5(September):183–191. https://doi.org/10.1016/j.hpj.2019.06.001 Wang Y, Li W, Xu X, Qiu C, Wu T, Wei Q, Ma F, Han Z (2019b) Progress of apple rootstock breeding and its use. Hortic Plant J 5(5):183–191. https://doi.org/10.1016/j.hpj.2019.06.001 Yu H, Li J (2022) Breeding future crops to feed the world through de novo domestication. 8–11. https://doi.org/10.1038/s41467-022-28732-8 Zha Q, Xiao Z, Zhang X, Han Z, Wang Y (2016) Cloning and functional analysis of MxNRAMP1 and MxNRAMP3, two genes related to high metal tolerance of Malus xiaojinensis. S Afr J Bot 102:75–80. https://doi.org/10.1016/j.sajb.2015.05.031 Zhu LH, Holefors A, Ahlman A, Xue ZT, Welander M (2001) Transformation of the apple rootstock M.9/29 with the rolB gene and its influence on rooting and growth. Plant Sci 160(3):433–439. https://doi.org/10.1016/S0168-9452(00)00401-5

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Genetic Improvement of Carnation Pooja Sharma

, Amarjit K. Nath, Akhil Kumar, and Anshul Shyam

Abbreviations ACC AP2/ERFBP bHLH bZIP C2’GT CHI CRISPR CSP DcHsfA1d DFR DREB Etr1-1 F3’5’H F3’H HSPs LEA MAPK

Amino cyclopropane carboxylic acid APETALA 2/ethylene-responsive element binding proteins Basic helix-loop-helix Basic leucine zipper domain THC 2′-glucosyltransferase Chalcone isomerase Clustered regularly interspaced short palindromic repeats Cold shock proteins Dianthus caryophyllus heat shock transcription factors Dihydroflavonol 4 reductase Dehydration-responsive element binding Arabidopsis ethylene receptor gene Flavonoid 3’5’-hydroxylase Flavonoid 3′-hydroxylase Heat shock proteins Late embryogenesis abundant proteins Mitogen-activated protein kinase

P. Sharma (✉) · A. K. Nath Department of Biotechnology, Dr YS Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India A. Kumar CSIR-IHBT, Palampur, Himachal Pradesh, India A. Shyam Department of Fruit Science, Dr YS Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_3

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NAC NO PA PDS SAMDC sgRNA TALENs WRKY ZFNs

3.1

NAM (no apical meristem), ATAF1 and –2, and CUC2 (cup-shaped cotyledon) Nitric oxide Polyamine Phytoene desaturase S-adenosylmethionine decarboxylase Single-guide RNA Transcription activator-like effector nucleases W-box binding protein Zinc finger nucleases

Introduction

The contribution of ornamental plants has revolutionized the horticultural industry in the past. Different ornamental plants are now widely used in subsistence agriculture, skilled landscaping, and as cut flowers. Breeders develop novel and alluring varieties every year. New characteristics like flower color, fragrance, modified flower facets, and lengthier shelf life are highly sought after by consumer. Along with this, improved agronomic behavior is equally important from the grower’s perspective. Therefore, yield and disease resistance also play a crucial part in the development of a successful commercial cultivar. Carnation (Dianthus caryophyllus L.) is a valuable commercial cut flower next to rose due to its diverse assortment of floral type and color. Carnation belongs to family Caryophyllaceae involving a total of 80 genera with 2000 species. The botanical name is derived from Greek, where “dios” signifies divine and “anthos” for flower. Dianthus is considered to originate from Mediterranean location and consists of around 300 species. The commercial important species includes carnation or clove pink (D. caryophyllus), sweet William (D. barbatus) and Indian pink or Japanese pink (D. chinensis). Carnation diploids (2n = 30) are a semi-hardy herbaceous perennial adapting more to cooler climatic conditions. Leaves are thick, narrow, linear, succulent, and color varies from green to gray blue or purple. Stems are hardy-bearing terminal flower. Inflorescence is terminal cyme, sometimes racemiform. Flowers are bisexual with color varying from white to pink or purple. It is an important cut flower excellent for bedding, pots, borders, edging, and rock gardens and widely used in bouquets and decorations (Sharma et al. 2022). However, this flower crop is adversely affected by several biotic stresses (bacterial wilt, bacterial leaf spot, aphid, thrips) and abiotic stresses (drought, salt, heat) which affect the plant growth leading to poor flower quality and reduced yield (Lim 2014; Zuker et al. 2001). High temperatures are typically deleterious to carnation plant growth and development resulting in flower wilting and decline in quality. The major biotic threat to carnations is wilt disease as a result of Fusarium oxysporum f. sp. dianthi and current methods for dealing with this soilborne fungus are

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Genetic Improvement of Carnation

Fig. 3.1 Improved traits in carnation through genetic engineering

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Biotic stress (Bacterial wilt, Fusarium wilt, Leaf spot, Rust) Improved productivity

Abiotic stress (Heat, salinity, drought, cold)

Delay senescence

Flower scent

Flower colour

extremely dangerous, inefficient, and expensive. In order to combat these stresses, plants develop a variety of defensive mechanism at the physiological, biochemical, and molecular levels and improve adaptation to adverse environmental conditions. As a result, understanding these stress conditions can aid in the development of new carnation varieties. Due to rising demand of carnations, efforts have been made to improve disease resistance, insect resistance along with ornamental attributes such as flower color, form, and longevity (Fig. 3.1). Previously, traditional breeding strategies are widely used to develop new plant lines; however, there are limitations such as degree of heterozygosity (DaSilva et al. 2011). Crossbreeding and mutagenesis approaches have resulted in the development of numerous cultivars, but they can only be applied to a minimum number of traits. Furthermore, because it is a vegetatively propagated crop, the gene pool is narrow and the chances of flower breeding are low. As a result, carnation is an ideal candidate for gene transfer technologies. Since the onset of the last decade, techniques such as genetic engineering and genome editing have been increasingly accepted as more viable ways to overcome the inherent limitations of traditional techniques. Genetic engineering techniques offer a tool for carnation breeders to improve existing cultivars or creating entirely new germplasm by inserting genes outside of the genus Dianthus, hence resulting in broadening the available gene pool. Therefore, it is very possible to introduce genes for disease resistance and stress tolerance in carnation. Likewise, plant characters such as flower color, form, fragrance, longevity, and floral architecture can also be improved through genetic engineering (Kamthan et al. 2016). Major transformation techniques used in carnation were developed in 1990s which includes Agrobacterium-mediated transformation, biolistic transformation, and protoplast transformation by Zuker et al. (1995) and Yantcheva et al. (1997). Modified blue

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color carnation (Moonseries) by Florigene was marketed in Canada, Japan, Russia, and USA is one such example of transgenic technology. Furthermore, current genome editing methods, such as RNA interference (RNAi) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, depend on transgenic technology to integrate mutations into any specific target gene and are projected to improve the rate of breeding programs as it is considered as a key tool for producing novel cultivars. This chapter summarizes the recent advances in carnation through genetic engineering, RNAi, and genome editing tools. According to the existing information, genetic engineering and genome editing techniques in carnation are efficient techniques for constantly improving horticulturally important traits in comparison to traditional breeding and widening the use of ornamentals to benefit society.

3.2

Genetic Transformation Methods

First report of genetic transformation in higher plants was given by Zambryski et al. (1983); however, the first successful example of change in flower color in ornamental plants (Petunia) by introduction of dihydroflavonol 4 reductase (DFR) gene was reported by Meyer et al. (1987). Carnation was the first world’s genetically manipulated cut flower which was successfully introduced in the market in 1997. The “Moon” transgenic carnation plants were sold in various countries mainly Australia, Northern America, Japan, and United Kingdom. Most of the genetic transformation in ornamental plant species is based on Agrobacterium-mediated transformation, biolistic transformation, and protoplast transformation. Agrobacterium-mediated transformation is the most stable and efficient system for gene transfer in plants. This method utilizes the bacterium Agrobacterium tumefaciens which is responsible for crown gall disease in plants. Agrobacterium transfers the T-DNA-encoding transgenes into the nucleus, integrates on chromosomes, and results in transgenic expression. Agrobacteriummediated transformation is mostly preferred for many plants as it results in stable transformation. Another widely used method for genetic transformation is biolistic transformation. This method employs a specifically designed instrument to deliver metal particles coated with target DNA under high pressure directly into the cell. This process results in transient expression and is relatively less efficient than Agrobacterium-mediated transformation. Protoplast transformation is also an effective way to produce transgenics. In this method, plant cell wall is mechanically or enzymatically digested to isolate protoplast and vectors are integrated by transfection. However, this method requires longer culture time and is more difficult for plant regeneration. Most of the genetic transformation methods in floriculture crops prefer Agrobacterium-mediated system. Zuker et al. (1995) and Yantcheva et al. (1997) attempted to transform carnation using Agrobacterium-mediated and biolistic transformation. Zuker et al. (1999) achieved successful transformation in carnation by wounding of stem explants using biolistic method followed by co-cultivation with

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Agrobacterium. A ten-fold increase in GUS expression was observed. Successful genetic transformation using stem segments and nodal explants was observed in carnation by Agrobacterium (strain AGL0) (Estopa et al. 2001; Nontaswatsri et al. 2004).

3.3

Genome Editing

Genetic transformation possesses opportunity to substantially lessen the breeding process. Transgenic plants, on the other hand, are strictly regulated in India and many other countries. As a result, breeders are reluctant to use them. Transgenic regulations necessitate a thorough risk assessment of potential effects on both the environment and human health. Development of transgenics entails exorbitant costs and precludes the production of genetically modified ornamental plants in several instances where the estimated market is insufficient to cover the additional costs of transgenic development. Trying to overcome these challenges, present genome editing techniques ideally allow the introduction of mutations into targeted genome sequence for creation of novel variants. CRISPR-Cas9 system is one of the prevalent genome editing system. It was invented in bacteria and archaea by Wiedenheft et al. 2012. Cas9 (nuclease) is directed by single-guide RNA (sgRNA) to bind and cleave a stretch of 20-nucleotide on directed DNA called protospacer. Repairing Cas9-induced double-stranded breaks in protospacer which results in mutation (insertion or deletion). As a result, the desired sequence can be inserted into vector for target-specific location. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are another two types of sequence-specific nucleases. Multiple-guide RNAs could be utilized at the same time, lowering the cost, and time required to generate plants with multitarget genetic variations (Cong et al. 2013). Unlike transgenic technology, genome editing system requires no integration of transgene to produce desired traits (Fauser et al. 2014). Modified plants could transfer the induced mutation to their progenies, similar to transgenics. Furthermore, genome editing produces a precise mutation in the template strand. As a result, it is anticipated to be more effective than traditional breeding in obtaining superior genetically mutated plants and represents a significant advancement in the breeding technology. The CRISPR/Cas9 system has popularized as the benchmark for precise genetic mutation in plants. However, its utilization in ornamentals is lower than in other crops. The first successful report of genome editing in an ornamental arises from Petunia hybrida in which the phytoene desaturase (PDS) gene was edited resulting in an albino mutant phenotype (Zhang et al. 2016). Few reports of genome editing in ornamentals, such as Japanese morning glory plants, petunias, and orchids have been well-documented in the past (Subburaj et al. 2016; Watanabe et al. 2017; Kui et al. 2017).

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Mechanism of Abiotic/Biotic Stress Response

Plant stress entails external environment that has a negative influence on development and growth. It causes various reactions in plants such as change in cell metabolism, productivity, gene expression, and crop yield. Biotic stress may be due to bacteria, viruses, fungi, insects, nematodes, or diseases, and abiotic stress predominately involves physical factors (UV radiation, heat, osmotic stress, salinity, temperature) and chemical factors (heavy metals, minerals, toxins) (Fig. 3.2). These stresses affect the plant germination, growth, and development and lead to poor quality and reduced yield. To cope with these stresses, plants develop a variety of

Fig. 3.2 Abiotic stress tolerance/resistance in plants

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defensive mechanisms at the morphological, physiological, biochemical, and molecular levels and improve adaptation to adverse environmental conditions. Plants primarily adapt to these stresses by avoidance and tolerance mechanisms. At the morphological and physiological levels, plants respond by changing leaf orientation, stomatal closure, transpirational cooling, expanding vascular elements and modifying lipid compositions in cell membrane. The presence of waxy coating in leaf or stem and curling of leaves also leads to avoidance of stress in the short term. However, tolerance mechanism includes long-term adaptations and requires major tolerance mechanisms in signaling cascades such as osmoprotectants (proline, alanine), ion transporters, antioxidants (superoxide dismutase, peroxidase, catalase, ascorbate, and glutathione reductase), late embryogenesis abundant proteins (LEA), transcriptional factors (DREB, WRKY, NAC), cold shock proteins (CSP 1, CSP2, CSP 3), and heat shock proteins (HSP 40, HSP 90). In general, the upregulation of these signaling molecules under stress conditions has been reported for long-term adaptation in plants (Tuteja 2009). Signaling molecules such as mitogen-activated protein kinase (MAPK), calcium-mediated signaling, hormonal signaling (salicylic acid, abscisic acid), and nitric oxide (NO) signaling together with transcriptional factors result in regulation of stressresponsible genes and their metabolic pathways. Knowledge about the signaling cascade and stress-responsive genes is crucial to develop stress-tolerant plants through genetic engineering. As a result, understanding these signaling mechanisms can aid in the development of new carnation varieties.

3.5

Genetic Alterations to Improve Abiotic Stress

To date, it has been observed that genetic engineering has offered numerous applications for the improvement of abiotic stress in carnation. During abiotic stress (heat, cold, salinity, drought, etc.), the various cellular, metabolic, and molecular pathways are regulated in the plants to cope with these stresses. Various genes are involved which encoding the biosynthesis pathways involving heat and cold shock protein (CSPs and HSPs) and several other transcription factors like AP2/ERFBP, bZIP, DREB, WRKY, MYB, and NAC are major gene families involved in the abiotic stress. Therefore, modification of the expression of these transcription factors through genetic engineering offers an advantage to create new abiotic stress-resilient varieties (Century et al. 2008; Yang et al. 2011). Previously a report shows that the heat stress transcription factor DcHsfA1d plays a positive regulatory role in heat stress and salt tolerance resulting in increased root and fresh mass in Arabidopsis (Wan et al. 2022). Similarly, under heat and drought conditions, DcHsf-A3, A7, A9a, A9b, and B3a were found upregulated in earlier flowering stages in response to salicylic acid (Li et al. 2019). In another study, the SAMDC gene involved in polyamine synthesis was overexpressed which resulted in increased polyamine content and enhanced tolerance to experienced environmental stress in tobacco (Wi et al. 2006). A recent study in carnation shows that the DcHSP17.8 withstand against heat stress in A. thaliana and improved H2O2, O2,

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photosynthesis, malondialdehyde content, antioxidant enzyme activity, and reduced electrolyte leakage (Sun et al. 2022). Nevertheless, the availability of genomic information helps in the creation of new carnation varieties which are abiotic stress-resistant.

3.6

Genetic Alterations to Improve Biotic Stress

Biotic stresses such as virus, bacteria, fungi, and insects are one of the main causes of crop loss globally. As an alternative to the application of chemicals, genetic engineering helps in altering the genetic composition to enhance resistance to biotic stress. Previously various conventional approaches have been applied for the improvement of carnation from biotic stresses. In a report by Shirasawa-Seo et al. (2002), ectopic expression of oat thionin with promoter of A. thaliana tryptophan synthase F1 subunit resulted in enhanced resistance to bacterial wilt. Very few reports on disease resistance in carnation are available. However, the available tissue culture protocol and genomic information on carnation are an advantage for the development of new disease-resistant varieties through genetic engineering.

3.7

Genetic Alterations to Improve Quality Characteristics

3.7.1

Flower Color Variation

Enhancement of anthocyanin biosynthesis in carnation by incorporation of MYB and bHLH family of transcription factor to modify flower color was attempted by Florigene Ltd. However, the downregulation of anthocyanin biosynthesis gene to develop white flowers in carnation was successfully engineered by Gutterson (1995). Transgenic violet carnations were successfully developed by Florigene Ltd. and Suntory Ltd. by introduction of DFR and F3’5’H gene from petunia (Mol et al. 1999). These transgenic carnations were commercially sold in Japan, Australia, UK, and Northern America under the trade name Florigene Moondust™ and Florigene Moonshadow™. Similarly, using the same approach Florigene Moonacqua™, Florigene Moonlite™ and Moonshade™ were marketed in similar countries. Brugliera et al. (2000) incorporated petunia cytochrome b5 gene and F3’5’H of carnation which led to enhancement in delphinidin level and resulted in variegated purple and mauve flower color from variegated red and pink. Zuker et al. 2002 observed that downregulation of F3’H not only changes flower color but also produces more fragrance as a result of increase in methylbenzoate in carnation. Yellow flower color in carnation was also produced by accumulation of THC 2′-glucosyltransferase (C2’GT) and lack of chalcone isomerase (CHI) activity (Itoh et al. 2002). Furthermore, genes encoding C2’GT activity were isolated from carnation (Okuhara et al. 2004) which is responsible for the accumulation of yellowcolored isosalipurposide. Zhang et al. (2014) introduced cyanidin, delphinidin glucosides, and pelargonidin-producing genes into the anthocyanin biosynthetic

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pathway which resulted in formation of red, pink, and violet flower color from white flowers in carnation.

3.7.2

Enhancing Vase Life

The post-harvest management in carnation is greatly affected by production of ethylene and colonization of microbes. Ethylene production in plants is responsible for flower senescence as well as petal drop and association of microbes leads to blockage of vascular elements resulting in wilting of flowers. Savin et al. (1995) and Kinouchi et al. (2006) silenced the gene ACC oxidase and ACC synthase to downregulate the ethylene production and enhanced the vase life of carnation. In a similar report, introduction of Arabidopsis thaliana Etr1-1 gene under CaMV35S as promoter which resulted in generation of ethylene-insensitive carnation lines and showed delayed senescence of 6–16 days (Bovy et al. 1999). Florigene Ltd. also attempted similar approach by employing Etr1-1 with CMB2 promoter for enhancing vase life of carnation. Delayed in flower senescence has also been achieved by co-suppression of ACC synthase (Acs) gene in carnation (Chandler 2007).

3.7.3

Modification in Plant Architecture

In carnation, attempts have been made to produce stem cuttings by integrating the roIC gene under 35C CaMV promoter in past (Zuker et al. 2001; Casanova et al. 2004). Another study in carnation resulted in pleiotropic morphological changes involving dwarfing and phyllotaxis alteration by incorporating PttKN1 gene (Meng et al. 2009). Wang et al. (2020) found DcaAG (AGAMOUS) gene to be involved in controlling number of petals, stamen, and carpel development in carnation. Similarly, the different expression patterns of the genes SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 6 (AGL6), and SEEDSTICK (STK) were found in the flower development in carnation (Zhang et al. 2018).

3.7.4

Alteration in Floral Fragrance

Flower fragrance plays a pivotal role to consumer choice as well as it is equally important in attracting pollinators. However, molecular knowledge for scent compounds is extremely limited. In carnation, eugenol, caryophyllene, and benzoic acid derivatives are the main compounds responsible for fragrance. Carnation cv. Eilat was transformed with Clarkia breweri S-linalool synthase which is a floral fragrance biosynthetic gene under the control of CaMV53S promoter to modify flower scent resulted in formation of linalool and its derivatives (Lavy et al. 2002). Biosynthesis of flower scent compounds in carnation is developmentally regulated; however, a very few reports on genetic transformation for floral scent are available.

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Conclusion

The increasing speed with which horticultural important genes have been isolated and identified encompasses the scope of genetic engineering in floriculture crops. Several efforts have been made in carnation to create more valued and novel variants to fulfill growing market demand. Genetic engineering and gene editing in carnation are more enticing than conventional breeding for improvement of valuable traits. Knowledge of the genetic and molecular mechanisms governing key traits is required along with the availability of genome information. To avoid legal restrictions, genome editing components must be segregated. It is still necessary to develop effective methods for deploying these technologies. Nonetheless, genetic engineering is clearly an effective technique for modifying horticulturally useful traits. The market for genetically engineered crops will undoubtedly rise in the coming years and the florist industry will profit society in the long run. Conflicts of Interest The authors declare that there is no potential conflict of interest.

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Wan X, Sun Y, Feng Y, Bao M, Zhang J (2022) Heat stress transcription factor DcHsfA1d isolated from Dianthus caryophyllus enhances thermo tolerance and salt tolerance of transgenic Arabidopsis. Biol Plant 66:29–38 Wang Q, Dan N, Zhang X, Lin S, Bao M, Fu X (2020) Identification, characterization and functional analysis of C-class genes associated with double flower trait in carnation (Dianthus caryophyllus L.). Plants 9:87. https://doi.org/10.3390/plants9010087 Watanabe K, Oda-Yamamizo C, Sage-Ono K, Ohmiya A, Ono M (2017) Alteration of flower colour in Ipomoea nil through CRISPR/Cas9-mediated mutagenesis of carotenoid cleavage dioxygenase 4. Transgenic Res 27:35–38. https://doi.org/10.1007/s11248-017-0051-0 Wi SJ, Kim WT, Park KY (2006) Overexpression of carnation S-adenosylmethionine decarboxylase gene generates a broad-spectrum tolerance to abiotic stresses in transgenic tobacco plants. Plant Cell Rep 25:1111–1121. https://doi.org/10.1007/s00299-006-0160-3 Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338. https://doi.org/10.1038/nature10886 Yang W, Liu XD, Chi XJ, Wu CA, Li YZ, Song LL, Liu XM, Wang YF, Wang FW, Zhang C, Liu Y (2011) Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways. Planta 233:219–229 Yantcheva A, Vlahova M, Todorovska E, Atanassov A (1997) Genetic transformation of carnation (Dianthus caryophyllus L.). Biotechnol Biotechnol Equip 11:21–25. https://doi.org/10.1080/ 13102818.1997.10818924 Zambryski P, Joos H, Genetello C, Leemans J, Montagu MV, Schell J (1983) Ti plasmid vector for the introduction of DNA into plant cells witt alteration of their normal regeneration capacity. EMBO J 2:2143–2150. https://doi.org/10.1002/j.1460-2075.1983.tb01715.x Zhang Y, Butelli E, Martin C (2014) Engineering anthocyanin biosynthesis in plants. Curr Opin Plant Biol 19:81–90. https://doi.org/10.1016/j.pbi.2014.05.011 Zhang B, Yang X, Yang C, Li M, Guo Y (2016) Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in Petunia. Sci Rep 6:20315. https://doi.org/10.1038/srep20315 Zhang X, Wang Q, Yang S, Lin S, Bao M, Bendahmane M, Fu X (2018) Identification and characterization of the MADS-box genes and their contribution to flower organ in carnation (Dianthus caryophyllus L.). Genes 9:193. https://doi.org/10.3390/genes9040193 Zuker A, Chang PL, Ahroni A, Cheah K, Woodson WR, Bressan RA, Watad AA, Hasegawa PM, Vainstein A (1995) Transformation of carnation by microprojectile bombardment. Sci Hortic 64:177–185 Zuker A, Ahroni A, Tzfira T, Ben-Meir H, Vainstein A (1999) Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.). Mol Breed 5:367–375. https://doi.org/10.1023/A:1009671131200 Zuker A, Tzfira T, Scovel G, Ovadis M, Shklarman E, Itzhaki H, Vainstein A (2001) RolC transgenic carnation with improved horticultural traits: quantitative and qualitative analyses of greenhouse-grown plants. J Am Soc Hortic Sci 126:13–18 Zuker A, Tzfira T, Ben-meir H, Ovadis M, Shklarman E, Itzhaki H, Forkmann G, Martens S, NataSharir I, Weiss D, Vainstein A (2002) Modification of flower colour and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol Breed 9:33–41. https://doi.org/10.1023/ A:1019204531262

4

Recent Advances in Genetic Improvement of Cotton Kajal Verma, Pooja Sharma, Kanchan Tripathi, Reena Yadav, and Surendra Pratap Singh

4.1

Introduction

Cotton (Gossypium spp.) is economically important and a model polyploid crop all over the world (Liu et al. 2017). Millions of cotton bales are needed yearly for the textile industry’s primary use of cotton fiber (Shahzad et al. 2019). The demand for cottonseed meals for livestock feed has risen along with using cotton vegetable oil (Shahzad et al. 2022). Polyploid evolution and domestication can also be understood through the diversity of Gossypium species. However, genomic research is hampered by the complex and large cotton genome (Yang et al. 2020). Multiple genes control the fiber quality characteristics of cotton with a minor effect, and traditional genetic improvement techniques require a long time to increase fiber quality. For cotton breeders, one of the biggest challenges is the genetic improvement of fiber quality (Ijaz et al. 2019). Plant biotechnology has allowed researchers to insert foreign genes that regulate many features like drought tolerance, fiber quality, herbicide resistance, CLCuV resistance, and pest resistance, which is crucial for preserving cotton yield (Razzaq et al. 2021). Increased economic potential in cottonseed and other economic features can help enhance profitability for cotton growers. Cotton breeders urgently aim to increase cotton production’s profit due to intense competition from other important crops (Ijaz et al. 2019). Besides being the most important natural fiber crop in the world, cotton provides a great environment for studying genome evolution, polyploidization, and cell elongation. Five cotton genomes were assembled, and an allopolyploidization K. Verma · K. Tripathi · R. Yadav · S. P. Singh (✉) Plant Molecular Biology Laboratory, Department of Botany, Dayanand Anglo-Vedic (PG) College, Chhatrapati Shahu Ji Maharaj University, Kanpur, India e-mail: [email protected] P. Sharma NUS Environmental Research Institute, National University of Singapore, Singapore, Singapore # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_4

69

70

K. Verma et al.

process was observed that united the A- and D-genomes (Huang et al. 2021). This recent advancement in cotton genomics will likely be useful for analyzing and genetically modifying cotton’s agronomic traits. Furthermore, it will contribute to understanding how allopolyploidization evolved in modern plants (Wang et al. 2018). Using data from the China National Germplasm Mid-term Gene Bank, Zaidi et al. (2018) have discovered genomic information on 419 tetraploid and 243 diploid cotton germplasms. Functional genomics has evolved from a futuristic concept to a recognized scientific field in the last decade. Our knowledge of fundamental plant biology is enhanced through cotton functional genomics, allowing us to improve cotton fiber quality and yield through the systematic use of genetic resources. Understanding how the cotton gene works is significantly more difficult and has not advanced quickly (Ashraf et al. 2018). Advances in cotton genomes have transformed functional genomics research. Map-based cloning and genome-wide association analyses (GWAS) are now possible for features crucial to cotton agriculture. Integrated multi-omics, homology-based cloning, and reverse genetics approaches can also be used to find breeding target genes (Yang et al. 2020). According to Guo et al. 2015, several molecular signatures (transcripts, transcription factors, and genes) and physiological and biochemical processes contribute to stress tolerance. Many transcription factors control the expression of different upstream and downstream genes related to stress tolerance in plants, ensuring stress signaling responses. Recently, transcriptome profiling has emerged as a crucial method for understanding gene activity through knowledge derived from sequencing data. Since high-throughput sequencing tools can directly sequence transcripts with RNA-Seq (Ashraf et al. 2018), it has demonstrated considerable potential for whole-genome transcriptome profiling. Among numerous species, genotypes, and biotic and abiotic conditions, RNA-Seq-based differential gene expression (DGE) for fiber-related genes has been documented in cotton (Etukuri et al.). Using sequencing data from other types of analysis, such as transcriptomics, proteomics, and epigenomics, could assist in identifying key candidate genes and biologically active DNA states involved in plant defense (Mahmood et al. 2019). The recent advances in the genetic improvement of cotton are discussed in this chapter (Fig. 4.1).

Structure variation

Gossypium spp.

Whole genome sequncing

Transcriptome

Epigenomics

SNPs, Indels, CNVs

Proteome

DNA-Methylation, Histonemodification

Fig. 4.1 Omics approaches deployed in the cotton genetic improvement

Metabolome

4

Recent Advances in Genetic Improvement of Cotton

4.2

71

Genetic Improvements in Cotton

In recent years, plant biotechnology has become one of the most important tools for improving crop plants, such as cotton. Since 1996, transgenic cotton has been planted on more than 70% and 50% of cotton lands in the United States and China, respectively (Zhang et al. 2008; Zhang 2019) Using genetic engineering to produce crops with beneficial agronomic and fiber traits will reduce production costs, increase yields, and enhance ecologically friendly farming practices. Due to advancements in gene identification and transformation technologies, recent advancements in these areas are likely to occur faster than in the past (John 1997). The invention of Bt cotton, the first genetically modified (GM) crop, has been one of the most significant contributions of plant biotechnology to farmers. It was successfully marketed during the previous 10 years and is currently used (Kumria et al. 2003). The following goals of cotton biotechnology include fiber enhancement, stress resistance, and male sterility and fertility for hybrid cotton. In various laboratories, most of the genes for fiber improvement and hybrid cotton are being tested (Zhang et al. 2000a, b, c). Even though transgenic cotton plants with disease resistance, abiotic stress tolerance, and enhanced fiber quality have been developed during the past few decades, insect-resistant and herbicide-tolerant cotton are the two most common cotton on the transgenic cotton market (Chakravarthy et al. 2014). The main cotton transformation techniques involve Agrobacterium, biolistic particle delivery systems, pollen-tube pathway-mediated approaches, etc. (Zhang et al. 2019). Currently, these techniques are used to obtain essentially all transgenic cotton plants (Zhang et al. 2019). Agrobacterium-mediated transformation of plants was reported for the first time in 1983 (Sidorov 2013). Agrobacterium-infected hypocotyl pieces were used to describe the first method of changing cotton genotypes of the “Coker” genotype, an easily regenerable genotype (Zhang and Jin 2007). Cotton was genetically improved in various ways (Fig. 4.2).

Genetic improvement in cotton

Biotic resistance

Abiotic resistanc

Insect resistance.

Heat resistance.

Herbicideresistance.

Salt resistance.

Disease resistance

Drought resistance.

Somatic embryogenes is

Protoplast culture. Anther & Ovule culture. Shoot & meristem culture

Fig. 4.2 Genetic improvement strategies in cotton

Epigenetic changes

Genome editing

Male sterility

Crisper cas9.

Hybrid seed

DNA methylation. Histone modification.

72

4.2.1

K. Verma et al.

Biotic Resistance

According to Kamburova et al. 2022, cotton productivity is reduced by insects and pathogens. Due to biotic stress, cotton production was reduced by up to 50–60%. However, traditional breeding is a time-consuming and expensive procedure for crop improvement. On the other hand, the breeding cycle is considerably shortened because of DNA recombinant technology (Hussain and Mahmood 2020). In order to effectively control pests, diseases, and insects and decrease crop losses, plant resistance to biotic factors must be improved (Kamburova et al. 2022). The main goal of cotton breeding against disease resistance remains among the several biotic variables (Ashraf et al. 2018). The transgenic approach has also improved qualities that are thought challenging or impossible to improve through conventional breeding (Hussain and Mahmood 2020). Using the transgenic methods, the resistance of cotton to biotic stresses is frequently increased (Kamburova et al. 2022).

4.2.1.1 Genetic Improvement for Insect Resistance in Cotton A major source of harm to the world’s commercially significant crops is insect pests (Estruch et al. 1997). Cotton crops are infested and damaged by 130 insect species. Table 4.1 lists several cotton transgenics developed using different insecticidal genes (Chakravarthy et al. 2014). During the last decade, transgenic cotton has drastically changed this crop’s pest control. Bacillus thuringiensis (Bt) insect and herbicideresistant (Ht) cotton was one of the earliest commonly grown transgenic plants. Over 300 transgenic cotton varieties are available to producers, including pyramided variants with herbicide tolerance and cultivars expressing single or dual-Bt proteins that target lepidopteron larvae (Torres et al. 2010). Cotton has been engineered to resist major insect pests by introducing genes from bacteria, insects, and plants (Chakravarthy et al. 2014). According to Naranjo, Naranjo 2011, more than 15 million hectares of transgenic cotton were planted in 11 countries in 2009 that produced Bacillus thuringiensis (Bt) insecticidal proteins, reducing the use of insecticides by almost 140 million kg from 1996 to 2008. According to Estruch et al. 1997, insecticidal proteins from Bacillus thuringiensis (Bt) served as the basis for the first generation of transgenic insect-resistant plants. A new generation of insectresistant plants is being developed that includes both Bt and non-Bt proteins with diverse modes of action and activity ranges against insect pests.

4.2.2

Genetic Improvement for Herbicide Resistance in Cotton

Herbicide-resistant weeds are a severe and rising agronomic issue widely distributed and rapidly evolving (Liu et al. 2019). Weed control has been a crucial component of cotton farming since weeds compete with crops for water, nutrients, and sunshine and raise the trash content of the cotton fiber collected. Several herbicides have been utilized for weed control in various crops, each with a different mode of action, toxicity, and environmental impact (Chakravarthy et al. 2014). Herbicide resistance management may be handled more effectively when herbicides are characterized

Insect resistance

Insect and weeds resistance Transgenic line GK12 and GK19 Insect resistance

Lectins inhibitor Insect pest resistance

Protease inhibitors Trypsin inhibitor

Transgenic cotton Bt cotton (insect)

B. thuringiensis

B. thuringiensis

BT and cp4epsps

cry1A

cry1Ia5

Bacillus sp.

Bacillus thuringiensis subsp. Israelensis (Bti) –

cry10Aa

cry1Ac, Vip1, or Vip2

–

Manduca sexta L. Cocculus hirsutus

Gene source Bacillus thuringiensis

GNA, P-Lec

CpT, SKTI, chTI

PI gene

Gene cryAc, cry1Ab

CaMV35S (RbcS)

CaMV35S

CaMV35S

CaMV35S

CaMV35S and uceA1.7

–

Agrobacterium

Agrobacterium

Agrobacterium

Biolistic (bombard5479)

Agrobacterium

Agrobacterium

– –

Agrobacterium

Transformation method Agrobacterium

CaMV35S

Promoter driven expression CaMV35S

Table 4.1 List shows genetic approaches for the insect resistance cotton

Defense against major target insects/pests, primarily including the bollworms Defense against western corn rootworm but did nothing to resist lepidopteran insects

Resistance against insect pests and herbicides

Resistance against insect pests and herbicides

Effect against homopteran pest B. tabaci. Resistance against bollworm, Helicoverpa armigera and Spodoptera litura Resistance against Aphids, bollworm Resistance against cotton boll weevil (CBW),

Effects Resistance against lepidopteran

Recent Advances in Genetic Improvement of Cotton (continued)

Noman et al. (2016a, b)

Noman et al. (2016a, b)

Bakhsh et al. (2011)

Butt and Awan (2016)

References Naranjo (2011), Liu et al. (2010) Thomas et al. (1995) Yadav et al. (2022), Zhang et al. (2000a, b) Zhang et al. (2000a, b) Ribeiro et al. (2017)

4 73

cry1Ac or cry1A

cry1Ac gene, modified cry1Ac gene, fusion cry1Ac/cry1Ab gene, cry1Ac and cry2Ab genes

Bacillus thuringiensis B. thuringiensis

Bacillus thuringiensis

cry1Ac + cry2A, GT

cry1Ac

B. thuringiensis

cry1Ac and CpTI

Transgenic cotton SGK321 CRSP-1 and CRSP-2 Insecticidal cotton

Bt cotton hybrid Bt cotton hybrid

B. thuringiensis subsp. Israelensis S1804 (Bti) B. thuringiensis

c

Insect resistance

B. thuringiensis

Gene source Bacillus sp.

cry10Aa

Gene cry1Ac, cry1F

CBW resistance

Transgenic cotton Widestrike®

Table 4.1 (continued)

–

–

–

Agrobacteriummediated or particle bombardment –

CaMV35S

CaMV35S

Agrobacterium

Baculovirus, pGemcry10Aa plasmid (pSynXIVVI+X3) Agrobacterium

Ppol

–

Biolistic transformation

Transformation method –

uceA1.7

Promoter driven expression CaMV35S

Resistance against sucking pests or bollworms Resistance against Earias spp.

Plants are more vigorous against insect pests

Resistance against lepidopteran

Resistance against insect and pest

Effects Resistance of Cabbage Loopers to Dual-Bt Toxin WideStrike Cotton Plants and B. thuringiensis (Bt) Toxin Cry1F Cry10Aa GM cotton plants can significantly impact the cotton industry and are a significant advancement in the fight against the destructive CBW insect pest. Resistance against insect and pest

Vennila et al. (2004) Shera et al. (2015)

Ali et al. (2016) Showalter et al. (2009)

Zhang et al. (2014a, b)

de Souza Aguiar et al. (2012)

Ribeiro et al. (2017)

References Kain et al. (2022)

74 K. Verma et al.

4

Recent Advances in Genetic Improvement of Cotton

75

according to their site of action herbicide. Transgenic plants have greatly helped humankind in boosting crop yields (Vats 2015). Transgenic cotton has proven to be highly effective against many herbicides. Cotton is highly susceptible to 2,4-D, even a small amount of spraying can result in significant harm. When 2,4-D is used on nearby crops, cotton is protected against 2,4-D damage by 2,4-D resistance (Zhang and Jin 2007). No herbicide can eliminate all potential weed issues in cotton, as shown in Table 4.2. By 1997 or 1998, the market should be able to purchase herbicide-resistant plants for Buctril, Roundup®, and several other herbicides. Carefully blending specific herbicides could offer season-long control of almost all problematic weed species (Wilkins et al. 2000; John 1997).

4.2.2.1 Genetic Improvement for Disease Resistance in Cotton Another significant factor is a disease that reduces the production of crops (Zhang et al. 2000a, b). The Cotton Disease Council calculates that illnesses cost the United States more than $1 billion between 1991 and 1997, causing a mean yearly loss of 12.3% from potential production. Other countries also suffer huge losses from cotton illnesses, frequently with little profit margins (Wilkins et al. 2000). During the attack of pathogens, plants produce a lot of pathogenesis-related proteins. Advancements in recombinant DNA technology and knowledge of plant-microbe interactions at the molecular level have made it possible to isolate and characterize the genes encoding such proteins, allowing for the development of transgenic crops resistant to various pathogen types (Cletus et al. 2013). For cotton breeding, genetic engineering is considered a great tool that plays a significant role in developing disease resistance cotton to improve net yield (Noman et al. 2016a, b). According to Shi et al. 2010, GhMPK7 regulates plant growth and development and may contribute to broadspectrum pathogen resistance. Table 4.3 (Chakravarthy et al. 2014) lists the different disease-resistant transgenic cotton.

4.3

Abiotic Resistance in Cotton

Abiotic factors such as cold, drought, and heat adversely affect cotton production. Excessive formation of activated oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydrogen radicals, results in oxidative stress when extreme temperatures or water stress are combined with high levels of sulfur dioxide, ozone, or intense light. (Kamburova et al. 2022; John 1997). Cotton production is limited by numerous abiotic factors, contributing to a 73% yield loss globally. Increasing cotton’s tolerance to these stresses has proven difficult (Ashraf et al. 2018). Transgenic methods frequently achieve increased cotton resistance to abiotic stressors. Transgenic approaches can also increase cotton’s tolerance to salinity and drought. Heat, drought, and salt tolerance in cotton are all controlled by various transcription factors that control the activity of functional genes (Kamburova et al. 2022; Hussain and Mahmood 2020). Previously, in cotton, a few stress-related genes, such as GhCIPK6, GbRLK, GhMKK1, and GhSnRK2, have been reported (Ashraf et al. 2018). Plant growth and development are affected by drought stress

dmo and bar

HPPDPfW336– 1 Pa and 2mEPSPS CYP749A16 (P450)

XtendFlex®

Isoxaflutoleresistant

Herbicide resistance Glufosinateresistance

Trifloxysulfuron sodium tolerance

GR79 EPSPS and GAT

– –

CaMV35S CaMV35S (P35S)

–

–

–

AHAS, A19

pat and bar

–

CaMV35S

–

Pseudomonas fluorescens

CaMV35S

Organism Alcaligenes eutrophus –

Promoter driven expression CaMV35S

Pseudomonas fluorescens

cp4EPSPS

Gene tfdA

Transgenic cotton 2,4-D resistance (herbicide) Glyphosateresistance Glyphosateresistance

A. tumefaciens strain GV3101 (VIGS) Agrobacterium and biolistic Agrobacteriummediated

Transformation method Agrobacteriummediated Agrobacterium (Pcambia1301) Agrobacterium (pBI 121 binary vector) Agrobacterium (PV-GHHT6997 vector) Agrobacterium

Table 4.2 List shows genetic approaches for herbicide resistance in cotton

Herbicide and weed control

Resistance against herbicide

Provides resistance to the HPPDinhibiting herbicide isoxaflutole (IFT) Acetolactate synthase (ALS) inhibiting herbicide

Dicamba-resistant

Controlling weeds

Controlling weeds

Effects Controlling weeds

Thyssen et al. (2018), Vulchi et al. (2022) Rajasekaran et al. (1996) Daud et al. (2009), Carbonari et al. (2016)

Vulchi et al. (2022)

Vulchi et al. (2022),

Liang et al. (2017a, b)

Latif et al. (2015)

References Bayley et al. (1992)

76 K. Verma et al.

Trichoderma virens

Endochitinase

Rhizoctonia solani resistant

Trichoderma virens

Endochitinase

CaMV35S

CaMV35S

CaMV35S

–

Agrobacterium-mediated

Agrobacterium-mediated

Using pathogen-derived resistance (PDR), virus-resistant plants can be produced. Disease resistance against the soilborne pathogen Rhizoctonia solani and the foliar pathogen Alternaria alternata was assessed in homozygous T2 plants of the high Endo chitinase-expressing cotton lines The transgenic plants’ biochemical and molecular investigations revealed rapid/greater induction of ROS, expression of numerous defense-related genes, activation of several PR enzymes, and activation of the terpenoid pathway.

βC1

(continued)

Kumar et al. (2009)

Emani et al. (2003)

Sohrab et al. (2016)

Rajasekaran et al. (2005)

Leaf curl virus resistant Fungal resistance

Agrobacterium-mediated

–

Fusarium verticillioides, Verticillium dahliae, and Thielaviopsis basicola Cotton leaf curl virus (CLCuV)

D4E1

References Kumar et al. (2013)

Black root rot

Effects AtNPR1-overexpressing transgenic plants exhibited stronger and faster induction of most of these defenserelated genes, particularly PR1, thaumatin, glucanase, LOX1, and chitinase Resistant to diseases and mycotoxin-causing fungal pathogens

Transgenes AtNPR1

Disease Black root rot

Transformation method Agrobacterium-mediated

Table 4.3 List shows genetic approaches for disease resistance cotton Promoter CaMV35S

Recent Advances in Genetic Improvement of Cotton

Organism Thielaviopsis basicola

4 77

CaMV35S CaMV35S

A. thaliana

CLCuV

Xanthomonas oryzae pv

CLCuV

Geminiviral

GhWRKY15

ACP,

Hpa1Xoo

βC1, AV2, NPTII AV1, ACP, NPTII

Viral and fungal resistance Virus resistance Transgenic cotton line (T-34) Disease resistance CLCuD resistance

AC1

Agrobacterium

–

Agrobacterium-mediated

Agrobacterium (vector pPZP)

–

Agrobacterium-mediated

Agrobacterium-mediated

Agrobacterium-mediated

CaMV35S

35S-GFP

CaMV35S

CaMV35S

Begomoviruses (CLCuKoV-Bur) Geminiviruses

V2

–

–

Xanthomonas citri pv. malvacearum

BSm and BDm

Bacterial blight resistance Virus resistance Virus resistance

Transformation method Agrobacterium (pCAMBIA2300 based)

Promoter CaMV35S

Organism Verticillium dahliae

Transgenes AtNPR1

Disease Fungal and nematode resistance

Table 4.3 (continued)

Disease resistance plant and viral causal agents Transgenic resistance to CLCuD

Resistance to Verticillium dahliae

Resistant against CLCuV

To increase virus resistance in cotton crops, breeding efforts for cotton can use transgenic cotton that expresses a portion of the CLCuV AC1 gene as a source of virus resistance. Resistance against disease and help in plant development

Resistance against CLCuV

Effects Verticillium dahlia isolates TS2, Fusarium oxysporum f. sp. vasinfectum, Rhizoctonia solani, and Alternaria alternate were all significantly resistant to NPR1expressing lines. Resistance against Bacterial blight disease

Rahman et al. (2017) Amudha et al. (2011)

Noman et al. (2016a, b) Miao and Wang (2012)

Yu et al. (2012)

Yasmeen et al. (2016) Hashmi et al. (2011)

Zhang et al. (2020)

References Parkhi et al. (2010)

78 K. Verma et al.

GhMPK16

Halimodendron halodendron and Populus euphratica Colletotrichum nicotiana, Alternaria alternata and P. solanacearum

GhWRKY39

HhERF2, PeDREB2a

CaMV35S

–

CkANN

Drought and wilt Disease and salt resistance

Disease and salt resistance Disease resistance and drought sensitivity

CaMV35S

Fusarium oxysporum

GhMPK7

Disease resistance

Through pollen-tube pathway

Agrobacterium strain GV3101

35SGhMPK16: GFP

A. tumefaciens strain LBA4404

RNAi-mediated resistance against Cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar Narasimha Agrobacterium-mediated

rd29A

CaMV35S

CaMV35S

Agrobacterium-mediated

Agrobacterium-mediated

CaMV35S

NaD1

Agrobacterium tumefaciens GV3101

Agrobacterium (plasmid vector pFGC5941)

CaMV35S

Fungal resistance

Fusarium oxysporum and Alternaria macrospora. Fusarium oxysporum f. sp. vasinfectum (Fov) and Verticillium dahliae –

Begomoviruses (CLCuMuV)

C4

CaMV35S

ChiII

CLCuKoV-Bu, CLCuMB

AC1, Βc1

Fungal resistance

Disease resistance to leaf curl Virus resistance

Resistance against disease and drought tolerance and wilt Increased GhWRKY39 expression may enhance plant defenses against pathogen invasion and salt stress. Transgenic cotton resistance against Vd, drought, and high-salt stresses GhMPK16 may participate in biotic and abiotic stress signaling pathways and other signal transduction pathways.

Resistance against pathogens and help in plant development

Transgenic cotton lines, resistance to CLCuMuV/CLCuMuB infection. Disease resistance against Fusarium wilt and Alternaria leaf spot NaD1 in transgenic cotton provides substantial resistance against fungal pathogen

Resistance against leaf curl

Recent Advances in Genetic Improvement of Cotton (continued)

Shi et al. (2011)

Zhang et al. (2011) Shi et al. (2014)

Shi et al. (2010)

Gaspar et al. (2014)

Ganesan et al. (2009)

Baig et al. (2021)

Ahmad et al. (2017)

4 79

Verticillium dahlias

V. dahlias, F. wilt

Verticillium dahliae

GbRLK

GAFP

PevD1

Wiltresistant Wiltresistant Wiltresistant

Verticillium dahlia

GbPMEI13, GbPG12 GhMKK6 and ghr-miR5272a

Verticillium dahlia

GhCDPK28-6

–

CaMV35S

A. tumefaciens strain GV3101

CaMV35S, pRI201AN-GUS vector CaMV35S

vector pBI35S-GAFP-BAR (pollen-tube pathway) pET30-TEV/LIC vector (E. coli BL21)

Agrobacterium

–

–

Agrobacterium (VIGS), pCAMBIA2300

Agrobacterium (VIGS)

–

Verticillium dahliae

35S promoter –

Agrobacterium tumefaciens GV3101 Agrobacterium (VIGS)

–

Verticillium dahliae

Verticillium dahlia

Agrobacterium-mediated

CaMV35S

Verticillium dahliae

Transformation method Agrobacterium-mediated

Promoter –

Organism CLCuRV

GbSOBIR1

CG02 and CG13 GhSNAT1, GhCOMT

Transgenes CLCuV genome GaRPL18

Wiltresistant Wiltresistant

Wiltresistant Wiltresistant

Disease Viral resistance Wiltresistant Wiltresistant Wiltresistant

Table 4.3 (continued)

Resistance against Verticillium wilt disease Strongly resistance to Verticillium wilt Resistance against V. dahliae

When the GhCDPK28-6 gene was silenced in cotton, ROS, lignin, and callose accumulation increased, and plant resistance increased Resistance against Verticillium wilt disease Resistance against disease

Enhance the gene expression of phenylpropanoid, mevalonate (MVA), and gossypol pathways Resistance against V. dahlia

Effects Resistance against CLCu viral disease Mechanism related to SAmediated pathway Resistance against V. dahliae

Jun et al. (2015) Wang et al. (2004) Bu et al. (2014)

Zhang et al. (2022) Wang et al. (2017a, b)

Zhou et al. 2019) Wu et al. (2021)

References Khatoon et al. (2016) Gong et al. (2017) Li et al. (2017a, b) Li et al. (2019a, b, c)

80 K. Verma et al.

Wilt resistance Wilt resistance

Wilt resistance Wilt resistance Wilt resistance

Wilt resistant Wiltresistant Wilt resistance Wilt resistance

Wiltresistant Wiltresistant Wiltresistant wilt resistant

Verticillium dahliae Kleb Verticillium dahliae

chitinase (chi)

Verticillium dahliae

Verticillium dahliae

Chi28, CRR1

GhJAZ2

GhDIR1 and GhDIR2

Phaseolus vulgaris

Verticillium dahliae Kleb Verticillium dahliae Kleb

Endochitinase

GbERF1-like

Mcchit1 (endochitinase) TMEM214

Xanthomonas oryzae pv. oryzicola (Xoc) Verticillium dahliae

CaMV35S

CaMV35S

CaMV35S

CaMV35S

Agrobacterium, vector (TRV: CRR1) Agrobacterium strain GV3101, vector(35S: GFP-GhJAZ2)

Agrobacterium

Agrobacterium plasmid (pBI121-CHI) Agrobacterium

Agrobacterium (VIGS)

Agrobacterium

– GhHCT1 and AtPAL3 promoter are used CaMV35S

Agrobacterium

CaMV35S

Agrobacterium

–

Verticillium dahliae

Gbve1

Hcm1

Verticillium dahliae

GST (chr-9)

– A. tumefaciens strain GV3101 (VIGS) Agrobacterium

CaMV35S

Verticillium dahliae

Agrobacterium (VIGS)

3CMV5S: GhGST

CaMV35S

Verticillium dahliae

GhNDR1, GhMKK2 GAFP

The JA production and signaling pathway is activated by GhbHLH171 overexpression in cotton, which also increases the

The cotton lignification process, which prevents the fungal disease V. dahliae from spreading, may be mediated by GhDIR1. Resistance against VW

Resistance against VW

GbERF1-like acts as a positive regulator in lignin synthesis and provide resistance against V. dahliae in plants Resistance against VW

Resistance against Fusarium and Verticillium wilts Resistance against Verticillium wilt disease Resistance against VW

Resistance against Verticillium wilt disease Resistance against Verticillium wilt disease Resistance against Verticillium wilt disease Resistant to Verticillium wilt

Recent Advances in Genetic Improvement of Cotton (continued)

Han et al. (2019) He et al. (2018a, b)

Tohidfar et al. (2012) Tohidfar et al. (2012) Shi et al. (2012)

Gao et al. (2011) Wang et al. (2016a, b) Li et al. (2019a, b, c) Zhang et al. (2012) Zhang et al. (2016) Xiao et al. (2007) Zhao et al. (2022) Munis et al. (2010)

4 81

Verticillium dahliae

Verticillium dahliae

Baculovirus

GLPs (GhABP19)

GbMPK3

GhLAC15

GhHB12

Tomato Ve1

Gbvdr5

p35 and op-iap

Wilt resistance

Wilt resistance

Wilt resistance

Wilt resistance

Wilt resistance Wilt resistance Wilt resistance

Verticillium dahliae

Verticillium and fusarium wilt

Verticillium and fusarium wilt

Verticillium and fusarium wilt

Verticillium dahliae

GhMYB4

Wilt resistance

Organism

Transgenes

Disease

Table 4.3 (continued)

Agrobacterium Agrobacterium

–

Agrobacterium

Agrobacterium

Agrobacterium strain GV3101

Agrobacterium (VIGS)

Agrobacterium strain GV3101

Agrobacterium

Transformation method

CaMV35S

CaMV35S

pGhHB12: GUS

CaMV35S

CaMV35S

CaMV35S

CaMV35S

Promoter

Resistance against VW

Resistance against VW

plant’s tolerance to the fungus V. dahliae. GhMYB4 downregulates lignin biosynthesis, which provides resistance against VW Activation or repression of JA-mediated signaling was caused by either GhABP19 overexpression or silencing, respectively. Silencing of GbMPK3 has a limited effect on cotton resistance to V. dahliae. GhLAC15 increases Verticillium wilt resistance by increasing defense-induced lignification and accumulating arabinose and xylose in the Gossypium hirsutum cell wall. GhHB12, a cotton stressresponsive transcription factor inhibiting JA-response genes, negatively controls cotton tolerance to V. dahliae. Resistance against VW

Effects

Song et al. (2018) Yang et al. (2015)

He et al. (2018a, b)

Zhang et al. (2019)

Long et al. (2020)

Pei et al. (2019)

Xiao et al. (2021)

References

82 K. Verma et al.

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due to changes in metabolic and biological processes. Plants respond to drought stress primarily by developing roots, closing stomata, producing hormones, and scavenging ROS (Mahmood et al. 2019). The following table lists various cotton transgenics that have been developed utilizing various abiotic resistance genes (Chakravarthy et al. 2014).

4.4

Somatic Embryogenesis and Cotton Regeneration

Somatic embryogenesis is preferred over organogenesis because regenerants are more susceptible to in vitro manipulation and likely have a single-cell origin (Kumria et al. 2003). The first regenerative plant was produced in 1986 by somatic embryogenesis using cotton somatic cell culture. In 2000, Chinese scientists grew regeneration plants from cotton tissue culture. Following gene transfer, regeneration is crucial for transgenic plants to recover (Wu et al. 2009). Gossypium hirsutum L. has developed an effective in vitro plant regeneration system characterized by the rapid and continuous synthesis of somatic embryos utilizing explants from the leaves and stems of aberrant seedlings. Embryogenic calluses and somatic embryos have been created from direct explants of defective cotton seedlings (Zhang et al. 2000a, b). In order to improve regeneration protocols, it was necessary to identify the genes involved in cotton somatic embryogenesis (Wu et al. 2009). Several genotypes and culture mediums have been examined for cotton to determine which Coker series cultivars would produce embryogenic calli in the presence of auxin. A Coker-310 pure line was established for strong regeneration potential, and the trait was then used in F1 hybrids with other recalcitrant kinds. By gradually selecting the ability for regeneration, extremely regenerable lines of the superior Acala cotton have also been created (Kumria et al. 2003). Certain Coker varieties reportedly have the most capacity for regeneration compared to other varieties. The China cultivar of Upland cotton YZ-1 demonstrated superior somatic embryogenesis ability to Coker lines with a high amount of somatic embryogenesis within 2 months. It produced more somatic embryos from 1 g of embryogenic calli (Zhang and Jin 2007). According to Sakthi et al. 2015, transgenic cotton plants produced by somatic embryogenesis and Agrobacterium-mediated transformation expressed a novel cry2AX1 gene with sequences from the cry2Aa and cry2Ac genes and driven by the CaMV35S or EnCaMV35S promoters were produced. In several plant species, AGAMOUS-LIKE15 (AGL15) encourages somatic embryogenesis. 2,4-D and kinetin strongly stimulated GhAGL15-1 and GhAGL15-3, while 2,4-D alone stimulated GhAGL15-4. The three GhAGL15s were overexpressed in cotton callus, which resulted in higher-quality cotton callus and a markedly higher rate of embryogenic callus development. The embryogenic capacity of transgenic calli is increased by GhAGL15s overexpression (Yang et al. 2014). miRNAs involved in SEG have been identified in recent years. MiR156, miR166a, miR162, miR167, miR171c, miR168, miR171a/b, miR393, miR397, and miR398 were particularly active during different stages of SEG (Siddiqui et al. 2019). Plantlets were produced from cotton cultivars

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(Gossypium hirsutum L.) by somatic embryogenesis (Nazilli and Nazilli 143; Aydin et al. 2004).

4.5

Epigenetics and Cotton Improvement

The term “epigenetics” commonly refers to a class of heritable molecular events involving a wide range of protein complexes and regulatory mechanisms that do not require changes in the DNA sequence. Changes are in gene expression patterns’ temporal, geographic, and abundance patterns due to epigenetic molecular processes. These alterations could result from morphological, physiological, and ecological effects (Rapp and Wendel 2005)—genome instability caused by the loss or gain of certain chromosomes. Recent research with plants has shown chromosome number changes brought on by whole-genome duplications (polyploidy). Creating the heteroploid genome results in rapid structural and epigenetic changes within a few generations (Matzke et al. 1999). DNA methylation influences various molecular processes in plant and animal development, such as gene imprinting, viral defense, and transposon silencing. Several mechanisms are used in plants for DNA methylation in the CG, CHG, and CHH (H = A, T, or C). METHYLTRANSFERASE1 (MET1) and CHROMOMETHYLASE3 (CMT3) maintain CG and CHG methylation in Arabidopsis. The Core Histones (H2A, H2B, H3, and H4) can undergo a variety of covalent changes, such as acetylation and methylation, at various sites of the amino-terminal tails (Song and Chen 2015). Salt stress affects cotton’s ability to respond to cytosine methylation (Gossypium hirsutum L.) and variations in its cytosine methylation pattern (Xue-Lin et al. 2009). The CRISPRCas9 gene editing technique makes many epigenetic alterations to crops to improve crop diversity and provide desired results (Khan et al. 2018). Three phenotypic marker genes, including two endogenous genes like phytoene synthase (PSY) and phytoene desaturase (PDS), were induced to have DNA methylation in their promoter regions in various cotton accessions to confirm the function of these genes (Khan et al. 2022). The transcriptional regulation of the DREB family of genes also involves epigenetic changes. Transgenic cotton with improved drought resistance was made more drought resistant by AtHUB2 overexpression, which altered H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus (Chen et al. 2019).

4.6

Genome Editing—Crisper cas9 in Cotton Improvement

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes has been successfully applied to many model plants. Three components of CRISPR-Cas9 recognize DNA cleavage sites using Watson-Crick base pairing: Cas9 protein, CRISPR-RNA (crRNA), and trans-activating crRNA (trancrRNA). Genetic editing is essential to gene functional studies and crop improvement (Gao et al. 2017). A recent breakthrough in CRISPR/ Cas9 technology that directs precise double-strand breaks in the genome using single

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guide RNA molecules (sgRNAs) has the potential to revolutionize agriculture (Gao et al. 2017). In Arabidopsis and rice, CRISPR/Cas9-based genome editing was applied for the first time in plant biology. Wheat and maize were added later. It is still early in developing CRISPR/Cas9 applications for cotton (Ashraf et al. 2018). In recent years, many studies have documented the use of this technology to provide tolerance against viral diseases caused by begomoviruses (Uniyal et al. 2019). Using genetic engineering, insect-resistant crops can be developed by modulating bacterial endotoxins, vegetative insecticidal proteins, and other plant genes such as lectins and protease inhibitors. CRISPR Cas9 and RNA interference provide new solutions for developing insect-resistant crops (Talakayala et al. 2020). CRISPR/Cas9-induced gene editing for GhCLA1 gene (Gao et al. 2017), EPSPS gene (Han and Kim 2019) MYB-15, and GhTST2 genes (Zhang and Rahman 2021) has been developed in cotton. Diverse cotton transgenics developed using different genes through gene editing are listed in Table 4.4 (Chakravarthy et al. 2014).

4.7

Male Sterility-Hybrid Seed Production Through Genetic Engineering in Cotton

Male sterility is a condition in which flowering plants produce abortive pollens or microspores. The first case of male sterility in flowering plants was reported by Koelreuter in 1763. In 1960, Justus and Leinweber reported the first evidence of male sterility in Gossypium hirsutum L. (Singh et al. 2002). Plant genetics has produced a fresh idea for male sterility and fertility induction for hybrid seeds (Singh et al. 2015; Zhang et al. 2000a, b). In cotton, male sterility is important in enhancing yield and fiber quality. The development of hybrid cotton produced by VIGS in the GhCYP450 gene has employed a complete male sterile line (ms5ms6) extensively globally (Mao et al. 2023). According to Li et al. 2021, high temperature (HT) reduces crop production and promotes male sterility. Male sterility was caused by GhMYB4 expression being increased in tetrad stage anthers because HT reduced siRNA-mediated CHH DNA methylations in the GhMYB4 promoter. The regulation of anther dehiscence and decreased pollen viability caused by glyphosate-induced male sterility in transgenic cotton (Yasuor et al. 2007; Chen et al. 2006). Cotton plants that overexpressed miR157 displayed increased susceptibility to HT stress, including microspore abortion and anther indehiscence, and repressed the auxin signal (Ding et al. 2017). As shown in Table 4.5, many cotton transgenics have been created through genetic/gene engineering for hybrid seed production (Chakravarthy et al. 2014) (Table 4.6).

4.8

Conclusions and Future Perspectives

Cotton is one of the most important sources of foreign exchange for many nations worldwide. Therefore, improving fiber yield and quality remains the primary objective. We wanted to highlight the necessity for several types of transgenic cotton in

–

GhCIPK6

Drought tolerance

Drought and Salt Stress Drought tolerance

GhPYL9-11A

GhNAC2

CaMV35S

Agrobacterium strain GV3101

Agrobacterium-mediated

Agrobacterium (VIGS)

–

CaMV35S

GhCIPK6a

35S: GhWRKY6 CaMV35S: GhNAC2

GhCIPK6a overexpression increased the expression of co-expressed genes induced by salt stress, which scavenged ROS and were engaged in MAPK signaling pathways, as shown by RNA-Seq analysis. Through the ABA signaling cascade, GhWRKY6 is a negative regulator of plants’ responses to abiotic stress. Compared to control plants, transgenic cotton that expresses GhNAC2 exhibits less leaf wilting and abscission when exposed to water stress. When exposed to drought, drought-tolerant cotton cultivars often express more GhPYL9-11A than droughtsensitive cotton cultivars do.

Agrobacterium-mediated

–

AsHSP70

GhWRKY6

Overexpression of AmDUF1517 improved cotton resistance to stress by maintaining ROS homeostasis by significantly increasing antioxidant enzyme activity and reducing reactive oxygen species (ROS) accumulation in trans-AmDUF1517 cotton. Resistance against heat stress

Tolerance to salt and osmotic stress in transgenic plants

Under salt and drought stress, GhCIPK6 is a positive regulator

Abiotic stress resistance

Resistance against abiotic stress

Effects Resistance against salt also delayed leaf senescence

Agrobacterium tumefaciens strain LBA4404

Agrobacterium, vector pCAMBIA2300-AnnBj1 Agrobacterium strain, GV3101, (vector pK2GW7.0) Agrobacterium-mediated

Agrobacterium-mediated

Transformation method Agrobacterium-mediated

CaMV35S

Heat stress tolerance Salt tolerance

CaMV35S

AnnBj1

CaMV35S

CaMV35S

SikCuZnSOD3

ABP9 (bZIP T.C.F) AmDUF1517

Promoter CaMV35S

Transgenes IPT

Abiotic stress Abiotic stress

Stress resistance Salt tolerance Abiotic stress Abiotic stress Abiotic stress

Table 4.4 List shows genetic approaches for abiotic stress resistance in cotton

Liang et al. (2017a, b)

Li et al. (2019a, b, c) Gunapati et al. (2016)

Batcho et al. (2021) Su et al. (2020)

Wang et al. (2017a, b) HAO et al. (2018)

Reference Liu et al. (2012) Zhang et al. (2021a, b) Divya et al. (2010) He et al. (2013)

86 K. Verma et al.

Abiotic stress Drought tolerance

Virus-induced gene silencing (VIGS) Agrobacterium-mediated

–

CaMV35S

GbNAC1

GhHUB2

Agrobacterium-mediated

Agrobacterium strain EHA107

Agrobacterium-mediated

CaMV35S CaMV35S

CaMV35S

Agrobacterium strain EHA106

Agrobacterium-mediated

EHA 101 and C 58

OsNAC6 or RCc3

GhCHR

Salt tolerance Drought and Salt Stress

SNAC1

AtSAP5

-

StDREB2

Drought and heat stress

CaMV35S

TaMnSOD

Drought tolerance Drought tolerance

CaMV35S

GhABF2D

Drought tolerance

The overexpression of these genes increases cotton’s resistance to drought and salt, enhances root development, and reduces transpiration. In both biotic and abiotic environments, GbNAC1 plays an important role. Through interaction with GhH2B1 directly, AtHUB2 overexpression enhanced the level of global H2B monoubiquitination (H2Bub1). -regulated droughtrelated gene expression in transgenic cotton plants

Through miRNVL5, developed salt resistance cotton plant

Endogenous ABF homologs greatly increase drought resistance in G. hirsutum, mostly through stomatal control, which has a detrimental effect on transpiration and photosynthetic output. Overexpression of these genes increases drought resistance Transgenic cotton lines show higher expression levels of antioxidant genes and stress-tolerant genes compared to wild-type plants The expression of AtSAP5 prevented photodamage to photosystem II complexes thoroughly after four days of dryness.

Wang et al. (2016a, b) Chen et al. (2019)

Gao et al. (2016) Liu et al. (2014)

Hozain et al. (2012)

Zhang et al. (2014a, b) El-Esawi and Alayafi (2019)

Kerr et al. (2018)

4 Recent Advances in Genetic Improvement of Cotton 87

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Table 4.5 List shows genome editing approaches for cotton improvement Cotton plants for Herbicide resistance

Target gene EPSPS, ALS, CESA3, SF3B1

Genome editing method CRISPR/Cas9

effects Generating herbicide-resistant plants by loss-offunction mutations Mutation produces STEMEs, W2125S herbicide resistance Resistance to abiotic and biotic stress

References Han and Kim (2019)

Herbicide resistance

ALS, ACCase, EPSPS

CRISPR/Cas9

Insect resistance & Herbicide Transgenic cotton Disease resistance

MYB-15, GhTST2

CRISPR/Cas9

GhALARP

CRISPR/Cas9

Fiber development

CLCuV genome

CRISPR/Cas9

Virus resistance in crops

Disease resistance Disease resistance

MIR482 family

CRISPR/Cas9 CRISPR/Cas9

Transgenic cotton

CLCuD-associated begomoviruses (CABs), TYLCV GhMYB-25 A DNA, GhMYB

Virus resistance in crops Resistance against CABs virus disease

Zhu et al. (2018) Mubarik et al. (2021), Khatodia et al. (2017) Zhu et al. (2022) Iqbal et al. (2016)

Verticillium wiltresistant cotton

Li et al. (2017a, b)

Transgenic cotton

GhPDS, GhCLA1, GhEF1

Gao et al. (2017)

Disease resistance

CLCuD CLCuV

Transgenic lines containing mutations in the target site resulting in an albino phenotype due to interference with chloroplast biogenesis. Tolerance against CLCuD, CLCuV

Stress resistance

cry1Ac + cry2A + GTG

Shahzad et al. (2022)

Disease resistance

Rep, βC1

Tolerance against bollworms, cotton leaf curl virus, heat, drought, and salt. Resistance against CLCuV

CRISPR/Cas9, Agrobacterium (strain EHA105) CRISPR/Cas9, Agrobacterium tumefaciens (GV3101 and LBA4404)

pYLCRISPR/ Cas9, Agrobacterium CRISPR/Cas9, Agrobacterium

CRISPR/Cas9, Agrobacterium gv 3101

Dong et al. (2021) Zhang and Rahman (2021)

Uniyal et al. (2019)

khan et al. (2020) (continued)

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Table 4.5 (continued) Cotton plants for Hybrid

Doublerecessive genic male sterility Viral disease resistance

Target gene GbFLA19-D, GhFLA19-A

Genome editing method CRISPR/Cas9, Agrobacterium (pRGEB32GhU6.7-NPT II expression vector)

CYP703A2-A, CYP703A2-D

CRISPR/Cas9

CLCuV

CRISPR/Cas9 vector (pHSE401/ pKSE401)

effects Genetic male sterile line photosensitive. Gossypium hirsutum and Gossypium barbadense were the parents of the hybrid plant known as CCRI9106, which was likely produced by HB. Uncovered functions in sporopollenin formation and fertility Resistance against CLCuD

References Zhang et al. (2021a, b)

Ma et al. (2022)

Binyameen et al. (2021)

our review. As a result of the use of a variety of gene sources, various forms of transgenic cotton have been developed that improve cotton quality and are resistant to both biotic and abiotic stress. Improvements to fiber length, strength, and uniformity using a variety of potential genes driven by promoters particular to the fiber would increase cotton’s productivity. Functional genomics of cotton will be advanced through the development of high-throughput technologies and the use of a wide range of methodologies, including genomics, transcriptomics, proteomics, epigenomics, and bioinformatics. When more crop species that express various cry genes are produced, the utilization of Bt crops will probably rise. However, some important pest species are still resistant to the Bt toxins that are now in use, and testing for active Bt proteins is ongoing. Further insecticidal proteins may be added to Bt toxins in the future, such as VIPs (vegetative insecticidal proteins of Bacillus), lectins, protease inhibitors, amylase inhibitors, chitinases, and cholesterol oxidases. Several genome editing technologies, such as CRISPR-Cas9, can be used to precisely change the genetic makeup of crops such as cotton efficiently and precisely. The cotton plant is one of the world’s most valuable cash crops, and genetic improvements are being conducted through the use of genome editing to improve yield, fiber quality, and stress tolerance while reducing the use of chemicals. In terms of cotton genome editing, there are a number of promising applications, including fiber quality improvement, herbicide tolerance, disease resistance, stress tolerance, generation of approaches for hybrid seed development, etc. Overall, genetic improvement of cotton using gene/genome engineering has tremendous potential to enhance crop yield, quality, and sustainability. Despite this, some regulatory,

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Table 4.6 Genetic engineering approaches in male sterility in Cotton Transgenic cotton Male sterility (CMS)

RF1 and QF1 hybrids (CMS)

Transgenes GhCYP450 gene encoding a cytochrome P450 (ms5ms6), Bt GST (CMS)

Transformation method virus-induced gene silencing (VIGS)

–

CMS line J4A

MAPKK6, AGL19, SNF1, WRKY28

-

Suvin (CMS)

CMS

-

Fuzzless lintless line hybrid (GMS)

GMS

-

Male sterile CMS-D2

PPR like gene

–

Male sterility hybrid

MNX GUS reporter gene

Agrobacteriummediated

Conclusion Enhance cotton fiber quality of hybrid

Reference Mao et al. (2023)

DES-HAF277 (a normal restorer) and Zheda strong restorer (transgenic restorer with GST gene) were crossed to develop the two cotton hybrids RF1 and QF1, which maintain the equilibrium between oxidative stress and scavenging enzymes The ghi-MIR7484-10/ MAPKK6 network and reduced glucose metabolism were suggested, and ghi-MIR7484-10/ MAPKK6 may be related to abnormal microspore meiosis and induction of excessive sucrose accumulation in anthers. Suvin restores were found to combine both high yield and superior fiber quality. The genetically male sterile line through backcrossing to develop a female parent for use in hybrid breeding programs. For molecular markerassisted selection (MAS) of restorer lines, a novel PPR-based candidate gene CAPS marker (CAPSR) co-segregating with Rf1 was developed. The male sterile plants typically have shorter internodes, smaller anthers, abortive pollen grains, unique leaves with deeper multi-lobes, and lower plant heights.

Bibi et al. (2014)

Li et al. (2021)

Manickam et al. (2010) Dagaonkar et al. (2007)

Wu et al. (2014)

Gan et al. (2010)

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ethical, and social issues still need to be addressed before it can be widely adopted in cotton. Ultimately, gene/genome engineering offers an exciting opportunity to improve cotton production. With continued research and development in this field, new cotton varieties will likely be developed with improved traits that benefit farmers and consumers in the future. Acknowledgments We are grateful to the Chhatrapati Shahu Ji Maharaj University, Kanpur, for the C.V. Raman Minor Research Project Grant (CSJMU/R&D/CVR/19/2022). KV is thankful to University Grant Commission (UGC) for the Junior Research Fellowship (JRF).

References Ahmad A, Zia-Ur-Rehman M, Hameed U, Qayyum Rao A, Ahad A, Yasmeen A, Akram F, Bajwa KS, Scheffler J, Nasir IA, Shahid AA, Iqbal MJ, Husnain T, Haider MS, Brown JK (2017) Engineered disease resistance in cotton using RNA interference to knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan beta satellite expression. Viruses 9(9):257 Ali A, Ahmed S, Nasir IA, Rao AQ, Ahmad S, Husnain T (2016) Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac+ Cry2A, GT gene and insect mortality. Biotechnol Rep 9:66–73 Amudha J, Balasubramani G, Malathi VG, Monga D, Kranthi KR (2011) Cotton leaf curl virus resistance transgenics with antisense coat protein gene (AV1). Curr Sci 101:300–307 Ashraf J, Zuo D, Wang Q, Malik W, Zhang Y, Abid MA, Cheng H, Yang Q, Song G (2018) Recent insights into cotton functional genomics: progress and future perspectives. Plant Biotechnol J 16(3):699–713 Aydin Y, Ipekci Z, Talas-Oğraş T, Zehir A, Bajrovic K, Gozukirmizi N (2004) High frequency somatic embryogenesis in cotton. Biol Plant 48:491–495 Baig MS, Akhtar S, Khan JA (2021) Engineering tolerance to CLCuD in transgenic Gossypium hirsutum cv. HS6 expressing Cotton leaf curl Multan virus-C4 intron hairpin. Sci Rep. 11(1): 14172 Bakhsh A, Shahzad K, Husnain T (2011) Variation in the spatio-temporal expression of insecticidal genes in cotton. Czech J Genet Plant Breed 47(1):1–9 Batcho AA, Sarwar MB, Rashid B, Hassan S, Husnain T (2021) Heat shock protein gene identified from Agave sisalana (As HSP70) confers heat stress tolerance in transgenic cotton (Gossypium hirsutum). Theor Exp Plant Physiol 33:141–156 Bayley C, Trolinder N, Ray C, Morgan M, Quesenberry JE, Ow DW (1992) Engineering 2, 4-D resistance into cotton. Theor Appl Genet. https://doi.org/10.1007/BF00226910 Bibi N, Yuan S, Zhu Y, Wang X (2014) Improvements of fertility restoration in cytoplasmic male sterile cotton by enhanced expression of glutathione S-transferase (GST) gene. J Plant Growth Regul 33:420–429 Binyameen B, Khan Z, Khan SH, Ahmad A, Munawar N, Mubarik MS, Riaz H, Ali Z, Khan AA, Qusmani AT, Abd-Elsalam KA, Qari SH (2021) Using multiplexed CRISPR/Cas9 for suppression of cotton leaf curl virus. Int J Mol Sci 22(22):12543 Bu B, Qiu D, Zeng H, Guo L, Yuan J, Yang X (2014) A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton. Plant Cell Rep 33:461–470 Butt SJ, Awan MF (2016) Transgenic cotton: harboring board term resistance against insect and weeds through Incorporation of CEMB double Bt and cp4EPSPS genes. Pak J Agric Sci. 53(3): 501–505 Carbonari CA, Latorre DO, Gomes GL, Velini ED, Owens DK, Pan Z, Dayan FE (2016) Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® cotton. Planta 243:925–933

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Zhang Y, Wang Q, Zhang X, Liu X, Wang P, Hou Y (2011) Cloning and characterization of an annexin gene from Cynanchum komarovii that enhances tolerance to drought and Fusarium oxysporum in transgenic cotton. J Plant Biol 54:303–313 Zhang B, Yang Y, Chen T, Yu W, Liu T, Li H, Fan X, Ren Y, Shen D, Liu L, Dou D, Chang Y (2012) Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae. PLoS One 7(12):e51091 Zhang DY, Yang HL, Li XS, Li HY, Wang YC (2014a) Overexpression of Tamarix albiflorum TaMnSOD increases drought tolerance in transgenic cotton. Mol Breed 34:1–11 Zhang YJ, Xie M, Peng DL (2014b) Effects of the transgenic CrylAc and CpTI insect-resistant cotton SGK321 on rhizosphere soil microorganism populations in northern China. Plant Soil Environ 60(6):285–289 Zhang Z, Zhao J, Ding L, Zou L, Li Y, Chen G, Zhang T (2016) Constitutive expression of a novel antimicrobial protein, Hcm1, confers resistance to both Verticillium and Fusarium wilts in cotton. Sci Rep 6(1):20773 Zhang Y, Wu L, Wang X, Chen B, Zhao J, Cui J, Li Z, Yang J, Wu L, Wu J, Zhang G, Ma Z (2019) The cotton laccase gene GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and lignin components in the cell walls of plants. Mol Plant Pathol 20(3):309–322 Zhang J, Bourland F, Wheeler T, Wallace T (2020) Bacterial blight resistance in cotton: genetic basis and molecular mapping. Euphytica 216:1–19 Zhang L, Tian W, Huang G, Liu B, Wang A, Zhu J, Guo X (2021a) The SikCuZnSOD3 gene improves abiotic stress resistance in transgenic cotton. Mol Breed 41:1–17 Zhang M, Wei H, Liu J, Bian Y, Ma Q, Mao G, Wang H, Wu A, Zhang J, Chen P, Ma L, Fu X, Yu S (2021b) Non-functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.). Plant J 107(4):1198–1212 Zhang L, Liu J, Cheng J, Sun Q, Zhang Y, Liu J, Li H, Zhang Z, Wang P, Cai C, Chu Z, Zhang X, Yuan Y, Shi Y, Cai Y (2022) lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt. Plant Physiol 189(1):264–284 Zhao J, Xu J, Wang Y, Liu J, Dong C, Zhao L, Ai N, Xu Z, Guo Q, Feng G, Xu P, Cheng J, Wang X, Wang J, Xiao S (2022) Membrane localized GbTMEM214s participate in modulating cotton resistance to verticillium Wilt. Plan Theory 11(18):2342 Zhou Y, Sun L, Wassan GM, He X, Shaban M, Zhang L, Zhu L, Zhang X (2019) Gb SOBIR 1 confers Verticillium wilt resistance by phosphorylating the transcriptional factor Gbb HLH 171 in Gossypium barbadense. Plant Biotechnol J 17(1):152–163 Zhu S, Yu X, Li Y, Sun Y, Zhu Q, Sun J (2018) Highly efficient targeted gene editing in upland cotton using the CRISPR/Cas9 system. Int J Mol Sci 19(10):3000 Zhu QH, Jin S, Yuan Y, Liu Q, Zhang X, Wilson I (2022) CRISPR/Cas9-mediated saturated mutagenesis of the cotton MIR482 family for dissecting the functionality of individual members in disease response. Plant Direct 6(6):e410

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Insights into Genetic Improvement of Cassava Joel Jose-Santhi and Rajesh Kumar Singh

5.1

Origin, Domestication, and History of Cultivation

Manihot esculenta, commonly known as cassava, yuca or manioc, is a major root crop believed to be originated from the neo-tropical realm (Olsen and Schaal 2001). The starchy roots produced by the plant are a major staple food for millions of people living in developing countries of sub-Saharan Africa, Southeast Asia, and South America (Fig. 5.1). The plant is a perennial woody shrub species that is usually grown as an annual crop in South & Central America. Spanish and Portuguese conquests later introduced Cassava to Sub-Saharan Africa and Southeast Asia, eventually becoming an important food crop for countries in these regions (Howeler et al. 2013). The roots of cassava have elevated levels of starch, and the energy yield per hectare is higher than that of cereals, making it an excellent source of energy (Latham 1997). The cassava plants can be easily established from stem cuttings, grown in marginal soil, require relatively little attention, and are well adapted to tropical climates. Depending on the variety and area of cultivation, cassava contains cyanogenic, a potential source of hydrogen cyanide poisoning. Roots, once harvested, are peeled, soaked in water, and boiled to reduce and release the volatile cyanogen gas. Apart from roots, leaves are also used as food (Latif and Müller 2015). Starch from cassava can also be used in several industries such as food, pharmaceuticals, textiles, feedstock, and to produce biofuel. Due to its restricted distribution in developing countries, less research and development have been conducted on cassava than on potatoes. But the importance of cassava in agriculture has changed dramatically over the years. Sub-Saharan Africa alone contributes to J. Jose-Santhi · R. K. Singh (✉) Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_5

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Fig. 5.1 Production quantities of Cassava, fresh by country average 1994–2021 in Tonnes (FAO 2023)

Fig. 5.2 Shows the Production share of Cassava, fresh by region, Average 1994–2021 (FAO 2023)

57% of global production and is mainly done by small-scale low-income farmers (Fig. 5.2). The region has seen an increase in cultivated area and yield in recent years but less than Southeast Asia. Nigeria is the largest producer of cassava tubers in the world and in Africa, followed by the Democratic Republic of Congo, Ghana, Angola, and the United Republic of Tanzania in terms of production and area of cultivation (Fig. 5.3). Asia accounts of about 29.5% of global production (Fig. 5.2) and cassava production increased from 45.9 million tonnes from 1980 to 84.2 million tonnes in 2021 even though the harvested area did not increase. Cassava growers in Asia are mainly small-scale crop holders and the crop is grown as reserve in case of shortfall of rice and as farm feed. Thailand is the highest producer of cassava in the region and exports cassava as dried pellets for livestock feed to Europe. China is the major consumer in the region with an import of dried cassava

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Fig. 5.3 Harvested area and Production in Tonnes of top ten cassava producers in 2021 (FAO 2023)

(Kaplinsky et al. 2011). Other major producers are Viet-Nam, India, and Indonesia. Cassava has been introduced as feedstock, and for biofuel in Asia, one tonne of dried cassava chips yields about 300 L of 96% pure ethanol (Aye 2012 CIAT). South America, Caribbean, and Oceania contribute about 13.5% of cassava production. Cassava in South America is generally grown in marginal soil and arid regions. The production in the region is dominated by Brazil followed by Paraguay, Colombia, and Peru. Cassava as a food crop has declined recently due to migration of rural to urban areas. This has been recognized by the Brazilian government through policies by mandatory blending of cassava flour with wheat to reduce the reliance of imported cereals. Despite important crop, yield improvement has received less attention (El-Sharkawy 2004). This is evident by the fact that average cassava yield per unit area did not significantly increase in Nigeria till present date. At the same time, maize yield increased by 129%, similar to 174% yield increase achieved the America (De Souza et al. 2017). Nigeria is the world’s largest producer of cassava (FAOSTAT 2019). Still, in terms of yield in tons per hectare, Nigeria’s production is less than 80% of the world average and threefold less than Laos, the highest producer per hectare (FAOSTAT 2017). Even though the area harvested doubled in Nigeria since 2007, storage root yield per hectare continued to fall (FAOSTAT 2019; (Otekunrin and Sawicka 2019). The gap between yield per harvested area and harvested area is huge among countries in sub-Saharan Africa. At the same time, South East Asian countries performed better.

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Botany and Physiology

Cassava belongs to the dicotyledon family Euphorbiaceae. Many species have been reported in the genus Manihot, and only Manihot esculenta Crantz is cultivated commercially. In plants propagated by seeds, primary taproot develops, and storage root is formed from adventitious roots originated from taproot. Roots are generally fibrous and adventitious in plants raised from stem cuttings. Only few fibrous roots develop into tuber (Carluccio et al. 2022) and tubers cannot be used for vegetative propagation due to the lack of buds which is common in sweet potato tubers (Alves 2001). Cassava storage root consists of different tissues like bark (periderm), peel (cortex), and parenchyma. The edible portion of the fresh root is parenchyma, contributing to 85% of the total weight, and xylem vessels are radially arranged in starch-containing parenchyma cell matrix (Wheatley and Chuzel 1993, Zierer, Rüscher et al. 2021). Stems are woody in nature cylindrical and have alternative nodes and internodes. The branching is mainly sympodial in nature; the main stem cutting grows and produces successive branches and flowers are produced in reproductive branches. Variations in branching patterns can be seen in different cultivars concerning climatic conditions. The leaves are simple and lobed with palmate veins; unlobed leaves are usually seen near inflorescence. Cassava plants are monoecious male and female flowers are arranged in upper and lower part of the inflorescence respectively. Cassava plants are cross-pollinated and fruit are trilocular capsule, ovoid, or globular. Each locule in the capsule contains a single seed. Cassava is a perineal plant which has an alternating period of vegetative growth, carbohydrate storage, and dormancy due to low temperature and water deficit. The storage root development initiates after 75 to 180 days of planting (Pereira et al. 1978).

5.3

Genetic Improvement of Cassava by Biotechnology Approaches

As the population increases in the coming decade, food demand can become greater than available supply. The need to identify and improve crops which are climateresilient and high yielding per area is very crucial. Cassava has a higher potential to become staple crop worldwide that can provide food security, withstand climate warming, and reduce hunger in developing nations. Unfortunately, the crop has to be improved in multiple areas and has some limitations like (1) low protein and nutrients, (2) antinutritional/toxins like cyanogenic glycosides, (3) pest and disease, (4) low yield, (5) postharvest crop deterioration, (6) abiotic stress. These limitations are targeted to be improved by biotechnology approaches. Due to the availability of whole-genome sequence (Prochnik et al. 2012) and standardized protocol for transgenic plant production, Zainuddin et al. (2012) make it easier for relevant problems to be addressed efficiently.

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5.3.1

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Enhancing Yield by Understanding Cassava Source–Sink Interactions

Carbohydrates are synthesized through the process of photosynthesis by plant leaf, generally called Source organs, and are partitioned to tissues that require carbohydrates in order to maintain ideal growth and metabolism or for storage in sink organs. Source-sink relations in plants are one of the major factors determining plant yield, as they govern the what percentage of resources are to be mobilized to edible plant parts, e.g., grains, cobs, fruits, or underground storage organs like stem/ root tubers, corms, bulbs, and taproots (Yu et al. 2015; Rodrigues et al. 2019). The foundation for improvement in yield and increased biomass allocation in staple grain crops such as wheat and rice over last decades can be credited to understanding that crop yield can be co-limited by both source and sink are often termed “source” and “sink” limitation (Sonnewald and Fernie 2018; Rosado-Souza et al. 2023). The two strategies to facilitate the increasing cassava yield are (1) improving photosynthetic carbon assimilation rate in source organs and (2) improving transport of assimilates and utilization for simultaneous growth and storage in sink tissues.

5.3.1.1 Enhanced Photosynthetic Carbon Assimilation in Leaves Early research on different cultivars advocates that cassava as a C3-C4 intermediate (El-Sharkawy and Cock 1987; El-Sharkawy 2016). However, a subsequent detailed analysis showed that cassava is rather a C3 crop (Edwards et al. 1990; Angelov et al. 1993; Arrivault et al. 2019). Even though cassava is reported to have higher photosynthetic rates and low rates of photorespiration, data from several studies shows the net photosynthetic rate average is considerably lesser than the highest rates reported, raising doubt to the view that cassava has a higher rates photosynthesis for a C3 species (De Souza et al. 2017). One of commonly explored strategies for improving photosynthesis and yield has been demonstrated in tobacco by the increased expression of sedoheptulose-1,7bisphosphatase (SBPase) and fructose-1,6-bisphosphate aldolase (FBP aldolase) enzymes within the Calvin cycle (Lefebvre et al. 2005; Zhu et al. 2007; Raines 2010; Uematsu et al. 2012; Simkin et al. 2015). Rubisco content in cassava was 1.6/ g2 which is low compared to rice and wheat, and in vivo rubisco activity and mesophyll conductance were reported to be accounted for 84% of the limitation for photosynthesis (De Souza et al. 2020). Improving RubP regeneration capacity and Rubisco amount also shows potential to withstand an increased atmospheric CO2 and elevated temperature identifying varia of Rubisco, SBPase, and FBP aldolase between cassava cultivars and these genes can be manipulated in cassava for improving cassava photosynthesis, given the high-temperature conditions of the tropics and increase in temperature due to upcoming climate change (GALMÉS et al. 2014; Carmo-Silva et al. 2015). Diverting chloroplastic glycolate from photorespiration can be a good strategy to improve the productivity of cassava, as synthetic photorespiratory bypass systems have been shown promising in decreasing CO2 loss, and increase photosynthesis in Arabidopsis thaliana (Kebeish et al. 2007). Another way of improving

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Fig. 5.4 Figure shows strategies for achieving improved yield by ideal source-sink interaction. In source organs, photosynthetic efficiency can be achieved by RuBp Regeneration, Rapid NPQ Relaxation , increase expression of sedoheptulose-1,7-bisphosphatase (SBPase) and fructose-1,6bisphosphate aldolase (FBP aldolase) enzymes within the Calvin cycle and by Synthetic photorespiratory pathway by upregulation of glycolate dehydrogenase (GDH), glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR). In sink organs, active growth can be maintained by vascular cambium associated genes and starch synthesis by SUS (Sucrose Synthase) and ADP-Glc pyrophosphorylase (AGPase)

photosynthesis and yield is through manipulating non-photochemical quenching (NPQ) mechanism to relax rapidly. In high light intensities, synthesis of pH gradient in trans-thylakoid region and de-epoxidation of the xanthophyll violaxanthin to zeaxanthin is known to dissipate excess excitation energy as heat; this photoprotective mechanism is termed non-photochemical quenching (NPQ). It prevents reactive oxygen species (ROS) generation and thereby protects photosynthetic apparatus from damage. However, on gradual transfer to shade conditions it takes substantial time for these processes to stop or relax. Therefore, even though plants are experiencing low-light conditions, huge proportion of the absorbed light energy keeps on dissipated as heat rather than being used for photosynthesis. Study using reverse ray-tracing algorithm predicted loss of 12.8% and 30% of total potential carbon gain due to this delay in recovery from the photoprotected state (Zhu et al. 2004). Accelerated recovery from photoprotection or rapid NPQ relaxation has been proven to improving photosynthesis and crop productivity in Nicotiana (Tobacco) (Kromdijk et al. 2016) and has been revalidated in soybean with an average yield increase of 24.5% compared with wild-type plants and 11 to 23% faster NPQ relaxation during the sun-to-shade transitions (De Souza et al. 2022). Engineering traits in cassava for faster relaxation of NPQ like, overexpression of AtVDE, AtPsbS, and AtZEP (VPZ Cassette) genes or simply overexpressing ZEP

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gene alone since these genes are responsible for NPQ relaxation (Fig. 5.4) (De Souza et al. 2017; Ghosh et al. 2023; Kromdijk and Long 2016).

5.3.1.2 Improved Transport of Assimilates and Storage in Sink Tissues Increasing photosynthetic rate in cassava alone cannot contribute to yield, since limited sink capacity can limit any photosynthetic enhancements due to negative feedback. This negative feedback loop can only be nullified by increased sink demand and transport capacity. Strategies to increase sugar transport from source (phloem loading) to sink (phloem unloading) and reduced carbohydrate accumulation in leaves may include elevated expression of sucrose transporters like SUC/SUT (sucrose–proton cotransporters) and SWEET (SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER) (Ainsworth and Bush 2010; Chen et al. 2015). The process of sugar partition from source to sink can be explained by following steps: (i) carbohydrates loading from mesophyll cells into companion cells, (ii) longdistance transport through the phloem sieve elements to sink tissues, (iii) unloading of the assimilates into sink cells, and (iv) post-phloem transport and starch synthesis in storage cells. Among two types of adventitious roots with different cellular origin, only some roots develop into storage root, what determine this differentiation and the signals that received for secondary growth of tuberous roots is still elusive (Chaweewan and Taylor 2015). In Cassava, sucrose produced during photosynthesis is mobilized from the mesophyll into the apoplast. From apoplast SUC/SUT-family, transports carry sucrose into the phloem companion cells. Sucrose then diffuses through plasmodesmata into the sieve elements for transport to sink organs. Sucrose is mobilized into the phloem parenchyma cells via companion cells and then diffuses into phloem and different parts of the tuberous root; however, most of sucrose moves from the phloem towards xylem parenchyma through vascular ray cells and is stored as starch. The fusiform cambial initials are isolated from the tuberous root symplast and most likely fed with sugars through the cell wall invertases (CWINV) activity and monosaccharide transport proteins. This is in contrast with cambial ray initials, which are connected to phloem and xylem via symplast (Mehdi et al. 2019; Zierer et al. 2021). Bacterial AGPase (glgC) with reduced feedback inhibition by fructose1,6-bisphosphate under the control of a Class I patatin promoter was overexpressed in cassava (Fig. 5.4). The transgenic plants had up to 70% higher AGPase activity, 2.6-fold increase in total Starchy root biomass as well as, plants with the high tuberous root AGPase activity had considerable increases in canopy biomass, probably due to reduction in feedback inhibition on photosynthesis. According to recent research on the morphological and transcriptional changes that take place during the early stages of storage root development, auxin-related transcripts significantly increase, gibberellin-related transcripts significantly decrease, active cell wall biosynthesis and secondary growth factors showed significantly activate transcription. KNAT1, PENNYWISE, and POUND-FOOLISH, three KNOX/BEL genes linked with xylem parenchyma, showed elevated expression concurrently with activation of starch storage metabolism. The activation of starch storage in cassava occurred only after the development of the cambium revealing the importance of cambium activation and associated signal, which can be utilized for storage root

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improvement in cassava (Rüscher et al. 2021). Cassava storage root yield can be increased by creating plants with excellent source-sink relationships through genetic manipulation of photosynthesis, starch and sugar metabolism, phloem transport, cambial activity, and sink activity.

5.3.2

Reduction of Cyanogenic Glycosides

One of the main concerns with cassava is its high levels of cyanogenic glycosides, which can release cyanide when the root is damaged or eaten raw. In Cassava plant, leaves and roots may accumulate between 200 & 1300 mg CN equivalents/kg dry weight and over 100 mg HCN/kg fresh peeled tuber is considered dangerously poisonous (Balagopalan 2001; Siritunga and Sayre 2004). The primary cyanogenic glycoside in cassava is linamarin, which is mostly kept in vacuoles while its degrading enzyme like β-glucosidase and linamarase are confined to the cell wall and laticifers. To produce acetone cyanohydrin, linamarin is hydrolysed by—glucosidase, linamarase, in response to tissue disruption or herbivory-induced damage. Acetone cyanohydrin can spontaneously dissociate to generate cyanide and acetone at ideal pH and temperature or can be hydrolysed by the enzyme hydroxynitrile lyase (HNL), which is present only in cassava leaves and stems and absent in roots. High levels of toxic and antinutritional compound laminarin in cassava starchy roots require processing before consumption due to its potential to be lethal if not removed to safe levels. Chronic to even moderate level exposure of linamarin in cassavabased diets may result in hyperthyroidism, neurological disorder like tropical neuropathy (Osuntokun 1980; Rosling 1994), permanent paralysis of the leg, and a disease known as konzo (Nzwalo and Cliff 2011). Sulphur amino acid concentrations in blood are low in those with these illnesses because the available sulphur is primarily utilized in cyanide detoxification (Adamolekun 2010). Early biochemical and later molecular analyses revealed that linamarin (>95%) is the predominant CG in cassava, with Lotaustralin (methyl linamarin) accounting for the remainder (McMahon et al. 1995; Balagopalan 2001). Both being synthesized in leaf with utilizing valine and isoleucine and are transported via stem to roots and stored in vacuole or provide reduced nitrogen for amino acid and protein synthesis (Koch et al. 1992; Siritunga and Sayre 2003; Jørgensen et al. 2005; Narayanan et al. 2011). Linamarin biosynthesis starts with cytochrome P450 monooxygenases (CYP79D1/ D2) sequentially hydroxylating and dehydrating valine or isoleucine to produce N-hydroxyamino acids, N,N-dihydroxyamino acids, and finally an E/Z oxime. The E,Z oxime is then dehydrated and C-hydroxylated by CYP71E11(cytochrome P450 monooxygenase) to produce acetone cyanohydrin. A UDP-glycosyltransferase then glycosylates the cyanohydrin to create linamarin (Fig. 5.5). This pathway shows high degree of similarity in the biochemical pathways for dhurrin a cyanogenic glycoside seen in sorghum, indicating plants of both the monocot and dicot clades maintain similar CG biosynthesis. (Andersen et al. 2000; Jørgensen et al. 2010; McMahon et al. 2021). The reduction of cyanogenic glycosides in cassava is an active area of research, with the goal of making it a safer food source for human

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Fig. 5.5 Figure shows linamarin biosynthetic pathway, protein assimilation pathway, and cyanide detoxification pathway

consumption. Techniques include breeding for low-cyanide varieties (Iglesias et al. 2002), using post-harvest processing methods to reduce cyanide levels, and genetic engineering becoming a promising tool to reduce or eliminate the production of cyanogenic glycosides. CYP79D1/D2 gene were targeted for RNAi-mediated suppression in leaves, the initial step of the biosynthetic pathway. However, the transgenic plants with lower linamarin did not survive in the soil without ammonia to replace lost nitrogen which has been provided by linamarin (Andersen et al. 2000; Siritunga and Sayre 2003; Jørgensen et al. 2005; McMahon et al. 1995, 2021). Another approach was to develop cyanogenic glycoside-free roots by metabolic engineering to reduce linamarin sink strength and to divert towards cyanogen assimilation by overexpressing CG catabolic enzymes like hydroxynitrile lyase (HNL), β-cyanoalanine synthase (CAS), and nitrilase (NIT). Root-specific overexpression of Arabidopsis AtCAS and AtNIT4 with class I patatin promoters resulted in 50% increase in root total amino acids and 9% increase in root protein accumulation but this came with a trade-off between overall growth and root development. The activity of AtCAS was correlated to reduced growth and root development (Zidenga et al. 2017). Overexpression of hydroxynitrile lyase led to twofold increase in root total free amino acids in transgenic plants (Narayanan et al. 2011). Manipulation of linamarin transporters and reduction of sink strength in vacuole can reduce CG levels in cassava roots. NPF (nitrate transporter/peptide transporter) proteins, a cyanogenic glycoside transporter in cassava encoded by MeCGTR1, can be used as a target for CRISPR/Cas gene editing and tissue-specific expression and localization studies will give more insight into role of NPF

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transporter in CG transport in Cassava (Jørgensen et al. 2017). Genome-wide association (GWAS) studies between cultivars with low and high CG showed control of vacuolar linamarin is due to differential expression of tonoplast MATE (multidrug and toxic compound extrusion) transporter. An ATP-dependent plasma membrane proton pump might be responsible for linamarin transport to vacuole which is similar to SbMATE2 in Sorghum bicolor involved in transport of CG dhurrin (Darbani et al. 2016; Ogbonna et al. 2021). Reduction of vacuolar sink strength by downregulation of MATE transporter will be promising for reduction of CG and diverting it into amino acid and protein synthesis (McMahon et al. 2021). There are currently no commercially available non-cyanogenic genetically modified cassava varieties on the market. Traditional breeding could not produce cyanogenfree roots probably due to the crucial function that linamarin plays in protein synthesis, nitrogen metabolism, and cyanide toxicity against herbivory. To develop non-cyanogenic cassava storage roots, genetic engineering that simultaneously decreases linamarin storage and diverts linamarin towards improving protein synthesis may be a more effective option.

5.3.3

Reduction in Post-Harvest Physiological Deterioration (PPD) of Roots

One of the major challenges in large-scale cassava cultivation is low shelf life of cassava roots due to post-harvest physiological deterioration (PPD). Major cause of PPD is by mechanical damage occurring while harvesting and leads to the storage roots becoming inedible within 2–3 days. PPD causes black streaking of the xylem vascular tissues, unpleasant odour, colour, and flavour (Iyer et al. 2010). PPD is also characterized by increase accumulation of secondary metabolites, like scopoletin and its oxidation yields a black colour (Uarrota and Maraschin 2015; Liu et al. 2017). PPD symptoms can be aggravated by environmental factors such as temperature, humidity, and oxygen. Modifying these environment conditions like storing at 10 °C and 80% relative humidity, waxing, and careful reducing of physical damage can delay PPD (Rickard 1985; Wenham 1995). Several studies pointed out increased ROS accumulation as one of the initial events in PPD in cassava. Oxidative burst generally occurs within minutes of harvest, and increased activity of ROS-modulating enzymes was reported. (Buschmann et al. 2000; Reilly et al. 2004; Sánchez et al. 2006; Iyer et al. 2010). Later, the events triggering oxidative burst were found to be cyanide released at the time of mechanical damage. To elevate this problem, alternative oxidase (AOX), a cyanide-resistant oxidase seen in plants, was overexpressed in cassava storage roots which subsequently reduced the ROS accumulation and delayed PPD by 21 days. AOX provides an alternative path in the electron transport chain (ETS) which is insensitive to cyanide thereby reducing oxygen, and prevents over-reduced complexes I and III being oxidized, which generates ROS following cyanide-mediated inhibition of cytochrome c oxidase. Low levels of cyanogen in roots had reduced levels of ROS buildup while supplementation of cyanide to low cyanogen root varieties confirmed a relation between

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cyanogenesis in cassava and ROS production. However, delayed PPD lead to reduced biomass in transgenic compared with controls; this trade-off in biomass might be caused by heterologous expression of AtAOX affecting mitochondrial activity during storage root development (Maxwell et al. 1999; Zidenga et al. 2012). More evidence that support the role of ROS-mediated oxidative stress can be seen in cassava varieties that showing increased levels of beta-carotene, a scavenger of ROS, and are less vulnerable to PPD, beta-carotene accumulating cassava storage roots displayed delayed onset of PPD. (Sánchez et al. 2006; Morante et al. 2010; Beyene et al. 2018). Overexpression of ROS scavenging enzymes such as MeCu/ZnSOD (copper/zinc superoxide dismutase) and MeCAT1 (catalyse) has also led to the increased post-harvest shelf life. Compared with wild-type storage roots transgenic lines show a delay in onset of PPD up to 10 days, followed by less mitochondrial oxidation and hydrogen peroxidase accumulation (Xu et al. 2013). N-acetyl-5-methoxytryptamine, commonly known as melatonin, has also been reported to reduce cassava PPD, by scavenging ROS. Exogenous application of melatonin lowered ascorbic acid and degradation of starch during post-harvest physiological deterioration. Transcriptome analysis showed the significance of calcium, melatonin, and ROS scavenging in PPD of cassava (Hu et al. 2018; Li et al. 2023). Exogenous CaCl2 also reduced post-harvest physiological deterioration leading to the knowledge of calcium-mediated induction of melatonin biosynthesis and its role in reducing PPD and yield loss in cassava (Hu et al. 2018). Exogenous melatonin application was found to cause significant delay in PPD and increase the CAT and peroxidase activities as well as hydrogen peroxide reduction. Melatonin biosynthesis genes in cassava can be targeted for transgenic and cisgenic approaches to a melatonin-led PPD reduction in cassava by keeping in mind that trade-off in yield should be expected and minimized. The link between cyanogenic glycosides and PPD can be utilized together for production of varieties with low cyanogen and less PPD (Fig. 5.6).

5.3.4

Achieving Biotic Stress Tolerance

Cassava is vegetative propagated through stem cutting which makes easy for the crop to achieve quick growth and uniformity in sprouting. The negative side of this clonal propagation is the susceptibility to disease. Cassava is prone to a long range of diseases caused by bacteria, virus, fungi, mealybug, and green mite (FAO 2005). The effect of cassava diseases on socio-economic levels is huge in developing countries due to less availability of insecticides and counter measures. Disease can cause storage root yield losses, reduction in leaf growth, and food shortages and even famine. This scenario can cause major threat to food and nutritional security as well as socio-economic development of millions of people in sub-Saharan Africa, South America, and Southeast Asia (McCallum et al. 2017). The productivity of cassava is reduced by major viral diseases like cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). These two viral diseases together estimated to cause

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Fig. 5.6 Figure shows molecular mechanism of post-harvest physiological deterioration (PPD) of cassava roots caused by hydrogen cyanide and strategies for PPD reduction. AOX alternative oxidase, SOD superoxide dismutase, CAT catalase

are an annual loss worth US$1 billion and significantly affect entire regions food security (Legg et al. 2011; Patil et al. 2015).

5.3.4.1 Cassava Mosaic Disease (CMD) Cassava mosaic disease (CMD) is caused by cassava mosaic geminiviruses (CMGs) belonging to the genus Begomovirus and family Geminiviridae. Cassava mosaic geminiviruses are spread by insect vector Bemisia tabaci: commonly known as whitefly. (Maruthi et al. 2002, 2005; PATIL and FAUQUET 2009; McCallum et al. 2017). CMD-affected leaves show symptoms of leaf twisting with chlorosis mosaic pattern (Thresh and Cooter 2005). Both African cassava mosaic virus and East African cassava mosaic virus—Uganda are reported to cause an average root yield loss of 82% (Owor et al. 2004). There are three genetic sources for host plant resistance known and currently exploited to combat CMD (1) CMD1 believed to be introgressed from Manihot glaziovii, characterized by multigenic and recessive nature, (2) CMD2 is monolocus, dominant in nature, and are identified from varieties in Nigeria (3) CMD3 which include CMD2 locus and an additional QTL, but the underlying genes and molecular system of resistance to CMD is still unknown (Okogbenin et al. 2012; Beyene et al. 2016; Chauhan et al. 2018). Genetic engineering approaches can be used to understand genes involved in CMD resistance.

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5.3.4.2 Cassava Brown Streak Disease (CBSD) Cassava brown streak disease (CBSD) is a viral disease caused by cassava brown streak viruses (CBSVs), belonging to genus Ipomovirus and family Potyviridae. CBSV consist of two distinct species CBSV and Ugandan CBSV (UCBSV) (Winter et al. 2010). The symptoms of CBSD are chlorosis, necrotic streaks, and stem die-back in leaves while starchy roots show browning, necrosis, and a reduction of starch as well as cyanide content (Jennings 2003). CSBVs are also transmitted by white flies similar to CMGs (Maruthi et al. 2005). RNA interference (RNAi) technology has been utilized for managing CBSD (Patil et al. 2015). Transgenic cassava-expressing RNAi construct targeting full-length coat protein of CBSUV showed 100% resistance to CBSUV and this resistance was replicated across graft inoculation experiments (Yadav et al. 2011). Cassava plants were modified to produce small interfering RNAs (siRNA) from truncated portions of the UCBSV coat protein sequence showing promising results in managing CBSD in confined trials at Namulonge, Uganda (Ogwok et al. 2012). RNAi construct of coat protein (CP) sequences from CBSV and UCBSV fused in tandem form (Beyene et al. 2017), where transformed into East African and Nigerian cassava cultivar NASE 13, NASE 14, and TMS 98/0505, respectively, which are CMD resistant, the cultivar retained functional CMD resistance (Narayanan et al. 2021). Grafting experiments conducted in elite cultivars of cassava resistant to CSBVs, KBH 2006/18, and KBH 2006/26 showed that the scion was symptom-free during a 16-week time period of virus graft inoculation, and CBSV replication was inhibited in the transgenic line KBH2006/18 and conformed in protoplast-based assays. The identified CBSD resistance might be through inhibition of virus replication (Anjanappa et al. 2016). Understanding the molecular mechanism causing inhibition of viral replication can be utilized and targeted for introducing CBSD resistance to other cultivars via genetic engineering. 5.3.4.3 Cassava Bacterial Blight (CBB) Cassava bacterial blight (CBB) in cassava is caused by Xanthomonas phaseoli pv. manihotis and is a common bacterial disease that affects cassava cultivation which leads to a significant yield loss (López and Bernal 2012; Constantin et al. 2016; McCallum et al. 2017). The mode of Xam infection in cassava is through hijacking the transcriptional control of SWEET10a (sugars will eventually be exported transporters) a sucrose transporter (Cohn et al. 2014). Through a type III secretion system (T3SS), Xam pathogens transfer transcription activator-like effectors (TALEs) into infected cassava cells (Timilsina et al. 2020). TALE contains repeat variable residues (RVDs) which is recognized by the effector-binding elements (EBEs) on promoters of SWEET10a gene and activate SWEET10a gene expression leading to transport of sucrose from the plant cells to the apoplast. This sugar provides carbon sources for the bacteria and facilitates bacterial growth and aggravates disease symptoms (Boch and Bonas 2010; Bart et al. 2012; Cohn et al. 2014). Xam-cassava pathosystem has been targeted for genetic engineering for better understanding the process of pathogenesis and achieving disease resistance (Veley et al. 2020). In Cassava cultivar SC8, MeSWEET10a Gene was hijacked by TALE20Xam11 from Xam11 strain by binding to the EBE (effector-binding elements)

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in the promoter region of MeSWEET10a. The ability of TALE20 Xam11 to activate the MeSWEET10a promoter and inability to activate the MeSWEET10a promoter lacking the EBETALE20 region have been demonstrated and proved by dual luciferase reporter assay. Disruption of EBETALE20 Sequence in the Promoter of MeSWEET10a Via CRISPR/Cas9 leads to mutants showing significant decrease in MeSWEET10a expression, enhanced resistance towards CBB, and showed no yield penalty (Wang et al. 2022). Same results were achieved thought methylating the EBE (effector-binding elements) in the promoter region of MeSWEET10a making the gene inaccessible to the transcription activator-like effectors (TALEs). The RNA-directed DNA methylation (RdDM) proteins like DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) are known perform de novo 5-methylcytosine methylation in plants (Law and Jacobsen 2010). Transgenic plants were produced expressing a DMS3-fused artificial zinc-fingers (ZFs) that can target EBE in MeSWEET10a promoters. MeSWEET10a expression was significantly reduced due to methylated EBE region in the promoter blocking TAL20 and leads to a decrease in CBB disease symptoms. The methylation was found to be stable in plants derived from clonal propagation (Veley et al. 2023).

5.3.4.4 Cassava Anthracnose Disease (CAD) Cassava anthracnose disease (CAD) is caused by different species of fungus belonging to genus Colletotrichum. Major causative organism of CAD is found to be Colletotrichum gloeosporioides f.sp. manihotis (Fokunang et al. 2002; Liu et al. 2019). The plants affected with the disease are characterized by symptoms like lesions (necrotic) on the leaf, stem, and leaf petiole base, leading to leaf wilt, shoot-tip die-back, and loss of leaves (Owolade 2009). Pseudotheraptus devastans is considered as a possible vector of CAD transmission (Fokunang et al. 2000). Two cassava cultivars, Hanatee-HN & Huay Bong 60-HB60, are widely used to study CAD, due to their varying response to C. gloeosporioides. HN is CAD-susceptible and HB60 is CAD-resistant. After CAD infection, resistant cultivar (HN) showed defensive measures such as deposition of callose, hydrogen peroxide accumulation, and elevated expression of the miRNAs. Elevated expression of two miRNA, miR160 and miR393, led to low transcript levels in their targets, ARF10 and TIR. But susceptible cultivar (HB60) exhibited the opposite pattern of expression. Two miRNAs, miR156 and miR164, were able to cross to the invading fungal cells. Several fungal gene targets were identified but whether these miRNAs play role in disease resistance is still not known (Pinweha et al. 2015; Pinweha et al. 2022) (Fig. 5.7).

5.3.5

Biofortification of Cassava for Food and Nutrition Security

Even though cassava roots are a valuable source of food, it lacks sufficient proteins and nutrients to meet minimum nutritional requirements. Biofortification of cassava can be an efficient approach to fulfil the nutritional requirements of consumers who mostly prefer cassava as major component in their diet especially sub-Saharan

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Fig. 5.7 Figure shows strategies employed for achieving biotic stress resistance in cassava. CMD Cassava mosaic disease, CBSD cassava brown streak disease, CBB cassava bacterial blight

African population, where deficiency of iron, zinc, and vitamin A is widely reported. Less than 10% of required iron and zinc are received by the consumption of cassava in West African human populations. (Montagnac et al. 2009; Narayanan et al. 2019; Sayre 2022). The cassava root protein content is considered lowest among of all major crops. Recommended caloric and protein intake for an adult daily is 2300 kCal and 69 g, respectively, but cassava normally contains 0.7 and 3% protein to dry weight, respectively. Cassava meals of 500 g can only provide 77% and 8% of the daily caloric intake and daily protein requirement, respectively (Sayre 2022). Biofortification of food crops through genetic engineering is one of the excellent strategies for improving micronutrients in foods (Hefferon 2015). Consumption of cassava starchy roots is increasing and one-third of the sub-Saharan African population depend on cassava almost 50% of daily caloric needs (Howeler et al. 2013). Cassava is an important source of starch, but the storage roots provide lower levels of iron and zinc that are bioavailable for absorption (Gegios et al. 2010). A genetic engineering approach is appealing and efficient to improve nutrition in cassava. To improve protein content in cassava tubers, aspartic proteinase (ASP1) gene was overexpressed, which is an artificial storage protein rich in essential amino acids (Zhang et al. 2003). Transgenic cassava-expressing beta-carotene in roots using rootspecific patatin inducible promoter was developed by Telengech et al. (2015) overexpressing 1-deoxy-D-xylulose-5-phosphate synthase (DXS), and bacterial phytoene synthase (crtB) in cassava. Improved expression of transgenes like phytoene synthase (crtB) that were upregulated by the patatin-type 1 promoter CYP and DXS leads to biofortified cassava roots having 20 times increase in carotenoids when compared to wild-type cassava roots (Beyene et al. 2018).

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AtIRT1 (major iron transporter) and AtFER1 (ferritin) were overexpressed in cassava which eventually achieved nutritionally relevant levels of iron and zinc in cassava storage roots. The accumulated iron and zinc levels were 7 to 18 times, 3 to 10 times higher, respectively, than those in non-transgenic controls. (Narayanan et al. 2019; Narayanan et al. 2021).

5.3.6

Abiotic Stress Tolerance

Cassava plants are known to have high resistance to drought and well adapted to marginal nutrient environment, due to these reasons cassava can be widely grown in semi-arid regions, making it ideal to combat future climate change. But persistent water deficit condition can negatively affect the growth and development of cassava plants and subsequently reduce root tuber yield. Under water deficit, cassava plants shed its leaves (Narayanan et al. 2021) resulting in significantly reduced productivity. Drought resistance in cassava is more like drought avoidance and comes with yield trade-offs. (El-Sharkawy 2004; Daryanto et al. 2016; Wei et al. 2020). HSP90mediated drought tolerance mechanism in cassava has been elucidated. MeHSP90.9 along with MeWRKY20 and MeCatalase1 converses drought stress resistance in cassava. MeHSP90.9 and MeWRKY20 interact together and bind to promoter region of NCED5 and activated ABA synthesis while MeHSP90.9 and MeCatalase1 interact and regulate endogenous H2O2 concentration thereby providing drought resistance (Wei et al. 2020). Cassava cultivation is restricted to tropical warm regions since the crop does not prefer to grow in climates where temperature goes below 16 °C and is cold-sensitive (El-Sharkawy 2004). A comparative study between cold-treated and normal cassava, a cold-responsive intergenic lncRNA 1 (MeCRIR1), was significantly induced by cold treatment. MeCRIR1 ectopic expression leads to enhance cold tolerance of transgenic plants. MeCRIR1 interacts with cold-shock protein MeCSP5 an RNA chaperone which might be providing cold resistance by enhancing translation efficiency at low temperatures (Li et al. 2022). But the experiment was conducted in 4-week-old seedling and in controlled condition. MeCRIR1 OE lines showed to be evaluated for any yield penalty and whether they can recover from cold treatment at tuber bulking stage.

5.4

Conclusion and Future Perspectives

As the global population is expected to increase and climate change possessing huge treat to food and health security, it is important to improve yield as well as make crop climate resilient through modern genetic engineering approaches. Cassava becomes an ideal candidate for climate-resilient crop due to its ability to grow in marginal and semi-arid regions, flexible harvesting time, high yield per hectare than cereals, drought tolerance, etc. Due to these characters, cassava is widely cultivated by small-scale farmers in developing countries of South-East Asia, sub-Saharan Africa, and South America and contributes to 2.6% of the global caloric intake. Cassava is

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most the important staple food in the world after rice, maize, and wheat. Even though cassava is an ideal climate resilient crop, it requires improvement in multiple areas like (1) achieving true yield potential by improving source and sink interactions, (2) pest and disease resistance, (3) biofortification (4) removal of antinutritional compounds, and (5) reducing post-harvest deterioration. The Availability of wholegenome sequence and efficient tissue culture as well as transgenic protocol paved way for addressing these issues. Through genetic engineering approaches, cassava has achieved increased nutritional content like increased beta-carotene (Telengech et al. 2015; Beyene et al. 2018), increased zinc and iron (Narayanan et al. 2021), increased biotic stress tolerance against viral and bacterial diseases (Narayanan et al. 2021; Veley et al. 2023), reduced cyanogenic levels and post-harvest deterioration (Narayanan et al. 2011). Along with translational research, fundamental research has also being carried out to understand the true yield potential, sink and source strength, long-distance transport of photoassimilate from source to sink through phloem (De Souza et al. 2017; Mehdi et al. 2019; Rüscher et al. 2021). Genetically modified organism is highly regulated by governments; only a few countries allow the use of GMOs (Turnbull et al. 2021). Global legislative regulation on GM crops should change in order to introduce these improved varieties after fast field trails. Projects like cassava source-sink (CASS) project (https://cass-research.org/) funded by Bill & Melinda Gates foundation, which brings various scientists from Computational Biology, Plant breeding, and Plant Science to achieve a common goal of improving crop yield and food security. This project aims at elucidating cassava biology which will be crucial to achieve increases in yield and sustainable food supply to developing countries.

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Owolade OF (2009) Line X Tester analysis for resistance to cassava anthracnose disease. Elec J Env Agricult Food Chem Title 8:52–60 Owor B et al (2004) The effect of cassava mosaic geminiviruses on symptom severity, growth and root yield of a cassava mosaic virus disease-susceptible cultivar in Uganda. Ann Appl Biol 145(3):331–337 Patil BL, Fauquet CM (2009) Cassava mosaic geminiviruses: actual knowledge and perspectives. Mol Plant Pathol 10(5):685–701 Patil BL et al (2015) Cassava brown streak disease: a threat to food security in Africa. J Gen Virol 96(5):956–968 Pereira A et al (1978) Evaluation of natural cross pollination in the cassava cultivar Branca de Santa Catarina. Bragantia Pinweha N et al (2015) Involvement of miR160/miR393 and their targets in cassava responses to anthracnose disease. J Plant Physiol 174:26–35 Pinweha N et al (2022) Cross-kingdom microRNA transfer for the control of the anthracnose disease in cassava. Trop Plant Pathol 47(3):362–377 Prochnik S et al (2012) The cassava genome: current progress, future directions. Trop Plant Biol 5(1):88–94 Raines CA (2010) Increasing photosynthetic carbon assimilation in C3 plants to improve crop yield: current and future strategies. Plant Physiol 155(1):36–42 Reilly K et al (2004) Oxidative stress responses during cassava post-harvest physiological deterioration. Plant Mol Biol 56(4):625–641 Rickard JE (1985) Physiological deterioration of cassava roots. J Sci Food Agric 36(3):167–176 Rodrigues J et al (2019) Source–sink regulation in crops under water deficit. Trends Plant Sci 24(7): 652–663 Rosado-Souza L et al (2023) Understanding source–sink interactions: progress in model plants and translational research to crops. Mol Plant 16(1):96–121 Rosling H (1994) Measuring effects in humans of dietary cyanide exposure from cassava. International Workshop on Cassava Safety. p 375 Rüscher D et al (2021) Auxin signaling and vascular cambium formation enable storage metabolism in cassava tuberous roots. J Exp Bot 72(10):3688–3703 Sánchez T et al (2006) Reduction or delay of post-harvest physiological deterioration in cassava roots with higher carotenoid content. J Sci Food Agric 86(4):634–639 Sayre RT (2022) Biofortification of cassava: recent progress and challenges facing the future. In: Biofortification of staple crops. Springer, Cham, pp 417–438 Simkin AJ et al (2015) Multigene manipulation of photosynthetic carbon assimilation increases CO2 fixation and biomass yield in tobacco. J Exp Bot 66(13):4075–4090 Siritunga D, Sayre RT (2003) Generation of cyanogen-free transgenic cassava. Planta 217(3): 367–373 Siritunga D, Sayre R (2004) Engineering cyanogen synthesis and turnover in cassava (Manihot esculenta). Plant Mol Biol 56(4):661–669 Sonnewald U, Fernie AR (2018) Next-generation strategies for understanding and influencing source–sink relations in crop plants. Curr Opin Plant Biol 43:63–70 Telengech PK et al (2015) Gene expression of beta carotene genes in transgenic biofortified cassava. 3 Biotech 5(4):465–472 Thresh JM, Cooter RJ (2005) Strategies for controlling cassava mosaic virus disease in Africa. Plant Pathol 54(5):587–614 Timilsina S et al (2020) Xanthomonas diversity, virulence and plant–pathogen interactions. Nat Rev Microbiol 18(8):415–427 Turnbull C et al (2021) Global regulation of genetically modified crops amid the gene edited crop boom – a review. Front Plant Sci 12:630396 Uarrota VG, Maraschin M (2015) Metabolomic, enzymatic, and histochemical analyses of cassava roots during postharvest physiological deterioration. BMC Res Notes 8(1):648

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Uematsu K et al (2012) Increased fructose 1,6-bisphosphate aldolase in plastids enhances growth and photosynthesis of tobacco plants. J Exp Bot 63(8):3001–3009 Veley KM et al (2020) Visualizing cassava bacterial blight at the molecular level using CRISPRmediated homology-directed repair. bioRxiv: 2020.2005. 2014.090928 Veley KM et al (2023) Improving cassava bacterial blight resistance by editing the epigenome. Nat Commun 14(1):85 Wang Y et al (2022) Engineering bacterial blight-resistant plants through CRISPR/Cas9-targeted editing of the MeSWEET10a promoter in cassava. bioRxiv: 2022.2003.2002.482644 Wei Y et al (2020) The chaperone MeHSP90 recruits MeWRKY20 and MeCatalase1 to regulate drought stress resistance in cassava. New Phytol 226(2):476–491 Wenham JE (1995) Post-harvest deterioration of cassava: a biotechnology perspective. Food & Agriculture Org, Rome Wheatley C, Chuzel G (1993) Cassava encyclopedia of food science, food technology and nutrition, vol 1. Academic Press, Cambridge, MA, pp 734–743 Winter S et al (2010) Analysis of cassava brown streak viruses reveals the presence of distinct virus species causing cassava brown streak disease in East Africa. J Gen Virol 91(5):1365–1372 Xu J et al (2013) Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots. Plant Physiol 161(3):1517–1528 Yadav JS et al (2011) RNAi-mediated resistance to Cassava brown streak Uganda virus in transgenic cassava. Mol Plant Pathol 12(7):677–687 Yu S-M et al (2015) Source–sink communication: regulated by hormone, nutrient, and stress crosssignaling. Trends Plant Sci 20(12):844–857 Zainuddin IM et al (2012) Robust transformation procedure for the production of transgenic farmerpreferred cassava landraces. Plant Methods 8(1):24 Zhang P et al (2003) Transfer and expression of an artificial storage protein (ASP1) gene in cassava (Manihot esculenta crantz). Transgenic Res 12(2):243–250 Zhu XG et al (2004) The slow reversibility of photosystem II thermal energy dissipation on transfer from high to low light may cause large losses in carbon gain by crop canopies: a theoretical analysis. J Exp Bot 55(400):1167–1175 Zhu X-G et al (2007) Optimizing the distribution of resources between enzymes of carbon metabolism can dramatically increase photosynthetic rate: a numerical simulation using an evolutionary algorithm. Plant Physiol 145(2):513–526 Zidenga T et al (2012) Extending cassava root shelf life via reduction of reactive oxygen species production. Plant Physiol 159(4):1396–1407 Zidenga T et al (2017) Cyanogen metabolism in cassava roots: impact on protein synthesis and root development. Front Plant Sci 8:220 Zierer W et al (2021) Tuber and tuberous root development. Annu Rev Plant Biol 72(1):551–580

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Genetic Improvement of Eggplant: Perspectives and Challenges Pallavi Mishra, Shailesh K. Tiwari, and Kavindra Nath Tiwari

6.1

Introduction: Eggplant

Solanum is a large genus with approximately 1500 species, comprising seven distinguished subgenera, which is further recognized into 13 major clades like Potatoe, Leptostemonum, Allophyllum, Thelopodium, Normania, Archaesolanum, Regmandra, Brevantherum, African non-spiny, Geminata, Dulcomaroida, Morelloida, and Cyphomandra (Bohs 2007, 2005; D’arcy 1979). Eggplants belong to leptostemonum clade with three common widespread forms namely common eggplants (S. melongena complex), scarlet eggplants (S. aethiopicum L.), and gboma eggplants (S. macrocarpon L.) (Daunay et al. 1991; Collonnier et al. 2001; Plazas et al. 2014a; Plazas et al. 2016). This genus contains a number of species that originate from the new world, but eggplant and its relatives are native to the Old World due to their phylogenetically distinct origins (Sękara et al. 2007). Among the Solanum family, eggplant (Solanum melongena L.) is the third most popular vegetable crop after tomatoes and potatoes (Yang et al. 2014). The eggplant is a member of the Magnoliophyta division, class Magnoliopsida, subclass Asteridae, and order Solanales of family Solanaceae. This family also includes P. Mishra Division of Crop Improvement, ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India Department of Botany, Mahila Maha Vidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India S. K. Tiwari (✉) Division of Crop Improvement, ICAR-Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India K. N. Tiwari Department of Botany, Mahila Maha Vidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_6

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Fig. 6.1 Multiple morphological forms of egg plants

potatoes, tomatoes, and peppers, tobacco, tomatillo, and petunia all of which are economically important crops. The FAOSTAT data show that eggplant yields have increased up to threefold over the last two decades. India is the world’s second largest producer of eggplants after China (40.96 tons/ha) and established an enhancement in the eggplant yield of 18.90 tons/ha (http://www.fao.org/faostat/ en/). The eggplant chromosome is an autogamous diploid (2n = 2x = 24), which is similar to that of the genome of tomato and pepper. The eggplant chromosome is slightly bigger than the tomato genome with an approximate size of 11 Mb (Doganlar et al. (2002a). Fruits of eggplant are commercially equal in importance with other vegetables of the family Solanaceae and offer reliable source of several vitamins, minerals, antioxidants, phenolic compounds, and dietary fibers important for human health (Gebhardt et al. 2008; Prabhu et al. 2009; Stommel and Whitaker 2003; Cao et al. 1996; Gramazio et al. 2014; Meyer et al. 2014). The ancient history of eggplant domestication also reveals that the eggplant fruits were used for therapeutic purpose against injuries or insect bites, which proves that this vegetable crop is an important source both for nutraceutical and pharmaceutical reasons. Their multiple morphological forms make them ideal for phenotyping and agronomical studies (Fig. 6.1). The European Eggplant Genetic Resources Network (EGGNET; van der Weerden and Barendse (2006)) and the International Board for Plant Genetic Resources have together developed various morphological descriptors to characterize the eggplant to be used in breeding. In addition to the traditional morphological descriptors, newly emerging phenomics tools such as Tomato Analyzer (Rodríguez et al. 2010) are potentially useful for detailed morphological classification and characterization of diversified forms of eggplant and their genetic resources. Phenotyping of 12 wild species of eggplant and their interspecific hybrids was done using Tomato Analyzer morphological descriptor to provide their utilization in breeding (Kaushik et al. (2016). The development of genomics and transcriptomics studies in the past few years has enabled biologists to elucidate the mechanism underlying the large-scale developmental and morphological diversity at both the generic and species levels and evolution of unique plant features among different members of this family (Wang et al. 2018; Evans 2015; Roux et al. 2015). This has also enabled biologists to understand the developmental changes that might

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have occurred in putative wild progenitors to produce variable and fleshy comestible fruits. Important horticultural crops in the family Solanaceae including eggplant are under subsequent modifications by introgression of beneficial alleles from their wild relatives (Koenig et al. 2013; Paran and van der Knaap 2007). However, the genome of several cultivated and wild species of eggplant is not thoroughly characterized despite its high nutritional perquisites and economic importance.

6.1.1

Nutritional Benefits

The Charaka and Sushruta Samhitas written about 100 BC in India describe the health and medicinal benefits of eggplant. Nutritionally, eggplant fruits possess less calories, but are vital source to dietary fibers, vitamins, minerals, polyphenol derivatives (Plazas et al. 2014b), anti-oxidant compounds (Gramazio et al. 2014), and phyto-active ingredients of human health benefit such as chlorogenic acid (CGA) (Gramazio et al. 2014), caffeic acid, phenylpropanoids (Docimo et al. 2016), and hydroxycinnamic acid (Whitaker and Stommel 2003). The berries are relished in variety of dishes both cooked and raw. One hundred gram of raw eggplant fruit supplement in human diet contained carbohydrates (5.7 g), protein (1 g), lipids (0.19 g), dietary fiber (3.40 g), vitamin A and beta-carotene equivalents (27 IU), vitamin B1 (0.037 mg), vitamin B6 (0.084 mg), vitamin C (2.2 mg), vitamin E (0.30 mg), vitamin K (3.5 mcg), niacin (0.649 mg), pantothenic acid (0.281 mg), folate (22.00 mcg), choline (6.9 mg), and minerals like calcium (9.00 mg), copper (0.082 mg), iron (0.24 mg), magnesium (14.00 mg), phosphorus (24.00 mg), potassium (230.00 mg), manganese (0.250 mg), zinc (0.16 mg), and sodium (2.00 mg). Researchers confirmed the most important phenolic compound in eggplants as CGA, which possesses free-radical scavenging activity (Cao et al. 1996), and have antimutagenic and anti-LDL attributes. Anthocyanin along with phenolics have multiple benefits for human health (Plazas et al. 2013a; Braga et al. 2016). Apart from gustative characteristics, eggplant fruits are also rich source of water-soluble flavonoids such as nasunin, solanine, and solasonine. Nasunin is known to protect the cell membranes from rupture against oxidative stress (Bliss and Elstein 2004). Research upon animals has confirmed that nasunin prevents the brain cell damage by increasing the concentration of thiobarbituric acid reactive substances (TBARS) and protecting fatty layer in the brain (Kimura et al. 1999).

6.1.2

Progenitor Species

Multiple evidences for the possible progenitor species of S. melongena often make it controversial to decide the exact progenitor of eggplant. Evidences from laboratory studies and DNA sequencing suggest S. linnaeanum as the possible parent of S. melongena as this species was well-dispersed throughout the Middle East and was most commonly domesticated along with the cultivated type in Asia, Africa, and European countries. In other studies, single-nucleotide polymorphism (SNP)

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examination to establish relationships among eggplant and related species has confirmed S. insanum to be the progenitor species of S. melongena (Page et al. 2019; Ranil et al. 2017; Meyer et al. 2012; Knapp et al. 2013; Plazas et al. 2013b). According to Daunay et al. (2001)), the eggplant complex is likely to be descended from S. incanum (which is very similar to S. insanum), a semi-wild weedy variety native to North Africa and eastwards to India. S. incanum is popularly known by other names such as bitter apple, thorn apple, and bitter garden egg, and is a rich source of diosgenin, many glycoalkaloids with antibiotic properties, and has been consistently domesticated in India since ancient times for its close relatedness and introgressive breeding with the cultivated eggplant. However, as confirmed by the AFLP analysis to establish genetic relationships among cultivated and wild landraces, all three eggplant forms (S. melongena, S. insanum, and S. incanum) are single species and likely to have common origin (Meyer et al. 2012).

6.1.3

Common Forms of Eggplant

The three common widespread forms are the common eggplants, scarlet eggplants, and gboma eggplants (Daunay et al. 1991; Collonnier et al. 2001; Plazas et al. 2014a; Plazas et al. 2016), all representing the Old world crop. The scarlet and gboma eggplants are popular African eggplants and are grown locally, while the common eggplant (also called brinjal) is generally considered Indo-Asian in origin and is cultivated worldwide as an economically important vegetable crop. Due to edible attributes of the fruits, common and gboma eggplants are usually referred as Melongena Dunal (Lester and Daunay 2003), while scarlet eggplant is included under the section Oliganthes Bitter Dunal (Lester 1985). High morphological diversity is characteristic to common and scarlet eggplants, while comparatively less phenotypic variability has been reported in gboma eggplants (Plazas et al. 2014a; Lester and Hasan 1991a; Prohens et al. 2012). Further, the taxonomic treatment of common eggplant is quite more challenging compared to scarlet and gboma eggplants due to narrow genetic background but high morphological diversity, and high level of genetic resemblance with other members of the “eggplant clade,” which includes closest wild-related species along with the progenitor species of cultivated eggplant (Knapp et al. 2013).

6.1.4

Global Occurrence of Cultivated and Wild Species of Eggplant

Crop wild relatives (CWRs) or in general, the wild relatives of agricultural crops, exhibit enormous diversity compared to the domesticated form and possess the capacity to develop more productive, disease-resistant, and resilient varieties. Breeders can draw essential alleles required in breeding programs from wild relatives. For the crops with narrow genetic base, the wild relatives act as a valuable source for transfer of genes of desired traits of interest to be used in breeding and broadening the genome of lesser diverse lines. CWRs of eggplant are richly

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distributed along the globe toward Africa, Asia, India, and eastwards to Pakistan. Eggplants are believed to be originated and domesticated in these regions (Vavilov (1926). Farming practices, breeding efforts, changing climatic factors, and increasing pressures of the market demands lead to subsequent alteration in the morphological and genetic individuality of CWR of eggplants. The primitive plant and fruit characters include broad, spiny foliage, small and large number of fruits, bitter taste, tough peel, and thick flesh. Systematic efforts are made by breeders for improvement of the nutritive value, flavor, aroma, and yield of fruits with better quality and soft flesh with fewer seeds. Wild relative of eggplants exhibited abnormally greater number of flowers per inflorescence and subsequent modification of this trait to obtain a reduction in number of flowers in their domesticated species resulted in an increased uniformity of fruit shape and size as compared to their wild form (Sękara and Bieniasz (2008). In self-pollinated crops like eggplant, fertilization process stimulates the pollen tube elongation and anthesis after which the number of genes starts to express in floral induction. Eggplant cultivars, particularly wild species, exhibit relatively greater differences in terms of the length of the style stigma position. The fruit setting pattern in eggplant was affected by stylar differences and the stigma position more than the number of flowers per inflorescence (Nothmann et al. (1983). Style length and the adjacency of stigma to the anther also regulate the fruit shape and seed quality (Passam and Bolmatis 1997). Long-styled flowers favor the development of fruits with long peduncle, which is the most preferred form. The above studies indicate that wild relatives are useful source for evolution and variation among eggplant germplams. Characterization of different forms of weedy and wild eggplant based on their morphological description and distribution reveals that S. incanum, S. insanum, S. macrocarpon, S. torvum, and S. aethiopicum are common wild eggplant species closely related to S. melongena and frequently used as a natural source of variation in eggplant breeding program (Lester and Hasan (1991a). It is found that the interspecific hybrids developed between cultivated eggplant and the wild species are fully fertile (Plazas et al. 2016). Although comprising of undesirable wild features such as small inedible fruits with tough peel, bitter taste, and multi-seeded, some of them are rich source of CGA and other biologically active ingredients, which may be of potential human health benefit, and, hence, are a subject with interest to breeding.

6.1.5

Common Diseases of Cultivated Eggplant

Being a warm-season crop with longer growth period, eggplant is exposed to wide range of pests, nematodes, and pathogens hampering its yield and productivity. Common pests include aphids, whiteflies, beetles, red spider mite, leafhopper, leaf roller, stem borer, and fruit and shoot borer (Rotino et al. 1997a; Medakker and Vijayaraghavan 2007). Eggplant cultivation is also subject to attack by various soilborne pathogens of bacterial and fungal origin causing common eggplant diseases like bacterial wilt, fusarium wilt, verticillium wilt, damping-off, phomopsis blight,

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phytophthora blight, leaf spot, fruit rot, and little leaf of brinjal (Rotino et al. 1997a). Due to lack of resistance in the genome of cultivated eggplant and insufficient characterization of its wild relatives, breeding strategies are often limited in the crop. Therefore, there is a need to improve farming practices and develop more resistance eggplant varieties through utilization of conventional breeding strategies blended with modern biotechnological techniques to overcome these challenges. Eggplant breeding program for disease resistance mainly focuses on utilization of F1 hybrids, which are of great significance due to high morphological variability, good shelf-life, and better quality fruits with well-developed internal resistance mechanisms (Rotino et al. 1997a; Muñoz-Falcón et al. 2009).

6.2

Genetic Improvement by Conventional Breeding

Initial breeding efforts were focused particularly in the massive production of the cultivated variety, i.e., S. melongena in its center of origin and countries where their fruits were preferred for therapeutic and culinary reasons. For example, Africans grew S. aethiopicum and S. macrocarpon cultivars locally for large-scale production, but today, the breeding objectives in the eggplant are used in much more broader sense (Sękara et al. 2007). Eggplant and their wild relatives attract interest of breeders for both agricultural and economic reasons. Since the wild species offer an efficient gene pool for controlled resistance to diseases and pests and intercrossable with many cultivated species (Plazas et al. 2016), S. melongena genotypes are crossed with the wild species for improved resistance to pests hampering eggplant cultivation. Due to the high morphogenetic potential of its tissues, eggplant genotypes are also used in cytological, developmental, and biotechnological research for production of improved varieties. Techniques such as tissue culture, embryo rescue, genetic transformations, somatic hybridization, and in vitro regeneration are useful for overcoming the problems of sexual incompatibilities arising during crossing with wild ancestors. In vitro regeneration of eggplant species is easy and fast approach offering efficient protocols for genetic transformation and gene regulation. Kashyap et al. (2003) reported the application of biotechnology for improvement of eggplant genetics by introgressing alleles from wild species. Frary et al. (2003) developed a population by crossing S. linnaeanum and S. melongena for mapping genes for disease resistance and abiotic stress and introducing into the cultivated eggplant. S. torvum, one of the wild relative tolerant to Verticillium wilt was used to transfer its resistance to S. melongena, and protoplast fusion between S. torvum and S. melongena was successfully carried out (Jarl et al. (1999)). The methanolic extracts of S. torvum fruits are traditionally used in the treatment of bacterial and fungal infections, making it an efficient model for pharmacological studies and drug development (Balachandran et al. 2012). The comparative transcriptome analysis of S. torvum and S. melongena for exploiting the genome of S. torvum identifies the scope for transfer of promising disease resistance traits of economic importance into the cultivated type (Yang et al. (2014)). S. aethiopicum and S. macrocarpon are intensively used in the breeding program for introgression

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of traits of agronomic importance (Alba et al. 2005). Anaso (1991) reported the breeding of homozygous lines of S. aethiopicum for improved quality and yield of the fruits. S. aethiopicum is also reported to be useful for transfer of resistance to Pseudomonas solanacearum into S. melongena. The dihaploid plants developed through the anther culture of somatic hybrids between these two species are reported to be completely resistant to the fungal wilt caused by Fusarium oxysporum (Rizza et al. 2002). Successful hybridization of S. melongena and S. macrocarpon and possibilities of transfer of genes resistance to two-spotted spider mite from S. macrocarpon to S. melongena was reported (Schaff et al. (1982)). Interspecific hybrids of these two species were also used (Gowda et al. (1990)) to obtain resistant hybrids against Leucinodes orbonalis(brinjal fruit and shoot borer). Few genetic linkage maps and QTLs associated with disease resistance have also been documented in eggplant (Sunseri et al. 2003; Barchi et al. 2010; Lebeau et al. 2013; Miyatake et al. 2016; Salgon et al. 2017), which may be cloned in future to develop highly resistant varieties and provides solution to severe agricultural problems faced by farmers during eggplant cultivation. Besides these, Bt-brinjal (eggplant) varieties expressing cry1Ac gene and having in-built mechanism of resistance against the brinjal fruit and shoot borer has been developed as the first genetically modified (GM) vegetable crop from India, but unfortunately, it sought a moratorium by the Parliamentary Committee on Agriculture in 2012 in view of nutritional security and toxicity assessment upon human health. The above studies make it very clear that the breeding objectives in eggplant are mainly focused to (i) detect QTLs associated with traits of agri-horticultural importance; (ii) exploit heterosis in breeding strategies for enhanced productivity; (iii) to develop eggplant cultivars with better quality and yield; (iv) to develop hybrids with multiple resistance to insects and pests; and (v) to develop disease resistance lines by identifying candidate genes directly involved in transcriptional regulation.

6.3

Plant Regeneration and Somaclonal Variation

Studies have been conducted on the eggplant’s response to in vitro culture, particularly the regeneration ability (Collonnier et al. 2001). Plants could be easily regenerated with in vitro organogenesis in case of eggplant. Shoot regeneration has been successfully reported in many wild relatives of common eggplant such as S. sisymbriifolium, S. indicum, S. khasianum, S. xanthocarpum, S. aethiopicum group gilo, and S. torvum (Bhatt et al. 1979; Gleddiei et al. 1985). Regeneration in eggplant can also be used to select resistant lines by growing cell cultures or regenerating calli on abiotic stress media (Asao et al. 1994). It has been shown that adjusting polyamine concentrations within cells and the ratio between diamine putrescine and triamine spermidine in eggplant helps to improve somatic embryogenesis and plant regeneration (Yadav and Rajam 1997; Singh Yadav and Venkat Rajam 1998). The genetic variation in eggplant lines of both embryogenic and androgenic origin has been found to be useful for agronomic traits in field trials (Rotino and Gleddie 1990; Rotino et al. 1997b).

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Somatic Embryogenesis and Haploidization

Several sources of somatic embryogenesis have been reported in eggplant, including stems, hypocotyls, leaves, cell suspensions, protoplasts, and roots isolated from the plant, but somatic embryogenesis induction was found to be higher in leaves and cotyledons than in the hypocotyls. According to the studies conducted on somatic embryogenesis up to till date, naphthaleneacetic acid (NAA) best enhances the differentiation of somatic embryos. Plantlets obtained with NAA also exhibited greater somaclonal variation than those obtained with 2,4-D using morphological features of somatic embryogenic raised plants. The eggplant somatic embryogenic responses are found to be genotype-dependent (Sharma and Rajam 1995). In addition, anther culture technique has primarily been used for obtaining double haploid parents for conventional breeding of cultivated eggplant (Rotino 1996). It has been demonstrated that double haploid plants can produce pure lines faster than selfed inbreds in conventional eggplant breeding programs. Eggplants lines with double haploid parents have been found extensively useful for breeding of wide variety of agronomic traits, such as disease resistance, high yield, earliness, and tolerance to various abiotic stresses.

6.5

Marker-Assisted Breeding in Eggplant

Characterization of genetic diversity is fundamental to follow an efficient breeding program, particularly for crops exhibiting high level of heterogeneity. With advancement in genomics studies, modern plant breeders have adopted marker-based breeding methodology in the crop improvement programs as the traditional phenotyping methods are not adequate enough to evaluate a particular trait. The markers are representative of a specific DNA segment that can be correlated with a trait of interest (Collard et al. 2005). The cost stability, reliability, and reproducibility of these markers let the large-scale use of these markers for construction of genetic linkage maps, QTL detection, MAS, assessment of genetic diversity, and gene pyramiding (Varshney et al. 2007; Wu et al. 2009b; Stuber et al. 1999) and offer prompt solutions to the problems such as undesirable wild characteristics and unfavorable scenarios like environmental stress (Thapa et al. 2015). In case of eggplant, molecular diversity have been studied using array of DNA-based markers, like randomly amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), restriction fragment length polymorphisms (RFLPs), inter-simple sequence repeats (ISSRs), simple sequence repeats (SSRs) including sequence-tagged microsatellite sites (STMS) and single-nucleotide polymorphism (SNPs), which have been extremely useful for germplasm characterization (Isshiki et al. 1994; Karihaloo et al. 1995; Karihaloo and Gottlieb 1995; Isshiki et al. 2003; Isshiki et al. 1998; Isshiki et al. 2008; Tiwari et al. 2009; Furini and Wunder 2004; Nunome et al. 2003b, 2009; Tiwari 2007; Baysal et al. 2010), assessment of genetic diversity (Ansari et al. 2015; Behera et al. 2006; Sakata and Lester 1994; Karihaloo et al. 2002; Singh et al. 2006; Koundal et al. 2006), establishment of

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taxonomy-based genetic relationships and phylogeny among diverse eggplant accessions (Sakata et al. 1991; Isshiki et al. 1998; Lester and Hasan 1991a), and QTL mapping studies (Nunome et al. 2001; Doganlar et al. 2002a; Nunome et al. 2003b, 2009; Sunseri et al. 2003; Wu et al. 2009a; Barchi et al. 2012; Fukuoka et al. 2012; Miyatake et al. 2012; Lebeau et al. 2013; Gramazio et al. 2014; Salgon et al. 2017; Frary et al. 2014; Doğanlar et al. 2014).

6.5.1

Molecular Marker-Based Assays in Eggplant

With advancement in genomics studies, marker-assisted breeding approach is increasingly used by plant breeders because traditional phenotypic tests are insufficient to assess a particular trait. Using the markers, one can directly correlate phenotypes with a particular DNA segment (Collard et al. 2005). In eggplants, molecular markers like chloroplast DNA markers (Sakata et al. 1991; Sakata and Lester 1994; Isshiki et al. 1998), isozyme-based markers (Karihaloo and Gottlieb 1995; Lester and Hasan 1991b; Isshiki et al. 1994), seed protein markers (Karihaloo et al. 2002), mitochondrial DNA markers (Isshiki et al. 2003), RFLPs (Doganlar et al. 2002a; Isshiki et al. 1998; Isshiki et al. 2003), RAPD markers (Koundal et al. 2006; Singh et al. 2006; Nunome et al. 2001; Karihaloo et al. 1995), ISSRs (Isshiki et al. 2008; Tiwari et al. 2009), AFLPs (Nunome et al. 2003a; c; Ansari et al. 2015; Furini and Wunder 2004), and microsatellite SSR markers (Behera et al. 2006; Nunome et al. 2003a, 2009; Stàgel et al. 2008) are consistently deployed in assessment of genetic diversity, breeding, and genetic improvement program. Behera et al. (2006) used STMS markers to analyze diversity in a limited set of accessions including few related species in eggplant. An interspecific F2 population of S. melongena x S. linnaeanum was used to locate QTLs underlying morphological traits of interest in eggplant (Frary et al. 2003). ISSR and RAPDs have been utilized in characterization of Fusarium oxysporum in S. melongena (Baysal et al. 2010). SSR, RAPD, and ISSR markers were utilized in S. aethiopicum and S. melongena for assessment of genetic diversity (Ansari et al. 2015). Gene-targeted molecular markers such as start codon targeted (SCoT) are a good alternative to the conventional molecular markers and have been reported in many crops like peanut (Xiong et al. 2011), durum wheat (Etminan et al. 2016), citrus (Mahjbi et al. 2015), ramie (Satya et al. 2015), rice (Collard and Mackill 2009), dendrobium (Bhattacharyya et al. 2013), jatropha (Mulpuri et al. 2013), mango (Luo et al. 2012), and tomato (Shahlaei et al. 2014) for assessment of diversity and genetic relationships among cultivars. However, SCoT markers are still not applied for analysis of genetic diversity and assessment of polymorphism in eggplant. Overall, the studies from previous reports show that molecular marker technique is a worthy tool to identify key genotypes with contrasting alleles in a population and selecting the genotypes with desirable traits of interest to enhance the efficiency of marker-assisted selection.

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Genetic Linkage and QTL Mapping Studies in Eggplant

Unlike tomato, potato, and pepper, which are the extensively studied fruit crops for molecular and genetic analysis (Tanksley et al. 1992; Livingstone et al. 1999), the genetic linkage and QTL mapping studies in eggplant and their wild species are rather limited. A genetic linkage map and QTL mapping studies in eggplant are essential to identify key genes regulating the expression of qualitative and quantitatively inherited characters between the different germplasms (Doganlar et al. (2002a). Additionally, a genetic map also provides worthful source for biotechnological tools for enhancing the methodologies in eggplant improvement such as haploid production, somatic hybridization, somaclonal variations, and transgenic development. Due to dramatic advancement in the area of genomics in the last decade, emergence of modern technologies like next-generation sequencing (NGS), and reduced costs of molecular marker development and sequencing platforms, QTL identification for wide range of agri-horticultural and economic traits becomes far easier and increases the eggplant breeding efficiency and development of amended varieties with enhanced yield and productivity in eggplant. It was only in 2000s when the first genetic linkage map of eggplant was developed by Nunome et al. (1998) to locate the QTL for fruit shape in eggplant using RAPD markers on an F2 population derived by EPL1 x WCGR112-8. Nunome et al. (2001) utilized a set of 181 dominant markers (88 RAPDs and 93 AFLP markers) and reported another intraspecific genetic linkage map using an F2 population of 168 individuals to identify genomic regions associated with development of fruit shape and color in eggplant. However, the results were obscured since the dominant markers have tendency to form clusters (Alonso-Blanco et al. 1998; Nilsson et al. 1997) and exhibit low degree of genetic polymorphism in species like eggplant (Doganlar et al. 2002a; Wu et al. 2009a), resulting into generation of large number of LGs without significant correlation with the basic number of eggplant chromosome. An interspecific genetic map was developed with the objectives of overcoming the situations like low degree of polymorphism and to establish synteny between tomato and eggplant by comparing the rearrangements between the genetic maps of the two species (Doganlar et al. (2002a). In this study, S. melongena and S. linnaeanum were used as the parents and tomato genomic DNA, tomato singlecopy cDNA, and a tomato conserved orthologous set (COS) RFLP markers, which were previously used to establish a synteny between the potato and tomato genome (Fulton et al. 2002; Tanksley et al. 1992) were accessed during the studies. Another genetic map with high collinearity between tomato and eggplant could be reported (Doganlar et al. (2002b)). These QTLs were reported to be associated with 22 domestication traits of eggplant (plant prickliness, fruit color, shape, and size), and high resemblance with the QTLs related to 18 eggplant phenotypic traits such as leaf morphology and fruit shape, size, color, and appearance was reported (Frary et al. (2003)). Sunseri et al. (2003) developed another interspecific genetic map to identify the molecular markers linked to tolerance to the Verticillium wilt in eggplant.

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Nunome et al. (2003b) added a set of seven SSR markers by screening a dinucleotide genomic library of eggplant and reported an improved version of his genetic map with LGs reduced from 21 to 17, while map length and the marker density remaining approximately same. In the same year, characterization and screening of tri-nucleotide microsatellites motifs for development of an eggplant genomic SSR enriched library was also done (Nunome et al. 2003c). It was found to be highly polymorphic within and across different eggplant species and exhibited higher values of polymorphic information content (PIC) than the arbitrary markers (Kalia et al. 2011). In fact, the SSR markers are highly reproducible, robust, co-dominant, and well-dispersed across the genome (Varshney et al. 2005). With the advances in sequencing platforms and time, identification and characterization of genomic SSRs and expressed sequence tag SSRs (EST-SSRs) became effortless, and cost-effective techniques and some other genomic libraries in eggplant also became available. An intraspecific genetic map using F2 population (305E40 x 67/3) was developed by Barchi et al. (2010) in order to map the gene Rfo-sa1 for resistance to Fusarium oxysporum. This map was composed of 212 AFLP and 22 SSR markers, 1 RFLP, and 3 cleaved amplified polymorphic sequence (CAPS) marker of Rfo-sa1 and spanned about 718.7 cM along the 12 LGs having an average marker density 3.0 cM (Stàgel et al. (2008)). This F2 population was also used to locate QTLs for anthocyanin content using restriction site-associated (RAD) tag-derived markers obtained from RAD tag sequencing library (Barchi et al. (2012)), which was earlier used to develop a large set of SNP and SSR markers from eggplant (Barchi et al. 2011). This F2 population was also used to identify QTLs associated with fruit yield and morphological traits and to map QTLs affecting morphological and biochemical attributes of the eggplant fruit (Portis et al. (2014); Toppino et al. (2016)). An integrated genetic linkage map using F2 populations (LWF2 and ALF2) and 952 SNP and SSR markers developed from the solanum orthologous (SOL) gene sets of eggplant was reported (Fukuoka et al. (2012)). This map was highly illustrious that covered 1.5 times greater the eggplant genomic regions presented by Nunome et al. (2009) and spread along 12 LGs spanning 1285 cM of the chromosomal length (Rafalski (2002)). These SNPs and SSRs were used to construct two intraspecific genetic maps from two F2 populations (ALF2 and NAF2), in which two major QTLs associated with parthenocarpy in eggplant, Cop3.1 in LG 12 and Cop8.1 in LG 15, spanning in length 1414 cM and 1153 cM, respectively, were identified in both maps (Miyatake et al. (2012)). The lists of mapping studies performed till date for locating QTLs underlying the fruit morphological variations and other traits in eggplant are reported in Table 6.1.

Eggplant genotypes EPL-1 (S. melongena) x WCGR112-8 (S. melongena) MM195 (S. linnaeanum) x MM738 (S. melongena) S. sodomeum [=S. linnaeanum] x S. melongena EPL-1 (S. melongena) x WCGR112-8 (S. melongena) EPL-1 (S. melongena) x WCGR112-8 (S. melongena) MM195 (S. linnaeanum) x MM738 (S. melongena) 305E40 (DH from S. melongena and S. aethiopicum) x 67/3 (S. melongena) 156 AFLPs 117 RAPDs 101 RAPDs 54 AFLPs 7 SSRs 245 SSRs

Verticillium wilt tolerance

Fruit shape and color development

SSR development

Synteny with tomato

F. oxysporum resistance

48 Interspecific F2

120 Intraspecific F2

94 Intraspecific F2

58 Interspecific F2

141 Intraspecific F2

232 RFLPs 110 COSII 5 tomato-derived markers 212 AFLPs 22 SSRs 1 RFLP 3 CAPS

232 RFLPs

Fruit domestication traits

58 Interspecific F2

Markers 93 AFLPs, 88 RAPDs

Traits Fruit shape and color

Population 168 Intraspecific F2

Table 6.1 Genetic linkage and QTL mapping studies reported in eggplant

12, 718 cM

12, 1535 cM

14, 959 cM

17, 716 cM

12, 736 cM

12, 1480 cM

Linkage groups and map length 21, 779 cM

3.0 cM

6.1 cM

4.3 cM

4.9 cM

2.7 cM

7.6 cM

Average marker density 4.9 cM

Barchi et al. (2010)

Wu et al. (2009a)

Nunome et al. (2009)

Nunome et al. (2003b)

Sunseri et al. (2003)

Doganlar et al. (2002b)

Reference Nunome et al. (2001)

134 P. Mishra et al.

305E40 (DH from S. melongena and S. aethiopicum) x 67/3 (S. melongena) LS1934 (S. melongena) x WCGR112-8 (S. melongena) LS1934 (S. melongena) x AE-P03 (S. melongena) LS1934 (S. melongena) x AE-P03 (S. melongena) NakateShinkuro (S. melongena) x AE-P03 (S. melongena) MM738 (S. melongena) x MM960 (S. melongena) Eggplant accessions from USA, India,

Genetic control of resistance to R. solanacearum Association analysis for

178 Intraspecific F6 RILs

141 Association

132 SNPs 118 SSRs 125 SSRs 49 SNPs

QTLs associated with parthenocarpy

135 Intraspecific F2 (ALF2) 93 Intraspecific F2 (NAF2)

105 SSRs

91 AFLPs 26 SSRs 2 SRAPs

639 SNPs 313 SSRs

Development of Solanum orthologous (SOL) sets

90 F2 Intraspecific (LWF2) 93 F2 Intraspecific (ALF2)

339 SNPs 33 SSRs 27 COSII 11 RFLPs 3 CAPS 2 HRM

Anthocyanin content of fruit

156 Intraspecific F2

–

–

Genetic Improvement of Eggplant: Perspectives and Challenges (continued)

Ge et al. (2013)

Lebeau et al. (2013)

Miyatake et al. (2012)

– –

8.8 cM

Fukuoka et al. (2012)

Barchi et al. (2012)

1.4 cM

3.8 cM

18, 884 cM

12, 1414 cM 15, 1153 cM

12, 1285 cM

12, 1390 cM

6 135

–

–

191, Association mapping (GWA)

4.4 cM

12, 1085 cM

99 SSRs 88 AFLPs 42 COSII 9 CAPS 4 SNPs, and 1 morphological marker 314 SNPs

Mapping CGA biosynthesis pathway and genes for polyphenol oxidase (PPO) Fruit, plant, and leaf morphological traits

91 Interspecific BC1

Eggplant breeding lines, old varieties, and landraces from Asia and Mediterranean

1.8 cM

12, 1518 cM

400 AFLPs 348 RFLP 116 COSII

Synteny with tomato

–

108 Interspecific F2

–

314 SNPs

Anthocyanin pigmentation and fruit color development

191 Association mapping (GWA)

Average marker density

fruit morphological traits

Linkage groups and map length

mapping (GWA)

Markers

Japan, Italy, Malaysia, Arab Emirates, Thailand, Korea Eggplant breeding lines, old varieties, and landraces from Asia and Mediterranean MM195 (S. linnaeanum) x MM738 (S. melongena) MM577 (S. incanum) x AN-S-26 (S. melongena)

Traits

Population

Eggplant genotypes

Table 6.1 (continued)

Portis et al. (2015)

Gramazio et al. (2014)

Doğanlar et al. (2014)

Cericola et al. (2014)

Reference

136 P. Mishra et al.

MM738 (S. melongena) x MM960 (S. melongena)

180 Intraspecific F2

Genetic control of resistance to R. solanacearum

867 SNPs 139 AFLPs 28 SSRs 1 SRAP

14, 1518 cM

1.4 cM

Salgon et al. (2017)

6 Genetic Improvement of Eggplant: Perspectives and Challenges 137

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P. Mishra et al.

Transcriptome Assemblies of Eggplant and Their Wild Relatives

Variety of RNA sequencing platforms like Illumina HiSeq 2000, Illumina MiSeq, and Roche 454 are popular and proved to be very useful to provide a comprehensive study of the gene expression and metabolic pathways controlling the defense and developmental and reproductive stages in many species of eggplant. For example, deep RNA sequencing for identification of miRNAs from the seedlings of S. melongena (SRA accession: SRR833801 and SRR833802) against V. dahlia was reported (Yang et al. (2013)). Comparative transcriptome analysis and phylogenomic study were carried out in two accessions of eggplant (SRA accession: SRR1104129) and a wild relative, turkey berry (SRA accession: SRR1104128) (Yang et al. (2014)). Further characterization of the WRKY TFs in these two eggplant species was reported (Yang et al. (2015)). RNA-Seq technology has also been exploited to characterize the NAC transcription factor family in S. melongena for its response against the bacterial wilt (Na et al. 2016). Zhou et al. (2016) performed the de novo sequencing and transcriptome analysis of wild species of eggplant, S. aculeatissimum (SRA accession: SRS1383901 and SRS1383902) in response to V. dahliae. In another study, de novo assembly of S. melongena transcriptome (SRA accession: SRR1291243) was carried out for identification of putative allergens and their epitopes (Ramesh et al. 2016). The transcriptome assembly of S. aethiopicum (SRA accession: SRR2229192) and S. incanum (SRA accession: SRR2289250) was reported (Gramazio et al. 2016). It was observed that both of them are close relatives of the common eggplant, and it is very important source of transfer of beneficial alleles for biotic and abiotic stress tolerance. Transcriptome profiling of S. melongena to provide insights into the molecular mechanisms underlying parthenocarpic fruit development was reported (Chen et al. (2017)). Recently, the transcriptome profiling of S. melongena genes related to light-induced anthocyanin biosynthesis before the purple color development after exposure to light was reported (Li et al. (2018)). As of today, the de novo transcriptome assembly of Ramnagar Giant (S. melongena), a local cultivar of Uttar Pradesh region, and W-4 (S. incanum) have also been reported (unpublished), and sequences were submitted at the National Centre for Biotechnology Information portal (NCBI; accession numbers: GAYR00000000 and GAYS00000000, respectively), but still the genomic resources in eggplant is quite less explored as only a total of around 300,000 sequences are available in the NCBI database, majority of which comprises the ESTs generated by Fukuoka et al. (2010)). The metrics of transcriptome assemblies of eggplant and related species is covered in Table 6.2.

Transcriptome Assembly, Root Transcriptome Assembly, Leaf, floral bud, and fruit Identification of miRNA, Pistil Transcriptome Assembly, Flower buds

S. aculeatissimum

S. melongena

S. melongena

S. aethiopicum S. incanum

Identification of putative allergens, whole fruit

Assembly type and plant tissue Identification of miRNA, seedlings Transcriptome Assembly, Leaves, root, and stem Draft Genome Assembly, Leaves, roots, fruits, and flowers Genome Assembly, Leaves

S. melongena

S. melongena

S. melongena

S. melongenaS. torvum

Species S. melongena

Illumina HiSeq 2000

Illumina HiSeq 2000 PE (300 bp) Small RNA library

Illumina HiSeq 2000 PE (100 bp) Illumina HiSeq 2000

Roche 454 GS FLX PE (200–300 bp) Illumina

Sequencing platform Illumina MiSeq Illumina HiSeq 2000 PE (72 bp)

Table 6.2 Whole genome and transcriptome studies in eggplant

SRR3479276 SRR3479277 SRP085349

1592 differentially expressed genes related to parthenocarpy

SRR2229192 SRR2289250

686 miRNAs

87,084 unigenes 83,905 unigenes

SRS1383901 SRS1383902

69,824 unigenes

Genetic Improvement of Eggplant: Perspectives and Challenges (continued)

Chen et al. (2017)

Wang (2018)

Gramazio et al. (2016)

Zhou et al. (2016)

Barchi et al. (2012) Ramesh et al. (2016)

– SRR1291243

Hirakawa et al. (2014)

Reference Yang et al. (2013) Yang et al. (2014)

DRR014074 DRR014075

NCBI accession SRR833801 SRR833802 SRR1104129 SRR1104128

149,224 transcripts

12 pseudo-molecules

33,873 scaffolds

Assembly statistics 5940 miRNA 38,185 unigenes 34,174 unigenes

6 139

Species S. melongena

Assembly type and plant tissue Transcriptome Assembly, Eggplant peel

Table 6.2 (continued) Sequencing platform Illumina HiSeq 2000 Assembly statistics 32,629 transcripts 1956 differentially expressed genes

NCBI accession SRR5650714 SRR5651526 SRR5658205 SRR5658226

Reference Li et al. (2018)

140 P. Mishra et al.

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141

The Whole-Genome Sequence of Eggplant: Paving a New Route with Implications to Molecular Breeding

Barchi et al. (2011) carried out the first restriction site-associated DNA (RAD)-tag sequencing of the two eggplant accessions, i.e., S. melongena and S. aethiopicum on Illumina platform in which 10,089 SNPs, 2000 putative SSRs, and 874 Indels were identified for fingerprinting a panel of eggplant germplasms and their mapping parents. Additionally, a number of high-quality informative SSR markers were developed using the eggplant genomic libraries by Vilanova et al. (2012) to cover the genome of this species. Recently, the first draft genome sequence of an Asian eggplant variety named “Nakate-Shinkuro” has been released. The assembly of this draft genome (SME_r2.5.1) was carried out using Illumina HiSeq 2000 sequencer by Hirakawa et al. (2014) in which the HQ reads were assembled into 33,873 scaffolds using SOAP de novo v1.05 that covered ∼75% (833.1 Mb) of the eggplant genome with 64.5 Kb N50 parameter. In this study, a large number of SSR motifs and repeats were also found along with 4536 SNPs, which are available at the NCBI with the SRA accessions DRR014074 and DRR014075. Portis et al. (2018) provided a comprehensive characterization of the high-quality SSRs (both perfect and imperfect) from the whole genome of eggplant and found 2449 perfect SSRs distributed in 2086 genes across the eggplant whole genome. Using these data, a microsatellite database has been developed, namely Eggplant Microsatellite Database or EgMiDB, which enables quick browsing, identification, and retrieval of the repeat type, motif type, complete sequence, and location of the SSR markers. This dataset comprises 42,035 genes, of which 4018 genes are exclusive to eggplant. In addition to this, the SSRs detailed in EgMiDB can also be used for the construction of linkage and QTL map and DUS-testing for marker-based variety identification. Overall, a detailed study of eggplant whole-genome assembly will facilitate the better understanding of the eggplant genome to make one forward move toward elucidating the molecular mechanisms underlying developmental, physiological, and defense responses in this species.

6.8

Conclusion

Literature survey revealed that the availability of genetic resources and genomic tools for eggplant is confined, delimiting the molecular characterization of this species. Therefore, advancement in the areas such as NGS is necessary to embark marker-enhanced breeding, molecular marker discovery, and mapping studies to untangle the complex traits controlling large phenotypic variation in eggplant. Applied areas such as bioinformatics shall assist in identification of candidate genes associated with physiological and biochemical attributes in eggplant. It shall also assist in decoding the unique genome sculptures of different cultivars having useful traits with interest to breeding for enhanced MAS in eggplant and related wild species.

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Advances in Chilli Pepper (Capsicum spp.) Improvement Using Modern Genetic Tools Ratna Kalita, Priyadarshini Bhorali, Manab Bikash Gogoi, and Bornali Gogoi

7.1

Introduction

The chilli peppers, also known as the Capsicum, are one of the most significant crops used as spice and vegetable worldwide. The global cultivation of chilli peppers can be attributed to their vast range of uses, including as an essential spice in food and for therapeutic and industrial purposes (Pawar et al. 2011; Duranova et al. 2022). These crops are endowed with various natural phytometabolites rendering the plant a valuable commodity. Chilli peppers are members of the Solanaceae family and grow well in tropical, subtropical, as well as temperate zones (Motbaynor et al. 2022). The genesis of the term “Capsicum” is the Greek word “Kapsimo,” which implies “to bite” or “to swallow.” Most of the species of chilli peppers are diploid with chromosome number x = 12, except one species, C. annuum var. glabriusculum, which is a tetraploid (Jindal et al. 2020). Another species with triploid chromosome number has also been reported (Bhutia et al. 2019). The color and pungency level of Indian chillies are acknowledged throughout the world (Karim et al. 2021). Only six out of approximately 38 varieties of chilli peppers, namely C. annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens, and C. assamicum, are grown commercially (Colney et al. 2018). The most extensively grown species is C. annuum, whereas C. chinense has the most potent flavor. Assam and other northeastern states of India are home to a unique domesticated species designated as C. assamicum (Purkayastha et al. 2012). R. Kalita (✉) · P. Bhorali · M. B. Gogoi Department of Agricultural Biotechnology, College of Agriculture, Assam Agricultural University, Jorhat, India e-mail: [email protected] B. Gogoi Department of Fruit Science, College of Horticulture & FSR Nalbari, Assam Agricultural University, Jorhat, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_7

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Although it shares a close relationship with C. frutescens and C. chinense, it can be distinguished by specific morphological characteristics. As reported by the Food and Agricultural Organization (FAO), India produced 1.7 million tons (MT) of dry chilli pepper in 2021, which was much more than the production in Thailand (0.32 MT) and China (0.31 MT). However, China (16.7 MT) led the chilli producers for the fresh market, succeeded by Mexico (2.8 MT) and Indonesia (2.7 MT). Vietnam, India, and the United States consumed the most pepper with 166K, 86K, and 68K tons. Bulgaria (7641 kg per 1000 persons), Singapore (5288 kg per 1000 persons), and Vietnam had the most significant per capita consumption rates of pepper (1724 kg per 1000 persons). The production of chilli peppers worldwide surged during 2007–2018, and a further increase is anticipated in the coming years (World Pepper Market Report 2020). However, statistics show that in India, the production volume of dry chillies fluctuated from the fiscal year 2015 to 2021, with a remarkable decline in 2019 and 2020 (Statista Research Department 2022). Harsh climatic conditions like elevated temperature, drought, and salinity along with various socioeconomic causes have driven the low development of chilli pepper varieties, resulting in a decline in production volume. On the other hand, as reported by the FAO in the year 2021, fresh and dry chilli pepper production has increased globally. By 2050, there will be more than 10 billion people on the planet, necessitating increased production of chilli peppers to meet the rising demand (Pandey et al. 2021). Since the advent of the Green Revolution, there has been a rapid advancement in plant breeding techniques. And over the last four decades, post-Green Revolution, genetic and protein engineering developments have taken plant biotechnology to the next level. Modern high-throughput genomics and molecular breeding tools along with multiomics-driven technologies could efficiently be utilized for genetic improvement of chilli peppers (Fig. 7.1).

7.2

Chilli Pepper: Origin and Evolution

Capsicum spp. are one of the earliest cultivated crops and are regarded as the first spice ever consumed by humans. With their ancestral roots in the Central and South America, the domestication of the chilli peppers dates back to more than 6000 years. There are currently 38 known species of Capsicum; however, only C. annuum, C. frutescens, C. chinense, C. baccatum, and C. pubescens are regarded as domesticated (Andrews 1984; Eshbaugh 1993). Every species has undergone a unique domestication process and emerged from a core centre of divergence where the most closely related wild species still exist. Phylogeographical studies use correlations between the spatial distribution of alleles and their genealogical links to infer details about the history of species divergence (Avise 2000). In the Americas, peppers are among the earliest domesticated plants, and archeological evidence suggests that C. annuum was cultivated even before the rise of agriculture (Pickersgill 1969). Through a comparative study of karyotypes of wild and domesticated C. annuum, Pickersgill (1971) recognized Mexico as the primary place of domestication of Capsicum. It is believed that these five species were

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Fig. 7.1 Modern genetic tools for genetic improvement of chilli pepper

independently domesticated in two parts of the neotropics: C. annuum and C. frutescens in Mesoamerica and C. chinense, C. baccatum, and C. pubescens in South America (Pickersgill 2007). Following Christopher Columbus’ first journey, peppers were brought from the West Indies to Europe in 1493. Afterward, they were disseminated throughout the Mediterranean nations and then subsequently to Africa, India, and China (Andrews 1984; Bosland and Votava 2000; Nicolai et al. 2013). While the Portuguese are credited with bringing the chilli pepper to India (Basu and De 2003), it is said that the Christian Missionaries brought them to the country’s northeastern region (Dhaliwal 2007). The most extensively grown species in the world is C. annuum, probably because it was the first Capsicum to reach Europe (Andrews 1984). While C. chinense and C. frutescens also gained prominence in Africa and Asia, C. pubescens and C. baccatum resided in South America and the Andes (Bosland and Votava 2000). In these secondary regions of divergence, various species were chosen by farmers throughout generations for cultivation across different agroclimatic regions and to meet the consumption habits of indigenous people, leading to the development of local lines or landraces. As a result, pepper cultivars today exhibit a wide range of phenotypes such as larger, pungent-free fruits with varying shapes and greater fruit mass (Stewart Jr et al. 2005; Paran and van der Knaap 2007; Djian-Caporalino et al. 2007; Pickersgill 2007). Today, the most economically significant varieties of C. annuum are the large-fruited blocky pepper or bell pepper cultivars, which are used mostly as spices and condiments (Bosland and Votava 2000). Over the past

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century, with the advent of modern plant breeding approaches, the traditional landraces were replaced by commercial cultivars and hybrids with better and consistent yields, and carrying disease-resistant traits (Lanteri et al. 2003). Consequently, it resulted in a substantial decrease in the genetic variability of chilli peppers, posing a risk to their cultivation and leading to genetic erosion (Hammer et al. 2003). The wild, related species and landraces are important genetic divergence repositories, even if they have not always been used in breeding programs. Various methods for evaluating genetic variability have been employed in crop diversity research, including advanced phenotypic and genetic tools. Since the beginning of the twentieth century, conserving diversity through maintaining germplasm collections has been a significant focus of private, national, and international organizations (Gonzalez-Perez et al. 2014). The Asian Vegetable Research and Development Center (AVRDC) in Taiwan houses a vast number of chilli pepper germplasms, which comprises a total of 8170 accessions from both wild and domesticated Capsicum species collected from all around the world. The National Bureau of Plant Genetic Resources (NBPGR), situated in New Delhi, India, has been maintaining 2774 chilli germplasms. The United States Department of Agriculture (USDA) has 6067 accessions of chilli germplasms, while the New Mexico Capsicum Accession (NMCA) has a collection of 2100 accessions of 22 species, from geographically diverse parts of the world (Lozada et al. 2022).

7.3

Genetic Improvement of Chilli Pepper

At the dawn of the twentieth century, Darwin and Mendel’s fundamental discoveries founded the modern science of plant breeding and genetics. Likewise, recent biotechnology and genomic research developments and genetic tools integrated with conventional plant breeding techniques have transformed crop improvement for the twenty-first century. Following the advent of the Agrobacterium-mediated transgenic plant production in the early 1980s, the development of molecular markers and genomics-led technologies gained momentum, resulting in the development of contemporary technologies that boosted the growth of the “omics” approaches (Moose and Mumm 2008). The omics-led approaches along with advanced genetic tools are being extensively employed to improve agronomic attributes related to yield, quality, flavour, and resilience to biotic/abiotic challenges in chilli pepper (Fig. 7.2). The following sections discuss the key developments and advances in chilli pepper improvement using modern genetic and omics-led technologies.

7.3.1

Genetic Diversity Studies in Chilli Peppers

Genetic diversity is a prerequisite for successful selection within a population or the population resulting from hybridization. The possibility of developing improved characteristics in offsprings is higher if the parents are more diverse (Bhutia et al. 2019). Hence, it is necessary to assess the genetic similarity and diversity in order to

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Fig. 7.2 Primary objectives of chilli pepper improvement using genetic tools

pinpoint the source of the genes for a certain trait within the available genotypes (Tomooka 1991). Accessing and utilizing the enormous genetic diversity are one of the critical challenges in plant breeding. Genetic variations can be identified by analyzing the nucleotide sequence variations using various molecular markers, such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), single-nucleotide polymorphism (SNP), and cleaved amplified polymorphic sequence (CAPS). Because of their stability and analytical efficiency, molecular markers have been extremely useful to assess the genetic variability in plants (Zhong et al. 2021). In a study using 10 RAPD (random amplified polymorphic DNA) markers, genetic diversity analysis of Capsicum landraces revealed two main clusters, with the genotypes of C. frutescens and C. chinense clustered together beside the genotypes of C. annuum. Compared to C. annuum genotypes, C. frutescens and C. chinense genotypes had higher average genetic diversity (Sanatombi et al. 2010). Assessment of genetic variability in chilli pepper has been done from various geographical regions, including Asia, Europe, America, and Africa (Taranto et al. 2016). In a recent study, genetic divergence of 147 accessions of C. frutescence from 25 different nations was analyzed using SSR markers, which then grouped the populace into seven main divisions by their geographic origins (Zhong et al. 2021). Furthermore, Tripodi et al. (2021) performed a genomic assessment on 10,038 C. annuum accessions from international gene banks using SNP markers generated from genotyping by sequencing (GBS). Barka and Lee (2020) reviewed a vast collection of chilli pepper germplasms that showed resistance to diseases like fungi, bacteria, and viruses. In a recent study, the genetic and population structure of 54 Capsicum accessions was analyzed by Haq et al.

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(2022) using ISSR markers. This study provides us information about germplasm characterization, genetic arrangement, and population structure of chilli germplasms, which could be utilized for planning, improvement, and devising management strategies for chilli breeding. The northeast India boasts of a rich biological divergence because of its unique geographical locus and has been labeled as one of the 12 “Genetic Epicentres” for the genesis of the global flora (Bhutia et al. 2019). Dubey et al. (2015) compared the phytochemical composition and antioxidant potential of 25 genotypes of chillies from northeast India. They revealed differences in the amounts of capsaicin, oleoresin, phenolics, carotenoids, and other antioxidants. A significant amount of genetic variation was found in the germplasms with respect to fruit shape and colour in a genetic variability study of the Naga King Chilli with morphological parameters (Bhagowati and Changkija 2009). According to a genetic diversity study of 53 Indian landraces of chillies from the northeast, the pun1 gene was found to have 79 SNPs and 3 indels (Yumnam et al. 2012). Moreover, the study found 3–9 alleles for every SSR locus (mostly 5.36 alleles/locus) when morphological attributes and SSR markers were examined together. Furthermore, the erect and campanulate fruit types were grouped into separate clusters.

7.3.2

Genome-Wide Mapping Studies in Chilli Peppers

The development of high-density genetic maps has been boosted by genomic advancements. In case of chilli pepper, the construction of highly dense genetic linkage maps was accelerated by the abundance of large-scale genomic resources. Linkage analysis and genome-wide association study (GWAS) are two techniques that can investigate the genetic basis of complex characters in plants. Investigating molecular markers associated with important genes or quantitative trait loci (QTL) controlling various agronomic characters has been made easier by analyzing the genetic maps combined with phenotypic data from relevant segregating mapping populations. A high-density genetic map provides an essential basis for QTL cloning and mapping. Due to the advent of multiple high-throughput technologies, the identification and utilization of SNPs have become faster and economically feasible in crops. Tanksley et al. (1988) created a genetic map with extensive genome coverage using 85 RFLP (restriction fragment length polymorphism) markers, for an interspecies hybrid of C. annuum and C. chinense. Later, using a cross of the same C. chinense parent with a different C. annuum parent, Prince et al. (1993) developed a more comprehensive map with 192 markers. Ben-Chaim et al. (2006) constructed the most detailed genetic map using the F2 population of the C. annuum and C. frutescence cross, which consisted of 728 molecular markers (489 SSRs, 195 AFLPs, 8 specific PCR based and 36 RFLP markers) and covered an overall length of 1358.7 cM. This map contains some of the candidate genes for the suggested model of the capsaicin biosynthesis pathway, including the pAMT, COMT, and Bcat genes (Stewart Jr et al. 2005).

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Various QTLs/candidate genes have been discovered in chilli peppers that impact fruit size, shape, color, pungency, and disease resistance. Detection of QTLs associated with disease resistance found in various chilli pepper germplasms would assist in faster breeding for resistance. Phytophthora capsici, which affects the entire plant, is the most harmful pathogen to pepper plants (Barka and Lee 2020). P. capsici resistance in pepper results from a single dominant gene working in concert with thousands of QTLs associated with partial resistance (Truong et al. 2012). Zhang et al. (2013) identified and described the P. capsici-resistant gene CaRGA2 from the cultivar CM334 of C. annuum. The short arm of chromosome P5 is a key genetic hotspot that contains QTL for P. capsici resistance (Du et al. 2021). Lee et al. (2017) mapped the Cvr1 gene, responsible for resistance to Chilli Veinal Mottle Virus (ChiVMV), using SNP markers in C. annuum. Using a customized sliding window technique and GBS, two disease-resistant loci were mapped on chromosomes 6 and 10. The detection of SNP markers and their genomic location assist fine-mapping of genes for breeding of disease-resistant pepper cultivars. Leveillula taurica, a significant pathogen in pepper, is the causal organism of powdery mildew. A key locus, PMR1, for resistance to powdery mildew was discovered by mapping QTLs on chromosome 4 using BC1F2 (Kim et al. 2017), F2, and F2:3 mapping populations (Jo et al. 2017). The genome of chilli pepper has also been used to map QTLs associated with a number of viral diseases, including pepper mottle virus, cucumber mosaic virus, and pepper mild mottle virus (Choi et al. 2018; Venkatesh et al. 2018). Again, chromosomes 9 and 10 have been found to have loci related to root-knot nematode and bacterial wilt resistance, respectively (Changkwian et al. 2019; Du et al. 2019). Capsaicinoids are a unique and important class of compounds exclusively found in pepper fruits that have been thoroughly researched for many years. Five SCAR (sequenced characterized amplified region) markers were developed based on the capsaicinoid synthase gene sequence and their use in the early detection of pungent genotypes (Lee et al. 2005). Ben-Chaim et al. (2006) discovered six QTLs for the three capsaicinoids, viz. capsaicin, dihydrocapsaicin, and nordihydrocapsaicin, which can be used for chilli pepper breeding. Moreover, linkage mapping has discovered multiple genomic areas and potential genes associated with the generation of capsaicinoids on chromosomes 1, 2, 3, 4, and 10 (Han et al. 2018). Molecular markers may be developed and utilized in the breeding programs for marker-assisted breeding and selection after these critical loci have been recognized using association mapping. In addition, recent developments in high-throughput sequencing technologies such as RNA sequencing (RNA-seq) can help in the identification of the more informative function-associated specific trait (FAST) SNP markers for accurate trait prediction, genetic effect estimation, and parental line selection (Fu et al. 2017). Essential nutrients such as vitamins A, E, and C, carotenoids, and a few minerals are abundantly found in the fruits of chilli peppers (Olatunji and Afolayan 2018). Genome-wide mapping approaches can enhance these nutritional contents in chilli pepper. One significant locus linked to carotenoid metabolism in C. annuum is the phytoene synthase 1 (CaPSY1) gene, which can be a plausible focus of molecular

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breeding to boost the carotenoid content in pepper fruits (Lozada et al. 2022; Wei et al. 2020). A study discovered 8 diverse genotypes containing higher levels of vitamin A than in sweet potato and 16 different genotypes containing higher vitamin C than in kiwi (Kantar et al. 2016), signifying chilli pepper to be an acceptable alternative in tackling nutritional deficiencies. Furthermore, Shu et al. (2022) identified the candidate genes for fruit color in pepper. They studied the quality of two C. chinense germplasms, HNUCC16 (dark green immature and yellow ripe fruit) and HNUCC22 (light green immature and red ripe fruit), and developed an F2 population. Two dCAPS markers were developed based on the SNP locus that provides a new way to recognize different genotypes and study the genetic determination and marker-assisted selection of fruit colour for pepper breeding (Shu et al. 2022).

7.3.3

The Omics Approach for Trait Dissection in Chilli Peppers

Along with genomics-assisted breeding, omics technologies like transcriptomics, metabolomics, proteomics, and epigenomics can be applied to investigate the genetic basis of different quality characters of chilli pepper. Using omics tools and data generated from whole-genome sequencing, modern genetic technologies have boosted the improvement of important agronomic traits in crops (Hao et al. 2020). This comprehensive strategy is expected to speed up the creation of superior lines through “precision breeding” by identifying crucial genes and their pathways controlled by multiple genetic and epigenetic factors (Weckwerth et al. 2020). Several stress-tolerant genes/loci have been identified in Capsicum, some of which have shown response against biotic and abiotic stresses. Hong and Kim (2005) discovered that the gene Ca-DREBLP1 in chilli pepper quickly gets activated by dehydration and high salinity. Numerous findings indicate its dual involvement in response to both biotic and abiotic stimuli. Using differential-display reverse transcription PCR, Yi et al. (2004) identified an ERF/AP2-type transcription factor CaPF1 in pepper leaves infected with Xanthomonas axonopodis pv. glycines. Additionally, studies on heat-tolerant and heat-sensitive pepper cultivars revealed differentially expressed transcripts and metabolites under heat stress (Wang et al. 2019). The expression of NAC transcription factor CaNAC064 under cold tolerance was studied by Hou et al. (2020). The characterization of the amino acid sequence of CaNAC064 revealed that its downregulation decreased tolerance to cold stress, while its overexpression increased tolerance to cold stress. Moreover, Yao et al. (2021) studied the effect of exogenous glutathione (GSH) on chilling injury in postharvest bell pepper fruits stored at low temperature and explored the mechanism of this treatment from the perspective of the ascorbate–glutathione (AsA-GSH) cycle. This study revealed that the GSH treatment was associated with upregulated AsA-GSH cycle genes, namely CaAPX1, CaGR2, CaMDHAR1, and CaDHAR1 and enzymes APX, GR, and MDHAR. The SBP-box (squamosa-promoter binding protein) genes are specific to plants and play important roles in plant growth, signal transduction, and stress response. Zhang et al. (2020a) studied one of the pepper SBP-box gene,

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CaSBP12, and indicated that this transcription factor negatively regulates salt stress tolerance in pepper and may relate to ROS signaling cascades. Comprehensive analyses of the transcriptome and proteome of two different pepper cultivars demonstrated the temporal specificity of protein expression during fruit development (Liu et al. 2019). Moreover, metabolome and transcriptome investigations revealed variations in cutin levels across two cultivars and showed that the production of cutin requires the overexpression of specific genes (Natarajan et al. 2020). Jasmonic acid (JA) induction during mite infection was further established by combined metabolomic and transcriptomic studies in chilli pepper (Zhang et al. 2020b). The study showed that when plants and arthropods interact, JA initiates a strong defensive response. Prior research using RNA-seq revealed that the genes CA00g9220 and CA00g96010 are significantly expressed in a resistant landrace CM-334, during P. capsici infection (Kim et al. 2019). According to a recent research, the P. capsici resistance in chilli peppers may be influenced by DNA methylation, chromatin remodeling, and histone acetylation (Du et al. 2021). Thus, to identify and breed P. capsici-resistant lines, it may be beneficial to analyze the chilli pepper epigenome using methods such as DNA methylation studies, PCR-based bisulfite sequencing, and chromatin immunoprecipitation tests (Li 2021). Fruit development and ripening of wild and a cultivated pepper variety over two developmental stages, namely green (immature stage) and red fruit (mature stage), were studied through Illumina-based transcriptome analysis (Razo-Mendivil et al. 2021). According to the study, the two peppers C. annuum cv. tampiqueno 74 and C. annuum var. glabriusculum Chiltepin revealed similar gene expression patterns. The difference in expression patterns of genes related to shape, size, ethylene, and secondary metabolite biosynthesis suggest that changes produced by domestication of chilli pepper could be very specific to the expression of genes related to traits desired in commercial fruits (Razo-Mendivil et al. 2021). Furthermore, transcriptome analysis in wild and cultivated species of chilli pepper demonstrated significant variation in the expression patterns of genes associated with fruit development, notably those involved in cell cycle and division (Martinez et al. 2021). In a recent transcriptome study of the cracking-prone cultivar “L92,” several genes related to cell wall metabolism and production of lignin were discovered, indicating their involvement in the fruit cracking process (Liu et al. 2022).

7.3.4

Genetic Transformation of Chilli Peppers

Although transgenic development in several plant species such as tobacco, petunia, and potato, gained momentum about four to five decades before, chilli pepper lagged in stepping into the era of advanced biotechnology and genetic transformation (Steinitz et al. 1999). Plant tissue culture and recombinant DNA technology are potent biotechnological tools that can supplement traditional breeding to improve chilli pepper. However, despite being economically important, the crop’s pace of improvement in regeneration and transformation is relatively slower compared to

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other members of the Solanaceae family because of its extremely recalcitrant nature and genotypic dependence (Kothari et al. 2010). Nevertheless, the improvement of chilli lineages has been significantly enhanced by the combination of plant tissue culture with genetic modification. Kim et al. (1997) expressed cucumber mosaic virus (CMV) coat protein and CMV satellite RNA in transgenic chilli plants, albeit with poor regeneration and transformation rate. Agrobacterium-mediated transformation is an effective technique for genetic modification of pepper to add crucial agronomic features and create functional genomic pools (Heidmann et al. 2011). Liu et al. (1990) published the first study on Agrobacterium-mediated transformation of chilli pepper using leaves, cotyledons, and hypocotyls as explants, using two strains of Agrobacterium. However, they were not successful in developing functional transgenic pepper. Subsequently, several reports of transformation in various chilli cultivars were published. A well-defined procedure to genetically transform C. annuum var. Pusa Jwala from cotyledonary leaves, utilizing the Agrobacteriummediated approach was reported by Manoharan et al. (1998). They used a binary vector called “pBI121” for the purpose. In 1993, DNA Plant Technology Co. (US 5262316) received a patent on “Genetically transformed pepper plants and methods for their production” (Steinitz et al. 1999). Later, Shivegowda et al. (2002) carried out successful regeneration and transformation of two chilli varieties, Pusa Jwala and G4 using A. tumefaciens strain C58 bearing binary vector pGV1040 containing the marker gene nptII and GUS reporter gene. Likewise, Mahto et al. (2018) reported a highly effective protocol for transforming two local Indian varieties of chilli peppers, Pusa Sadabahar and Pusa Jwala, utilizing the Agrobacterium strain LBA4404. During the late twentieth century, in the infant stage of transgenic breeding of chilli peppers, the major objective was to generate transgenic varieties that were resistant to CMV by adopting cDNA from a viral satellite or the viral coat protein, and most of these studies were carried out in C. annuum and C. frutescens (Steinitz et al. 1999). Since the turn of the century, transgenic varieties with disease resistance and tolerance to various abiotic stresses have been reported. Shin et al. (2002) developed transgenic C. annuum cv. Nockwang by transferring the Tsi1 (tobacco stress-induced 1) gene, which regulates genes related to stress response and parthenogenesis, via Agrobacterium-mediated gene transfer. The transformed chilli plants displayed improved resistance to P. capsici, Xanthomonas campestris pv. vesicatoria, CMV, as well as pepper mild mottle virus. Later, Cai et al. (2003) developed transgenic Capsicum resistant to CMV and tobacco mosaic virus (TMV). Moreover, transgenic chillies (C. annuum) have been shown to have increased resistance to anthracnose disease when PepEST gene was expressed constitutively under the CaMV35S promoter (Ko et al. 2016). The PepEST genes are known to promote resistance against various diseases in fruits through the production of H2O2 and the expression of pathogenesis-related (PR) genes. The level of PR gene expression in transgenic lines was much higher than in the non-transgenic lines when infected with the anthracnose fungus Colletotrichum gloeosporioides. Agrobacterium-mediated genetic modification has also been used to develop transgenic chilli for abiotic stress tolerance. A study by Subramanyam et al.

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(2011) revealed substantial enhancement in salt tolerance in C. annuum cv. Aiswarya 2103. The transgenic chilli varieties showed higher levels of relative water content, glycine betaine, chlorophyll, ascorbate peroxidase, proline, as well as superoxide dismutase and glutathione reductase. Similarly, for the purpose of enhanced resistance against salinity stress, Bulle et al. (2016) developed genetically transformed C. annuum expressing the wheat Na+/H+ antiporter gene (TaNHX2). Under salinity stress conditions, the transformed varieties showed enhanced relative water content, proline, chlorophyll, superoxide dismutase, and ascorbate peroxidase as compared to the untransformed plants. Moreover, H2O2 and malondialdehyde were found to be decreased in the transformed varieties. The transcription factor DREB1A, which is known to confer multiple resistance to drought, cold, and salinity stress, was employed to genetically transform the capsicum variety G4 (Maligeppagol et al. 2016). Most transformation experiments in Capsicum have been focused on the Agrobacterium-mediated gene transfer method due to the limited morphogenetic potential of chilli plants, which renders genetic engineering challenging (Chee et al. 2018). However, there are certain reports on the direct genetic transfer of genes through particle bombardment. Gilardi et al. (1998) utilized the biolistic method to introduce the pepper mild motile virus’s coat protein into C. chinense. They succeeded in transiently expressing the purple moderate motile virus’s coat protein in the leaves of pepper plants via biolistic co-bombardment with a plasmid encoding the β-glucuronidase gene. In another study by Gilardi et al. (2004), the biolistic approach was utilized to introduce Tobamovirus coat protein into C. frutenscens. Nianiou et al. (2002) employed a biolistic handheld gene gun to insert the reporter gene β-glucuronidase into chilli plant. In C. frutescens, Chee et al. (2018) devised an improved procedure for biolistic-based direct transformation.

7.3.5

Gene Editing in Chilli Peppers

Gene editing is a technology that enables precise modification of genes by employing a single guide RNA (gRNA or sgRNA). The CRISPR/Cas9 system consisting of the Cas9 protein and one sgRNA has emerged as the most potent alternative to generate accurate and predictable targeted point mutations (Jinek et al. 2012). It has a tremendous chance of improving the breeding of chilli peppers, especially for enhancing disease resistance (Lozada et al. 2022). Using CRISPR/ Cas9 technology, it may be feasible to introduce changes in susceptible chilli peppers that will provide resistance to P. capsici and other serious diseases. In a study, the NAC72 locus in chilli pepper was precisely edited utilizing a CRISPR/ Cas9-fused cytidine base editing (CBE) system employing an Agrobacteriummediated transformation technique resulting in anthracnose resistance with accurate base edition efficiency of up to 69% (Joshi 2019). Another study revealed genomewide CRISPR/Cas9 editing sites based on the “Zunla-1” reference genome. Wholegenome alignment was then used to assess the specificity of the editing sites, and 29,623,855 highly specific NGG-PAM sites were detected (Li et al. 2020).

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Furthermore, the efficacy of a DNA-free, genome editing method was studied in hot and sweet cultivars of C. annuum “CM334” and “Dempsey,” using preassembled SpCas9 or LbCpf1 with a single guide RNA RNP (ribonucleoprotein), CRISPR/ Cas9-RNP, or CRISPR/LbCpf1-RNP (Kim et al. 2020). Depending upon the applied CRISPR/RNPs, the targeted CaMLO2 gene was differentially edited in both the cultivars. Moreover, the study also indicated that protoplasts could be used as an useful system for efficient guide RNA screening (Kim et al. 2020). Recently, Mishra et al. (2021) carried out a single transcript CRISPR/Cas9 alteration of CaERF28, a significant susceptibility gene, which resulted in enhanced resistance to anthracnose in C. annuum against Colletotrichum spp. They have reported this system to be a rapid, efficient, and versatile approach for generating anthracnose resistance in chilli as well as other solanaceous crops. Thus, with the rapid development of technologies for efficient in vitro regeneration and genetic transformation of Capsicum species, the CRISPR-based gene editing systems are soon likely to become a powerful tool for gene functional analysis and genetic improvement of chilli pepper.

7.4

Conclusion

Currently, a plethora of information regarding the genome of Capsicum is available and accessible due to the expeditious progress in genomic technologies. Access to novel genes and QTLs has become possible due to the advancement of genome sequencing tools and multiomics-based resources. In addition, comprehensive metabolomics platforms have helped explore various metabolites and bioactive substances in chilli peppers, which will be helpful in breeding programs for enhancement of biochemical traits in addition to the previously studied characters of pungency and carotenoid content. These resources and technologies offer significant scope for genetic improvement in the context of chilli pepper breeding programs, but more work and research need to be done to utilize their full potential.

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8.1

Introduction

Vegetable crops especially those belonging to the large Solanaceae family, such as potato, tomato, peppers, and others, constitute a major portion of our daily food consumption and agriculture revenue in both developed and developing countries. Within this family, the genus Capsicum contains about 22 wild species and five domesticated pepper species (including C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens) (Pickersgill 1997). Capsicum annuum L. is the most cultivated pepper species containing both pungent (such as Capsicum annuum L. var. annum; chili or hot pepper) and nonpungent peppers (such as Capsicum annuum L. var. grossum Sendt.; sweet or bell pepper) (Chaudhary et al. 2015), which are native to the Central and South America, with primary center of highest diversity in Peru and Bolivia (Zonneveld et al. 2015). It usually grows as herbaceous annual in temperate areas and perennial shrub in tropical areas (OECD 2006) and consumed both as a vegetable and as a spice (Islam et al. 2022). It possesses several important medicinal (such as antioxidant, anti-inflammatory, anti-obesity, and anticancer) and nutritional properties (such as vitamins, carotenoids, flavonoids, and others) (Manivannan et al. 2018). It has a great variability in colors and shapes of the fruits (such as erect, blocky, pendant type, cherry, and jalapeños), and flavors (such as fiery hot and sweet) (Islam et al. 2022). China is the largest producer of pepper followed by India, the United States, and Turkey (Ateş and Yilmaz 2020). C. annuum L. is a diploid, self-pollinated crop, with chromosome number of 24 (2n = 24), and considerable ability of outcrossing (11–64%) (Chhapekar et al. 2018). The target traits for Capsicum breeding programs are biotic and abiotic stress S. Kumar · A. Singh (✉) Department of Biotechnology, Dr. Y. S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_8

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resistance, improved yield, and enhanced nutritional quality. Introgression of genes conferring resistance or tolerance against various diseases in commercial cultivars from wild relatives is considered the most efficient and simplest strategy for disease control. Recently, the genomes of two C. annuum lines including a Mexican landrace Criollo de Morelos 334 (CM334) known for resistance against Phytophthora diseases (Kim et al. 2014b) and Zunla-1, an inbred line (F9 generation) (Qin et al. 2014) along with its progenitor and wild relative Chiltepin (C. annuum var. glabriusculum), a north-central Mexican wild selection landrace, were completely sequenced using whole-genome shotgun approach. The average size of CM334 genome is about 3.48 Gb with 34,903 genes of average coding sequence 1009.9/ 35.2 Mb and 80% repetitive sequences (76.4% long terminal repeats (LTR) transposable elements) (Kim et al. 2014b). In case of Zunla-1 genome, there are about 70.3% LTR elements and 35,336 protein coding genes (Qin et al. 2014). These sequence information aids in elucidating the molecular mechanisms associated with evolution and domestication of C. annuum L. and its dynamic interactions with different pathogens and pests. It also facilitates identification of various genetic loci or genes with possible implications on disease resistance and linked molecular markers such as SNPs, useful in pepper improvement (Ahn et al. 2016).

8.2

Major Diseases of Peppers

The cultivated area of peppers is steadily increasing in different ecological conditions globally due to their high economic demands; however, it exposes them to different biotic and abiotic stresses without any prior exposure. Biotic stresses comprising pathogens and pests indeed are much more consistent with prolonged exposure and devastated impacts on yield and quality (Tables 8.1 and 8.2). The wide and overlapping plant host range for different pathogens and emergence of new highly virulence strains or races attributed to the breakdown of plant resistance that increases the chances of coinfection as well as disease severity along with several frequent unpredictable pathogen outbreaks (Chowdhury et al. 2020). The majority of the pepper pathogens share their hosts with other crops of the Solanaceae and other crop families, which make their managements very difficult through conventional control measures including cultural practices and chemicalbased remedies.

8.2.1

Advances in Disease Resistance Against Major Pathogens of Capsicum annuum L.

8.2.1.1 Phytophthora capsici P. capsici is one of the most destructive and economically important soil-borne polycyclic oomycete-based pathogen that infects all parts of the plants in all growth stages and has diverse host range for crops belong to the Cucurbitaceae, Rosaceae,

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Table 8.1 Different diseases in Capsicum annuum L. caused by fungi, bacteria, and nematodes Diseases Powdery Mildew

Causal agent Leveillula taurica

Phytophthora Blights Anthracnose/ fruit rot

Phytophthora capsici Colletotrichum spp.

Damping-off

Pythium spp.

Root rot and reductions

Bacterial wilt

Rhizoctonia solani

Fusarium wilt

Fusarium spp.

Browning of roots and lower part of stem leading to wilting of plant and damping off Leaf chlorosis, vascular discoloration, and wilting

Alternaria rot

Alternaria alternate

Pepper gray mold

Botrytis cinerea

White mold

Sclerotinia sclerotiorum

Chili leaf spot/gray leaf spot

Stemphylium solani Cercospora capsici

Leaf and vascular wilt

Verticillium dahliae

Bacterial spot

Xanthomonas campestris pv. vesicatoria (Xcv) Meloidogyne incognita (Rootknot nematodes (RKN)

Root knot

Symptoms Grayish white patches on the undersides of leaves and light green– yellow lesions on the upper leaf surface, and shedding Affects root and lower portion of the stem leading to wilting Water soaked and sunken lesions with characteristic rings of acervuli in concentric rings

Gray and watery-soaked lesion on fruit Leaves first appear as a brown, blighted area and progress up the petiole and into the stem, infected fruit turn gray–white, soft, and rot Water-soaked lesions with brownish color on leaves and fruits, rotten stem, and wilting White spots and sunken red or purple lesions on leaves and necrosis Infected leaves, stem, petiole, and peduncle turn dark brown with a distinctive sporulating gray center, known as “frogeye” spot Stunting and yellowing of leaves leading to leaf shedding, permanent wilt, and damping off Water-soaked lesions on leaves, brown, patches on fruits and stem

Root dysfunction, reduced efficiency in water and nutrient uptake, stunting, premature wilting

References McGrath et al. (2001)

Saxena et al. (2016) Than et al. (2008); Saxena et al. (2016) Chellemi et al. (2000) Sandani and Weerahewa (2018) Ramdial and Rampersad (2010) Soomro et al. (2019) Roberts (2006)

Jeon et al. (2006) Zheng et al. (2008) Heald and Wolf (1911)

Reusche et al. (2012) Abbasi et al. (2002)

Noling (2019)

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Table 8.2 Viral diseases in C. annuum L. Virus Symptoms Thrips-transmitted orthotospovirus Tomato spotted wilt Stunting, leaf distortion, ringspots on leaves, lesions virus (TSWV) on stems, bronzing of leaves, wilting, deformed fruit with mottling and ringspots Tomato chlorotic Chlorosis, necrosis, mottle/mosaic, bronzing spot virus (TCSV) Chlorotic and necrotic ringspot, leaf mottling Capsicum chlorosis virus (CaCV) Distortion of leaves and fruits, chlorotic and necrotic Groundnut ringspot spots on newly developed leaves, terminal necrosis, virus (GRSV) and mottle Aphid-transmitted potyvirus Potato virus Y (PVY) Leaf mosaic or mottling, vein clearing, dark green vein banding, small and deformed fruit Tobacco etch virus Vein clearing, chlorotic, mottling or distortion of foliar tissues, stunting of plants, and necrosis of roots. (TEV) Pepper yellow Leaf curling, yellow–green mosaic, fruit deformation mosaic virus (PepYMV) Leaf mottling, mosaic, mottle, yellow vein banding, Chilli veinal mottle and wilting virus (ChiVMV) Pepper veinal mottle Mosaic, vein mottling, and stunted growth virus (PVMV) Aphid-transmitted cucumovirus Cucumber mosaic Curling, mosaic, vein banding, leaf mottling, and virus (CMV) malformation Contact-transmitted tobamoviruses Pepper mild mottle Mottling, puckering, malformed leaves, small and virus (PMMoV) deformed fruit marked by off-colored sunken areas, stunted growth Pepper severe mottle Mosaic, leaf deformation, stunted growth, necrotic virus (PepSMoV) streaks, and spots on fruits, stem, and leaves Leaf chlorosis, mosaic leaves, leaf distortion, and Tobacco mosaic arrested growth accompanied with small-sized fruits virus (TMV) Whitefly-transmitted geminivirus Pepper leaf curl virus Stunted growth, upward leaf curling, crowding of (PepLCV) leaves, swelling of veins, puckering of intravenous regions, blistering Tomato yellow leaf Curling and yellowing (a dead-end host) curl virus (TYLCV) Interveinal chlorosis and wrinkle of young leaves, Pepper golden apical necrosis, and stunting growth mosaic virus (PepGMV)

Reference Gitaitis et al. (1998) Almeida et al. (2014) McMichael et al. (2002) Webster et al. (2011)

Mijatovic et al. (2002) Murphy et al. (2021) Inoue-Nagata et al. (2002) Gao et al. (2016) Skelton et al. (2018) Deloko et al. (2022) Choi et al. (2004) Ahn et al. (2006) Kumar et al. (2011) Rai et al. (2010) Morilla et al. (2005) Brown et al. (2005)

Fabaceae, Liliaceae, and Solanaceae families including Capsicum species (Arpaci and Karatas 2020; Zhang et al. 2020; Ateş and Yilmaz 2020). The disease syndrome

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is commonly known as Phytophthora root rot, fruit rot, and foliar and stem blight that results up to 100% yield losses in most cases. P. capsici reproduces both sexually to produce the oospores as main survival propagules that remain viable for more than 10 years under extreme conditions and asexually to produce the mobile zoospores that spread the pathogen easily through wind, water, seeds, and soils. It makes almost impossible to control this pathogen though traditional disease control measures, and hence, P. capsici has emerged globally fifth most dangerous oomycete (Moreira-Morrillo et al. 2022). The resistance mechanism in C. annuum L. against P. capsici is not fully understood due to its complex physiological, genetic, and molecular properties (Quirin et al. 2005; Rabuma et al. 2022). A thick cell wall and high contents of the phenolic compounds and flavonoids serve as the initial barriers to pathogens (Piccini et al. 2019), followed by pathogen inhibition by antimicrobial compounds such as phytoalexins, hydrolytic enzymes (chitinase and glucanase), and proline- and hydroxyproline-rich proteins, reactive oxygen species (ROS), and capsidiol (Stoessl et al. 1972; Egea et al. 1996). Silencing of the CaChiVI2 gene related to the Chitin-binding protein (CBP) family increased susceptibility of peppers to heat and P. capsici infection (Ali et al. 2020). In addition, CaSBP08, CaSBP11, CaSBP12, and CaSBP13 are Squamosa promoter-binding protein (SBP) box (CaSBP) genes that regulate plant growth, development, stress responses, and signal transduction in pepper. The virusinduced gene silencing of these genes in Capsicum annuum L. revealed that the silencing of CaSBP08 gene in particularly increased resistance against P. capsici infection, reduced malondialdehyde content, peroxidase activity, and disease index percentage, and increased expression of other defense-related genes including CaBPR1 and CaSAR8.2. Significantly, the overexpression of CaSBP08 gene in Nicotiana benthamiana plants increased their susceptibility to P. capsici infection (Zhang et al. 2020). Pepper cultivars with different degree of resistance during P. capsici infection exhibited differential expression of defense-related genes including those encoding for basic PR protein (CABPR1), 1, 3-basic glucanase (CABGLU), a peroxidase (CAPO1), and a cyclase sesquiterpene (CASC1). The expression of the PR-1, peroxidase, and cyclase sesquiterpene genes was always higher in resistant cultivars in comparison with susceptible ones (Silvar et al. 2008). Similarly, higher expression of phenylalanine ammonia-lyase (PAL), a key enzyme of the phenylpropanoid biosynthesis, (1.57 times) was observed in infected resistant plants (CM-334) than susceptible plants (NMCA10399), suggesting their involvement in the resistance responses to P. capsici infection (Li et al. 2020). The plant host resistance for different P. capsici isolates has evolved separately and independently, as different resistance genes found associated against different isolates (Monroy-Barbosa et al. 2008). The Phytophthora resistance reported to be controlled by either a single dominant gene (Kim et al. 1990) or many genes with additive or epistatic effects (Lefebvre et al. 1996) in different resistant cultivars of C. annuum. Serrano Criollo de Morelos (CM334), a Mexican landrace that exhibits much higher resistance response to various ecologically different P. capsici isolates

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(Quirin et al. 2005), possesses major QTL on chromosome 5 (Pc5.1) closely linked to robust and broad-spectrum resistance in different genetic backgrounds (Mallard et al. 2013). Furthermore, there were reported of several candidate resistance genes such as CaDMR1, CaPhyto, Capana05g000764, Capana05g000769, PhR10 (Xu et al. 2016), and CaRGA2 (Rehrig et al. 2014; Zhang et al. 2013), and six linked chromosomal regions including Phyto4.1, Phyto5.1, Phyto5.2, Phyto6.1, Phyto11.1, and Phyto12.1 on different chromosomes (Majid et al. 2016). Zhang et al. (2013) found rapid increase (fivefold) in CaRGA2, a resistance gene analog, expression in CM334 (fivefold) than susceptible cultivar at 24-h post-P. capsici inoculation. Its silencing using virus-induced gene silencing (VIGS) induced disease symptoms in resistant plants after P. capsici infection, which indicated CaRGA2 as a potential resistance gene. Furthermore, it was also reported a resistance inhibitor gene, Ipcr, for P. capsici in a pepper variety NMCA10399 (Reeves et al. 2013). Kang et al. (2022) studied nine receptor-like proteins (RLPs) in C. annuum L. (CaRLPs) using VIGS of which knockdown of three genes including CaRLP264, CaRLP351, and CaRLP277 inhibited race-specific and non-race-specific resistance responses and consistently increased susceptibility to Ralstonia solanacearum and Phytophthora capsici as indicated by the lower hypersensitive response (HR). The functional characterization of these candidate resistance elements would help in understanding the molecular and cellular aspects of various mechanisms underlying resistance response in pepper plants to Phytophthora diseases (Choi and Hwang 2015). There are only few pepper varieties and landraces, which show consistent and similar resistant to all races of pepper pathogens and disease symptoms such as CM334 (Rabuma et al. 2022; Candole 2012). Various molecular markers linked to the resistance loci in chili pepper, especially CM334, are commonly used in molecular breeding to incorporate P. capsici resistance into different cultivars of chili pepper (Kumar et al. 2022) and bell pepper (Thabuis et al. 2004).

8.2.1.2 Colletotrichum Species Anthracnose disease is a seed-borne pepper disease of peppers caused by five different species of the genus Colletotrichum worldwide including C. capsici, C. acutatum, C. gloeosporioides, C. coccoides, and C. graminicola, (Than et al. 2008), which inhabit as hemibiotrophic or facultative biotrophic pathogens and responsible for more than 70% losses of pepper yields (Sahitya et al. 2014). In India, three main species such as Colletotrichum capsici (or C. truncatum), C. acutatum, and C. gloeosporioides were found to be associated with anthracnose of which C. truncatum is most lethal during ripening stage of the fruits (Kiran et al. 2020). The typical symptoms shown by infected pepper plants are dark sunken necrotic lesions with concentric rings, starting as sunset yellow and then turn as gray spots, and produce conidial masses in fruits. It leads to the rotting of fruits at both preharvest and postharvest stages (Rao and Nandineni 2017) and further reduces their dry weight and quantity of capsaicin and oleoresin. Apart from fruit rot, the pathogen also induces leaf spots, dieback on stem, seedling blight, or damping off in infected plants (Kiran et al. 2020).

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The potential genetic elements encoding factors such as subtilisins, carbohydrateactive enzyme (CAZyme) family of pectinases, FAD oxidases, pectin lyases, and cutinases and other proteases showed significant response to Colletotrichum infection (Baroncelli et al. 2016). Several transcription factors such as WRKY33, CaNAC, bZIP10, and CaMYB (Mishra et al. 2017), and genes encoding allene oxide synthase (AOS), lipoxygenase 3 (Lox3), chitinase (CcChiIII2), and ACC synthase 2 (ACS2) responsible for ethylene biosynthesis, and plant defensins 1.2 (PDF 1.2) responsible for JA biosynthesis confer resistance against anthracnose caused by C. truncatum (Ali et al. 2021). Antimicrobial peptides (AMPs) in pepper accession UENF1381 suppress activities of trypsin and amylase and significantly reduce C. scovillei proliferation (Da Silva et al. 2021). Recently, 18 of 79 C2H2 zinc finger transcription factors identified in C. annuum were differentially expressed during C. truncatum infection (Sharma et al. 2021). CaChi2, a pepper basic class II chitinase gene, is constitutively expressed in pepper’s fruit, leaf, and root endodermis upon invasion by C. coccodes (Hong and Hwang 2002). The etiology of the disease is quite complex as a single Colletotrichum species can infect many different plant hosts belong to the Malvaceae, Brassicaceae, Fabaceae, and Solanaceae families (Jayawardena et al. 2016), and various C. species can infect a single plant host at different growth stages, with varying pathogenicity and mechanisms with respect to the plant host (Freeman et al. 1998). This leads to poor understanding of genetic and molecular processes in relation to the anthracnose resistance in pepper (Kiran et al. 2020), which in turn make anthracnose management very difficult through traditional disease control measures (Saxena et al. 2016). Different Capsicum annuum L. cultivars show different resistance levels for different etiological agents of anthracnose, such as 83–168 breeding line resistant to Colletotrichum capsici 158ci, which is governed by a single dominant gene and chungryong variety resistance to Colletotrichum dematium, which is governed by partially dominant gene (Lin et al. 2002; Sahitya et al. 2014). The thaumatin-like protein encoded by pepper gene, PepTLP, improved resistance for Phytophthora blight and anthracnose, apart from its role in fruit ripening (De Souza et al. 2011). Five lines of C. annuum from AVRDC, Taiwan (AVPP0513, AVPP0207, AVPP1102-B, AVPP1004-B, and AVPP0719), are most promising pepper lines for various agronomic characters and anthracnose tolerance (Hasyim et al. 2014). Apart from these, Jinda, Acchar lanka, BS-20, CA-4, BS-35, BS-28, Punjab Lal, Taiwan-2, IC-383072, Bhut Jolokia Bangchang, 83–168 Pant C-1, and Lankamura Collection are resistant varieties that are employed in developing resistant pepper cultivars through breeding, and in genetic studies and mapping of the responsible resistance genes or loci (Mahasuk et al. 2016). Recently, various molecular markers including SNP markers closely linked to the anthracnose resistance QTLs designed from Capsicum annuum “Bangchang” × C. chinense “PBC932” and C. baccatum “PBC80” × “CA1316” populations. In former cross, the parents are clearly discriminated and identified two anthracnose resistance QTLs (RA932 g and RA932r) using 288 SNPs, whereas in later cross, three major resistance QTLs

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(RA80rP2, RA80rP3.1, and RA80rHP1) were identified using 510 polymorphic SNPs (Mahasuk et al. 2016).

8.2.1.3 Pythium spp. Pythium spp. are another soil-borne fungal pathogens that infect pepper hosts extensively in their younger or juvenile stages leading to the damping off and causing root rot in their mature stages (Arora et al. 2021). The major causal Pythium spp. are Pythium aphanidermatum, P. myriotylum, P. helicoides, and P. splendens (Chellemi et al. 2000). The associated disease development is stimulated by numerous carbohydrate active enzymes (CAZymes) such as carbohydrate esterases, polysaccharide lyases, proteases, and glycoside hydrolases, which promote penetration into plant cell wall and further colonization (Zerillo et al. 2013; Lévesque et al. 2010). Unlike Phytophthora and other pathogens, Pythium spp. do not have avirulence (Avr) factors such as RXLR effectors, indicating presence of only nonhost specificity and necrotrophic lifestyle (Adhikari et al. 2013; Arora et al. 2021). The complex etiology, absence of race-specific resistance, and high dispersion rate of the Pythium spp. prevent effective disease control measures, and only quantitative partial resistance is the more consistent mean for developing resistance against these pathogens (Klepadlo et al. 2019). 8.2.1.4 Leveillula taurica Leveillula taurica is an obligate biotrophic ascomycete, causing powdery mildew disease in pepper and many other crops including legumes, cereals, and model plants (Arabidopsis and tobacco). The leaf of the affected plants turns grayish white in patches underside and yellowish green lesions on opposite side with defoliation that leads to the photosynthetic rate reduction and slowing growth (Islam et al. 2022). The H3 and H-V-12 (H3 x Vania (susceptible)) are the two known resistant C. annuum varieties. (Anand et al. 1987), and the resistance is controlled by QTLs on chromosome 6 (Lt 6.1) with epistatic interactions explaining more than 50% of the genotypic variance (Lefebvre et al. 2003). At least three resistance genes in H3 cultivar (Daubèze et al. 1995), a single dominant locus, PMR1, on chromosome 4 and six molecular markers including SCAR and five SNPs, were found associated with powdery mildew resistance (Jo et al. 2017). 8.2.1.5 Virus Diseases Peppers are known to be the host of over 40 different viruses causing significant yield losses (Çelik et al. 2018) in which the major viruses are cucumber mosaic virus (CMV), potato virus Y (PVY), pepper mottle virus (PepMoV), and tobacco mosaic virus (TMV) (Villalon 1981; Özdemir 2021). The transmission of most of these viral pathogens is mediated through insect vectors such as aphids, thrips, and whiteflies (Kenyon et al. 2014), and the common symptoms in infected plants include chlorosis, abnormal foliar tissues, and necrosis (Murphy and Bowen 2006). For CMV, the resistance-associated mechanisms include viral multiplication inhibition, cell-to-cell inhibition, and others (Caranta and Palloix 1997; Manivannan et al. 2018), which show polygenic and incompletely dominant inheritance patterns.

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Three linked SNP markers and two genes Tm-1 (tomato mosaic resistant-1) and Cmr1 (cucumber mosaic resistance 1) were reported for CMV resistance in C. annuum “Bukang” of which a SNP marker named CaTm-int3HRM is located very close (2 cM) to the Cmr1 gene. These SNP markers are useful for identifying CMV-resistant pepper cultivars in a germplasm collection (Kang et al. 2010). Moreover, perennial, an Indian CMV-resistant hot pepper line, shows high resistance to CMV and commonly used in developing highly resistant pepper varieties (Lapidot et al. 1997). In case of PVY, the major resistance genes in C. annuum L. include pvr1 in Avelar, CM334, Yolo Y, and PI264281 I5491 (Dato et al. 2015; Yeam et al. 2005); pvr2 in PI264281, Yolo Y, Florida VR2135, and SC46252; pvr3 in Avelar; and pvr4, pvr5, and pvr8 in CM334 (Janzac et al. 2008). In addition, various QTLs attributing full and partial resistance to some PVY isolates such as To72 and Son41 identified near to the pvr2 and pvr6 (Caranta and Palloix 1997). The recessive alleles of pvr21 and pvr22 genes contribute to the resistant against PVY-0 and PVY-1 strains of PVY. These genes encode a translation eukaryotic initiation factor 4E (eIF4E) in pepper (Ruffel et al. 2002), which interact with the potyviral genomelinked protein (VPg) leading to the viral multiplication and PVY resistance breakdown (Léonard et al. 2000), whereas the mutations in the concerned genes undergo incompatible host–virus interaction, leading to viral resistance (Lellis et al. 2002). Furthermore, two genes, Pr4 (dominant) and pr5 (recessive), providing resistance against all known PVY strains have been identified in SCM334 (Dogimont et al. 1996; Parisi et al. 2020). A dominant potyvirus resistance gene, Pr4 (Pvr4), induces durable resistance against many different potyviruses including all known isolates of potato virus Y (PVY) and pepper mottle virus (PepMoV) (Dogimont et al. 1996) in Capsicum annuum. In addition, several molecular markers tightly linked to the L locus genes (L3 and L4) known for resistance against PMMoV were identified in the pepper and useful in PMMoV resistance breeding (Kim et al. 2008; Matsunaga et al. 2003). The resistance against Japanese strain is linked to the temperature-insensitive resistance allele L1a in bell pepper (Sawada et al. 2005), which elicits by the viral coat protein of the P0 pathotype of tobamoviruses (Matsumoto et al. 2008). Microarray analysis revealed molecular aspects of cytosolic pyruvate kinase 1 (CaPK(c)1) gene induction and its association with the TMV resistance response. This gene stimulates hypersensitive response (HR), salicylic acid (SA), ethylene, and methyl jasmonate (MeJA) production in C. annuum cv. Bugang during TMV (TMV-P (0)) infection (Kim et al. 2006). Various transcription factors include CaWRKYd that bind to the W-box containing promoters of PR genes involved in the positive regulation of immune response to TMV-P0 infection. In addition, basic transcription factor 3 (CaBtf3) regulates the PR-related gene expression during HR upon TMV infection in C. annuum L. (Huh et al. 2012). Several breeding programs initiated for incorporation of resistance genes from resistant varieties into commercial pepper varieties such as two recessive alleles (pvr11 and pvr12) of the pvr1 locus that linked to the susceptibility for viral infection encode elF4E homologs that unable to bind to the VPg and induced resistance to potyviruses including TEV (Kang et al. 2005). It was also developed a superior

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pepper line being resistant to three viruses PVY, TSWV, and PMMoV, using molecular markers linked to the Pvr4, Tsw, and L4 locus (Özkaynak et al. 2014). These markers are useful in selection of the resistant pepper genotypes (Dato et al. 2015). Recently, it was identified that TSWV and PMMoV isolates can break resistance to Tsw and L3 in pepper such as a Tsw resistance breaking strain TSWV-P1 in a commercial C. annuum variety in South Korea (Yoon et al. 2021). PMMoV coat protein containing substitution of two amino acids reversed L3 (Hamada et al. 2002) and L4 r mediated resistance in C. annuum L. varieties (Genda et al. 2011). A well-adopted antiviral resistance mechanism in bell pepper is the mature plant resistance or age-related resistance to CMV (Garcia-Ruiz and Murphy 2001) because resistance induced in early growth stage in infected plants is readily overcome by evolution of different resistance breaking isolates. As a consequence, a more dangerous CMV pathotype Ca-P1-CMV was identified that can break the resistance of the P0-CMV-resistant pepper cultivar variety (Islam et al. 2022).

8.2.1.6 Xanthomonas campestris pv. vesicatoria (Xcv) Xanthomonas campestris pv. vesicatoria (Xcv) is an etiological agent of the bacterial leaf blight in Capsicum. CaPO2 is a putative resistance gene against Xcv (Choi et al. 2007) for which the loss-of-function mutation enhanced susceptibility to the X. campestris, leading to cell death and increased ROS (Zheng et al. 2013). On contrary, CaMLO2, a membrane-bound amphiphilic Ca2 + -dependent calmodulinbinding protein, interacts with CaCaM1 and transports it from cytoplasm to plasma membrane and induced rapid Xcv growth by disrupting downstream signaling in pepper and Arabidopsis to inhibit resistance response (Kim and Hwang 2012). Silencing of the CaMLO2 gene by VIGS induced ROS production, cell death, and defense response against Xcv, and increased expression of the CaPR1 (PR-1), CaCaM1, and CaPO2 (peroxidase) (Kim et al. 2014a, b, c). CaLOX1 is another potential defense-related gene encoding nine specific lipoxygenase, which upon overexpression induced tolerance against Pseudomonas syringae pv. tomato, Hyaloperonospora arabidopsidis, and Alternaria brassicicola (Hwang and Hwang 2010), and could be used for generating stress tolerance pepper varieties. Other resistance genes in peppers include BS4C that recognizes Xanthomonas campestris TALE protein AvrBs4 (Schenke et al. 2020) and Bs3 that recognizes effector avrBS3 through TALE binding site in their promoters (Boch et al. 2014). There are many genes in pepper that involved defense responses against multiple pathogens such as genes encoding different lipid transfer proteins I and II, thionin, osmotin (PR-5), SAR 8.2, stellacyanin, leucine-rich repeat protein, chitinase, auxinrepressed protein, and β-1,3-glucanase that are induced by Xanthomonas campestris or P. capsici (Jung and Hwang 2000a; b). Similarly, CALRR1 gene is another defense gene that encodes leucine-rich repeat proteins (LRRs) and predominantly expressed only in the pepper plants infected by Colletotrichum coccodes, P. capsici, and X. campestris. A transcriptional factor encoded by CABGLU gene was highly expressed during incompatible interactions and transcribed only in the roots (Majid et al. 2016).

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Table 8.3 List of genetic elements/genes conferring pepper resistance to different pathogens Gene CaRGA2

Product Blight resistance proteins Hypersensitiveinduced reaction (HIR) protein Polygalacturonaseinhibiting proteins (PGIPs) Peroxidase (POD) β-1,3-glucanase Capsicum annuum pathogenesis-protein 4 SAR8.2 protein

Resistance P. capsici

CATHION1

Gamma-thionin 1 precursor

CAOSM1

Osmotin-like protein

CALRR1

Leucine-rich repeat protein Methionine sulfoxide reductase B2 Homeodomain– leucine zipper I RD receptor-like kinase Chitinase Cysteine-rich receptor-like kinase (CRK) WRKY transcriptional factor Zinc finger (ZNF) transcription factor Basic helix-loop-helix transcription factor N-methyltransferase

P. capsici; Xanthomonas campestris pv. vesicatoria P. capsici; Colletotrichum coccodes P. capsici and X. campestris P. capsici

CaHIR1

CaPGIP2

CanPOD CaBGLU CaPR4

CASAR82A

CaMsrB2 CaHDZ27 CaLRR-RLK1 ChiIV3 CaCRK5

CaWRKY22 CaZNF830 CabHLH113 CaASHH3 cmv11.1 Cucumber mosaic resistance 2 (cmr2)

Reference Zhang et al. (2013) Jung et al. (2008)

Wang et al. (2013b)

Wang et al. (2013a) Yang et al. (2014) Yang et al. (2014)

Ralstonia solanacearum

Lee and Hwang (2006) Lee et al. (2000)

Hong et al. (2004) Kim et al. (2014a) Hong Truong et al. (2013) Mou et al. (2017) Mou et al. (2019) Liu et al. (2019) Mou et al. (2021)

Husssain et al. (2018) Noman et al. (2018)

Bacterial pathogens Cucumber mosaic virus

Husssain et al. (2021) Husssain et al. (2022) Yao et al. (2013) Choi et al. (2018)

(continued)

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Table 8.3 (continued) Gene Cmr1 pvr’s (4E (eIF4E));

Product

Resistance Potato virus Y (PVY)

PVY, PepMoV, PMMoV PVMV-HN pepy-1 L1a a

Pepper yellow leaf curl Tobamoviruses

Hk L Me4, Mech1, Mech2 N.

Paprika mild mottle virus (PaMMV) Root-knot nematodes

CaMi Me1 to Me7 CaChitIV

Class IV chitinase

Bs

Bacterial spot

Bs5 CaPO2

CYSTM protein Peroxidase

CaMLO2

Mildew resistance locus O

CaCaM1 CaChi2

Calmodulin 1 Class II chitinase

Xanthomonas campestris pv. vesicatoria (Xcv) Xanthomonas campestris

Fusarium oxysporum

Reference Kang et al. (2010) Ruffel et al. (2002); Hwang et al. (2009a, b) Rubio et al. (2008) Gao et al. (2014) Pohan et al. (2022) Matsumoto et al. (2009) Sawada et al. (2005) Tomita et al. (2011) Djian-Caporalino et al. (2001, 2007) Thies and Ariss (2009) Chen et al. (2007); Fazari (2012) Changkwian et al. (2019) Kim et al. (2015)

Romer et al. (2010); Vallejos et al. (2010) Jones et al. (2002) Choi and Hwang (2012) Kim and Hwang (2012); Zheng et al. (2013) Kim et al. (2014a) Ferniah et al. (2018)

Different reported resistance and susceptible genes that serve as targets for attributing resistance in C. annuum L. for traditional and genetic engineeringbased approaches of pepper improvement are summarized in Tables 8.3 and 8.4.

8.2.2

Exploring Genetic Components of Disease Resistance in Crop Plants

The plant defense system mainly constitutes of inherent physical barriers and innate pattern-triggered immunity (PTI; first layer of defense) and effector-triggered immunity (ETI; second line of defense), and does not have any adaptive immune response

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Table 8.4 List of some of the susceptibility genes in C. annuum L. Susceptibility gene CaWRKY58

Product WRKY transcription factor

Pathogen Ralstonia solanacearum

Expansin

Xanthomonas Campestris P. capsici

CaWRKY40b UPA7 Ipcr UPA20 pvr2

pvr6

bHLH transcription factor, induces expression of UPA7 Eukaryotic (translation) initiation factor eIF4E Eukaryotic (translation) initiation factor eIF4E and eIF (iso)4E

Potyviruses (TEV, PepVMV, ChiVMV, PVY) Potyviruses (PVMV, ChiVMV)

Reference Wang et al. (2013a, b, c) Khan et al. (2018) Kay et al. (2007) Reeves et al. (2013)

Charron et al. (2008) Hwang et al. (2009a, b)

such as animals. During pathogen invasions, both local responses and systemic signaling activate in plant to induce disease resistance. Local defense responses lead to the localized hypersensitive response (HR) to prevent pathogen spread, whereas the systemic defense signaling primarily involves several phytohormone signaling pathways and reprogramming of the transcriptomes, proteomes, and metabolomes to protect noninfected plant parts (Dangl and Jones 2001; Choi and Hwang 2015). Thus, coordinated actions of PTI and ETI are essential for successful plant protection against pests and pathogens. Furthermore, latest findings revealed the transgenerational passage of immunological memory associated with SAR (systemic acquired resistance) (Backer et al. 2019). Hypersensitive response (HR) is an intrinsic programmed cell death mechanism activated by recognition of the pathogen’s effector proteins by intracellular receptors, that is, resistance (R) proteins. It is more effective against biotrophic pathogens than necrotrophic pathogens and can benefit the plant hosts only during early stage of the infection by hemibiotrophic pathogens (Münch et al. 2008; Jupe et al. 2013). Pepper plants utilize HR to respond effectively to various pathogen attacks, and thus, the genes or DNA elements triggering HR should be characterized at structural and functional levels to define their roles and delineate molecular and biochemical basis of the HR-mediated cell death in pepper (Choi and Hwang 2015). Disease resistance in plants is broadly classified into two categories: qualitative and quantitative resistances. Qualitative resistance is driven by individual resistance (R) genes that encode R proteins to interact with pathogen-specific effectors to initiate effector-triggered immunity (ETI) and induce complete resistance against a particular strain or race of the pathogen. In this case, the novel resistance genes are originated by duplication and diversification of the existing resistance genes to acquire the distinct functional specificity for recognizing newly evolved effector proteins secreted by pathogens (Kim et al. 2017a, b). Such kind of resistance is race-

182 Fig. 8.1 Functional genomic approaches to detect and map the defense responsive elements

S. Kumar and A. Singh Forward Genetics

Reverse Genetics

Mutagenesis

Selection of mutant phenotype

Alter gene expression pattern

Mapping and identification of the mutated DNA sequence or gene

Analyzing phenotypic effects

specific and not durable, and easily overcome by pathogens through even single or multiple mutations in the avirulence genes. On contrary, quantitative resistance is non-race-specific resistance with higher durability and complex inheritance as it is controlled by many genes or QTLs with moderate to small effects. It is driven by multiple nonspecific cellular phenomenon including thickening of cell wall, production of reactive oxygen species (ROS), phytoalexins, pathogenesis-related (PR) proteins, antimicrobial peptides (AMPs), pathogen enzyme inhibitors, and detoxification of mycotoxins (Khaliluev and Shpakovskii 2013). However, quantitative resistance is only a partial resistance that actives against all genotypes of a pathogen or even against different pathogens. It is highly affected by the ecological conditions and physiological state of the host plants (Miedaner et al. 2020). Genetic variations in relation to disease resistance in plants, even those of the same species, describe implications of different genetic backgrounds on resistance response. Frequent interactions between plants and pathogens could shape the host plant genome through coevolution processes as selection pressures imposed by infecting pathogens produced certain specific genomic signatures in infected plants. Hence, genetic resistance is considered a most feasible mean of controlling different diseases in crop plants (Dolatabadian and Fernando 2022). The potential genetic elements conferring disease resistance in plants can be identified through: 1. Effector-based identification of R genes: Nowadays, plant resistance genes can be identified and functionally characterized with the help of concerned pathogen’s effectors, which also facilitate the deployment of resistance genes using conventional breeding and genetic engineering. Significantly, the catalogues of entire effector repertoires for many economically important plant pathogens have been developed such as the pathogen causing potato late blight disease. In this case, 54 effectors containing a signal peptide and a RXLR motif that activate innate immune response in potato were analyzed in wild Solanum species to identify tentative Avr candidate genes, and their cognate R genes in potato (Vleeshouwers et al. 2008).

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2. Functional genomic approaches to determine the genetic basis of disease severity and resistance in plants such as forward genetics (positional cloning, mapping by sequencing (MBS), exome sequencing, candidate gene approach, etc.) and reverse genetics (TILLING, transposon/T-DNA-mediated insertional mutagenesis, virus-induced gene silencing (VIGS), RNA interference, and genome editing) (Fig. 8.1). Next-generation sequencing (NGS) is extensively used in both effector-based and functional genomic-based identification of the resistance loci and closely linked DNA markers in plants. It enables to identify differentially expressed genes in plant–pathogen interaction, and also, the small RNAs of host and/or pathogen origin with significant implications on the disease development and defense responses (Dolatabadian and Fernando 2022). It helps to establish possible regulatory molecular mechanisms in resistant and susceptible pepper genotypes and other crops, and also determines the potential breeding targets and predicts the possible implications of their manipulation to improved resistance (Schenke et al. 2020).

8.2.2.1 Modern Approaches of Introducing Disease Resistance in Capsicum annuum L. Wild relatives of domesticated crop species harbor multiple, diverse disease resistance (R) genes that could be used to engineer plants using conventional and nonconventional crop improvement approaches (Alemayehu 2017). Conventional or traditional plant breeding approaches rely on hybridization and selection and allow incorporation of many resistance genes simultaneously from wild resistant lines into susceptible elite cultivars. However, they have very low success rate because of the polygenic nature of the disease resistance, high evolutionary rate, and broader host range of the pathogens with complex pathogenicity mechanisms (Islam et al. 2022). It often takes 5–15 years and several generations to develop a resistant variety with useful agronomic traits (such as high yielding) without any linkage drag (Alemayehu 2017). Furthermore, these methods are restricted to only sexually compatible crops and limited by very low frequency of natural variation for resistance trait and phenotypic selection. They are laborious, time-consuming, and costly (Schenke et al. 2020). Several newly emerging techniques outweigh the demerits of conventional breeding such as target-induced local lesions in genomes (TILLING) that increase the frequency of genetic variation and identifying allelic polymorphisms in the candidate resistance genes, and the marker-assisted selection that allows indirect selection of desirable resistance alleles (Islam et al. 2022). Molecular markers allow identification of the undesirable genotypes in the early stage of plant life cycle as they are not affected by the environment and physiological states of the plants, and applicable even when resistance trait has recessive or polygenic inheritance. It enhances both precision and efficiency of the selection for developing diseaseresistant varieties in a short duration (Chowdhury et al. 2020). Recent advances in genetic engineering speed up modifications of the plant structural barriers and other defense components, and create genetic diversity and

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discover new allelic variants with beneficial consequences on stress tolerance (Xu et al. 2022). These nonconventional techniques include RNA interference (RNAi), gene editing, activation tagging, and enhancer trapping, which all are based on either knock-up or knockdown or knockout approach to identify the phenotypic relevance of all the genetic elements with respect to the disease severity and tolerance in plants and pathogens. Genome editing is the latest advancement in genetic engineering that allows target-specific modifications of genomic regions in almost all kinds of eukaryotic organisms using engineered or bacterial nucleases such as meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (Park et al. 2021). They modify endogenous plant DNA by small deletions, insertions, and replacements, and introduce double-stranded breaks at predetermined sites that repair through either errorprone nonhomologous end joining (NHEJ) or error-free homologous recombination (HR). The resulted genetically modified plants may or may not be transgenic (Dong and Ronald 2019) and have more acceptable rate than traditional transgenic plants. Therefore, these genetic engineering methods provide ecologically sustainable and economically viable alternatives that address the issues of rapidness and precision and create new allelic forms for the same resistance gene or to introduce specific resistance genes into elite crop cultivars from any source across species regardless of the sexually incompatibility (Koseoglou et al. 2021).

8.2.2.2 Genetic Targets to Engineering Plants for Disease Resistance 1. Pathogen factors such as effectors and other proteins associated with pathogen colonization and pathogenicity. CRISPR/Cas9 constructs containing single guide RNA (sgRNAs) specific to viral Rep, or IR region or CP were used to attribute resistance against CLCuMuV (Yin et al. 2019) and tomato yellow leaf curl virus (TYLCV) in both transgenic tomato and Nicotiana benthamiana lines (Tashkandi et al. 2018; Shingote et al. 2022). 2. Plant-associated factors such as deployment of specialized cell surface immune sensors and/or intracellular receptor traps, disabling of susceptibility genes (Barka and Lee 2022) and production of pathogen-related proteins (PR) or antimicrobial peptides (AMPs) (Majid et al. 2016). For example, editing of plant-/host-specific translation factors such as eIF4E, eIF4G, and their isoforms (pro-viral factors) was associated with virus protein translation. 3. Insect vector-associated proteins: the transformed plant host with whiteflyspecific insecticidal proteins such as Tma12 restricted spread of the vectors and ultimately control the ChiLCV infection (Shingote et al. 2022; Dong and Ronald 2019). Peppers are recalcitrant to in vitro genetic transformation and thus rely heavily on classical and molecular breeding approaches for genetic improvement (Kumar et al. 2008). However, suitable vector, transformation mediator, and protocol standardization required for C. annuum L. transformation have been well

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established. In a study, pepper explants were inoculated with Agrobacterium tumefaciens carrying pCAMBIA1301 with hygromycin phosphotransferase (hpt) and uidA marker genes to undergo stable transformation of hot pepper. Southern and northern hybridization validated the stable integration and expression of hpt and uidA genes in transgenic pepper plants (T0) and their inheritance to subsequent progeny (T1). The T1 progenies obtained by selfing were segregated in the 3:1 ratio for hygromycin resistance, indicating the presence of one copy of the T-DNA in transgenic lines and homozygous transgenic progenies (Ko et al. 2007). Bentgrass (Agrostis stolonifera) transformed with Pepper esterase (PepEST) gene which gets stably integrated into host genome and expressed as confirmed by Southern and Northern blot analysis. The transformed plants suppressed the growth of Rhizoctonia solani, mycelia as indicated by low disease severity (10%) than non-transformed plants (50%) (Cho et al. 2011). Pathogen-derived resistance (PDR) is another effective approach to develop virus resistance crops (Powell et al. 1990). It is based on the post-transcriptional gene silencing (PTGS) mechanism or RNAi that employs virus-derived genes such as viral coat protein, defective-interfering RNA (DI-RNA), and replicase viral satellite RNA (Brummell and Pathirana 2007), to generate resistance against them. RNAi is an innate antiviral defense system in plants and other eukaryotes guided by doublestranded RNA (dsRNA) or small RNAs to facilitate sequence-specific viral RNA cleavage by RISC complex. PDR has been successfully used in peppers to restrict the spread of ChiLCV infection through knocking up the expression of AC1-/AC2-/ βC1-specific dsRNA, which had targeted various ChiLCV species (Sharma et al. 2015). Furthermore, Mishra et al. (2020) identified miRNAs specific to important ChiLCV genes in chili using in silico tools, which target CP (V1) and Rep (C1) genes of ChiLCV to develop resistance against it. The major limitations of RNAi are its inability to completely silence the target genes and off-target effects that make it less feasible under natural conditions having high viral titer and rapid evolution of new virulent viral strains such as ChiLCV variants that can escape easily sequence-specific recognition of RNAi. Furthermore, the use of RNAi is further limited by strict government policies in many countries and unfavorable public perception toward transgenic or genetically modified (GM) crops (Shingote et al. 2022). Alternatively, genome-engineering tools are precise and can produce transgene-free crops. It has potential to deploy recessive resistance as for potyvirus resistance mediated by eukaryotic translation initiation factor 4E (eIF4E) variants in several resistant crops including pepper (Capsicum annuum L.), wild tomato (Solanum habrochaites), and lettuce (Lactuca sativa) (Van Esse et al. 2020; Ruffel et al. 2002). In particular, the CRISPR/Cas system is more convenient due to its simplicity to design construct as compared to the transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFN). Therefore, it is frequently used for engineering plants for biotic and abiotic stress tolerance (Shingote et al. 2022). Agrobacterium-based CRISPR/Cas gene editing system has been successfully utilized to modify MLO gene (CaMLO2) in both callus-derived protoplasts and leaf protoplasts from CM334 and bell pepper cultivar Dempsey (Kim et al. 2020). In this

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Table 8.5 Examples of genetic engineering in disease management of Capsicum annuum L. Transgene/modified gene Coat protein

Pathogen Chilli leaf curl virus (ChLCuV)

Coat protein (cmv_cp) CMV satellite RNA CMV-CP CMV-CP + TMV-CP CMV-CP + ToMV-CP TMV-CP RB (from Solanum bulbocastanum) Chitinase

Cucumber mosaic virus CMV

Me1 Pepper esterase (PepEST) J1-1 (defensin) Coat protein (CP) Viral sequence

Multiple virus Resistance TMV P. capsici Alternaria alternate and Colletotrichum capsici RKN (root-knot nematodes) Anthracnose fungus (Colletotrichum gloeosporioides) Cucumber mosaic virus (CMV) Pepper mild mottle virus (PMMoV)

Reference Raghunathachari et al. (2012) Oerke (2006) Kim et al. (1997) Zhu et al. (1996) Cai et al. (2003) Shin et al. (2002) Lee et al. (2004) Bagga et al. (2019) Mythili et al. (2015) Toth et al. (2021) Ko et al. (2016) Seo et al. (2014) Chen et al. (2003) Tenllado and DıazRuız (2001)

study, complexes of CRISPR/Cas9 or Cpf1 containing ribonucleoproteins (RNPs) and endonuclease, and single guide RNA in binary vector (pBAtC) delivered through PEG-mediated genetic transformation in C. annuum cvs. CM334 Dempsey. The efficacy was highest for sweet pepper cultivar with GV3101 strain of A. tumefaciens, while no variation was observed in CM334 for all bacterial strains (AGL1, EHA101, and GV3101) as indicated by the number of calli induced by different A. tumefaciens strains. Roy et al. (2019) designed multiplexed sgRNA targeting ChiLCV genome for genetic modification of chili against devastating ChiLCD disease. The overexpression of Tma12 gene has insecticidal activity against whitefly and derived from an edible fern (Tectaria macrodonta), in transgenic cotton lines induced resistant to whitefly attack and cotton leaf curl viral disease without affecting yield component. Such biopesticides can be expressed in chili to prevent whitefly-borne ChiLCV (Shukla et al. 2016). Mishra et al. (2021) employed CRISPR/Cas9 system to obtain T-DNA-free homozygous mutant lines containing desired modification in a susceptibility gene CaERF28 of susceptible chilli genotype Arka Lohit. The modified genotype showed improvement in expression of the defense responsive genes and exhibited strong resistance to Colletotrichum truncatum and associated disease. The resulted allelic mutants of C-ERF28 governed resistant trait that inherited to the subsequent generations while following Mendelian inheritance, as shown by the segregation patterns of T1 and T2 generations. It also led to identify T-DNA-free and markerfree C-ERF28 mutant lines and five homozygous mutants of C-ERF28, with improved anthracnose. Some of the reports of using genetic engineering to prevent

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the spread of different pathogens and developing resistance C. annuum L. varieties are mentioned in Table 8.5.

8.3

Conclusions

Genetic engineering-based crop improvement methods are more precise and efficient to utilize different genetic targets present in the resistant cultivars, wild relatives of C. annuum L. genetically distant plants (wild or cultivated), or even other organisms including animals and microorganisms, to produce disease-resistant pepper varieties. This can be achieved in a shorter duration, without or with minimal undesirable effects arising due to the linkage drag. Furthermore, it can be used to identify and conquer the functions of all the potential genetic elements in plant hosts and pathogens associated with resistance response in peppers, using targeted genetic knockdown, knockout, and knock-up approaches. It could be useful in dissecting the quantitative resistance or QTLs associated with resistance against P. capsici, Colletotrichum species, and other pathogens with similar resistant response in peppers.

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Jung HW, Hwang BK (2000a) Isolation, partial sequencing and expression of pathogenesis-related cDNA genes from pepper leaves infected by Xanthomonas campestris pv. vesicatoria. Mol Plant-Microbe Interact 13:136–142 Jung HW, Hwang BK (2000b) Pepper gene encoding a basic b-1,3- glucanase is differentially expressed in pepper tissues upon pathogen infection and ethephon or methyl jasmonate treatment. Plant Sci 159:97–106 Jung HW, Lim CW et al (2008) Distinct roles of the pepper hypersensitive induced reaction protein gene CaHIR1 in disease and osmotic stress, as determined by comparative transcriptome and proteome analyses. Planta 227(2):409–425 Jupe J, Stam R et al (2013) Phytophthora capsici-tomato interaction features dramatic shifts in gene expression associated with a hemi-biotrophic lifestyle. Genome Biol 14:R63 Kang BC, Yeam I et al (2005) The pvr1 locus in Capsicum encodes a translation initiation factor elF4E that interacts with Tobacco etch virus VPg. Plant J 42:392–405 Kang WH, Hoang NH et al (2010) Molecular mapping and characterization of a single dominant gene controlling CMV resistance in peppers (Capsicum annuum L.). Theor Appl Genet 120: 1587–1596 Kang WH, Lee J et al (2022) Universal gene co-expression network reveals receptor-like protein genes involved in broad-spectrum resistance in pepper (Capsicum annuum L.). Hortic Res 5:9 Kay S, Hahn S et al (2007) A bacterial effector acts as a plant transcription factor and induces a cell size regulator. Science 318(5850):648–651 Kenyon L, Kumar S et al (2014) Virus diseases of peppers (Capsicum spp.) and their control. Adv Virus Res 90:297–354 Khaliluev M, Shpakovskii G (2013) Genetic engineering strategies for enhancing tomato resistance to fungal and bacterial pathogens. Russ J Plant Physiol 60(6):721–732 Khan I, Zhang Y et al (2018) CaWRKY40b in pepper acts as a negative regulator in response to Ralstonia solanacearum by directly modulating defense genes including CaWRKY40. Int J Mol Sci 19:1403. https://doi.org/10.3390/ijms19051403 Kim DS, Hwang BK (2012) The pepper MLO gene, CaMLO2, is involved in the susceptibility celldeath response and bacterial and oomycete proliferation. Plant J 72:843–855 Kim B, Hur J et al (1990) Inheritance of resistance to bacterial spot and to Phytophthora blight in peppers. J Korean Soc Hort Sci 31:350–357 Kim SJ, Lee SJ et al (1997) Satellite-RNA-mediated resistance to cucumber mosaic virus in transgenic plants of hot pepper (Capsicum annuum cv. Golden Tower). Plant Cell Rep 16: 825–830 Kim KJ, Park CJ et al (2006) Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum. Plant Cell Rep 25:359–364 Kim HJ, Han JH et al (2008) Development of a sequence characteristic amplified region marker linked to the L4 locus conferring broad spectrum resistance to tobamoviruses in pepper plants. Mol Cells 25:205–210 Kim DS, Choi HW et al (2014a) Pepper mildew resistance locus O interacts with pepper calmodulin and suppresses Xanthomonas AvrBsT-triggered cell death and defense responses. Planta 240(4):827–839 Kim DS, Kim NH et al (2014b) Glycine-rich rna binding protein interacts with receptor-like cytoplasmic protein kinase and suppresses cell death and defense responses in pepper (Capsicum annuum). New Phytol 205(2):786–800 Kim S, Park M et al (2014c) Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet 46(3):270–278 Kim DS, Kim NH et al (2015) The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses. J Exp Bot 66(7):1987–1999 Kim SB, Kang WH et al (2017a) Divergent evolution of multiple virus-resistance genes from a progenitor in Capsicum spp. New Phytol 213(2):886–899

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Kim S, Park J et al (2017b) New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication. Genome Biol 18:210 Kim H, Choi J et al (2020) A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum. BMC Plant Biol 20:449 Kiran R, Akhtar J et al (2020) Anthracnose of chilli: status, diagnosis, and management. In: Capsicum. IntechOpen, London Klepadlo M, Balk CS et al (2019) Molecular characterization of genomic regions for resistance to Pythium ultimum var. ultimum in the soybean cultivar Magellan. Theor Appl Genet 132:405– 417 Ko MK, Soh H et al (2007) Stable production of transgenic pepper plants mediated by Agrobacterium tumefaciens. HortScience 42(6):1425–1430 Ko M, Cho JH et al (2016) Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants. Planta 244(2):379–932 Koseoglou E, van der Wolf JM et al (2021) Susceptibility reversed: modified plant susceptibility genes for resistance to bacteria. Trends Plant Sci 27(1):69–79 Kumar AM, Reddy KN et al (2008) Towards crop improvement in bell pepper (Capsicum annuum L.): Transgenics (uid A:: hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach. Sci Hortic 119(4):362–370 Kumar S, Udaya AC et al (2011) Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR. Lett Appl Microbiol 53:359–363 Kumar M, Kambham MR et al (2022) Identification of molecular marker linked to resistance gene loci against Indian isolate of Phytophthora capsici L. causing root rot in chilli (Capsicum annuum L.). Australas Plant Pathol 51(2):211–220 Lapidot M, Paran I et al (1997) Tolerance to cucumber mosaic virus in pepper: development of advanced breeding lines and evaluation of virus level. Plant Dis 81:185–188 Lee SC, Hwang BK (2006) CASAR82A, a pathogen induced pepper SAR8. 2, exhibits an antifungal activity and its overexpression enhances disease resistance and stress tolerance. Plant Mol Biol 61(1–2):95–109 Lee SC, Hong JK et al (2000) Pepper gene encoding thionin is differentially induced by pathogens, ethylene and methyl jasmonate. Physiol Mol Plant Pathol 56(5):207–216 Lee YH, Kim HS et al (2004) Anew selection method for pepper transformation: callus-mediated shoot formation. Plant Cell Rep 23:50–58 Lefebvre V, Daubèze AM et al (2003) QTLs for resistance to powdery mildew in pepper under natural and artificial infections. Theor Appl Genet 107:661–666 Lefebvre V, Palloix A et al (1996) Both epistatic and additive effects of QTLs are involved in polygenic induced resistance to disease: a case study, the interaction pepper-Phytophthora capsici Leonian. Theo. Appl Genet 93:503–511 Lellis AD, Kasschau KD et al (2002) Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for elF(iso)4E during potyvirus infection. Curr Biol 12:1046–1051 Léonard S, Plante D et al (2000) Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J Virol 74:7730–7737 Lévesque CA, Brouwer H et al (2010) Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire. Genome Biol 11:R73 Li Y, Yu T et al (2020) The dynamic transcriptome of pepper (C. annuum) whole roots reveals an important role for the phenylpropanoid biosynthesis pathway in root resistance to P. capsici. Gene 728:144288 Lin Q, Kanchana Udomkarn C et al (2002) Genetic analysis of resistance to pepper anthracnose caused by Colletotrichum capsici. Thai J Agric Sci 35:259–264 Liu Z, Shi L et al (2019) ChiIV3 Acts as a Novel Target of WRKY40 to Mediate Pepper Immunity Against Ralstonia solanacearum Infection. MPMI 32. https://doi.org/10.1094/MPMI-11-180313-R

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Mahasuk P, Struss D et al (2016) QTLs for resistance to anthracnose identified in two Capsicum sources. Mol Breed 36(1):10 Majid MU, Awan MF et al (2016) Phytophthora capsici on chilli pepper (Capsicum annuum L.) and its management through genetic and bio-control: a review. Zemdirbyste-Agriculture 103(4): 419–430 Mallard S, Cantet M et al (2013) V. A key QTL cluster is conserved among accessions and exhibits broad-spectrum resistance to Phytophthora capsici: A valuable locus for pepper breeding. Mol Breed 32:349–364 Manivannan A, Kim JH et al (2018) Next-generation sequencing approaches in genome-wide discovery of single nucleotide polymorphism markers associated with pungency and disease resistance in pepper. Biomed Res Int 2018:7. (Article ID 5646213) Matsumoto K, Sawada H et al (2008) The coat protein gene of tobamovirus P0 pathotype is a determinant for activation of temperature-insensitive L 1agene-mediated resistance in Capsicum plants. Arch Virol 153:645–650 Matsumoto K, Johnishi K et al (2009) Single amino acid substitution in the methyltransferase domain of Paprika mild mottle virus replicase proteins confers the ability to overcome the high temperature-dependent Hk gene-mediated resistance in Capsicum plants. Virus Res 140(1–2): 98–102 Matsunaga H, Saito T et al (2003) DNA markers linked to Pepper mild mottle virus (PMMoV) resistant locus (L4) in Capsicum. J Jpn Soc Hortic Sci 72:218–220 McGrath MT, Shishkoff N et al (2001) First occurrence of powdery mildew caused by Leveillula taurica on pepper in New York. Plant Dis 85:1122 McMichael LA, Persley DM et al (2002) A new tospovirus serogroup IV species infecting capsicum and tomato in Queensland, Australia. Australas Plant Pathol 31:231–239 Miedaner T, Boeven AL et al (2020) Genomics-assisted breeding for quantitative disease resistances in small-grain cereals and maize. Int J Mol Sci 21(24):9717 Mijatovic M, Ivanovic M et al (2002) Potato virus Y (PVY) on pepper in Serbia. Acta Hortic 579: 545–549 Mishra R, Nanda S et al (2017) Differential expression of defense related genes in chilli pepper infected with anthracnose pathogen Colletotrichum truncatum. Physiol Mol Plant Pathol 97:1– 10 Mishra M, Kumar R et al (2020) In silico analysis of chili encoded miRNAs targeting chili leaf curl Begomovirus and its associated satellite. J Appl Biol Biotechnol 8:1–5 Mishra R, Mohanty JN et al (2021) A single transcript CRISPR/Cas9 mediated mutagenesis of CaERF28 confers anthracnose resistance in chilli pepper (Capsicum annuum L.). Planta 254(1): 5 Monroy-Barbosa A, Bosland PW et al (2008) Genetic analysis of Phytophthora root rot racespecific resistance in Chile pepper. J Am Hortic Sci 133:825–829 Moreira-Morrillo AA, Monteros-Altamirano Á et al (2022) Phytophthora capsici on Capsicum plants. A destructive pathogen in chili and pepper crops, IntechOpen, London. https://doi.org/ 10.5772/intechopen.104726 Morilla G, Janssen D et al (2005) Pepper (Capsicum annuum) is a dead-end host for Tomato yellow leaf curl virus. Phytopathology 95:1089–1097 Mou S, Liu Z et al (2017) CaHDZ27, a homeodomain leucine zipper i protein, positively regulates the resistance to Ralstonia solanacearum infection in pepper. Mol Plant-Micr Interact Mou S, Gao F et al (2019) CaLRR-RLK1, a novel RD receptor-like kinase from Capsicum annuum and transcriptionally activated by CaHDZ27, act as positive regulator in Ralstonia solanacearum resistance. BMC Plant Biol 19. https://doi.org/10.1186/s12870-018-1609-6 Mou S, Meng Q et al (2021) A cysteine-rich receptor-like protein kinase CaCKR5 modulates immune response against Ralstonia solanacearum infection in pepper. BMC Plant Biol 21:382 Münch S, Lingner U et al (2008) The hemibiotrophic lifestyle of Colletotrichum species. J Plant Physiol 165:41–51

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Murphy JF, Bowen KL (2006) Synergistic disease in pepper caused by the mixed infection of Cucumber mosaic virus and Pepper mottle virus. Phytopathology 96:240–247 Murphy JF, Hallmark HT et al (2021) Three strains of Tobacco etch virus distinctly alter the transcriptome of apical stem tissue in Capsicum annuum during infection. Viruses 13(5):741 Mythili JB, Rashmi HJ et al (2015) Transgenic chili possessing Baculovirus Chitinase gene exhibits in vitro fungal inhibition. J Crop Improv 29(2):159–187 Noling J (2019) Nematode management in tomatoes, peppers, and eggplant. UF IFAS Extension ENY-032 Noman A, Liu Z et al (2018) Expression and functional evaluation of CaZNF830 during pepper response to Ralstonia solanacearum or high temperature and humidity. Microb Pathog 118. https://doi.org/10.1016/j.micpath.2018.03.044 OECD (2006) Section 12 - Capsicum annuum complex. In: Safety assessment of transgenic organisms, OECD Consensus Documents, vol 1. OECD Publishing, Paris Oerke EC (2006) Crop losses to pests. J Agric Sci 144:31–43 Özdemir ÖF (2021) Phenotypic and genotypic characterization of pepper genotypes for tomato spotted wilt virus (TSWV) disease reaction and resistance (Master’s thesis, Niğde Ömer Halisdemir Üniversitesi/Fen Bilimleri Enstitüsü) Özkaynak E, Devran Z et al (2014) Pyramiding multiple genes for resistance to PVY, TSWV and PMMoV in pepper using molecular markers. Europ J Hort Sci 79:233–239 Parisi M, Alioto D et al (2020) Overview of biotic stresses in pepper (Capsicum spp.): Sources of genetic resistance, molecular breeding and genomics. Int J Mol Sci 21(7):2587 Park SI, Kim HB et al (2021) Agrobacterium-mediated capsicum annuum gene editing in two cultivars, hot pepper CM334 and bell pepper dempsey. Int J Mol Sci 22:3921 Piccini C, Parrotta L et al (2019) Histomolecular responses in susceptible and resistant phenotypes of Capsicum annuum L. infected with Phytophthora capsici. Sci Hortic 244:122–133 Pickersgill B (1997) Genetic resources and breeding of Capsicum spp. Euphytica 96:129–133 Pohan NS, Alfan G et al (2022) Pepper (Capsicum annuum) plants harboring the Begomovirus resistance gene Pepy-1 show delayed symptom progress and high productivity under natural field conditions. Hortic J. QH-015 Powell PA, Sanders PR et al (1990) Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences. Virology 175:124–130 Qin C, Yu C et al (2014) Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc Natl Acad Sci 111(14): 5135–5140 Quirin EA, Ogundiwin EA et al (2005) Development of sequence characterized amplified region (SCAR) primers for the detection of Phyto. 5.2, a major QTL for resistance to Phytophthora capsici Leon. in pepper. Theor Appl Genet 110(4):605–612 Rabuma T, Gupta OP et al (2022) Integrative RNA-Seq analysis of Capsicum annuum L.Phytophthora capsici L. pathosystem reveals molecular cross-talk and activation of host defence response. Physiol Mol Biol Plants 28(1):171–188 Raghunathachari P, Nivas SK et al (2012) Agrobacterium mediated transformation of a pure line variety of hot pepper. RCL 59M Int J Sci Res 2319–7064 Rai VP, Rai AC et al (2010) Emergence of new variant of chilli leaf curl virus in North India. Veg Sci 37:124–128 Ramdial HA, Rampersad SN (2010) First report of Fusarium solani causing fruit rot of sweet pepper in Trinidad. Plant Dis 94(11):1375–1375 Rao S, Nandineni MR (2017) Genome sequencing and comparative genomics reveal a repertoire of putative pathogenicity genes in chilli anthracnose fungus Colletotrichum truncatum. PLoS One 12(8):e0183567 Reeves G, Monroy-Barbosa A et al (2013) A novel Capsicum gene inhibits host-specific disease resistance to Phytophthora capsici. Phytopathology 103(5):472–478

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Rehrig WZ, Ashrafi H et al (2014) CaDMR1 cosegregates with QTL Pc5. 1 for resistance to Phytophthora capsici in pepper (Capsicum annuum). Plant Genome 7(2):plantgenome2014-03 Reusche M, Thole K et al (2012) Verticillium infection triggers Vascular-related NAC Domain7dependent de novo xylem formation and enhances drought tolerance in Arabidopsis. Plant Cell 24:3823–3837 Roberts P (2006) Disease management: Gray mold on tomato and ghost spot on pepper. UF/IFAS SWFREC2686 SR 29:105-6 Romer P, Jordan T et al (2010) Identification and application of a DNA-based marker that is diagnostic for the pepper (Capsicum annuum) bacterial spot resistance gene Bs3. Plant Breed 129:737–740 Roy A, Zhai Y et al (2019) Multiplexed editing of a Begomovirus genome restricts escape mutant formation and disease development. PLoS One 14:0223765 Rubio M, Caranta C et al (2008) Functional markers for selection of potyvirus resistance alleles at the pvr2-eIF4E locus in pepper using tetra-primer ARMS-PCR. Genome 51:767–771 Ruffel S, Dussault MH et al (2002) A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E). Plant J 32:1067–1075 Sahitya UL, Sri Deepthi R et al (2014) Anthracnose, a prevalent disease in capsicum. Res J Pharm, Biol Chem Sci 3:1583–1604 Sandani HB, Weerahewa HL (2018) Wilt diseases of tomato (Lycopersicum esculentum) and chilli (Capsium annum) and their management strategies: Emphasis on the strategies employed in Sri Lanka: a review. Sri Lankan J Biol 3(2). https://doi.org/10.4038/sljb.v3i2.24 Sawada H, Takeuchi S et al (2005) A New Tobamovirus-resistance Gene, Hk, in Capsicum annuum. Engei Gakkai Zasshi 74:289–294. https://doi.org/10.2503/jjshs.74.289 Saxena A, Raghuwanshi R et al (2016) Chilli anthracnose: the epidemiology and management. Front Microbiol 7:1527 Schenke D, Cai D et al (2020) Applications of CRISPR/Cas to improve crop disease resistance: beyond inactivation of susceptibility factors. Iscience 23(9):101478 Seo HH, Park S et al (2014) Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper. PLoS One 9(5):e97936 Sharma VK, Basu S et al (2015) RNAi mediated broad spectrum transgenic resistance in Nicotiana benthamiana to chili-infecting Begomoviruses. Plant Cell Rep 34:1389–1399 Sharma R, Mahanty B et al (2021) Genome wide identification and expression analysis of pepper C2H2 zinc finger transcription factors in response to anthracnose pathogen Colletotrichum truncatum. 3. Biotech 11(3):118 Shin R, Han JH et al (2002) The potential use of a viral coat protein gene as a transgene screening marker and multiple virus resistance of pepper plants coexpressing coat proteins of cucumber mosaic virus and tomato mosaic virus. Transgenic Res 11:215–219 Shingote PR, Wasule DL et al (2022) An overview of chili leaf curl disease: molecular mechanisms, impact, challenges, and disease management strategies in Indian subcontinent. Front Microbiol 13:899512 Shukla AK, Upadhyay SK et al (2016) Expression of an insecticidal fern protein in cotton protects against whitefly. Nat Biotechnol 34:1046–1051 Silvar C, Merino F et al (2008) Differential activation of defence-related genes in different pepper cultivars infected with Phytophthora capsici. J Plant Phsio 165:1120–1124 Skelton A, Uzayisenga B et al (2018) First report of Pepper veinal mottle virus, Pepper yellows virus and a novel enamovirus in chilli pepper (Capsicum sp.) in Rwanda. New Dis Rep 37:5 Soomro HU, Khaskheli MI et al (2019) Disease intensity and eco-friendly management of Alternaria alternata in chili (Capsicum annuum L.). Pure Appl Biol 8(4):2333–2342 Stoessl A, Unwin CH et al (1972) Postinfectional inhibitors from plants. I. Capsidiol, an antifungal compound from Capsicum frutescens. Phytopathology 74:141–152 Tashkandi M, Ali Z et al (2018) Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato. Plant Signal Behav 13:1525996

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Zhang YL, Jia QL et al (2013) Characteristic of the pepper CaRGA2 gene in defense responses against Phytophthora capsici Leonian. Int J Mol Sci 14(5):8985–9004 Zhang HX, Feng XH et al (2020) Identification of pepper CaSBP08 gene in defense response against Phytophthora capsici infection. Front Plant Sci 11:183 Zheng L, Huang J et al (2008) First report of leaf blight of garlic (Allium sativum) caused by Stemphylium solani in China. Plant Pathol 57:380 Zheng Z, Nonomura T et al (2013) Loss of function in Mlo orthologs reduces susceptibility of pepper and tomato to powdery mildew disease caused by Leveillula taurica. PLoS One 8(7): e70723 Zhu Y-X, Ou-Yang W-J et al (1996) Transgenic sweet pepper plants from Agrobacterium mediated transformation. Plant Cell Rep 16:71–75 Zonneveld MV, Ramirez M et al (2015) Screening genetic resources of Capsicum peppers in their primary center of diversity in Bolivia and Peru. PLoS One 10(9):e0134663

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Genetic Improvement of Poplar Deepika Singh and Rajesh Kumar Singh

9.1

Introduction

A variety of products are provided by trees for the benefits of human beings such as wood, energy fuels, food, and fiber. The genus Poplar has important role in forest production and for ecological environment. It is main species for artificial forest production due to its fast growth and high yield. Poplar, a deciduous forest species, belongs to Salicaceae family. It has about 20–30 species, especially in Northern Hemisphere. The two main species of poplar in India are Populus deltoides and Populus ciliata in which P. deltoid is found in plains of India, while P. cilita is found in hilly region. P. deltoides is now well-known and rapidly growing woody trees in Haryana and Punjab states of India. Genetic improvement is extremely important for plants to improve its yield and quality. For genetic and molecular improvement in woody plants, poplar has been selected as model plants (Lin et al. 2000; Lin and Zhang 2004). It represents as model plants for the following reasons. These are as follows: (1) Poplar plants have a small genome size, a short life cycle period, and are easy to propagate in vitro due to their rapid vegetative growth (Bradshaw et al. 2000; Brunner et al. 2004), (2) in P. trichocarpa, a popular species, genome size has been sequenced (Tuskan et al. 2006). (3) Also, gene transformation protocol has been established for P. trichocarpa (Song et al. 2006). However, in some laboratories, it is still difficult to propagate. Therefore, P. trichocarpa is used for gene sequences and expression analysis while to observe phenotypic traits, different hybrid poplar have been selected such as P. tremula and tremuloides (Ohtani et al. 2011), P. alba and D. Singh · R. K. Singh (✉) Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_9

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Biotic stress

Abiotic stress

Biofuels

Wood quality and fast growth

Phytoremediation

Future goals

Fig. 9.1 Genetic engineering in poplar to enhance desirable traits

grandidentata (Maloney and Mansfield 2010), P. alba and P. tremula (Cho et al. 2016), and P. simonii and P. nigra (Zhao et al. 2017). Poplar alba is also called white poplar, and it is very important for ecological and economic values (Eckenwalder 1996). This species of poplar is mostly found in Europe and Asia and hybridize with other poplar species such as P. tremula generating other hybrids plant species (Lazowski 1997; Lexer et al. 2005; Van Loo et al. 2008). According to various studies, P. alba is an easily transformable species, and one genotype of this poplar species can produce flower within 9 months of regeneration (Soliman et al. 2017; Wang et al. 2008; Meilan et al. 2004). Besides basic and applied studies, this chapter covers the uses of poplar in phytoremediation and biofuels industries, tolerance in stress conditions, and production of quality wood for various purposes by using genetic engineering system (Fig. 9.1). Also, what could be the future prospective of poplar to improve its quality and growth?

9.2

Genetic Transformation in Poplar Using Marker Genes

Genetic engineering in poplar species P. trichocarpa × P. deltoides was initiated by Parsons et al. in 1986 through Agrobacterium-mediated transformation. Later, the Agrobacterium-mediated protocol was modified in P. trichocarpa × P. deltoides and P. deltoides × P. nigra by the transformation of two reporter genes GUS and NPT-II (Han et al. in 2000). The beta-glucuronidase gene also known as GUS is used as a

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reporter gene in various plant species. GUS gene produces blue color upon incorporation into plant genome and is widely used in gene expression analysis. Another marker is NPT-II called neomycin phosphotransferase II enzyme provides resistance against neomycin and kanamycin drugs in transformed plant cell. These genes were transferred in a no of poplar species such as P. ciliate, P. deltoides clone G48, and P. trichocarpa using poplar leaf as explant through Agrobacteriummediated transformation methods (Thakur et al. 2005; Saraswat et al. 2016; Li et al. 2017). After transformation, both transgene expression levels and transformation frequency have been found to vary between genotypes. When GUS was attached to a matrix attachment region (MAR) derived from tobacco, its expression was increased tenfold in hybrid poplar clones P. tremula × P. alba and P. trichocarpa × P. deltoides (Han et al. 1997). In contrary, Meyer et al. in 2004 introduced RNAi technology to reduce the expression levels of GUS gene in transgenic poplar lines. Takata and Eriksson developed an in-planta transformation method in 2012 to determine the function of specific genes in P. tremula × P. tremuloides. Maheshwari and Kovalchuk used the LUC reporter gene in 2016 to enhance the Agrobacterium-mediated gene transfer method in P. angustifolia and P. balsamifera. Okumura et al. in 2006 also transformed the GFP, green florescent protein that produces florescent upon integration with genomic DNA in P. alba species. The species and names of certain poplar plants are given transformed with particular marker genes (Table 9.1).

9.3

Poplar Use as Biofuels

The need to lessen our reliance on petroleum and make fuels from renewable sources, like lingo-cellulosic biomass, and for this, a species poplar has been considered an alternative feedstock for biofuel production (Fulton et al. 2015; Mansfield et al. 2012; Ho et al. 2014; Cheng and Timilsina 2011; Ragauskas et al. 2006). In the United States, poplar is being grown for wood feedstock. Poplar is as an important feedstock due to a variety of factors, including its quick growth, high cellulose content, ability to grow on marginal land, low ash, and extractives content, as well as easy to harvest, handling, and storing of such biomass. However, recent studies have shown scarification efficiency must be improved in poplar to be an economically feasible feedstock (Sannigrahi et al. 2010; Nordborg et al. 2018; Shooshtarian et al. 2018; Porth et al. 2015; Chudy et al. 2019). Conversion of biomass into biofuels is called fermentation or scarification process. It involves mainly three steps where the first step is deconstruction of cell wall and enhancement of enzyme efficiencies to access the cellulose and hemicellulose. The second step involves scarification process in which polysaccharides are converted into fermentable sugars through using celluloses and other enzymes. Furthermore, yeast or bacteria can turn these fermented sugars into biofuels. Hence, one method to increase productivity throughout the conversion process is to change the characteristics of poplar’s cell walls. For this purpose, the various species of poplar genomes were sequenced to alter the cell wall’s properties (Tuskan et al. 2004; Tuskan et al. 2006; Ma et al. 2019). One of the obstacles in converting

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Table 9.1 Marker genes transformation in poplar

Species/hybrid names

Reporter genes names

References

P. tremula

GUS and NPT-II

Tzfra et al. (1998)

P.ciliate

GUS and NPT-II

Thakur et al. (2005)

P.deltoids

GUS and NPT-II

Saraswat et al. (2016)

P. tremula×P. alba

GUS

Studart et al. (2006)

P. trichocarpa×P. deltoids

GUS

Wang et al. (1995)

P. tremula×P. tremuloides

GFP

Takata and Eriksson (2012)

P. angustifolia and P. balsamifera

LUC

Maheshwari and Kovalchuk (2016)

P. alba

GFP

Okumura et al. (2006)

P. trichocarpa×P. deltoides

T-DNA

Parsons et al. (1986)

P. tremula×P. alba

NPT-II

Leple et al. (1992)

P. nigra×P. maximowiczii

GUS

Devantier et al. (1993)

P.tremula

GUS

Shani et al. (2000)

P.trichocarpa

GUS and NPT-II

Li et al. (2017)

P. tremula×P. alba

GUS and NPT-II

Tian et al. (1999)

P.species

GUS

Meyer et al. (2004)

lingo-cellulosic biomass into fermentable sugars is a recalcitrance property (Sindhu et al. 2016). Modification of gene expression in plants is common phenomenon for enhancing the desirable attributes (Jeong et al. 2010; Zhou et al. 2013; Chen et al. 2008; Zhu et al. 2012; Liping et al. 2017; Pasonen et al. 2004; Qingquan et al. 2018; Polle et al. 2013). Through recent efforts, it has been possible to increase the utility of poplar as a feedstock for bioenergy by altering the expression of genes in cell wall biosynthesis. For this, there have been ongoing efforts to alter the lignin biosynthetic pathway and modify the lignin traits to make poplar more appropriate for enzymatic hydrolysis, without changing its productivity (Cai et al. 2016). The 4-coumarate:coenzyme A (CoA) ligase (4CL), which converts p-coumaric acid to p-coumaroyl CoA, was initially used to modify the lignin content of tobacco and Arabidiopsis. This study was later applied in P. tremuloides (greenhouse grown) in which four transgenic lines showed at least 10% reduction in 4CL expression and had 40–45% reduction in

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lignin content (Hu et al. 1999). Similar results were observed in P. tomentosa (field grown) where 8% lignin contents were decreased by suppressing the 4CL enzyme expressions (Wang et al. 2012). However, despite the beneficial transgenic effects, there is no change in total sugar content or improved enzymatic digestion efficiency was observed. P. tremula × P. alba with downregulated 4CL (field grown) also showed reduced lignin content without increasing the release of sugar content (Voelker et al. 2010). In contrast to these results, Populus Trichocarpa which has been grown in greenhouse with reduced 4CL expression showed low lignin content similar to Wang et al. and Voelker et al., but the most interesting observation was that it had enhanced saccharification efficiency which leads to increased total sugar release (Min et al. 2012). Further, Xiang et al. in 2015 reported that only the reduced lignin content alone would not be able to enhance saccharification efficiency and total sugar release; it might be possible that reduced 4CL is prone to environmental changes. Another gene in lignin biosynthetic pathway that has been altered is CAld5H; in various studies, CAld5H simultaneously altered with 4CL in the same plants (Min et al. 2012; Xiang et al. 2015; Min et al. 2013). Overexpression of CAld5H and downregulation of 4CL affected both lignin content and syringaldehyde to vanillin (monolignols) ratio. Another gene in lignin pathway was 4-coumarate-3-hydroxylase (C3H) which affects both the lignin content and syringyl to guaiacyl ratio (monolignols) ratio. C3H gene downregulation in P. alba × P. grandidentata reduced lignin content and increased syringyl to guaiacyl ratio (Ralph et al. 2012). Another side, greenhouse-grown P. alba × P. glandulosa with downregulated C3H showed decreased lignin content and increased cellulose content. Another enzyme hydroxycinnamoyl transferase (HCT) downregulation in greenhouse-grown P. alba × P. glandulosa also exhibited reduced lignin content compared to wild type, but the cellulose content is not affected (Zhou et al. 2018). These studies suggested that lignin structure and quantity have ability to positively impact the biomass recalcitrance properties in poplar. Cellulose is the main component of the cell wall and has been the target of gene modification systems in order to make plants more amenable as bioenergy feedstock. It is the most abundant biopolymer and comprised of beta-1,4-linked D-glucose units (Li et al. 2014; Vandavasi et al. 2016; McNamara et al. 2015). Cellulose gives mechanical strength to plants and is used in textile industry, building materials, and renewable energy. The activity of enzyme invertase (INV) or sucrose synthase is a key pathway for the production of UDP-glucose, which serves as a substrate for the cellulose biosynthesis process (Rende et al. 2017). It has been shown that sucrose synthase gene SUSY increased transcript levels and affects cellulose contents in plants (Coleman et al. 2009). Similarly, overexpression of DUF266 gene enhances the cellulose content and decreases in lignin content was observed (Yang et al. 2017). The cellulose content increment increases the sugar release properties in poplar transgenic line of DUF266 as in altered lignin biosynthetic plants. Min et al. in 2013 suggested that a glycosyltransferase enzyme GT8D downregulation decreased the xylan content in plants which is associated with total sugar release in plants. A member of GAUT family gene GAUT12 downregulation in P. deltoides

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also decreased the xylan content and increased the sugar release without impacting lignin content (Biswal et al. 2015). This phenomenon demonstrates how enhanced biomass growth or conversion performance can be the result of combined factors. These findings denote that alteration of genes linked with cellulose or hemicellulose production can be as useful as alteration of genes related with lignin in enhancing the properties of poplar for use as a bioenergy feedstock.

9.4

Poplar Use in Phytoremediation

Poplar is an important plant for phytoremediation because of its high photosynthesis efficiency, which reduces environmental pollution (Soudek et al. 2004). The extensive root system of poplar enhanced the groundwater uptake having pollutants and its large canopy fixed the atmospheric CO2 using unique method. Poplar has been reported to intake various inorganic and heavy metals. Although, genetic transformation has been done to introduce exotic gene and improve its remedial activity (Bittsanszky et al. 2005). Gamma-ECS gene commonly known as gammaglutamylcysteine synthetase is one such bacterial gene that, when transferred into the poplar species of P. tremula and P. alba, improves the cadmium tolerance properties (Arisi et al. 2000). A ScYCF gene was transferred in a hybrid poplar clone glandulosa which imparts resistance to heavy metals (Shim et al. 2013). The importance of poplar species in this area opened up new possibilities for the planting of these trees as a method of phytostabilization in heavy metal-contaminated area.

9.5

Biotic Stress Regulation in Poplar

A number of bacterial and fungal pathogens infect the poplar plant and impact significant yield losses. Therefore, poplar lines incorporated with different antifungal and antibacterial genes were generated. Mentag et al. in 2003 reported that when a poplar hybrid P. tremula × P. alba transformed with D4E1 gene a synthetic antimicrobial peptide showed resistant to various types of pathogens. Poplar transgenic plants having ECH42 gene, a Trichoderma harzianum endochitinase gene, showed resistance against Melampsora medusa and leaf rust pathogen (Noel et al. 2005). A PtWRKY23 gene transformation in P. tomentosa × P. alba also found to enhance the tolerance against Melampsora infection (Levee et al. 2009). Ye et al. in 2014 also transformed P. tomentosa clone 741 with PtoWRKY60 for incorporation of antifungal trait against Dothiorella gregaria. Jia et al. in 2010 also reported that an antimicrobial LJAMP2 transcript accumulation in poplar species of P. tomentosa induced resistance against fungal pathogen. Transcript accumulation of two WRKY genes PtrWRKY18 and PtrWRKY35 in poplar transgenic lines also exhibited resistance against Melampsora rust. Besides fungal and bacterial infection poplar species are also susceptible to the various types of insect–pest which retarded plant growth. In order to enhance the resistance against insect, various types of genes have been incorporated in poplar species such as

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proteinase inhibitor genes, CRY gene from Bacillus thuringiensis, and insect toxin genes. McCown et al. in 1991 first reported about Bt gene transgenic lines in hybrid poplar species transformed through microprojectile bombardment technique. Sala et al. in 2000 found that P. nigra clones harboring Bt. endotoxin gene showed tolerance against Apocheima cinerarium. A Bt gene CRY3A transformation in P. alba × P. glandulosa resulted in the inhibitory effect against Anoplophora glabripennis larvae (Zhang et al. 2006). Guo et al. in 2011 also found that transgenic lines of poplar harboring Bt gene reduced the growth rate of larva Hyphantria cunea. In another study, CRY1AC and CRY3A gene transformation in P. × euramericana enhances the resistance against H. cunea (Yang et al. 2016). In addition to Bt genes, various other types of genes were studied in poplar which showed resistance to insect–pest. Likewise, Klopfenstein et al. in 1997 developed transgenic lines in clone P. alba × P. grandidentata expressing a proteinase inhibitor-II (PIN 2) gene exhibited resistance against lepidopteran insect. A PtdPP01 gene when incorporated with genomic DNA of hybrid poplar controlling resistance against Malacosoma disstria (Wang and Constabel 2004).

9.6

Abiotic Stress Regulation in Poplar

Abiotic stresses like oxidative stress, drought tolerance, and herbicide tolerance also impact the yield of poplar. Poplar is the important crop for agroforestry and weed infestation causes serious problem in poplar growth. Therefore, various strategies were developed to neutralize the harmful effects of weed. Out of various strategies, one is to introduce herbicide resistance gene in poplar such as in 2000 Meilan et al. generated transgenic hybrid poplar expressing AROA gene (responsible for glyphosate tolerance) which is found to be tolerant against herbicide Roundup Pro™. In another study, a BAR gene encodes enzymes phosphinothricin acetyltransferase was transformed in white poplar responsible for resistance against herbicide phosphinothricin (Confalonieri et al. 2000). Simultaneous transformation of CP4 and GOX genes developed resistance against glyphosate in transgenic of P. trichocarpa × P. deltoides and P. trichocarpa × P. nigra (Ault et al. 2016). GOX gene encodes glyphosate oxidoreductase enzyme which degrades glyphosate and CP4 binds with glyphosate. In various environmental stresses, plants produce reactive oxygen species also called ROS and the production of large amount of ROS in plant cells creates oxidative stress in plants. Foyer et al. in 1995 found that P. tremula × P. alba expressing glutathione synthetase was more tolerance to oxidative stress. Besides these, salt and drought stress are also a major abiotic factors that impact the yield of poplar. Various types of genes were introduced in poplar to enhance the salt and drought tolerance capacity such as MTLD (mannitol-1-phosphate dehydrogenase) gene introduction in P. tomentosa induced the salt stress tolerance in poplar species (Hu et al. 2005). Also, MnSOD (manganese superoxide dismutase) transformation in hybrid poplar improved salt tolerance properties (Wang et al. 2010). White poplar transformed with glutaredoxin-2 gene had higher peroxidase and phenoloxidase levels which enhance salt stress tolerance (Soliman et al.

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2017). Simultaneous transformation of CkAREB and JERF36 genes in P. × euramericana clone “Lingfeng 2” improved the drought tolerance (Zhang et al. 2018). Zhang et al. in 2017 reported that PtNDPK2 introduction in poplar increases the salt and drought tolerance.

9.7

Genetic Engineering in Poplar to Improve Wood Quality and Fast Growth

Poplar’s rapid growth is required for genetic engineering to incorporate desirable traits. Schwartzenberg et al. in 1994 reported that ipt gene transformation in poplar (P. tremula x P. alba) elevates the cytokinin levels. Transformed plants regenerated the callus without external supply of cytokinin. Overexpression of pine glutamine synthetase gene in poplar is associated with increase in soluble sugars, chlorophyll content, and height in transgenic plants compared to control (Gallardo et al. 1999). A “rol” gene expression isolated from A. rhizogenes expression in clones of P. tremula showed increase in growth rate and stem height in plants (Tzfra et al. 1999). However, P. tomentosa harboring rolB gene had higher root growth than control plants (Xiong et al. 2005). Transgenic P. alba lines expressing xyloglucanase gene exhibited internode elongation and high cellulose content. It also enlarged the stem size and increased the cellulose density in secondary xylem (Park et al. 2004). In another study, the expression of Vitreoscilla hemoglobin gene (Vgb) in poplar (P. alba x P. glandulosa) enhances the growth rate with respect to height and diameter (Zhang et al. 2005).

9.8

Conclusion and Future Goals

Poplar is known as the most economically important as it provides hardwood. Like other plants, Poplar trees also faced various environmental challenges. Genetic engineering and breeding are common methods to cope with these challenges in plants. Upon regulation of biotic stress, pesticide spray can kill economic friendly pathogens too. Therefore, genetic engineering techniques develop the resistance against particular pathogens. However, the genetically modified poplars will probably require in-depth investigations into gene flow and its effects on forest ecological system which are challenging to conduct. Poplar is also used widely in biofuel productions. Gene modification system is used to change the constituents of cell wall of poplar and induced the production of biofuels. Biofuel production requires sugars from plant fiber, and the competitive processing cost to obtain sugar from plants is a difficult task. The biochemical conversion of sugar to ethanol is simple, but obtaining sugar is difficult. In biofuel production sugar is obtained from plant fiber made up of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are sugars used for biofuel production, but lignin is hard to process. Although there are numerous known lignin biosynthetic pathways, the genes that control lignin production have not yet been identified. To uncover the function of these genes, we are

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required to examine the genome of poplar that participates in biofuel production. This method has already been used to identify various genes that control in making of cell wall constituents. Besides this, an alternative strategy could be the transformation of gene that increases the production of sugars in poplar plants. Higher sugar content directly affects the yield of biofuel production, which is a cost-effective and long-term strategy. Poplar is also best candidate for phytoremediation. Overexpression of YCF1 gene enhances cadmium uptake capacity in Arabidopsis (Song et al. 2006). Hence, the genetic approach could give the better result in model plants poplar for phytoremediation.

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Min D et al (2012) The cellulase-mediated saccharification on wood derived from transgenic low-lignin lines of black cottonwood (Populus trichocarpa). Appl Biochem Biotechnol 168: 947–955 Min Y et al (2013) The elucidation of the lignin structure effect on the cellulase-mediated saccharification by genetic engineering poplars (Populus nigra L. × Populus maximowiczii A.). Biomass Bioenergy 58:52–57 Noel A, Levasseur C, Van QL, Seguin A (2005) Enhanced resistance to fungal pathogens in forest trees by genetic transformation of black spruce and hybrid poplar with a Trichoderma harzianum endochitinase gene. Physiol Mol Plant Pathol 67(2):92–99 Nordborg M et al (2018) Energy analysis of poplar production for bioenergy in Sweden. Biomass Bioenergy 112:110–120 Ohtani M, Nishikubo N, Xu B, Yamaguchi M, Mitsuda N, Goue N, Shi F et al (2011) A NAC domain protein family contributing to the regulation of wood formation in poplar. Plant J 67: 499–512 Okumura S, Sawada M, Park YW, Hayashi T, Shimamura M, Takase H, Tomizawa KI (2006) Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts. Transgenic Res 15(5):637–646 Park YW, Baba K, Furuta Y, Tida T, Sameshima K, Arai M, Hayashi T (2004) Enhancement of growth and cellulose accumulation by overexpression of xyloglucanase in poplar. FFBS Lett 564(1/2):183–187 Parsons TJ, Sinkar VP, Steller RF, Nester W, Garden MP (1986) Transformation of poplar by Agrobacterium tumefaciens. Biotechnol J 4:533–536 Pasonen HL et al (2004) Field performance of chitinase transgenic silver birches (Betula pendula): resistance to fungal diseases. Theor Appl Genet 109:562–570 Polle A et al (2013) Poplar genetic engineering: promoting desirable wood characteristics and pest resistance. Appl Microbiol Biotechnol 97:5669–5679 Porth I et al (2015) Using Populus as a lignocellulosic feedstock for bioethanol. Biotechnol J 10:510 Qingquan L et al (2018) Lignins: biosynthesis and biological functions in plants. Int J Mol Sci 19: 335 Ragauskas AJ et al (2006) The path forward for biofuels and biomaterials. Science 311:484–489 Ralph J et al (2012) Effects on lignin structure of coumarate 3- hydroxylase downregulation in poplar. Bioenergy Res 5:1009–1019 Rende U et al (2017) Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood. New Phytol 214:796–807 Sannigrahi P et al (2010) Poplar as a feedstock for biofuels: a review of compositional characteristics. Biofuels Bioprod Biorefin 4:209–226 Saraswat A, Khan AA, Thakur AK, Gaur A, Srivastava DK (2016) Agrobacterium-mediated genetic transformation of Populus deltoides Marsh. clone G48 with gus and npt-II genes. Vegetos 29:4. https://doi.org/10.5958/2229-4473.2016.00097.5 Shani Z, Dekel M, Jensen CJ, Tzfra T, Goren R, Altman A, Shoseyov O (2000) Arabidopsis thaliana endo1,4-β-glucanase (cell) promoter mediates ‘uida’ expression in elongating tissues of aspen (Populus tremula). J Plant Physiol 156:118–120 Shim D, Kim S, Choi Y, Song WY, Park J, Youk ES, Jeong SC, Martinoia E, Noh EW, Lee Y (2013) Transgenic poplar trees expressing yeast cadmium factor 1 exhibit the characteristics necessary for the phytoremediation of mine tailing soil. Chemosphere 90(4):1478–1486 Shooshtarian A et al (2018) Growing hybrid poplar in western Canada for use as a biofuel feedstock: a financial analysis of coppice and single-stem management. Biomass Bioenergy. 113:45–54 Sindhu R et al (2016) Biological pretreatment of lignocellulosic biomass – an overview. Bioresour Technol 199:76–82 Soliman MH, Hussein MHA, Gad M, Mohamed AS (2017) Genetic transformation of white poplar (Populus alba L.) with glutaredoxin-2 gene. Biosci Res 14:464–472

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Song J, Lu S, Chen Z-Z, Lourenco R, Chiang VL (2006) Genetic transformation of Populus trichocarpa genotype Nisqually-1: a functional genomic tool for woody plants. Plant Cell Physiol 47:1582–1589 Soudek P, Tykva R, Vaneˇk T (2004) Laboratory analyses of 137Cs uptake by sunflower, reed and poplar. Chemosphere 55:1081–1087 Studart GC, Lacorte C, Brasileiro ACM (2006) Evaluation of heterologous promoters in transgenic Populus tremula×P. alba plants. Biol Plantarum 50(1):15–20 Takata N, Eriksson ME (2012) A simple and efficient transient transformation for hybrid aspen (Populus tremula×P. tremuloides). Plant Methods 8:30 Thakur AK, Sharma S, Srivastava DK (2005) Plant regeneration and genetic transformation studies in petiole tissue of Himalayan poplar (Populus ciliata Wall.). Curr Sci 89:664–668 Tian LN, Levee V, Mentag R, Charest PJ, Seguin A (1999) Green fluorescent protein as a tool for monitoring transgene expression in forest tree species. Tree Physiol 19(8):541–554 Tuskan GA et al (2004) Poplar genomics is getting popular: the impact of the poplar genome project on tree research. Plant Biol 6:2–4 Tuskan GA et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604 Tzfra T, Jensen CS, Wang W, Zuker A, Vincour B, Altman A, Vainstein A (1998) Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium-mediated transformation. Plant Mol Biol Rep 15:219–235 Tzfra T, Vainstein A, Altman A (1999) rol-gene expression in transgenic aspen (Populus tremula) plants results in accelerated growth and improved stem production index. Trees 14:49–54 Van Loo M, Joseph JA, Heinze B, Fay MF, Lexer C (2008) Clonality and spatial genetic structure in Populus 9 canescens and its sympatric backcross parent P. alba in a central European hybrid zone. New Phytol 177:506–516 Vandavasi VG et al (2016) A structural study of CESA1 catalytic domain of Arabidopsis cellulose synthesis complex: evidence for CESA trimers. Plant Physiol 170:123–135 Voelker S et al (2010) Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar. Plant Physiol 154:874–886 Wang JH, Constabel CP (2004) Polyphenol oxidase over-expression in transgenic Populus enhances resistance to herbivory by forest tent caterpillar (Malacosoma disstria). Planta 220(1):87–96 Wang YC, Emerick RM, Denchev PD, Conger RV, Tuskan GA (1995) A biolistic approach for the transient expression of a GUS reporter gene in callus cultures of hybrid poplar. In Vitro Cell Dev Biol Plant 31(4):226 Wang J, Zhu M, Wei Z (2008) Cotton laccase gene overexpression in transgenic Populus alba var. pyramidalis and its effects on the lignin biosynthesis in transgenic plants. Fen Zi Xi Bao Sheng Wu Xue Bao 41:11–18 Wang YC, Qu GZ, Li HY, Wu YJ, Wang C, Liu GF, Yang CP (2010) Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from Tamarix androssowii. Mol Biol Rep 37:1119–1124 Wang X et al (2012) Lignin modification improves the biofuel production potential in transgenic Populus tomentosa. Ind Crop Prod 37:170–177 Xiang Z et al (2015) Wood characteristics and enzymatic saccharification efficiency of field-grown transgenic black cottonwood with altered lignin content and structure. Cellulose 22:683–693 Xiong J, Liang J, Chen XY, Li W, Li H, Liu Y (2005) The rooting ability of rolB transformed clones of Populus tomentosa. J Beijing For Univ 27(5):54–58 Yang RL, Wang AX, Zhang J, Dong Y, Yang MS, Wang JM (2016) Genetic transformation and expression of transgenic lines of Populus×euramericana with insect-resistance and salt-tolerance genes. Genet Mol Res 15(2):15028635 Yang Y et al (2017) Overexpression of a domain of unknown function 266-containing protein results in high cellulose content, reduced recalcitrance, and enhanced plant growth in the bioenergy crop. Biotechnol Biofuels 10:74

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Genetic Engineering for Potato Improvement: Current Challenges and Future Opportunities

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Baljeet Singh, Vadthya Lokya, Priyanka Kaundal, and Siddharth Tiwari

10.1

Introduction

Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat in terms of human consumption (Campos and Ortiz 2020). Potato originated from the Andes of South America. It belongs to the genus Solanum of the nightshade family, Solanaceae. This family comprises 98 genera and about 2700 species (Manda et al. 2020). Extensive variations exist in the potato germplasm in terms of morphologic traits, nutrient content, and biotic and abiotic stresses (Singh et al. 2021a). The modern cultivated potatoes are tetraploid (2n = 2x = 48) with a basic chromosome number 12. However, in the wild tuber-bearing potato species, ploidy is varying from diploid to hexaploid (Gutaker et al. 2019). It is a staple crop of many countries with a global production of ~370 million tons per year (ten Den et al. 2022). China is the largest producer of potato (90.26 million tons), followed by India (48.53 million tons) (Tiwari et al. 2022a). It is a rich source of nutrients such as carbohydrates, vitamins, minerals, and dietary fibers (Singh et al. 2021a, 2022). Moreover, it is a short-duration crop and produces more dry matter per unit area, time, and money (Singh et al. 2021a). It is a versatile crop with high consumer acceptability and industrial value. Apart from eating, potato tubers have significant importance in other non-food-related industries such as pharmaceuticals, cosmetics, paper, and textiles (Sawicka et al. 2022). In the climate change scenario, potato crop could play a key role in feeding the ever-increasing human population. However, due to the complex genetics and autotetraploid nature of self-incompatibility at the diploid level it is difficult to B. Singh · V. Lokya · P. Kaundal · S. Tiwari (✉) Plant Tissue Culture and Genetic Engineering Lab, National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology, Ministry of Science and Technology (Government of India), Mohali, Punjab, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_10

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improve the potato yield and quality rapidly using traditional breeding (Jansky and Spooner 2018; Sood et al. 2020). Moreover, for decades potato breeding was focused to enhance agronomic traits that are why most of the modern potato varieties have a narrow genetic basis and have almost reached the plateau (Bradshaw 2009). Potato breeding programs have relied on a limited number of parent lines, which have been selected for their desirable traits and crossed repeatedly to create new varieties. As a result, the genetic diversity of the modern potato has been reduced, leading to concerns about its vulnerability to emerging pests and diseases and its ability to adapt to changing environmental conditions (Bradshaw 2009). To address this issue, efforts are underway to conserve and utilize the genetic diversity of wild and primitive potato relatives, which may hold valuable traits for future breeding programs. Further, they have lower resistance against biotic and abiotic stresses. For example, the late blight of potato caused by the Phytophthora infestans is the most devastating disease in potato. The annual yield loss and management cost for late blight potato account for about 3 to 10 billion USD, globally (Dong and Zhou 2022). There are so many biotic stress-resistant genes in the wild potato species (Tiwari et al. 2022b). Over the last two decades, several attempts have been made to improve potato crop using various approaches alternative to traditional plant breeding. These include the use of chemical mutagens, irradiations, T-DNA insertions, somaclonal variations, virus-induced gene silencing (VIGS), RNA interference (RNAi), transgenics, and gene editing technologies. The early genome editing techniques in plants involved the use of transposons and homologous recombination (HR). These require the introduction of a DNA molecule with homologous sequences to the target site in the genome, which allows for the precise insertion or replacement of DNA sequences. These techniques were powerful tools for the study of gene function in plants, but they were limited in their ability to target specific sites in the genome (Mohanta et al. 2017). Zinc finger nucleases (ZFNs) were the first genome editing technology that allowed for the precise targeting of specific DNA sequences in plants. These proteins are composed of a zinc finger domain that recognizes a specific DNA sequence and a nuclease domain that can cut the DNA. By fusing the two domains, scientists were able to create a chimeric protein that could recognize and cut specific DNA sequences in the plant genome (Weinthal et al. 2010). However, the production of these proteins was expensive, and they were challenging to design. Then, the transcription activatorlike effector nucleases (TALENs) were developed as an alternative to ZFNs, providing a more straightforward design process and better targeting efficiency. TALENs are composed of transcription activator-like effectors that can recognize specific DNA sequences and a nuclease domain that can cut the DNA (Sun and Zhao 2013). The TALEN system was more efficient than ZFNs and could be used in a wide range of plant species, including monocots and dicots. The clustered regularly interspaced short palindromic repeat (CRISPR) is the most recent development in genome editing technology and has revolutionized the field of plant genetics. The CRISPR-Cas system is based on the bacterial immune system, which uses RNA-guided nucleases to cut specific DNA sequences. In the CRISPR-Cas system, a single guide RNA (sgRNA) is used to direct the Cas protein to the target site in the

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genome, where it cuts the DNA. This system is highly efficient, cost-effective, and easy to design, allowing for the creation of precise modifications in a wide range of plant species. The CRISPR-Cas system has been used to edit various plant genomes, resulting in plants with improved characteristics such as disease resistance, yield, and nutritional content (Chincinska et al. 2023; Capdeville et al. 2023). Over the last decade, various attempts throughout the globe have been made to improve potato crop using CRISPR/Cas-mediated genome editing (Tiwari et al. 2022b). The journey toward sustainable potato production is a lengthy one, and there is still much work to be done in terms of improvement.

10.2

Need of Genetic Engineering in Potato

Potato is one of the world’s most important crops, providing vital nutrition to millions of people. However, the potato plant faces numerous challenges, including susceptibility to disease and pests’ abiotic stresses (Tiwari et al. 2022c). Rising temperatures, changing rainfall patterns, and extreme weather events such as droughts and floods are all affecting the growth, yield, and quality of potato plants (Handayani et al. 2019). Potato plants are sensitive to high temperatures, and prolonged exposure to heat can lead to reduced growth, lower yields, and decreased quality. Heat stress can also increase the incidence of disease and pests, which can further impact crop productivity (Singh et al. 2020a; Demirel 2023). Further, droughts can reduce yields and increase the risk of pests and diseases such as nematodes (Holgado and Magnusson 2012). Further, the human population is projected to reach 9.8 billion by 2050 and 11.2 billion by 2100 (Feltrin 2018). In addition, with modernization eating habits are changing, and nowadays, people prefer more nutritious food. Currently, about 2 billion people are suffering from micronutrient deficiencies as they cannot afford a diversified diet (Singh et al. 2020b; Singh et al. 2021a). To feed the ever-increasing human population and to reduce the global burden of hidden hunger in the scenario of climate change, new better potato varieties are required. However, it is difficult and time-consuming to further enhance the quality and yield of this crop using traditional breeding. Most commercial potato varieties are derived from a small number of ancestral lines, resulting in limited genetic diversity (Spanoghe et al. 2022). This can lead to reduced adaptability to changing environmental conditions and increased susceptibility to pests and diseases. The potato plant has a relatively long breeding cycle, taking up to 10 years to develop and release a new variety. This can limit the pace of progress in developing new varieties, as well as increase the cost and time required for breeding programs. Potato is a vegetatively propagated crop, which can make it difficult to maintain the genetic integrity of breeding lines. Many important traits in potato, such as tuber yield and quality, are complex and controlled by multiple genes and environmental factors. This complexity can make it challenging to identify and select desirable traits in breeding programs. However, the availability of potato reference genome sequence (PGSC, 2011) and low-cost high-throughput nextgeneration sequencing (NGS) opened various opportunities for potato

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improvements. Genomics tools and approaches such as genome-wide association studies (GWAS), transcriptomic profiling, RNA-Seq, and expressed sequence tags (ESTs) have produced ample amounts of highly accurate data. Based on these, a large number of putative candidate genes associated with different traits have been identified in potato. The function of these potential candidate genes can be studied by using reverse genetic approaches such as VIGS, RNAi, and T-DNA insertions or it could be studied by employing the modern gene editing technique CRISPR/Cas (Singh et al. 2018; Zhang et al. 2021). After validating their functionality, the candidate genes associated with different traits can be subjected to CRISPR/Casmediated gene editing in the most promising potato genotypes.

10.3

Genome Editing

Genetic engineering is a powerful tool that has the potential to improve various aspects of potato cultivation, including pest and disease resistance, yield and productivity, nutritional quality, and environmental sustainability. One of the key challenges associated with genetic engineering in potato is the complexity of the plant’s genome. The potato genome is large, highly heterozygous, and contains numerous repetitive sequences (Wang et al. 2022). These characteristics make genetic engineering in potato more challenging than in many other crops. However, recent advances in genetic engineering tools and techniques have made it possible to overcome these challenges and make significant progress in developing improved potato varieties.

10.4

CRISPR/Cas

CRISPR/Cas technology has rapidly evolved since its initial discovery, and it is now widely used in genetic engineering, including in plants. CRISPR/Cas technology allows scientists to precisely and efficiently edit the genome of plants, which has numerous applications in agriculture, horticulture, and other industries. The CRISPR/Cas system is a natural defense mechanism that bacteria use to protect themselves from viruses (Langner et al. 2018). The basic mechanism of CRISPR/Cas technology involves three main components: the Cas enzyme, a guide RNA (gRNA), and a target DNA sequence. The gRNA is designed to be complementary to the target DNA sequence, and it binds to the Cas enzyme, forming a complex that can recognize and bind to the target DNA sequence. Once the CRISPR/Cas complex has bound to the target DNA sequence, the Cas enzyme cuts the DNA, creating a double-strand break (DSB) at the site. This DSB can be repaired by one of two mechanisms: non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone mechanism that often results in small insertions or deletions (indels) at the site of the DSB, which can disrupt the function of the targeted gene. HDR, on the other hand, is a more precise mechanism that can be used to introduce specific changes to the DNA

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sequence, such as the insertion of a new gene or the replacement of a specific sequence. CRISPR/Cas technology has revolutionized the field of genome editing and has many potential applications in areas such as agriculture, biotechnology, and medicine.

10.5

Recent Advancements in Genome Editing

Since the initial discovery of CRISPR-Cas9, researchers have been developing new variations and applications of this technology. Some of the recent advancements in CRISPR technology include prime editing, base editing, CRISPR imaging, and CRISPR therapeutics. The development of new CRISPR tools and methods has further expanded the potential applications of this technology in plant science research. In recent years, various new Cas proteins have been identified or developed for genome editing (Table 10.1). Transgene-free editing has been performed successfully in potato for Phytoene Desaturase (PDS) gene (Bánfalvi et al. 2020; Siddappa et al. 2023). In this chapter, we will highlight some of the recent advancements in CRISPR technology in the field of plant science.

10.6

Prime Editing

A new CRISPR-based genome editing technique called “prime editing” allows for the precise insertion, deletion, or modification of DNA without creating doublestrand breaks. Prime editing involves the use of a Cas9 enzyme fused to reverse transcriptase, along with a prime editing guide RNA (pegRNA), which directs the Cas9 to a specific genomic site and provides a template for the desired edit. The pegRNA directs the Cas9-RT enzyme to the target DNA sequence, where the Cas9RT nicks one strand of the DNA at a specific location. The donor template contained within the pegRNA is then reverse-transcribed into the nicked DNA, using the other Table 10.1 Different types of Cas proteins are used in genome editing Cas Protein Cas9 Cas12a (Cpf1) Cas13

Function RNA-guided endonuclease RNA-guided endonuclease

Type Type II Type V

Reference Jinek et al. (2012) Zetsche et al. (2015a, b)

TypeVI

Abudayyeh et al. (2016)

Cas14

RNA-guided RNA endonuclease RNA-guided endonuclease

Harrington et al. (2018)

CasX FnCas9 CasPhi

RNA-guided endonuclease RNA-guided endonuclease RNA-guided endonuclease

Type IV Type V Type II Type V

Liu et al. (2019) Acharya et al. (2019) Richter et al. (2020); Pausch et al. (2020)

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strand as a template. This results in the replacement of the targeted DNA sequence with the desired sequence from the donor template. CRISPR prime editing can be used to introduce a wide range of modifications, including point mutations, insertions, and deletions, without the need for double-strand breaks or homologydirected repair. This technique has the potential to greatly expand the scope of genome editing applications, particularly in cases where conventional CRISPR/ Cas methods are not feasible (Anzalone et al. 2020; Veillet et al. 2020). Perroud et al. (2022) used prime editing to edit the StALS1 gene in tetraploid potato. This technique has the potential to greatly expand the scope of genome editing applications.

10.7

Base Editing

Another CRISPR-based genome editing technique called “base editing” allows for the precise modification of individual DNA bases without creating double-strand breaks. It involves the use of a Cas9 enzyme fused to a deaminase enzyme, along with a specific guide RNA, which directs the Cas9 to a specific genomic site and the deaminase to a specific base. It allows for precise nucleotide substitutions without creating double-stranded breaks or requiring the use of a donor template. This technique can potentially be used to correct single-point mutations that cause genetic diseases (Marx 2018; Molla and Yang 2019). This technology has been used to create new crop varieties with improved yield, disease resistance, and nutritional value (Liang et al. 2018). However, base editing has limitations in terms of the types of nucleotide substitutions that can be made and the potential for off-target effects. As such, further optimization and research are needed to fully realize the potential of this technique. Through the implementation of base editing techniques, the StGBSSI gene was precisely edited in tetraploid potato (Veillet et al. 2019).

10.8

CRISPR Imaging

A CRISPR-based imaging technique called “SHERLOCK” can detect specific RNA or DNA sequences in cells with high sensitivity and specificity. SHERLOCK involves the use of a Cas13 enzyme, which is guided to the target sequence by a specific guide RNA, and then activated to cleave a non-target RNA molecule, leading to a fluorescent signal. This technique has the potential to be used for a wide range of applications, including disease diagnosis and monitoring (Kellner et al. 2019). For example, CRISPR/Cas system was used to visualize endogenous mRNA in living Arabidopsis thaliana plants. In this study, a fluorescent RNA aptamer fused to a nuclease-deficient Cas9 protein was used to target specific mRNAs for visualization in real time (Wu et al. 2019). This technique can also be used for the visualization of multiple genomic loci at the same time (Singh and Jain 2022).

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Multiplex CRISPR-Cas9 System

The CRISPR-Cas9 system has been used extensively in plant research to generate targeted mutations. The recent development of multiplex CRISPR-Cas9 systems has allowed for the simultaneous editing of multiple genes in a single experiment. This approach has been used to create new crop varieties with multiple desirable traits (Xu et al. 2019). For instance, Abdallah et al. (2022) used a multiplex CRISPR-Cas9 system to simultaneously edit different genes involved in drought stress tolerance in wheat. Zhang et al. (2016) used a multiplex CRISPR-Cas9 system to generate mutants of six different genes involved in plant hormone signaling in Arabidopsis and demonstrated that these genes are essential for proper plant growth and development. Uranga et al. (2021) engineered potato virus X (PVX) to develop a vector having multiple sgRNAs for Solanaceous crops. Ly et al. (2023) used a multiplex CRISPR-Cas9 system to improve the potato tuber quality by reducing the tuber browning and acrylamide.

10.10 CRISPR Off-Target Detection A major concern with CRISPR technology is the potential for off-target effects. Recently, researchers have developed new CRISPR tools that can detect off-target effects in plant genomes. This approach has been used to validate the specificity of CRISPR-Cas9 editing in plants (Molla and Yang 2019).

10.11 CRISPR Epigenome Editing The recent development of CRISPR epigenome editing tools has allowed for the precise modification of epigenetic marks in plant genomes. This technology has been used to study the function of epigenetic marks in gene regulation and to create new crop varieties with improved agronomic traits. CRISPR genome engineering has been used to accelerate the domestication of wild plants. This approach involves the identification of genomic regions that differ between wild and domesticated plants, followed by the use of CRISPR to modify these regions in wild plants. This technology has the potential to create new crop varieties with improved yield and other desirable traits (Miglani and Singh 2020; Qi et al. 2023).

10.12 Applications of CRISPR for Potato Improvement CRISPR technology offers precise and targeted gene editing capabilities that can be used to enhance desired traits in potato varieties, such as abiotic stress tolerance, disease resistance, increased yield, herbicide tolerance, improved nutritional content, and enhanced storage life of potato tubers (Fig. 10.1).

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Fig. 10.1 Applications of CRISPR/Cas for the development of improved potato varieties. Some potential genes are mentioned that can be used to improve the specific traits in the existing highyielding potato varieties

10.13 Heat Tolerance Heat stress is a major challenge for potato production, as high temperatures can negatively impact plant growth, yield, and quality (Singh et al. 2020a). Genome editing technologies, including CRISPR/Cas, offer a promising approach to developing potato varieties with enhanced heat tolerance. Several studies have investigated the use of CRISPR/Cas to modify genes involved in heat stress response and thermotolerance in potato. One study targeted the expression of the DREB2A gene, which encodes a transcription factor that regulates stress-responsive genes in plants. Using CRISPR/Cas9, the researchers generated potato plants with mutations in the DREB2A gene that exhibited improved thermotolerance, as evidenced by higher photosynthetic rates and biomass accumulation under high-temperature conditions (Sarkar et al. 2019; Wang et al. 2020). Another study targeted the expression of the StCBF1 gene, which is part of the CBF/DREB1 regulation that controls cold and heat stress response in plants. CRISPR/Cas9 system can be used to develop potato plants with mutations in the StCBF1 gene to improve combined abiotic stress tolerance (Zhu et al. 2018; Song et al. 2021). CRISPR/Cas has also been used to modify other genes involved in heat stress response and

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thermotolerance in potato, including the HSP gene family and the WRKY transcription factor family (Zhang et al. 2017; Cheng et al. 2021; Shahzad et al. 2021). Overall, these studies demonstrate the potential of CRISPR/Cas to improve heat tolerance in potato, which could lead to the development of more resilient and productive potato varieties that are better adapted to changing climatic conditions. Tomar et al. (2021) validated the role of five genes namely StSSH2, StWTF, StUGT, StBHP, and StFLTP in potato tuberization under heat stress. Further, there are several positive and negative regulators of potato tuberization under elevated temperatures (Dutt et al. 2017; Singh et al. 2020a). All these candidate genes can also be targeted by using CRISPR/Cas to enhance heat tolerance in potato.

10.14 Drought Potato production is severely impacted by drought stress. During drought, potato plants may not be able to absorb the necessary nutrients from the soil, resulting in stunted growth and reduced yields. Drought can also increase the risk of diseases and pests, as stressed plants are more susceptible to these issues. In addition, prolonged drought can lead to soil degradation and erosion, which can have long-term effects on potato cultivation in the affected area (Nasir and Toth 2022). With global climate change leading to more frequent and severe drought events, there is an urgent need to develop new potato cultivars that are more resilient to drought. Genome editing technologies, such as the CRISPR/Cas system, offer a promising approach to address this challenge by targeting genes involved in drought stress response and improving potato drought tolerance. CRISPR/Cas systems have been used to target various genes involved in drought stress response in potato. For example, one study targeted the expression of the StERF3 gene, which encodes a transcription factor that regulates drought and salt stress response in plants (Wang et al. 2015). CYCLING DOF FACTOR 1 (StCDF1) and its lncRNA counterpart StFLORE link tuber development and drought response (Gonzales et al. 2021). So far not much work has been done on potato to improve drought stress using CRISPR technology. However, there are a number of candidate genes available in the literature from the model plants, cereals, and potato itself (Monneveux et al. 2013; Gervais et al. 2021). For example, the expression levels of StCIPK10 and StCIPK18 increase significantly under drought stress in potato and these genes interact with various other genes such as StCBL1, StCBL4, StCBL6, StCBL7, StCBL8, StCBL11, and StCBL12 in response to the stressed conditions (Ma et al. 2021; Yang et al. 2023).

10.15 Salinity Salt stress can have a detrimental impact on potato crop, as excessive levels of salt in the soil can damage the roots of the plants and inhibit their ability to absorb water and nutrients. This can result in reduced growth and yield. In addition, salt stress can

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lead to changes in the chemical composition of the potato tubers themselves, affecting their taste, texture, and nutritional value. It can lead to reduced yield and quality of potato tubers, and there is a need to develop new cultivars that are more tolerant to salt stress (Chourasia et al. 2021). The utilization of genome editing techniques, including the CRISPR/Cas system, offers a viable solution to enhance the salinity tolerance of potato crops. This could be achieved by focusing on specific genes that play a role in the plant’s response to salt stress. By targeting and modifying these genes, potato plants can become more resistant to the negative impacts of salt stress, which ultimately leads to improved crop yield and quality. CRISPR/Cas technology has been employed to study several genes related to the salt stress response in potato and various other candidate genes could be targeted in near future. For example, targeted expression of the StSOS1 gene, which encodes a plasma membrane Na+/H+ antiporter that regulates Na+ exclusion and K+ uptake under salt stress, can be studied through CRISPR/Cas9 to generate potato plants with mutations in the StSOS1 gene that might exhibit improved salinity tolerance, by increasing the growth, K+ content, and reduced Na + accumulation under salt stress (Li et al. 2020a, b, c, 2022a, b). In addition to these studies, other genes involved in salt stress response should also be targeted using CRISPR/Cas in potato, including the StNHX1 gene and the StWRKY transcription factor family (Wani et al. 2020; Akrimi et al. 2021). These studies collectively demonstrate the potential of CRISPR/ Cas to improve potato salinity tolerance by modifying genes involved in salt stress response.

10.16 Cold Stress Potato is a highly sensitive crop to frost stress. In regions where cold temperature events occur, frost damage can lead to significant yield loss and reduced quality of potato tubers (Yan et al. 2021). Traditional breeding methods have been used to improve frost tolerance in potato, but these approaches are time-consuming and rely on genetic variation within the available germplasm. The advent of genome editing technologies, such as the CRISPR/Cas system, provides a new and effective approach to improve frost tolerance in potato. CRISPR/Cas has been used to enhance frost tolerance in plants by targeting genes that regulate cold acclimation by which plants increase their freezing tolerance in response to low temperatures. For example, CRISPR/Cas-mediated mutations in the StCBF1 gene, which encodes a transcription factor that regulates cold acclimation in potato, could enhance frost tolerance and survival rates and reduce ion leakage under freezing conditions (Song et al. 2021; Li et al. 2022a; b). Kou et al. (2018) reported that ADC1-associated putrescine pathway regulates frost tolerance in potato by regulating the expression of CBF genes. It could also be a possible target to improve cold stress tolerance in potato using CRISPR/Cas technology. Another approach involves targeting genes involved in the production of protective compounds that help plants tolerate freezing stress.

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10.17 Biotic Stress Potato is a staple food crop that is susceptible to various biotic stresses, including diseases caused by fungi, bacteria, viruses, and nematodes. Potato varieties with better resistance to biotic stress have been developed through conventional breeding methods. However, these methods face limitations due to the genetic variability in the available germplasm. Genome editing technologies like CRISPR/Cas have emerged as a promising and powerful tool to enhance biotic stress tolerance in potato. This technique has been used to target various resistance and susceptibility genes involved in biotic stress tolerance in potato. Biotic stress tolerance can be increased by targeting genes that regulate disease resistance pathways. This can be achieved by enhancing the expression of resistant genes or knocking out susceptible genes (Moniruzzaman et al. 2020). In cases where these genes are not present in the crop germplasm, resistant genes can be transferred from other species via transgenic methods. In recent years, CRISPR/Cas-based editing has been used to improve resistance against various pathogens in crops. Potato virus Y (PVY) can cause significant yield loss, up to 80% (Quenouille et al. 2013), and CRISPR complexes that target RNA, such as Cas12a from Leptotrichia shahii (LshCas13a), have been used to enhance PVY resistance in potato. For instance, Zhan et al. (2019) targeted four conserved regions among three strains of PVY and showed that the most promising LshCas13a/sgRNA transgenic lines did not exhibit any infection symptoms when exposed to PVY. Similar multiplexing approaches can be used to develop multiple resistance against different potato viruses. Lucioli et al. (2022) demonstrated that targeted mutagenesis of eIF4E1 using CRISPR/Cas9 technology confers resistance against the PVYNTN strain in potato. Late blight caused by Phytophthora infestans is the most devastating disease among all potato diseases. Kieu et al. (2021) employed CRISPR/Cas-mediated techniques to generate knockout lines of StCHL1, StDND1, and StDMR6-1, which are susceptibility genes, and these edited potato lines showed enhanced resistance against late blight. Razzaq et al. (2022) used the CRISPR/Cas9 approach, to inhibit the functionality of the StERF3 gene, which resulted in the development of StERF3 gene edited potato plants. These plants displayed enhanced resistance against Phytophthora infestans, indicating that this method could be an effective strategy for controlling late blight disease in potato plants. Alternatively, many wild potato species have known resistance genes (R-genes) that can be introgressed into modern high-yielding potato cultivars (Paluchowska et al. 2022).

10.18 Tuber Quality Potato is one of the most important food crops in the world, providing a significant source of carbohydrates, vitamins, and minerals to millions of people. The quality of potato tubers is an important aspect of their overall value and is determined by a range of traits such as starch content, texture, color, and flavor (Ahmad et al. 2022).

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Potato varieties with better tuber quality were created through traditional breeding methods, but these methods faced constraints due to the genetic variability and loss of other traits which improved one. However, genome editing techniques such as CRISPR/Cas provide a new and efficient way to enhance tuber quality in potatoes. Several attempts have been made to improve potato tuber quality through CRISPR/Cas by targeting various genes. The major focus has remained on the genes involved in the regulation of starch biosynthesis. Consumption of potato tubers with a high amylose content is associated with a low glycemic index. For example, the Starch Branching Enzyme (SBE) gene family, which encodes enzymes involved in the biosynthesis of amylopectin, has been targeted using CRISPR/Cas to generate potato plants with altered starch composition and improved tuber quality. The resulting potato plants exhibited improved texture, cooking quality, and digestibility (Tuncel et al. 2019; Zhao et al. 2021). Further, Veillet et al. (2019) developed transgene-free knockouts through base editing of the GBSS gene via CRISPR/Cas9 targeting. Storing potato tubers in cold temperatures results in the transformation of sucrose into reducing sugars. If these cold-stored potato tubers are cut and processed at high temperatures, acrylamide can be produced, which is considered a carcinogenic substance. Potato processors prefer to use cultivars that accumulate fewer reducing sugars, which results in lower levels of acrylamide during processing. Ly et al. (2023) used CRISPR-Cas9 to mutate the genes encoding vacuolar invertase (VInv) and asparagine synthetase 1 (AS1) to lower the accumulation of reducing sugars and asparagine production during cold storage. The findings of this study suggest that editing of these genes can lighter the tuber color due to less production of acrylamide. Another approach involves targeting genes involved in the regulation of tuber color and antioxidant content. For instance, the R2R3-MYB transcription factor gene, which regulates the production of anthocyanin pigments responsible for fruit color (Allan et al. 2008), could be a possible target to generate potato plants with altered skin color and improved antioxidant content using CRISPR/Cas. The R2R3-MYB transformants exhibited increased levels of anthocyanin pigments and improved antioxidant activity in potato (Jung et al. 2009). Further, Singh et al. (2021b) mentioned potato periderm as a protective barrier against various biotic and abiotic stresses and suggested various candidate genes involved in the periderm formation and suberization that could be used to further reinforce this protective layer. In one such study, CRISPR/Cas9-mediated genome editing of the Caffeoyl-CoA O-methyltransferases gene (StCCoAOMT) in potato resulted in a single nucleotide polymorphic (SNP) mutation that facilitated quantitative resistance. StCCoAOMT is responsible for catalyzing the methylation of caffeoyl-CoA to feruloyl-CoA in the phenylpropanoid pathway, with the latter being a crucial substrate for the synthesis of various defense-related metabolites in plants. Deposition of these metabolites leads to secondary cell wall thickening (Hegde et al. 2021). In addition to these studies, other genes involved in tuber quality could also be targeted using CRISPR/ Cas in potato, including genes involved in the regulation of tuber size, shape, and texture.

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10.19 Nutrition At present, there are approximately 2 billion people worldwide who are experiencing nutritional deficiencies, also known as “hidden hunger.” This issue is particularly prevalent in developing countries, where micronutrient deficiencies are widespread. Solanaceous crops have a high potential for both industrial use and as food, and their biofortification could be an effective way to combat global hidden hunger. These crops are widely consumed around the world and are generally well-accepted by consumers. Previous research has suggested that solanaceous crops have the potential to reduce the risk of hidden hunger (Singh et al. 2021a; Vats et al. 2022), and efforts have been made to improve their nutritional value through breeding, agronomic practices, and transgenic methods, with several promising candidate genes identified in the literature. However, there is still a long way to go, and modern genome editing techniques such as CRISPR/Cas may prove to be a valuable tool for enhancing crop biofortification. Potato is already an important staple food crop worldwide and a major source of carbohydrates, vitamins, and minerals (Singh et al. 2021a). However, the nutritional quality of potato can be further improved through the use of genome editing technologies such as CRISPR/Cas. In recent years, significant progress has been made in the application of genetic engineering methods for potato nutrition. Transgenic potatoes have been developed to increase their nutritional value by expressing a seed protein gene Amaranth Albumin 1 (AmA1) in the potato tubers. This modification was made in seven different genotypic backgrounds, allowing for potato cultivation in climatic conditions. These transgenic tubers showed a significant increase of up to 60% in total protein content compared to the wild type (Chakraborty et al. 2010).

10.20 Future Perspectives Genetic engineering has been a valuable tool for improving crop yield, disease resistance, and nutritional value. One of the crops that have been extensively studied in genetic engineering is potato. The use of genetic engineering techniques can improve potato production and address some of the challenges that potato producers face, such as disease susceptibility and environmental stress. CRISPR/Cas technology has been used in potato to improve traits such as yield, disease resistance, and nutritional content. The use of CRISPR/Cas for potato nutrition also holds great promise for reducing hidden hunger. The targeted manipulation of genes involved in amino acid biosynthesis, vitamin and mineral accumulation, and other metabolic pathways can result in the production of nutritionally enhanced potato varieties. Furthermore, CRISPR/Cas provides a more precise and efficient gene editing method than traditional breeding methods. Despite the promising results of recent studies, there are still challenges that need to be addressed in the use of CRISPR-Cas technology for potato improvement. One of the major challenges is the potential for off-target effects, which could result in unintended changes in the genome. In polyploid crops such as potato, the presence

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of multiple homologous genes in the genome increases the risk of off-target effects, where the CRISPR/Cas system may cleave unintended regions of the genome. This could have negative consequences on the plant’s overall health, yield, and quality. Therefore, it is important to carefully design and test guide RNAs to minimize off-target effects. Further, due to gene duplication, knocking out a single copy of a gene may not result in a noticeable phenotype, as the redundant copies can compensate for the loss. The genome of the potato crop is large and complex, with multiple copies of genes and regulatory elements. This complexity can make it difficult to design and deliver CRISPR/Cas components to the desired target sites. Another challenge is the regulatory landscape surrounding genome-edited crops. While many countries, including the United States, have taken a more permissive approach to regulation, other countries, such as those in the European Union, have implemented more stringent regulations. This can create uncertainty for growers and breeders and may limit the adoption of CRISPR technology. Despite these hurdles, there are also significant opportunities for CRISPR-based potato improvement. For example, CRISPR can be used to rapidly introduce or knock out specific genes associated with desired traits, such as disease resistance or improved nutritional content. This can lead to faster and more precise breeding, and ultimately, more resilient and higher-quality potato varieties. In addition, the ability to precisely control the genetic makeup of plants through CRISPR can enable the development of more customized and tailored potato varieties for specific growing conditions and end uses, such as processing or fresh consumption. Overall, while there are certainly challenges associated with the use of CRISPR for potato improvement, the technology also offers significant opportunities for enhancing the sustainability, resilience, and quality of potato crops.

10.21 Conclusion In conclusion, the potato crop plays a crucial role in ensuring global food and nutrition security, but it is facing numerous challenges such as disease, pests, and abiotic stresses, in addition to the need for developing more nutritious and productive varieties to feed the ever-growing human population. Traditional breeding methods have limitations in addressing these challenges, given the limited genetic diversity in most modern potato cultivars. Genome editing techniques, particularly CRISPR/Cas-mediated genome editing, offer a promising solution to overcome these challenges. With the availability of the potato reference genome sequence and the low cost of high-throughput next-generation sequencing, numerous candidate genes have been identified for various traits in potato, which can be studied using reverse genetic approaches or CRISPR/Cas-mediated gene editing. CRISPR/ Cas technology has shown great potential in editing the potato genome precisely and efficiently, opening up new opportunities to improve the potato crop in terms of pest and disease resistance, yield and productivity, nutritional quality, and environmental sustainability. This technology is also evolving rapidly, and various new Cas proteins and transformation methods have been used during the last few years.

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Therefore, genetic engineering tools such as CRISPR/Cas-mediated genome editing can be considered an essential tool to further enhance the quality and yield of potato crops, particularly in the context of global food security and climate change. Acknowledgements The authors express their gratitude to the National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology (DBT) and Biotechnology Industry Research Assistance Council (BIRAC), Government of India for research support and facilities. The present work is also supported through the externally funded project “Accredited Test Laboratory (ATL) under National Certification System for Tissue Culture Raised Plants (NCS-TCP)” (No. BT/AB/03/ 02/2021) by Department of Biotechnology (DBT), Government of India. Authors acknowledge to DBT-eLibrary Consortium (Del-CON) for providing access to online journals.

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Zhao X, Jayarathna S, Turesson H, Fält AS, Nestor G, González MN, Olsson N, Beganovic M, Hofvander P, Andersson R, Andersson M (2021) Amylose starch with no detectable branching developed through DNA-free CRISPR-Cas9 mediated mutagenesis of two starch branching enzymes in potato. Sci Rep 11(1):1–13 Zhu W, Shi K, Tang R, Mu X, Cai J, Chen M, You X, Yang Q (2018) Isolation and functional characterization of the SpCBF1 gene from Solanum pinnatisectum. Physiol Mol Biol Plants 24: 605–616

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11.1

Introduction

The global population, which is projected to be around 10 billion by 2050, will require at least another 50% increase in food supply to meet the demand of the projected population of 2050. Global rice production in 2018 was 782 million tons from 167.1 million hectares, with an average productivity of 4.68 t/ha (FAOSTAT 2020). The rice crop productivity is severely affected by both biotic and abiotic factors due to various factors, including climate change. Among the biotic constraints, insect pests are considered the major factor causing a reduction in rice yield (Behura et al. 2011). Chemical pesticides are the predominantly preferred mode of pest control. Chemical pesticides are less effective in controlling most insect pests due to their feeding nature, wherein either they live inside the stem or inside folded leaf nests or in the soil, avoiding direct contact with the applied pesticides. Moreover, the indiscriminate use of pesticides in rice is becoming a major concern for the agricultural ecosystem affecting the beneficial insects (Waddington et al. 2010). The development of insect-resistant rice varieties by using conventional breeding techniques is challenging as it is time-consuming to develop a variety with insect resistance. The diversity of rice germplasms with insect resistance traits is scarce, and the existing resistant rice varieties could not provide sufficient levels of insect resistance. Advances in plant biotechnology have resulted in the development of transgenic crop plants expressing novel traits, including insect resistance. Genetically engineered insect-resistant crops are reported to provide environmental benefits as there is a potential reduction in overall pesticide usage and soil conservation (Fernandez-Cornejo and Caswell 2006). Genetically engineered crop varieties are G. Rajadurai · S. Varanavasiappan · L. Arul · E. Kokiladevi · K. K. Kumar (✉) Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_11

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approved for commercial cultivation in 29 countries in 2019 (ISAAA 2020). The Bt transgenic plants engineered with the different cry genes belonging to the Cry 1 and 2 group are protected against lepidopteran pests. In this review, we focus on candidate genes with the potential to be deployed in transgenic plants and assess the future potential of this technology for making host plant resistance an effective weapon in pest management.

11.2

Genetic Transformation of Rice for Insect Resistance

Cereal crops have been the primary target for improvement by genetic transformation because of their worldwide importance for human consumption. Over the past centuries, the improvement in cereals was achieved mostly by conventional breeding. Genetic engineering of cereals has provided new opportunities for the introduction of agronomically useful traits (Kumlehn et al.2009). Genes for resistance to insect, fungal, viral diseases, and nematodes have been utilized in rice transformation. To evolve crop plants with insect resistance, genes from bacteria such as Bacillus thuringiensis, B. subtilis (Ehrenberg) Gohn, and B. sphaericus Meyer and Neide, as well as genes derived from genes such as protease inhibitors, plant lectins ribosomeinactivating proteins, and small RNA viruses, have been used alone or in combination. Since their introduction in 1996, genetically engineered crops with cry genes (Bt genes) have been widely used in agriculture worldwide (Abbas 2018). Other strategies for protecting plants from insect attacks have also been investigated. Lectins, found in various plants, bind to carbohydrates in the midguts of phytophagous insects, causing the digestive system to malfunction (Vandenborre et al. 2011). Transgenic techniques have also been used to deploy protease inhibitors and alphaamylase inhibitors, which prevent insects from digesting the ingested food (Singh et al. 2020) to impart resistance to insects belonging to Lepidoptera, Coleoptera, Diptera, and Hemiptera. The bioefficacy of various insecticidal genes expressed in crops plants is discussed below.

11.2.1 Insecticidal Toxin Proteins of Bacillus thuringiensis B. thuringiensis (Bt), a gram-positive, was discovered in 1901 from diseased silkworm (Bombyx mori Linnaeus) larvae by Ishiwata. Further research on Bt by Steinhaus (1951) led to renewed interest in biopesticides, which led to the development of potent biopesticides such as Thuricide and Dipel (Karthikeyan et al. 2012). The HD-1 strain identified by Dulmage (1981) is one of the most important Bt strains available worldwide. Bt is a spore-forming bacterium that synthesizes parasporal crystalline proteins called δ-endotoxins, which are well known for their insecticidal properties (Leopoldo et al. 2014). Most δ-endotoxins belong to the Cry (crystal) family of proteins, but they also include members of the Cyt (cytolytic) family, a group of proteins found in diptericidal strains of Bt (Crickmore et al. 1998).

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Fig.11.1 Mode of action of Bacillus thuringiensis Cry proteins expressed in rice plants

Insecticidal crystalline proteins (ICPs) are extremely toxic to specific classes of pests (Panwar et al. 2018). To date, 81 classes of cry genes (cry1 to cry81) were included in a database maintained by Crickmore et al. (2021) (http://www.sussex.ac.uk/ lifesci/btlab/toxinnomenclature), with individual toxins showing well-documented toxicity against lepidopterans, coleopterans, hemipterans, dipterans, and nematodes (Rajadurai et al. 2022;Torres-Quintero et al. 2022;de Oliveira et al. 2023). The crystal contains a protoxin protein that is solubilized in the larval midgut by alkaline pH and then cleaved enzymatically to form an active toxin. The toxin diffuses through the peritrophic membrane that covers the gut and binds to receptors present in the midgut epithelium, causing pores to form in the midgut epithelium, then the insect gut becomes paralyzed, stops feeding, and dies within 2–3 days (Fig.11.1). The first transgenic rice plant with insect-resistant Bt protein was reported by Fujimoto et al. (1993). Thereafter, many rice varieties have been transformed with cry genes and shown to resist major lepidopteran pests (Table 11.1). Scented rice cultivars, viz., Basmati370 and M7, have been transformed with cry2A, conferring resistance to the yellow rice stem borer (YSB), Scirpophaga incertulas, and the rice leaf folder (RLF), Cnaphalocrosis medinalis (Maqbool et al. 1998). Microprojectile bombardment and protoplast systems were used to introduce a truncated cry1Ab gene into several indica and japonica rice cultivars (Datta et al. 1998). Khanna and Raina (2002) developed Bt transgenic plants expressing synthetic cry1Ac gene in the genetic background of elite indica rice lines (IR64, Pusa Basmati 1, and Karnal Local). Bt events of IR64 and Pusa Basmati 1 expressing Bt proteins at 0.1% of total

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Table 11.1 Bacillus thuringiensis cry genes for insect resistance in rice S. no. 1.

Transgenes used cry1Ab

2.

cry1Ab

IR58 indica rice

CaMV35S

3.

cry1Ab

Rice actin 1

4.

cry1A

5.

cry1AC

Taipei 309 japonica rice Taipei 309 and Taipei 85-93 (japonica rice), Minghui 63, and Qingliu Rai (indica rice) IR64 (indica rice)

6.

cry1Aa, cry 1Ac, cry2A, cry1C

Indica, japonica

7.

cry1Ab

Aromatic rice, Tarommolaii

8.

cry1Ab

9.

cry2A

Vaidehi (indica rice) Basmati 370 and M7 (indica rice)

10.

cry1Ab

Indica and japonica rice

11.

cry1Ab

Maintainer line IR68899B

12.

cry1Ab

PR16 and PR18

Rice variety Basmati 370 and M7

Promoter used CaMV35S

Target insects Chilo suppressalis, Cnaphalocrocis medinalis Scirpophaga incertulas, Chilo suppressalis, Cnaphalocrocis medinalis Scirpophaga incertulas

References Fujimoto et al. (1993)

–

Scirpophaga incertulas

Wu et al. (1997b)

Maize ubiquitin 1 promoter –

Scirpophaga incertulas

Nayak et al. (1997)

Scirpophaga incertulas, Chilo suppressalis Scirpophaga incertulas and Chilo suppressalis

Lee et al. (1997)

Scirpophaga incertulas Scirpophaga incertulas, Cnaphalocrocis medinalis Scirpophaga incertulas

Alam et al. (1998) Maqbool et al. (1998)

Maize C4 PEP carboxylase gene promoter CaMV35S promoter CaMV35S promoter

35S from CaMV and actin 1 from rice 35S constitutive promoter

Scirpophaga incertulas and Cnaphalocrosis medinalis Scirpophaga incertulas

Wünn et al. (1996)

Wu et al. (1997a)

Ghareyazie et al. (1997)

Datta et al. (1998)

Alam et al. (1999)

Ye et al. (2000) (continued)

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Table 11.1 (continued) S. no.

Transgenes used

13.

cry1Ab, cry1Ac

14.

cry1Ab

15.

cry1Ab

16.

cry1Ab

17.

Rice variety

Minghui 63 (indica CMS restorer line) and its derived hybrid rice Shanyou 63 KMD1 (japonica elite line)

Promoter used Maize ubiquitin promoter Rice actin 1 promoter

Target insects

References

Scirpophaga incertulas and Cnaphalocrosis medinalis

Tu et al. (2000)

Scirpophaga incertulas, Chilo suppressalis, and Cnaphalocrosis medinalis Scirpophaga incertulas Scirpophaga incertulas and Chilo suppressalis Scirpophaga incertulas and Cnaphalocrosis medinalis

Shu et al. (2000)

Scirpophaga incertulas Chilo suppressalis

Maiti et al. (2001) Zeng et al. (2002)

Scirpophaga incertulas

Khanna and Raina (2002)

–

Scirpophaga incertulas

Slamet et al. (2003)

Maize ubiquitin promoter

Scirpophaga incertulas

Raina et al. (2003)

PEPC promoter and PB 10 (pollen-

Scirpophaga incertulas

Husnain et al. (2003)

Maize ubiquitin promoter

Pusa Basmati 1 (indica rice) KMD1 and KMD2 of Xiushui 11

–

cry1Ac, cry2A

M7 and Basmati 370 (indica rice varieties)

18.

cry1Ab

19.

cry1Ac

IR64 (indica rice) Minghui 81

Maize ubiquitin-1 promoter, CaMV 35S promoter –

20.

cry1Ac

21.

cry1Ab

22.

cry1Ac

23.

cry1Ac, cry2A

Pusa Basmati1, IR64, and Karnal Local (indica rice) Rajalele (javanica progenies) IR64, Pusa Basmati-1, and Karnal Local (indica rice) Basmati (indica rice)

Maize ubiquitin-1 promoter

Maize ubiquitin-1 promoter Maize ubiquitin-1 promoter

Gosal et al. (2000) Ye et al. (2001a)

Maqbool et al. (2001)

(continued)

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Table 11.1 (continued) S. no.

Transgenes used

Rice variety

Promoter used specific) promoter Maize ubiquitin I promoter Maize ubiquitin promoter

24.

cry1Ac

Pusa Basmati1 (indica rice)

25.

cry1Ab, cry1Ac

26.

cry1B, cry1Aa

IR58025A, IR58025B, and Vajram (indica rice) Ariete and Senia

27.

cry1Ab, cry1Ac, cry1C, cry2A, cry9C

In vitro

28.

cry1Ac cry2A

Indica basmati rice (B-370)

Maize ubiquitin promoter CaMV 35S promoter

29.

cry2A

30.

cry1Ac, cry2A

Minghui 63 (indica restorer line) Basmati line B-370 (indica rice)

Maize ubiquitin promoter –

31.

cry1Ac, cry2A

Basmati 370 (indica rice)

33.

cry2Ab

34.

cry1Ab

35.

cry1Ab

Minghui 63 (indica restorerline)/T (1Ab)-10 Korean varieties, P-I, P-II, P-III Khazar, Neda, and Nemat

Ubiquitin promoter and CaMV 35S promoter –

36.

cry1Ac, cry2A, cry9C

ubi 1 promoter or mpi promoter –

Maize ubiquitin promoter –

Target insects

References

Scirpophaga incertulas

Gosal et al. (2003)

Scirpophaga incertulas

Ramesh et al. (2004)

Chilo suppressalis

Breitler et al. (2004)

Scirpophaga incertulas and Chilo suppressalis Scirpophaga incertulas, Cnaphalocrosis medinalis, and Pelopidas mathias (rice skipper) Scirpophaga incertulas

Alcantara et al. (2004)

Scirpophaga incertulas and Cnaphalocrosis medinalis Scirpophaga incertulas

Bashir et al. (2005)

Scirpophaga incertulas and Cnaphalocrosis medinalis Scirpophaga incertulas

Tang and Lin(2007)

Chilo suppressalis Scirpophaga incertulas and

Kiani et al. (2008) Chen et al. (2008)

Bashir et al. (2004)

Chen et al. (2005)

Riaz et al. (2006)

Kim et al. (2008)

(continued)

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Table 11.1 (continued) S. no.

Transgenes used

Promoter used Maize ubiquitin promoter rbcS promoter

37.

cry1C

38.

cry1Ab, cry1Ac, cry1C, cry2A

Rice variety Minghui 63 (elite indica restorer line) Zhonghua 11 (Oryza sativa L. subsp. japonica)/RJ5 line Minghui 63 (elite indica restorer line)

39.

cry1Ab

Mfb-MH86

Ubiquitin promoter

40.

cry1Ac, cry1lg

Xiushui 134

41.

Loop replacements with gut-binding peptides in cry1Ab domain II cry2A

In vitro assay

Maize ubiquitin promoter (pUBi)/ modified cauliflower 35S promoter –

42. 43.

cry64Ba, cry64Ca

44.

OsNCED3 overexpression

Maize ubiquitin promoter

Bg 94-1 indica variety –

CaMV35S

Zhonghua11

–

–

Target insects

References

Chilo suppressalis Scirpophaga incertulas, Chilo suppressalis, and Cnaphalocrosis medinalis

Ye et al. (2009)

Scirpophaga incertulas, Chilo suppressalis, and Cnaphalocrosis medinalis Chilo suppressalis and other lepidopteran pests Chilo suppressalis and Cnaphalocrosis medinalis

Yang et al. (2011)

Nilaparvata lugens

Shao et al. (2016)

Cnaphalocrosis medinalis Laodelphax striatellus, Sogatella furcifera Nilaparvata lugens

Gunasekara et al. (2017) Liu et al. (2018)

Wang et al. (2014)

Zhao (2015)

Sun et al. (2022)

soluble protein exhibited 100% mortality of yellow stem borer larvae in a detached stem bit assay as well as in whole-plant assays. Transgenic cry1Ac rice was also shown to be effective against YSB, RLF, rice green caterpillar (Melanitis leda ismene), and rice skipper (Pelopidas mathias) (Kim et al. 2009). A field experiment

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Fig. 11.2 Agrobacterium-mediated transformation using a co-integrate vector to produce markerfree transgenic plants

with transgenic rice Basmati 310 lines expressing cry1Ac or cry2A genes exhibited a high level of toxicity against YSB, RLF, and rice skipper (Bashir et al. 2004). In a multilocation field testing conducted in Iran with Bt rice, genotypes expressing cry1Ab showed that the transgenic rice were resistant to stem borer and had lower yield loss than their non-transgenic counterpart (Dastan et al. 2020). Transgenic Shanyou 63, a popular hybrid in China, expressing cry1C gene exhibited a high level of resistance to stem borers and leaffolders throughout the growth period in field experiments (Tang et al. 2006). The leaffolder-resistant transgenic rice (Bt- T event) was developed by expressing a toxic protein mCry1Ac1 (Amin et al. 2020). A synthetic cry1C gene was transformed into a japonica rice variety, Jigeng 88, and showed higher resistance to SSB than non-transgenic plants (Jin et al. 2021). Retention of selectable marker genes (SMG) poses a major barrier to the development of consumer-friendly genetically engineered rice because of the perceived risk of horizontal gene transfer from plant to bacteria or from plant products consumed as food to intestinal microorganisms, with the possibility of the emergence of antibiotic resistance in them (Ramessaret al.2007). Therefore, the production of marker-free transgenic crops is a dire need of the hour to promote their commercial deployment. Co-transformation has been reported to be an efficient and simple strategy for generating marker-free transgenic plants. The co-transformation followed by rounds of segregation creates marker-free plants (Fig. 11.2) (Yau and Stewart 2013). Agrobacterium-mediated co-transformation is an efficient strategy for generating marker-free transgenic plants. Three marker-free homozygous lines of Minghui86 containing cry2A* were generated using a co-transformation strategy

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against lepidopteran insects (Ling et al. 2016). The marker-free transgenic indica rice (ASD16) was developed by expressing a chimeric cry2AX1 gene driven by green tissue-specific rbcS promoter. For rice transformation, Agrobacterium strain C58C1 carrying cry2AX1 and hphII genes in two independent T-DNAs was used. Markerfree transgenic rice lines showed moderate toxicity levels to rice yellow stem borer and leaf folder (Rajadurai et al. 2018b). Similarly, Chakraborty et al. (2016) demonstrated the cry2AX1 gene by introducing it into JK1044R, the restorer rice line, and tested it against yellow stem borer, leaf folder, and oriental armyworm. Biosafety of the transgenic rice, Xiushui 134Bt (a highly insect-resistant transgenic rice expressing CryIAc1 gene), was assessed by Wistar rat feeding experiments and resulted in no adverse dose-related effects on the growth and development (Yang et al. 2023). Despite the successful application of cry gene technologies in crops to achieve resistance against various insect pests, some insects frequently develop resistance to insecticidal toxins. Other issues that limit transgenic crops’ utility for insect control include secondary pest outbreaks, the evolution of new biotypes, effects on nontarget organisms, environmental influences on gene expression, perception of the general public on biosafety of food derived from transgenic crops, and socioeconomic and ethical concerns.

11.2.2 Vegetative Insecticidal Protein Genes Vegetative insecticidal proteins (Vip) are widely regarded as an excellent alternative to Cry protein for insect control. In addition to Cry proteins found in the parasporal inclusions, insecticidal toxins designated Vip are secreted into the medium during the vegetative phase of growth (Estruch et al. 1996). They differ from Cry proteins on their receptor sites and sequence homology; Vip is one of the best-known families of Bt proteins. The Vip protein family consists of three subfamilies. The heterodimer toxins Vip1 and Vip2 are effective against pests from the Hemiptera and Coleoptera orders, whereas the Vip3 family is toxic to lepidopterans and other insects (Estruch et al. 1997). These proteins have acute toxicity activity comparable to that of Cry proteins. They cause intestinal paralysis, which is followed by complete lysis of the gut epithelium cells, resulting in larval death. Vip proteins are also known as secondgeneration insecticidal proteins that can be used alone or in combination with Cry proteins to control various pest insects (Gupta et al. 2021). They are reported to be toxic to pests, which are not susceptible to Cry proteins. Expression of a Vip3A toxin in sugarcane imparted superior resistance to the sugarcane stem borer, Chilo infuscatellus, with 100% mortality (Riaz et al. 2020). Besides, the Bt isolates with cry1, cry2, and vip3 gene combinations produced 100% toxicity in all the tested lepidopteran insects, as reported by Maheesha et al. (2021) and Karuppaiyan et al. (2022). These proteins are promising candidates for further development of insectresistant plants because they have broad toxicity ranges, particularly against lepidopteran pests.

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11.2.3 Lectins Plant lectins are carbohydrate-binding proteins found in many plant species with a high affinity for specific sugar components found in glycoproteins and glycolipids in the cell membrane of insects (Camaroti et al. 2017). They deter predation by harming various insects and animals that eat plants. They are most commonly found in plants belonging to the families of Solanaceae, Fabaceae, and Poaceae. Some leguminous seeds are known to have a high concentration of lectins. Plant lectins from various sources have previously been reported to be toxic to important members of insects (Rauf et al. 2019). Concanavalin A was the first lectin to be discovered and commercially available, and it is now the most extensively studied lectin for insect pest control (Powell et al. 1993). Using lectins in transgenic plants has produced promising results, especially for crops expressing Bt Cry toxins, which provide resistance to sap-sucking insects. In addition, lectins in artificial diets and their expression in transgenic plants have been shown to reduce performance in insects of various orders, including Lepidoptera, Coleoptera, and Hemiptera (Vandenborre et al. 2011). Resistance to major sap-sucking insects such as brown planthoppers (BPH), white-backed planthoppers (WBPH), and green leafhoppers (GLH) was imparted in transgenic rice by expressing Allium sativum leaf agglutinin (ASAL) or Galanthus nivalis lectin (GNA) (Sengupta et al.2010; Rao et al. 1998 and Sudhakar et al. 1998). Transgenic rice plants expressing GNA showed significant resistance to sap-sucking pests (Sudhakar et al. 1998). Similar observations in sap-sucking pests of rice were made by Yoshimura et al. (2012) when they expressed Dioscorea batatas tuber lectin 1 under the control of phloem-specific rice-sucrose synthase promoter. Fitches et al. (2004) and Down et al. (2006) reported increased toxicity of GNA-spidervenom toxin I (SFI1) fusion protein to rice brown planthopper, BPH (Nilaparvata lugens). Lectins have been shown to control insect pests of various orders and stages of development, preventing growth, survival, nutrition, development, and reproduction (Napoleao et al. 2019). Insect resistance was demonstrated by the expression of various lectin genes in rice plants (Table 11.2). Progress in lectin research has been hampered due to toxicity on higher animals, despite the fact that no adverse effect was observed in rats fed transgenic rice containing GNA for 90 days (Poulsen et al. 2007). However, due to their known toxicity to mammals and humans, they should be used with caution in transgenic plants.

11.2.4 Protease Inhibitors Protease inhibitors (PIs) are plant-derived inhibitors that prevent insect pests from digesting their food by inhibiting the activity of digestive proteases (Zhu-Salzman and Zeng 2015). It is known that PIs inhibit insect digestive proteases by preventing proteolysis, which causes a reduction in fecundity, an increase in mortality, and a lengthening of the developmental period due to a lack of essential amino acids.

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Table 11.2 Other toxins for insect resistance in rice S. no. 1.

2.

3.

4.

Transgenes used pinII (potato proteinase inhibitor) Cowpea proteinase inhibitor

Snowdrop lectin (Galanthus nivalis agglutinin; GNA) GNA (Galanthus nivalis agglutinin)

Rice variety Nipponbare, Tainung67, Pi4 Taipei 309 and Taipei 85-93 (japonica rice), Minghui 63, and Qingliu Rai (indica rice) ASD 16, M5, M12, FX 92

ASD 16, M5, M12, FX 92

5.

GNA

ASD16/M12

6.

Snowdrop lectin gene GNA

M7 and Basmati 370 (indica rice varieties)

7.

Spider insecticidal gene

8.

Snowdrop lectin gene GNA Snowdrop lectin gene GNA

Xiushuill and Chunjiang 11 –

9.

10.

GNA

Rajalele (javanica progenies) Chaitanya and Phalguna, indica cultivars

Promoter used pin2 promoter

Target insects Mythimna separata

References Duan et al. (1996)

–

Scirpophaga incertulas

Wu et al. (1997b)

Maize ubiquitin 1 promoter

Sap-sucking pests

Sudhakar et al. (1998)

Phloemspecific ricesucrose synthase, CaMV35S Sucrose synthase/ maize ubiquitin Maize ubiquitin1 promoter, CaMV 35S promoter –

Nilaparvata lugens

Rao et al. (1998)

Nilaparvata lugens and Nephotettix virescens Nilaparvata lugens

Foissac et al. (2000)

– –

Phloemspecific ricesucrose synthase

Chilo suppressalis and Cnaphalocrosis medinalis Eoreuma loftini (Dyar) Planthoppers

Nilaparvata lugens, Nephotettix virescens, and Sogatella furcifera

Maqbool et al. (2001)

Huang et al. (2001)

Setamou et al. (2002) Slamet et al. (2003) Nagadhara et al. (2003), (2004)

(continued)

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Table 11.2 (continued) S. no. 11.

Transgenes used Snowdrop lectin gene GNA

12.

GNA

13.

mpi gene (maize proteinase inhibitor) GNA-spidervenom toxin I (SFI1) ASAL (Allium sativum agglutinin)

14.

15.

Rice variety Indica rice lines

Zhuxian B, an indica rice Senia and Ariete

Promoter used Ricesucrose synthase promoter

–

–

Maize ubiquitin 1 promoter –

IR64

CaMV35S

Pusa basmati-1 and Tarori Basmati (indica rice) and TNG67 (japonica rice) Chaitanya and BPT5204, indica cultivars

Pin2 woundinducible promoter

16.

PINII (potato proteinase inhibitor)

17.

ASAL

18.

ASAL

IR64

CaMV35S

19.

ASAL and GNA

T49 X OU-1

–

20.

DB1/ G95AmALS (Dioscoria

Tachisugata

Phloemspecific rice-

CaMV35S

Target insects Nilaparvata lugens, Nephotettix virescens, and Sogatella furcifera Nilaparvata lugens Chilo suppressalis

References Ramesh et al. (2004)

Li et al. (2005) Vila et al. (2005)

Nilaparvata lugens

Down et al. (2006)

Nilaparvata lugens and Nephotettix virescens Scirpophaga incertulas

Saha et al. (2006)

Nilaparvata lugens, Nephotettix virescens, and Sogatella furcifera Nilaparvata lugens and Nephotettix virescens Nilaparvata lugens, Sogatella furcifera, and Nephotettix nigropictus Nilaparvata lugens

Bhutani et al. (2006)

Yarasi et al. (2008)

Sengupta et al. (2010)

Bharathi et al. (2011)

Yoshimura et al. (2012) (continued)

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Table 11.2 (continued) S. no.

Transgenes used

21.

batata tuber lectin) ASAL

IR-64

Overexpression of Csu-miR-14

Japonica rice variety ZH11

22.

Rice variety

Promoter used sucrose synthase Phloemspecific ricesucrose synthase Maize ubiquitin promoter

Target insects

References

Nilaparvata lugens

Chandrasekhar et al. (2014)

Chilo suppressalis

He et al. (2019)

Serpins and cystatins are the most studied plant PIs against pests. Serpins, with a molecular mass of approximately 39–43 kDa, are irreversible serious inhibitors of serine proteases. Serine proteases have been found in the insect orders Diptera, Lepidoptera, Orthoptera, Coleoptera, and Hymenoptera (Irving et al. 2002). The activity of cysteine proteases, which are the main proteases used for digestion in Coleopterans and Hemipterans, is inhibited by cystatins, a PI protein with a molecular mass of 12–16 kDa. Legume trypsin inhibitors inhibit a wide range of proteases and have insecticidal activity against several important insect pests (Sharma 2015). Protease inhibitor genes were introduced in rice cultivars to develop resistance against stem borers (Duan et al. 1996). Constitutive expression of CpTi in rice was shown to confer resistance to rice striped stem borer (SSB) Chilo suppressalis and rice pink stem borer, Sesamia inferens Walker (Xu et al. 1996). Mochizuki et al. (1999) reported that the growth of C. suppressalis larvae in rice was significantly retarded by a synthetic gene (mwti11b) encoding a winged bean trypsin inhibitor (WTI-11B). Additionally, rice with a high concentration of Kunitz-type SBTI has N. lugens resistance (Lee et al. 1999). In China, the field investigation revealed that two transgenic rice lines, both expressing Cry1Ac and CpTI proteins, are extremely resistant to natural C. suppressalis infestations and give significantly higher control efficacy than chemical pesticide control treatments (Han et al. 2006). In another experiment, the japonica rice variety “Minghui86” was transformed with the cry1Ac gene and CpTI, resulting in highly resistant transgenic lines against rice leaf folders (Han et al. 2007). The expression of the protease inhibitors genes against rice insect pests is provided in Table 11.2. PIs have limited commercial application due to the enormous adaptive potential of insect pests and their long coevolutionary relationship with host plants. The resolution of these issues may pave the way for future research.

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11.2.5 Insect Chitinase Insect chitinases are hydrolytic enzymes that have the ability to inhibit or degrade chitin. Chitin is the primary component of the exoskeleton and peritrophic membrane in insects. It safeguards against harsh environmental conditions, external mechanical disruption, and natural enemies (Chen et al. 2018). The hydrolysis of chitin is essential for ecdysis (periodic shedding of the old cuticle). Chitinases are expressed in various organisms, including those lacking chitin, such as plants, to recognize and degrade chitin in insects (Oyeleye and Normi 2018). The role of chitinase enzyme in insect pests management has been studied in several insects such as silkworm, B. mori, rice brown planthopper, N. lugens, cotton mealybug, P. solenopsis, and rice striped stem borer C. suppressalis (Pan et al. 2012;Xi et al. 2015;Su et al. 2016;Omar et al. 2019). Due to their ability to prevent the growth and development of insects, insect chitinases have been developed as biopesticides and transgenes for crop protection. Their use in biotechnological processes will be accelerated by a better understanding of their structure and biochemistry.

11.2.6 Gene Pyramiding Due to the extensive cultivation of transgenic plants expressing Bt genes, the insects have evolved to resist the crystal proteins expressed in the host plant. Techniques such as refugia strategy and gene pyramiding were recommended to prevent insects from becoming completely resistant and increase the time taken to evolve such resistance. This strategy of gene pyramiding or stacking is based on the assumption that a single mutation in a pest is unlikely to confer simultaneous resistance to two different Bt toxins, and thus two-toxin rice cultivars would have the potential to delay the development of resistance more effectively than single-toxin cultivars (Carrière et al. 2016). Thus, the chances for the evolution of resistant insects are quite low compared to plants with single genes (Liu et al. 2017). Maqbool et al. (2001) introduced three insecticidal genes (cry1Ac, cry2A, and snowdrop lectin gene gna) into an indica rice varieties (M7 and Basmati 370) and reported that the pyramided genes expressed stably and the triple-transgenic plants had enhanced and broad insecticidal action against S. incertulas, C. medinalis, and N. lugens. Another study found that rice plants co-transformed with cry1Ac and cry2A genes were more effective against S. incertulas and C. medinalis (Riaz et al. 2006). Pyramiding a fused cry1Ab/1Ac gene conferring resistance to lepidopteran insects and a Xa21 gene providing resistance to bacterial blight disease resulted in desirable target phenotypes in rice, and the pyramiding genes exhibited a yield-stabilizing effect on the recipient line and its hybrids (Jiang et al. 2004). Liu et al. (2016) pyramided two foreign genes, cry1Ac driven by the rice actin 1 promoter, and lysinerich protein (LRP), driven by the endosperm-specific GLUTELIN1 (GT1) promoter, into the elite indica cultivar 9311 and found resistance against C. suppressalis and C. medinalis in the pyramided lines, along with increased lysin content in the seeds.

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Transgenic rice lines expressing cry1Ac + ASAL provided long-lasting resistance to major insects, viz., yellow stem borer, leaf folder, and brown planthopper (Boddupally et al. 2018). A significant level of protein expression was recorded in transgenic rice plants containing cry2Aa + cry1Ca genes toxic to Asiatic rice borer, C. suppressalis (Qiu et al. 2019). The use of the pyramiding strategy for insect resistance in rice plants is given in Table 11.3.

11.2.7 Fusion Protein Fusing two or more genes is a sustainable strategy for efficiently managing different groups of insect pests. Transgenic rice lines expressing fusion proteins of Cry1Ac and Cry1I (Yang et al. 2014) and Cry1Ab and Cry1Ac (Cheng et al. 1998) were reported to be toxic against rice leaf folder (RLF) and striped stem borer (SSB). Cry2A confers resistance against both lepidopteran and dipteran insects. Cry2A is toxic to several of the major lepidopteran pests such as YSB, SSB, and two species of RLF (Marasmia patnalis and C. medinalis) when the insects are fed with the toxin in an artificial diet (Karim and Dean 2000;Alcantara et al. 2004). Furthermore, biochemical studies showed that cry2A did not share binding sites with cry1A in brush border membrane vesicles (BBMV) from rice leaf folder, striped stem borer, or yellow stem borer, indicating that it would be effective to combine cry2A with cry1Ab or cry1Ac (Karim and Dean 2000; Alcantara et al. 2004). A novel chimeric Bt gene cry2AX1 (fusion gene) was developed by using the sequences of cry2Aa and cry2Ac cloned from indigenous isolates of Bt (Udayasuriyan et al. 2010) and reported to be toxic against different lepidopteran pests. Transgenic rice lines expressing cry2AX1 gene showed resistance to rice leaf folder (C. medinalis), yellow stem borer (S. incertulas), and oriental army worm, Mythimna separata (Chakraborty et al. 2016;Manikandan et al. 2016;Rajadurai et al. 2018a). Resistance to both the Asian rice borer and the rice leaf folder was demonstrated by the fusion of cry1Ab and vip3A (C1V3 protein) (Xu et al. 2018). The engineered fusion protein was demonstrated to be more effective against both chewing (lepidopteran) and sucking (Hemiptera) insects, holding tremendous potential for future pest management challenges. The uses of fusion proteins in transgenic rice plants for pest management are shown in Table 11.4.

11.2.8 RNAi Approach for the Management of Rice Insect Pests RNA interference (RNAi) approach is an alternative approach to rice insect pest control, particularly for control of sap-sucking insects for which Bt toxins are not effective. RNAi is an evolutionary conserved homology-based gene-silencing found in all eukaryotic organisms. The RNAi is triggered by double-stranded RNAs (dsRNA), which are processed by the RNase-III-like Dicer protein to produce small interfering RNAs (siRNAs). The guide strand of siRNA directs an

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Table 11.3 Gene pyramiding for insect resistance in rice S. no. 1.

Transgenes used cry1Ab and cry1Ac

Rice variety Nipponbare, Zhong8215, 93VA, ZAU16, 91RM, T8340, Pin92-528, T90502, Kaybonnet Indica rice varieties M7 and Basmati 370

Promoter used Maize ubiquitin, CaMV35S, and Brassica Bp10 gene promoters

References Cheng et al. (1998)

Scirpophaga incertulas, Cnaphalocrosis medinalis, and Nilaparvata lugens Chilo suppressalis Rice leaf folders

Maqbool et al. (2001)

2.

cry1Ac, cry2A, and snowdrop lectin gene gna

3.

Cry1Ac and CpTI Cry1Ac and CpTI cry1Ac and cry2A

Minghui86

–

Minghui86

–

Basmati 370

Maize ubiquitin, CaMV35S

1Ab/1C, 1C/1Ab, 1Ab/2A, 2A/1Ab, 1Ac/1C, 1C/1Ac, 1Ac/2A, 2A/1Ac, 1C/2A and 2A/1C cry1Ac and CpTI

Minghui63

–

Minghui86

–

Chilo suppressalis

Sbk (modified from Cry1A (c)) and sck (modified from CpTI) Cry1Ac and lysine-rich protein (LRP)

Nanjing 45

Maize ubiquitin promoter, rice actin promoter

Chilo suppressalis

Indica cultivar 9311

Rice actin 1 and glutelin 1 promoters

Chilo suppressalis and Cnaphalocrosis medinalis

4. 3.

4.

5.

6.

7.

Maize ubiquitin, CaMV35S

Target insects Chilo suppressalis, Scirpophaga incertulas, and Cnaphalocrosis medinalis

Scirpophaga incertulas and Cnaphalocrosis medinalis Chilo suppressalis, Scirpophaga incertulas, and Cnaphalocrosis medinalis

Han et al. (2006) Han et al. (2007) Riaz et al. (2006)

Yang et al. (2011)

Zhang et al. (2011) Zhang et al. (2013)

Liu et al. (2016)

cry1Ab/ cry1Ac fusion gene

Cry1Ab/Cry9Aa fusion protein

mpi-pci fusion gene

cry2AX1 (cry2Aa and cry2Ac fusion) Fusion proteins of Cry1Ac and Cry1I

7.

8.

9.

10.

12.

11.

cry2AX1 (cry2Aa and cry2Ac fusion)

cry1Ab and vip3H fusion gene

JK1044R

Xiushui-134

ASD16

Ariete

Xiushui 110

Shanyou63

Xi-ushui 110

Pusa Basmati-1

Maize ubiquitin 1 promoter Maize polyubiqutin-1 promoter, pGreen promoter Chrysanthemum rbcS1 promoter

mpi promoter

Maize polyubiquitin1 promoter

Maize ubiquitin promoter –

Chilo suppressalis and Cnaphalocrosis medinalis

Cnaphalocrosis medinalis

Mythimna separata, Cnaphalocrocis medinalis, and Chilo suppressalis Chilo suppressalis

Chilo suppressalis and Sesamia inference Chilo suppressalis

Scirpophaga incertulas

Scirpophaga incertulas

(continued)

Chakraborty et al. (2016)

Quilis et al. (2014) Manikandan et al. (2014) Yang et al. (2014)

Kumar et al. (2010) Chen et al. (2010) Zhang et al. (2011) Jianhua et al. (2011)

Ho et al. (2006)

6.

5.

– Scirpophaga incertulas and Cnaphalocrosis medinalis

cry1Ab-1B (translationally fused gene) and cry1A/cry1Ac (hybrid Bt gene) cry1b and cry1Aa fusion gene

IR68899B and IR68897B (maintainer lines), MH63 and BR827- 35R (restorer lines) Elite Vietnamese cultivars

IR72 (indica rice) TT-9 35S and PEPC promoters; actin 1 promoter Maize ubiquitin promoter and rice actin 1 promoter PEPC promoter

4.

3.

2.

References Tu et al. (2000) Ye et al. (2001b) Balachandran et al. (2002)

Transgenes used Cry1Ab and Cry1Ac fusion protein Bt fusion gene (cry1AB/ cry1Ac) Chimeric Bt gene, cry1Ab; cry1Ab/cry1Ac fusion gene

S. no. 1.

Target insects Scirpophaga incertulas and Cnaphalocrosis medinalis Scirpophaga incertulas

Table 11.4 Fusion protein for insect resistance in rice Promoter used Rice actin 1 promoter

Insect Pest Management in Rice Through Genetic Engineering

Rice variety Minghui 63

11 249

Cry1Ac::ASAL fusion protein

cry1Ab + vip3A fusion protein

15.

16.

A1L3

Pusa Basmati (PB1)

ASD16 indica rice

MH63

TT51 (fusion of cry1Ab and cry1Ac) cry2AX1 (cry2Aa and cry2Ac fusion)

13.

14.

Rice variety

Transgenes used

S. no.

Table 11.4 (continued)

Rice actin 1 promoter, maize polyubiqutin-1 promoter

Maize ubiquitin promoter, rbcS promoter CaMV35S promoter

Rice actin 1

Promoter used

Scirpophaga incertulas, Cnaphalocrocis medinalis, and Nilaparvata lugens Ostrinia furnacalis, Chilo suppressalis, and Cnaphalocrocis medinalis

Scirpophaga incertulas and Cnaphalocrosis medinalis

Target insects Scirpophaga incertulas, Cnaphalocrocis medinalis, and Mythimna separata Chilo suppressalis

Xu et al. (2018)

Niu et al. (2017) Rajadurai et al. (2018a, b) Boddupally et al. (2018)

References

250 G. Rajadurai et al.

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Fig.11.3 Mechanism of RNAi approach

RNA-induced silencing complex (RISC) to the target mRNA (Fig.11.3). The RNAi pest control strategy is based on ingesting dsRNA into the target pest system. The double-stranded RNAs specific to key insect genes can be stably expressed in plant tissues fed on by the insect and that in turn can trigger the RNAi pathway to degrade the mRNAs transcribed by the key insect genes (Price and Gatehouse 2008;Agarwal et al. 2012). The use of the RNAi tool for insect resistance in rice is still in its early stages. The majority of reports on RNAi in rice have focused on BPH (Wang et al. 2018) and YSB (Renuka et al. 2017). Yu et al. (2014) demonstrated significant downregulation of target gene expression and decreased the number of offspring produced by BPH using feeding assays and stable expression of NlEcR (ecdysone receptor gene) targeting dsRNAs in rice. Pan et al. (2018) demonstrated that 32 CPs (chitin and cuticular protein) are required for normal egg production and the development of BPH by using RNAi to knock down 135 CP genes by injecting specific dsRNAs. Kola et al. (2016) revealed that the feeding by YSB larvae with dsRNA of cytochrome P450 derivative (CYP6) and amino peptidase N (APN) decreased target gene expression and resulted in increased larval mortality. Kola et al. (2019) observed reduced larval length and weight of yellow stem borer within 15 days when fed on rice lines with knockdown of the acetylcholine esterase gene (AChE). Zeng et al. (2018) knocked down three chemosensory protein (CSP) genes in rice leaf folder (C. medinalis) through injection of dsRNAs, which downregulated insect response to the specific chemicals. He et al. (2019), in contrast,

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Table 11.5 Transgenic rice for insect resistance through RNAi S. no. 1.

Silenced gene dsRNA-spray

2. 3.

dsRNA designed from two genes cytochrome P450 derivative (CYP6) and aminopeptidase N (APN) Aminopeptidase N genes APN1 + APN2

4.

AchE-acetylcholine esterase

5.

Midgut V-ATPase subunit A gene

Target insects Lepidopteran Scirpophaga incertulas Chilo suppressalis Scirpophaga incertulas Chilo suppressalis

References Li et al. (2015) Kola et al. (2016) Qiu et al. (2017) Kola et al. (2019) Qiu et al. (2019)

overexpressed striped stem borer-derived miR-14 microRNA in rice to target Spook (Spo) and Ecdysone receptor (EcR) in the ecdysone signaling network, which resulted in high resistance against the pest. Two methods have been used to demonstrate the generation and transport of dsRNAs into target insects. The first method is the host-induced gene silencing (HIGS), which involves the transgenic expression of dsRNAs derived from the crop genome. In this method, while the insects feed on transgenic crops, they also uptake dsRNA expressed in the transgenic plant. In the second strategy, dsRNAs are synthesized in high concentrations and applied as a foliar spray to insect-infested crops termed spray-induced gene silencing (SIGS). The genes are silenced in both approaches in the target species (Christiaens et al. 2020a). There are still numerous challenges. While some insects rapidly absorb dsRNA, resulting in high mortality rates, other species have low dsRNA uptake and nuclease degradation, resulting in inefficient outcomes (Shaffer 2020;Christiaens et al. 2020b). Success is also determined by the amount of dsRNA that accumulates in the tissues on which the insects feed. Several studies on rice insect pest management use the RNAi tool as an effective method for insect control (Table 11.5).

11.3

Genome Editing in Rice to Develop Insect-Resistant Varieties

Plant gene editing is a site-directed mutagenesis method employed for making specific alteration in target genes. Gene editing, also called genome editing, is a technique that involves inserting, deleting, or replacing DNA bases in a specific target DNA sequence of the genome for effectively altering the function of a gene (Bortesi and Fischer 2015). CRISPR/Cas9 has recently emerged as a technically simple, effective, and efficient tool for developing insect pest resistance (Anastacia Books 2019). Most herbivory insects use plant volatiles, gustatory signs, visual appearance, oviposition sites, and collaborations to recognize host plants (Larsson et al. 2004). Genome editing can be utilized to change plant volatile mixtures, which

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could be an alternative pest management strategy. However, caution should be taken to ensure that the alteration has no negative consequences for the nontarget organisms. We can efficiently delete the genes that cause insect susceptibility using CRISPR-Cas9 genome editing to develop insect-resistant cultivars. Insectresistant rice plants with CYP71A mutations, which catalyze the conversion of tryptamine to serotonin, accumulated high levels of salicylic acid but lacked serotonin, which reduced planthopper growth. Lu et al. (2018) modified rice OsCYP71A1 using CRISPR-Cas9 technology to make it resistant to the striped stem borer, C. suppressalis, and brown plant hoppers, N. lugens. Although CRISPR-based genome-editing technology is used for functional genomics of insect genes, its use for pest resistance in crops is yet to be fully realized. There is a dearth of information about insect-susceptibility genes of host plants, specifically in rice. The recessive resistance genes identified so far are likely to represent nonfunctional susceptibility genes, hence the need for more studies to characterize such candidate genes, which represent ideal targets for genome editing to develop new sources of resistance.

11.4

Conclusions

Rice insect pest control is mainly mediated by the use of chemical pesticides. The indiscriminate use of synthetic pesticides has a negative impact on nontarget organisms and the environment. From this perspective, developing insect-resistant rice varieties offers a solution for the control of insect pests with no harmful effect on nontarget organisms and the environment. Rice breeding efforts resulted in developing varieties tolerant to certain insect pests. However, due to the lack of resistance sources for many insects such as yellow stem borer, an alternate approach for pest control is needed. Transgenic rice expressing insecticidal genes, mainly the cry genes, is successful in giving protection against major rice lepidopteran pests such as yellow stem borer and rice leaf folder. Most of the available Bt proteins cannot control insects belonging to orders other than the lepidopteran order. Hence, RNAi technologies can be used for sucking pests’ control by expressing the dsRNA of the target gene. In recent times, genome editing in rice resulted in stem borer resistance, offering the possibility to impart resistance against major pests of rice. We conclude that transgenic technologies can be effectively implemented for eco-friendly insect pest management in rice.

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Yang R, Piao Z, Tang J, Wan C, Lee G, Bai J (2023) Subchronic toxicological evaluation of Xiushui 134Bt transgenic insect-resistant rice in rats. Agronomy 13(3):826. https://doi.org/10. 3390/agronomy13030826 Yarasi B, Sadumpati V, Pasalu IC, Reddy DV, Rao KV (2008) Transgenic rice expressing Allium sativumleaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests. BMC Plant Biol 8:102. https://doi.org/10.1186/1471-2229-8-102 Yau YY, Stewart CN (2013) Less is more: strategies to remove marker genes from transgenic plants. BMC Biotechnol 13:36. https://doi.org/10.1186/1472-6750-13-36 Ye G, Shu Q, Cui H et al (2000) A leaf-section bioassay for evaluating rice stem borer resistance in transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner. Bull Entomol Res 90:179–182. https://doi.org/10.1017/s0007485300000298 Ye G, Shu Q, Yao H et al (2001a) Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers. J Econ Entomol 94:271–276 Ye G, Tu J, Hu C, Datta K, Datta SK (2001b) Transgenic IR72 with fused Bt gene cry1AB/cry1Ac from Bacillus thuringiensis is resistant against four lepidopteran species under field conditions. Plant Biotechnol 18:125–133 Ye R, Huang H, Yang Z, Chen T, Liu L, Li X, Chen H, Lin Y (2009) Development of insectresistant transgenic rice with Cry1C-free endosperm. Pest Manag Sci 65:1015–1020 Yoshimura S, Komatsu M, Kaku K et al (2012) Production of transgenic rice plants expressing Dioscorea batatas tuber lectin1 to confer resistance against brown planthopper. Plant Biotechnol 29:501–504 Yu R, Xu X, Liang Y et al (2014) The insect ecdysone receptor is a good potential target for RNAi based pest control. Int J Biol Sci 10:1171–1180. https://doi.org/10.7150/ijbs.9598 Zeng Q, Wu Q, Zhou K et al (2002) Obtaining stem borer-resistant homozygous transgenic lines of Minghui 81 harboring novel cry1Ac gene via particle bombardment. Yi Chuan Xue Bao 29: 519–524 Zeng F, Liu H, Zhang A et al (2018) Three chemosensory proteins from the rice leaffolderCnaphalocrocis medinalis involved in host volatile and sex pheromone reception. Insect Mol Biol 27:710. https://doi.org/10.1111/imb.12503 Zhang Y, Li Y, Zhang Y et al (2011) Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker). Environ Entomol 40:1323– 1330 Zhang QJ, Li C, Liu SK, Lai D, Qi QM, Lu CG (2013) Breeding and identification of insectresistant rice by transferring two insecticidal genes-sbk and sck. Rice Sci 20(1):19–24 Zhao Q (2015) Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene. J Zhejiang Univ Sci B 16(10): 824–831 Zhu-Salzman K, Zeng R (2015) Insect response to plant defensive protease inhibitors. Annu Rev Entomol 60:233–252. https://doi.org/10.1146/annurev-ento-010814-020816

Genetic Engineering of Squash for Food and Health Security

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T. R. Usha Rani, R. N. Yashwanth Gowda, H. Kavya, and R. Pooja

12.1

Introduction

The demand for genetically modified organism (GMO) foods toward human consumption is gradually increasing due to their nutritive value and safety. GMO foods are often referred to as the future of our food supply. GMO crops are systematically engineered to incorporate novel trait(s) for food and nutritional security, viz., increased vitamin availability, biofortifications of zinc, Fe, and tolerance to biotic and abiotic stresses (Table 12.1). Genetically modified (GM) foods support the food production system by increasing yields, supporting better nourishment, and building environment-friendly methods (Sharma et al. 2022). Fruits and vegetables belonging to the Cucurbitaceae family, namely cucumber, watermelon, melon, and squash, that contribute enormously to the human diet are being genetically engineered for food and nutrition security (Kumari et al. 2021). Among these, summer squash (Cucurbita pepo L.), an annual crop that is grown in tropical and subtropical areas, are native to the American countries, especially Mexico. The world's total production of squash and gourds in 2021 was approximately 36.5 million tons (http:// faostat.fao.org). The largest producers of squash and gourds in 2021 were China, Turkey, Iran, Egypt, and India, which together accounted for around 62% of the world's total production. Squash serves as food and medicine for the presence of vitamins and antioxidants with several varieties and landraces. The fatty acids in C. moschata seeds revealed the highest linoleic acid content (48.5%) among the 528 GenBank accessions (Jarret et al. 2013). Although squash exhibits a great diversity, natural resistance to viruses is lacking among the Cucurbita pepo germplasm. Hence, transgenic pepo tolerant to various biotic and abiotic stresses combined with nutraceutical enhancement is the need of the hour. This review T. R. Usha Rani (✉) · R. N. Yashwanth Gowda · H. Kavya · R. Pooja ICAR-IIHR, Division of Basic Sciences, Bengaluru, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_12

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Table 12.1 List of GM crops approved for consumption S, no. 1

Crop Alfalfa

2

Apples

3

Canola

4

Corn (maize) Papaya Potatoes Summer squash Sugar beet Soybean

5 6 7 8 9

Trait(s) Resistance to herbicide (glyphosate) and less lignin Nonbrowning apples by suppressing polyphenol oxidase enzyme Resistance to herbicides and produce less phytate Resistance to insects and tolerance to herbicides Against papaya ring spot virus Reducing acyrlamide formation Resistant to zucchini yellow mosaic virus

Developer Monsanto and Forage Genetics Okanagan Specialty Fruits Monsanto and Bayer Crop Sciences Monsanto

Herbicide resistance Herbicide tolerance and improved quality

Monsanto and KWS Saat Monsanto

Hawaii University J R Simplot Asgrow Seed Co

summarizes the regeneration and transformation protocols for developing transgenic pepo and future scenarios of modern genome engineering methods in achieving these targets to supplement the traditional breeding program. This chapter provides an outline of the summer squash improvement program by engineering traits of interest through transformation, post-transcriptional gene silencing, and pathogenderived resistance. Additionally, a discussion of CRISPR/Cas-mediated genome editing in squash is provided. The widespread use of genome engineering technologies has augmented characterization of gene function and crop improvement for nutritional security.

12.2

Plant Regeneration and Transformation

An efficient regeneration protocol of crop plants is a prerequisite for the successful transformation protocol. The somatic embryogenesis pathway of regeneration was consistently reported for C. pepo (Gonzalves et al. 1995). A reproducible organogenic regeneration pathway for C. pepo from seedling material was established (Ananthakrishnan et al. 2003; Kathiravan et al. 2006). The Agrobacterium-mediated genetic transformation of shoot tip explants with C-repeat binding factor 1 (CBF1) was successful in Cucurbita (Shah et al. 2008). The transformation efficiency in C. moschata Duch cv. Heiankogiku was enhanced by the addition of aluminum borate whiskers at 1% (w/v) (Nanasato et al. 2011). The whiskers are needle-like crystals that are capable of inducing wounds to facilitate entry of Agrobacterium and increase the percentage of transformation (Çürük et al.

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Fig. 12.1 Food and nutritional traits of squashes addressed in the current scenario

2005). Further, the vacuum infiltration was employed to boost the transformation efficiency of the wounded explants in Cucurbita pepo wherein the Agrobacterium was able to penetrate the deeper layers of the plant tissue (Nanasato et al. 2013). In another study, Ilina et al. (2012) suggested the use of two A. rhizogenes strains, R1000 and MSU440, to insert DR5-driven Egfp-gusA or DR5-guided gusA into Cucurbita pepo plants to study the development of roots. Although the crop responds to the Agrobacterium-mediated transformation, very few traits have been addressed through genetic engineering of squash (Fig 12.1).

12.3

Genetically Modified for Nutrition

Carotenoids are antioxidants that play a vital role in human health as they are required as precursors of vitamin A but synthesized at insufficient levels in edible parts of several crops. In particular, vitamin A deficiency affects the lives of millions of people across the globe, which has led to the development of high provitamin A crops using genome engineering approaches (Potrykus 2001; Kaur et al. 2020) or through classical breeding. In squash, the development of an alternative strategy was demonstrated using the viral RNA vector derived from zucchini yellow mosaic virus (ZYMV) for carotenoid fortification. A bacterial phytoene synthase (crtB) expressed in the rind and flesh of the fruits resulted in the accumulation of substantial level of provitamin A in both rind and flesh (Houhou et al. 2021). However, significantly higher accumulation of α- and γ-tocopherol was particularly noticed in fruit rind (Houhou et al. 2022). It was also observed that the peel coloration was attributed to

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higher transcription level of PIF4, APRR2, bHLH128, ERF4, PSY1, LCYE2, and RCCR3 that correlated with carotenoid accumulation and chlorophyll degradation of the peel in yellow and orange zucchini varieties (Xu et al. 2021). Further, the squash gene B affects carotenoid and vitamin E accumulation in mesocarp, regulating the flux of the isoprenoid pathway products (Tadmor et al. 2005).

12.4

Genetically Modified for Biotic Stress

Cucurbit viruses are a major threat to squash cultivation, causing huge economic losses worldwide. The most common emerging plant virus belong to the families Potyviruses, Criniviruses, Cucumoviruses, Ipomoviruses, Tobamoviruses, and Begomoviruses (Martín-Hernández and Picó2020).

12.5

Post-Transcriptional Gene Silencing (PTGS)

In recent years, the mechanism of post-transcriptional gene silencing helps the plants to recover from viral pathogen. Jan et al. (2000) demonstrated squash transgenic lines hemizygous for the squash mosaic virus (SqMV) CP gene-depicted-resistant (SqMV-127), -susceptible (SqMV-22), or recovery (SqMV-3) phenotypes. The recovery phenotype was due to PTGS that was activated at a later developmental stage under field conditions. It was found that a number of plants with transgenes from the recovery and susceptible lines or the self-pollinated recovery line were not showing symptoms even at a young stage after inoculation. Northern analysis confirmed that resistance was due to PTGS. The resistance of the transgenic plants was affected by their developmental stage and the interaction of transgene inserts. In another study, Pang et al. (2000) developed transgenic squash lines with both coat protein (CP) genes of the melon strain of SqMV and crossed with nontransgenic squash. Results showed that the resistant line SqMV-127 displayed posttranscriptional silencing of the CP transgene as evidenced by high transcription rates but less accumulation of transgene transcripts. This was the first report on the development of transgenic squash that are resistant to SqMV.

12.6

Pathogen-Derived Resistance

Squash genotypes exhibit greater susceptibility to virus, with severe yield losses of up to 80%. Genetically modified plants expressing partial or complete viral sequences show resistance to particular virus (Lomonossoff1995). Single or multiple genes of one or many viruses over expressing squash lines were developed and field tested. Fuchs et al. (1998a) showed that the transgenic line CZW-3 expressing the coat protein genes from three viruses, namely CMV, ZYMV, and WMV 2, exhibited the highest level of resistance with a 50-fold increase in marketable yield compared to controls. Also, the transgenic line ZW-20 expressing the CP genes from two

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Potyviruses, namely ZYMV and WMV 2, displayed high levels of resistance with a 40-fold increase in marketable yield relative to controls. Fuchs et al. (1998b) developed transgenic melon and squash expressing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV). During field experiments, the transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 did not mediate the spread of aphid nontransmissible strains of CMV. Also, a single dominant gene Zym, or perhaps by two closely linked genes imparting resistance to WMV2 and zucchini yellow mosaic virus (ZYMV) in Cucurbita moschata “Menina,” was effective against eight strains from different geographic origins (Gilbert-Albertini et al. 1993). Further, Klas (2012) recorded marketable quality of fruits of transgenic squash lines ZW-20H and ZW-20B resistant to zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus (WMV) and of a susceptible nontransgenic line. The transgenic line ZW-20B expressing both ZYMV and WMV 2 CP genes showed excellent resistance in that none of the plants developed severe foliar symptoms, although localized chlorotic dots or blotches appeared on some leaves. In contrast, the two transgenic lines expressing only single CP genes, either the ZYMV or WMV 2 CP gene, developed severe symptoms by the end of the trial period, as did the nontransformed control lines (Fuchs and Gonsalves1995). Dual construct containing the CP genes from CMV and WMV 2 showed higher resistance to CMV and WMV 2 in transgenic squash inbreds. Similarly, a transgenic line ZW-20, which contained the CP genes from ZYMV and WMV 2, displayed complete resistance to ZYMV and WMV 2 (Tricoll et al. 1995). Additionally, ZYMV-infected plants induce a plant immune response mediated by salicylic acid, thereby addressing reduction in powdery mildew infection (Harth et al. 2018)

12.7

Genetically Modified for Abiotic Stress

Abiotic stresses such as drought, salinity, heat, chilling, and intense light often influence plant growth and development. An attempt has been made to introduce the winter squash (Cucurbita moschata, Cm) superoxide dismutase (SOD) CmSOD gene in Arabidopsis seedlings. The transformed plants displayed greater resistance to chilling and less oxidative injury by eliminating •O2-. than nontranformed plants under chilled conditions (Lin et al. 2019).

12.8

Genome Editing, a Novel Approach

Genome editing is the novel approach of targeted mutation in endogenous gene without introducing any transgene into the genome. Reports on CRISPR/Cas mediated editing are limited in squash; Xin et al. (2022) targeted homologs of the ERECTA family of receptor kinase genes using an “optimal infiltration intensity” strategy to create a compact plant architecture with shorter internodes in melon, squash, and cucumber. The optimized transformation method presented here enables

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stable CRISPR/Cas9-mediated mutagenesis and provides a solid foundation for functional gene manipulation in cucurbit crops.

12.9

Conclusion

Squashes are annual crops vulnerable to transformation. The scope for nextgeneration GE crops using advanced genome-editing tools is described in the chapter. The transgenic development in squashes for various biotic stresses, particularly for virus resistance and nutritional enrichment, is explained; however, the studies on abiotic stresses are lacking. Also, the product development for enhanced shelf life and other quality parameters is to be addressed in future studies. The CRISPR/Cas technology in squashes, except for altering plant architecture, is not yet utilized widely. Since genome-editing technology does not introduce foreign DNA into the crop and relaxed from regulatory approvals, the technique can be used for further crop improvement. The food security has been well addressed but nutritional security with respect to squashes can be explored further for crop improvement programs.

References Ananthakrishnan G, Xia X, Elman C, Singer S, Paris HS, Gal-On A, Gaba V (2003) Shoot production in squash (Cucurbita pepo) by in vitro organogenesis. Plant Cell Rep 21:739–746 Çürük S, Çetiner S, Elman C, Xia X, Wang Y, Yeheskel A, Zilberstein L, Perl-Treves R, Watad AA, Gaba V (2005) Transformation of recalcitrant melon (Cucumis melo L.) cultivars is facilitated by wounding with carborundum. Eng Life Sci 5(2):169–177 Fuchs M, Gonsalves D (1995) Resistance of transgenic hybrid squash ZW-20 expressing the coat protein genes of zucchini yellow mosaic virus and watermelon mosaic virus 2 to mixed infections by both potyviruses. Bio/Technology 13(12):1466–1473 Fuchs M, Tricoli DM, Carney KJ, Schesser M, McFerson JR, Gonsalves D (1998a) Comparative virus resistance and fruit yield of transgenic squash with single and multiple coat protein genes. Plant Dis 82(12):1350–1356 Fuchs M, Klas FE, McFerson JR, Gonsalves D (1998b) Transgenic melon and squash expressing coat protein genes of aphid-borne viruses do not assist the spread of an aphid non-transmissible strain of cucumber mosaic virus in the field. Transgenic Res 7:449–462 Gilbert-Albertini F, Lecoq H, Pitrat M, Nicolet JL (1993) Resistance of Cucurbita moschata to watermelon mosaic virus type 2 and its genetic relation to resistance to zucchini yellow mosaic virus. Euphytica 69(3):231–237 Gonzalves C, Xue B, Gonsalves D (1995) Somatic embryogenesis and regeneration from cotyledon explants of six squash cultivars. HortScience 30:1295–1297. Return to ref 1995 in article Harth JE, Ferrari MJ, Helms AM, Tooker JF, Stephenson AG (2018) Zucchini yellow mosaic virus infection limits establishment and severity of powdery mildew in wild populations of Cucurbita pepo. Front Plant Sci 9:792 HouhouF, CorderoT, AragonésV, MartíM, Cebolla-CornejoJ, deCastroAP, RodríguezConcepciónM, PicóB,DaròsJA(2021) Carotenoid and tocopherol fortification of zucchini fruits using a viral RNA vector. bioRxiv, pp.2021-02

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Houhou F, Martí M, Cordero T, Aragonés V, Sáez C, Cebolla-Cornejo J, Perez de Castro A, Rodríguez-Concepción M, Picó B, Daròs JA (2022) Carotenoid fortification of zucchini fruits using a viral RNA vector. Biotechnol J 17(5):2100328 Ilina EL, Logachov AA, Laplaze L, Demchenko NP, Pawlowski K, Demchenko KN (2012) Composite Cucurbita pepo plants with transgenic roots as a tool to study root development. Ann Bot 110(2):479–489 Jan FJ, Pang SZ, Tricoli DM, Gonsalves D (2000 Sep) Evidence that resistance in squash mosaic comovirus coat protein-transgenic plants is affected by plant developmental stage and enhanced by combination of transgenes from different lines. J Gen Virol 81(Pt 9):2299–2306 Jarret RL, Levy IJ, Potter TL, Cermak SC, Merrick LC (2013) Seed oil content and fatty acid composition in a genebank collection of Cucurbita moschata Duchesne and C. argyrosperma C. Huber Plant Genet Resour 11(2):149–157 Kathiravan K, Vengedesan G, Singer S, Steinitz B, Paris HS, Gaba V (2006) Adventitious regeneration in vitro occurs across a wide spectrum of squash (Cucurbita pepo) genotypes. Plant Cell Tissue Organ Cult 85:285–295 Kaur N, Alok A, Kumar P, Kaur N, Awasthi P, Chaturvedi S, Pandey P, Pandey A, Pandey AK, Tiwari S (2020) CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit. MetabEng 59:76–86 Klas FE (2012) On the impact of zucchini yellow and watermelon mosaic viruses on the production rhythms of transgenic ZW-20 and nontransgenic squash (Cucurbita pepo L.). Am JExp Agric 2(3):525 Kumari PH, Kumar SA, Rajasheker G, Jalaja N, Sujatha K, Kumari PS, Kavi Kishor PB (2021) Nutritional value, in vitro regeneration and development of transgenic Cucurbita pepo and C. maxima for stress tolerance: an overview. In: Genetically modified crops: current status, prospects and challenges, vol 1. Springer, Cham, pp 227–240 Lin KH, Sei SC, Su YH, Chiang CM (2019) Overexpression of the Arabidopsis and winter squash superoxide dismutase genes enhances chilling tolerance via ABA-sensitive transcriptional regulation in transgenic Arabidopsis. Plant Signal Behav 14(12):1685728 Lomonossoff GP (1995) Pathogen-derived resistance to plant viruses. Annu Rev Phytopathol 33(1): 323–343 Martín-Hernández AM, Picó B (2020) Natural resistances to viruses in cucurbits. Agronomy 11(1): 23 Nanasato Y, Konagaya KI, Okuzaki A, Tsuda M, Tabei Y (2011) Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschataDuch) induced by woundingwith aluminum borate whiskers. Plant Cell Rep 30:1455–1464 Nanasato Y, Okuzaki A, Tabei Y (2013) Improving the transformation efficiency of Cucurbita species: factors and strategy for practical application. Plant Biotechnol 30:287–294 Pang SZ, Jan FJ, Tricoli DM, Russell PF, Carney KJ, Hu JS, Fuchs M, Quemada HD, Gonsalves D (2000) Resistance to squash mosaic comovirus in transgenic squash plants expressing its coat protein genes. Mol Breed 6:87–93 Potrykus I (2001) The golden rice tale. Vitro Cell Dev Biol-Plant 37:93–100 Shah P, Singh NK, Khare N, Rathore M, Anandhan S, Arif M, Singh RK, Das SC, Ahmed Z, Kumar N (2008) Agrobacterium mediated genetic transformation of summer squash (Cucurbita pepo L. cv. Australian green) with cbf-1 using a two vector system. Plant Cell Tissue Organ Cult 95(3):363–371 Sharma P, Singh SP, Iqbal HM, Parra-Saldivar R, Varjani S, Tong YW (2022) Genetic modifications associated with sustainability aspects for sustainable developments. Bioengineered 13(4):9509–9521 Tadmor Y, Paris HS, Meir A, Schaffer AA, Lewinsohn E (2005) Dual role of the pigmentation gene B in affecting carotenoid and vitamin E content in squash (Cucurbita pepo) mesocarp. J Agric Food Chem 53(25):9759–9763 Tricoll DM, Carney KJ, Russell PF, McMaster JR, Groff DW, Hadden KC, Himmel PT, Hubbard JP, Boeshore ML, Quemada HD (1995) Field evaluation of transgenic squash containing single

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or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and zucchini yellow mosaic virus. Bio/Technology 13(12):1458–1465 Xin T, Tian H, Ma Y, Wang S, Yang L, Li X, Zhang M, Chen C, Wang H, Li H, Xu J (2022) Targeted creation of new mutants with compact plant architecture using CRISPR/Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants. Hortic Res 9. https://doi.org/10.1093/hr/uhab086 Xu X, Lu X, Tang Z, Zhang X, Lei F, Hou L, Li M (2021) Combined analysis of carotenoid metabolites and the transcriptome to reveal the molecular mechanism underlying fruit colouration in zucchini (Cucurbita pepo L.). Food Chem Mol Sci 2:100021

Genetic Improvement in Peanut: Role of Genetic Engineering

13

Riddhi Rajyaguru, Nataraja Maheshala, and Gangadhara K

13.1

Introduction

Groundnut (Arachis hypogaea L.) is an allotetraploid (AABB-type; 2n = 2x = 40) self-pollinating intermediate herbaceous legume mainly grown for its edible oil and protein. The parentage of cultivated groundnut includes A. duranensis (AA-type; 2n = 2x = 20) and A. ipaensis (BB-type; 2n = 2x = 20), wild groundnut species. The average productivity of groundnut is 1831 kg/ha, with a production of nearly 10.1 million tons from an area of 5.7 million ha (agricoop.nic.in). Globally, China tops the chart in production and consumption, but the area available for groundnut production is the highest in India. Traditionally, peanut is grown in the states of Andhra Pradesh, Karnataka, Tamil Nadu, Maharashtra, Gujarat, and Rajasthan. Rainfed cultivation of groundnut predominates in India such as all the Kharif groundnut areas. Groundnut kernels contain 40–60% oil, 20–40% protein, and 10–20% carbohydrates. They provide 567 kcal of energy from 100 g of kernels (USDA nutrient database). Additionally, they also contain several health-enhancing nutrients such as minerals, antioxidants, and vitamins (Janila et al. 2016).

R. Rajyaguru (✉) Department of Biotechnology, Junagadh Agricultural University, Junagadh, India N. Maheshala ICAR-Directorate of Groundnut Research, Junagadh, India Gangadhara K ICAR-CTRI Research Station, Kandukur, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_13

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13.1.1 Challenges for Peanut Cultivation Groundnut is mostly grown in arid and semi-arid tropical countries, where the majority of the cultivable area is highly prone to extreme weather conditions such as frequent droughts, erratic rainfall patterns, salinity, and extreme temperatures, leading to severe yield reductions. Drought is the major stress accounting for a significant loss of approximately 6–7 MT of groundnut (Bhatnagar-Mathur et al. 2014). The yield losses may vary from 5 to 75% depending on the timing, intensity, and duration of drought in different growth periods of crop. Various researchers reported that drought stress severely affects the nitrogen fixation (Pimratch et al. 2008) and rate of photosynthesis (Subramaniam and Maheswari 1990). Damage severity of drought stress depends on the stage of crop, the duration of stress, and the magnitude of drought stress (Nageswara Rao et al. 1985;Wright and Nageswara Rao 1994). Based on the incidence of drought stress during crop growth stages, it has been classified as early-season, mid-season, and end-of-season droughts. Although no detrimental effect was observed in vegetative and preflowering stage but at the reproductive phase, it could cause significant yield reduction from 15 to 88% (Nageswara Rao et al. 1985,1988;Nautiyal et al. 1999). Similarly, soil salinity is another devastating stress wherein excess of soluble and insoluble salts affects crop growth. According to estimates, the present area under salt-affected soils (6.73 million ha) in the country would almost triple to 20 million ha by 2050 owing to the rise in temperature, evaporation, and seawater inundation (Sharma et al. 2014). In India, 2% of the total geographic area (2.96 mha under saline and 3.77 mha under sodic soils) is affected by salinity (Tripathi 2011;NAAS 2012). Soil salinity seriously affects the crop productivity, water quality, and food security. In the case of groundnut, salinity severely affects germination and seedling growth, leading to low dry matter production (Nautiyal et al. 1989;Singh et al. 1989;Janila et al. 1999), and induces Ca, K, and Fe deficiencies in groundnut (Singh2004). Apart from abiotic stresses, insect pests alone can cause crop losses of around 47.3%, while losses by diseases range from 10 to 70% globally (Baskaran and Rajavel 2013). Defoliator pests such as tobacco caterpillar, Spodoptera litura, red-headed hairy caterpillar, Amsacta sp. and leafminer, Aproaerema modicella, cause significant damage to Kharif crops, while sucking pests such as thrips, aphids, and leafhoppers infest young peanut seedlings. Thrips and aphids are also vectors of peanut bud necrosis and peanut rosette diseases, respectively (Nataraja et al. 2014). In storage, bruchid (Caryedon serratus) inflict losses and also help in aflatoxin contamination, thus hampering the export potential of Indian peanuts to world markets (Harish et al.2014). In the case of diseases, soil-borne stem rot, collor rot, aflaroot, and dry root rots are significant, while rust, early and late leaf spots, and Alternaria leaf blight affect groundnut crop (Nataraja et al. 2014).

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13.1.2 Need for Genetic Improvement in Peanut The objective of groundnut breeding programs is based on the target product profiles involving different stakeholders consisting of growers, processors, and consumers. Peanut growers are looking for high-yielding and early-maturing varieties with saline, drought, insect, and disease resistance/tolerance traits. Peanut processors and consumers are interested in the quality of groundnut produce. Quality is determined by its physical, biochemical, sensory, and nutritional traits. Physical traits include seed size, seed shape, seed weight, and blanching efficiency. Sensory traits include flavor, texture, and color. Nutritional traits include protein content, oil content, vitamins, antioxidants, carbohydrates, fatty acid composition, and minerals (Janila et al. 2013). Groundnut genotypes with oleic acid content >75% are referred to as high oleic genotypes and possess the desired quality parameter to enhance the shelf-life of oil seeds by delaying the rancidity. The trait of high oleic-to-linoleic acid ratio (high O/L) in groundnut is favored over low O/L as it confers health benefits and oil stability. Also, groundnut occupies the prime status as a confectionery type for snack and food use because of its rich protein, low oil, and high sugars. Blanchability is one of the demanding properties because of the easy removal of the testa for processing groundnut in the preparation of peanut butter and other products.

13.1.3 Genetic Bottlenecks The genetic variation in groundnut is limited due to the origin of A. hypogaea from a single hybridization event and little introgression from diploid species to A. hypogaea since its inception (Kochert et al. 1996). Progress in breeding for resistance to soil-borne diseases, aflatoxin development, and virus diseases has been very difficult and slow. Research efforts in these directions probably are not adequate due to the dearth of qualitative sources of resistance to these biotic stresses. The wild species of groundnut offers a vast reservoir of novel genes that are not available within the cultivated species. Improving cultivated peanut by using diploid species has been extremely difficult and time-consuming due to the low success rate of pollinations that result in sterile hybrids and genomic incompatibilities. Introgression from Arachis species to A. hypogaea appears to be in large blocks (Garcia et al. 1995;Nagy et al. 2010), rather than as single genes or small chromosome segments. Thus, linkage drag of undesirable traits can restrict the use of progenies until the linkages can be broken. Further, many of the traits of biotic and abiotic stress tolerance are multigenic and complex environmental interaction effects. The transgenic stress-tolerant peanut varieties have a potential to be used as donor parents in conventional breeding approaches for developing superior varieties tolerant to biotic and abiotic stresses (Gantait and Mondal 2018).

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13.1.4 Genetic Resources In India, groundnut accessions are available from ICRISAT, Hyderabad, and ICARDirectorate of Groundnut Research, Junagadh. These gene banks also hold wild species of Arachis and can be utilized for peanut improvement programs.

13.2

Crop Improvement Techniques

With the increasing demand for agricultural produce, crop improvement has become necessary. To date, peanut improvement programs largely depended on traditional breeding methods, but the use of modern transgenic technology such as transgenic and gene editing is limited. In the last few years, many advanced biotechnology techniques speed up the process of crop improvement compared to traditional methods. The development of next-generation sequencing reveals the genetic makeup of numerous plant species. This is a potent tool for identifying domestication genes in crop plants and their wild relatives. The accessibility of peanut reference genomes helps in understanding genome architecture, trait mapping, gene discovery, and molecular breeding (Ojiewo et al. 2020). The availability of draft genome sequence of diploid and tetraploid species of peanuts is expected to functionally validate novel gene and can be effectively utilized for genome editing and transgenics. The majority of the transgenic studies have been focused on biotic and abiotic stress only; details of yield parameters, plant adaptation, and nutrition enhancement are limited. Since 2010, genetic codon can be rewritten using advanced gene or genome-editing techniques, viz., Meganulcease, ZFN, TALEN, and CRISPR. Crops can be customized using new gene editing tools in many ways to hold out against new diseases or pests, droughts, or different habitats that were often lost. Here, we will briefly describe the different methods used for genetic improvement and how to improve peanut quality.

13.2.1 Traditional Breeding Traditionally plants were developed via conventional breeding in which plants were crossed together with relevant characteristics and the offspring with the desired combination of characteristics were selected. Many peanut varieties/lines have been developed using conventional breeding approaches (Table 13.1). Using conventional breeding, peanut qualities were enhanced for biotic, abiotic, nutrition, and plant physiology. Several varieties were developed for biotic, abiotic, and quality traits. To address these abiotic stresses, considerable progress has been made in breeding groundnut for drought tolerance in India. Some of the drought-tolerant varieties released in India were Kadiri-3, K-134, ICGV-87119, ICGV-87121, ICGV-87187 and ICGV87141, ICGV-86031, and TAG-24, though these were not exclusively bred for drought tolerance but were found to possess tolerance also. Recently, a few

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Table 13.1 Groundnut varieties developed through traditional breeding Trait Large seeded

Drought tolerance

Salt tolerance

Heat tolerance Short duration Thrips tolerance High Zn and Fe content High oleic content High oil content Rust and LLS resistance PBND tolerance Kalahasti melody Low aflatoxin contamination

Groundnut varieties TG 1, Somnath, TPG 41, TLG 45, Mallika, M 548, HPS 2, ICGV 03137, Asha, TKG 19A, TG 39 K-5, DH 257, Ajeya, Greeshma, ICGV 87846, ICR 48, ICGV 00350, SRV 1-3, SRV 1-96 VRI 3, UF 70–103, TKG 19A, S 206, Tirupati 4, M 522, Punjab 1, BG 3, Somnath, ICGV 86590 Dheeraj, ICG 1236, ICGV 86021, ICGV 06420, ICGV 03043 Girnar 1, JL 24, TMV 2, GG 7 ICUG 9205, Dh 101, K 1319 ICGV 06099, ICGV 06040 Dh 245 GG 34, ICGV 03057, ICGV 03042, ICGV 05155, ICGV 06420, ICGV 03043 GPBD 4 Divya, GJG HPS 1, Vijetha, DRG 17, CSMG 884, ICGS 11, ICGS 44, ICGV 86325 Prasuna, Tirupati 3 J 11, ICG 7633, ICG 4749, ICG 1859, ICG 9610

References Badigannavar and Mondal (2007); Janila et al. (2012); Rathnakumar et al. (2014); Pal et al. (2021) Mayeux et al. (2003); Vindhiyavarman et al. (2014); Rathnakumar et al. (2014); Pal et al. (2021) Singh et al. (2010)

Craufurd et al. (2003); Rathnakumar et al. (2014); Pal et al. (2021) Patil et al. (1980); Rathnakumar et al. (2014); Pal et al. (2021) Rathnakumar et al. (2014); Pal et al. (2021) Janila et al. (2014) Rathnakumar et al. (2014); Pal et al. (2021) Rathnakumar et al. (2014); Pal et al. (2021) Gowda et al. (2002) Ghewande et al. (2002); Rathnakumar et al. (2014); Pal et al. (2021) Mehan et al. (1993); Rathnakumar et al. (2014); Pal et al. (2021) Nigam et al. (2009); Rathnakumar et al. (2014); Pal et al. (2021)

improved and drought-tolerant varieties have also been developed based on the targeted breeding approach and released for cultivation in different states. These include ICGV-91114, Kadiri-7, Abhaya, Prasuna, Co (Gn)-6, TMV-13 and VRI (Gn)-7, and ICGV-91114 and R-2001-3 (Rathnakumar et al. 2014). Similarly, VRI 3, UF 70–103, TKG 19A, S 206, Tirupati 4, M 522, Punjab 1, BG 3, Somnath, and ICGV 86590 were identified as salt-tolerant cultivars (Singh et al. 2010). The salttolerant mechanism includes salt exclusion, osmotic tolerance, and tissue tolerance (Munns et al. 2016). Singh et al. (2008) identified JNDS-2004-1, JNDS-2004-3, JNDS-2004-16, TG 28, TG 38C, TG 42, PBS 30031, PBS 30033, NRCG 6155, and ICGV 86031 as having moderate tolerance at 3 ds/m salinity level. To address biotic stresses, traditional breeding gave several varieties (Table 13.1). Thrips tolerance was reported in cultivars ICUG 9205, Dh 101, and K 1319 (Pal

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et al. 2021). For managing foliar fungal diseases such as rust and leaf spots, GPBD4 was released for cultivation (Gowda et al. 2002). Similarly, Prasuna and Tirupati 3 were released targeting Kalahasti malady caused by nematodes (Pal et al. 2021). Similarly, traditional breeding was also employed to produce varieties with traits targeting heat tolerance, large seed size, short duration, high Zn and Fe content, and high oil and oleic acid contents.

13.2.2 Genetic Transformation 13.2.2.1 Brief History of Genetic Transformation in Peanut A study of genetic transformation in peanut was started by Lacorte et al. (1991), where they transformed uidA reporter gene from Escherichia coli that encodes the enzyme b-glucuronidase. For the first time, plasmid pTiBo542 was transferred into peanut genotypes (Tatu, Tatu branco, Tatui, and Tupa) using Agrobacteriummediated transformation, and the result also reflects the successful application of ATC1 promoter in stem internode explant tissue. Since then, numerous efforts have been made to enhance the peanut cultivar using Agrobacterium-mediated transformation. In order to provide resistance against insects, viruses, fungi, herbicides, droughts, and salinity, multiple genes were transferred from various sources such as Arabidopsis thaliana, Bacillus thuringiensis, Brassica juncea, Medicago sativa, Pisum sativum, E. coli, and Macrotyloma uniflorum. To pull off successful transformation, different explants and different plasmids were used by a versatile group of scientists. Apical meristem, cotyledon, cotyledonary node, de-embryonated cotyledon, embryonic axes, immature cotyledons, immature embryo, leaf, and shoot apices were reported to be acceptable explants for genetic transformation (Yang et al. 1998; Anuradha et al. 2006;Athmaram et al. 2006;Entoori et al. 2008;Khandelwal et al. 2011;Vasavirama and Kirti 2012;Mehta et al. 2013;Prasad et al. 2013;Manjulatha et al. 2014). Among these, preeminent explant is immature cotyledon for genetic transformation, with 83–90% efficiency (Singsit et al. 1997;Yang et al. 1998). However, CaMV35S(r)PR1a, CaMV35S(EN4), CaMV35S(DE), Oleosin, rd29A, and SARK have also been reported to be suitable for the development of transgenic peanut plants, and CaMV35S is an extensively used promoter. Promoter is crucial for successful transformation, but only a small number of promoters were tested for peanut. To date, many genes were transferred to peanuts to develop resistance against insects, viruses, fungi, herbicides, droughts, and salinity (Fig. 13.1), even though these burning questions have not been solved. The progression of transgenic peanut has been slow due to the insufficient protocol of stable genetic transformation for cultivated peanut. Genetic transformation is a good genotype-independent tool to produce improved cultivars. Even though a few factors such as genotype, explant, and protocol dependency flag the process of peanut transformation, on the other hand, the allotetraploid nature of peanut genome acts as a barrier to target specific genetic modification. The use of explants from embryo axes and cotyledonary nodes

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cry8Ea1,cry8Ea1+MARs,Bt cry1A(c), cry1EC,cry1AcF,CpTIuidA,Bt cry1A(c)cry1X

Insect Resistance SbpPAX,AVP1,AtDREB1A, mtID,AtNHX1,RabG

Viral Resistance

Salinity Resistance

IPCVcp,TSV-CP,N +GUS,PStV CP,PStV CP4

Peanut Genetic Improvment Drought Resistance

Fungal Resistance

Zmpsy1,DREB1A,IPT PDH45 Herbicide Resistance

CHI, PStV CP2, RsAFP1,RCG-3,AdSGT1, EN4+RCG3

BclxL, HN,UreB,RVPH,RPVH

Fig. 13.1 Traits and their target genes for peanut genetic improvement

functionally dealt with resistance and accelerates the transgenic process, but there is still a need to develop PEG-mediated delivery methods. The delivery of novel genes using Agrobacterium is widely accepted for peanut, but the only downside of using this method is the use of a binary vector in which an alien gene (markers genes, viz., kanamycin, hygromycin, and herbicides) is incorporated into the plant genome. PEG-mediated delivery is useful for vector-free high-frequency transformation and also multiple plasmids can be delivered. So far, there is not a single successful protocol available for regeneration of peanut plants from protoplasts. Even after transgene incorporation via either Agrobacterium or particle bombardment methods, the conversion rate of somatic embryos into normal plantlets in peanut remains low. There is a dire need to develop plant tissue culture-free delivery methods for peanut such as floral-dip, nanoparticle-mediated, and pollen magnetofection-mediated. Not a single effort was made to establish these cost-effective and straightforward methods for peanut genetic transformation.

13.2.3 Research Status on Genetic Transformation of Peanut In spite of the key role played by conventional breeding, the progress has been slow. The complex polygenic nature with significant Genotype × Environment

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interactions of abiotic stresses poses serious challenges. The major challenges include low genetic variability, difficulty in foreground and background selection, linkage drag, and unfavorable traits in breeding climate-resilient varieties (Varshney et al. 2005;Feng et al. 2012;Kishor et al. 2018). Therefore, application of modern technologies such as genetic engineering and genome editing will play a key role in sustainable food production under vagarious weather conditions. Genetic engineering approaches have been successfully employed in groundnut to overcome the incompatibility of wild species, recovery of hybrids, and linkage of detrimental traits (Garcia et al. 2006). Agrobacterium-mediated transformation method was successfully used to develop transgenic groundnut plants for various abiotic stresses. Bhatnagar-Mathur et al. (2007) were the first to attempt and develop transgenics in groundnut using AtDREB1A, transcription factor, which improved water use efficiency. The overexpression of isopentenyl transferase gene involved in cytokinin biosynthesis has enhanced drought tolerance as well as higher seed production and biomass in groundnut (Qin et al. 2011). A variety of transcription factors and regulatory genes (DREB1A, PDH45, MuNAC4, AtDREB1A, AtHDG11, and AhKCS1) responsible for drought tolerance have been effectively used with a different promoter system to develop transgenics; eventually there was a significant improvement in drought tolerance by increasing water use efficiency-related traits along with growth and yield in groundnut. The overexpression of regulatory genes/ transcription factors (ATNHX1, SbpAPX, SbNHXLP, and AhWRKY75) by various workers successfully showed higher chlorophyll content, increased superoxide dismutase and catalase activity, accumulation of more proline and K+, followed by lower Na+ accumulation and higher photosynthesis rate under artificially induced salinity levels (Asif et al. 2011;Singh et al. 2014;Kandula et al. 2019;Zhu et al. 2021). Cry1AcF, BjNPR, and Tfgd genes were transferred to introduce resistance to insect pests/diseases. To test the effectiveness of Cry1AcF against tobacco caterpillar (Spodoptera litura), numerous primary transgenic events were produced in groundnut using an Agrobacterium-mediated, in planta transformation method (Keshavareddy et al. 2013). In 2016, Sundersha and coworkers offered fungal resistance to peanut by co-overexpression of NPR1 and defensin gene from Brassica juncea and Trigonella foenum-graecum, respectively. Successfully transformed peanut cultivar showed field resistance to Aspergillus flavus and Cercospora arachidicola (early leaf spot). Oxalate oxidase gene from barley that encodes for oxalic acid was transferred into peanut using biolistic transformation (PartridgeTelenko et al. 2011). Transgenic plants from T0 generation reported resistance as opposed to Sclerotinia minor under field conditions. Peanut leaf spot resistance was evolved by Whisker-mediated transformation of EN4 + RCG3 gene (Akram et al. 2016). Another emerging tool to genetically modify plants is intragenesis, where a new combination of genes was achieved by transferring genes and regulatory sequences belonging to that particular species. Intragenic peanut lines were developed by transferring SGT1, Ara h 2, AhKCS1, and AhWARKY75 from wild type to cultivated variety (Table 13.2). To date, not even a single cisgenesis event has been reported,

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Table 13.2 Genetic transformation-mediated peanut improvement Improvement technique Transgenic

Mode of transformation Agrobacteriummediated

Transgene AtNHX1 IPT AtNHX1 AtDREB1A

Salinity and drought stress Drought stress

AtDREB1A

Drought stress

mtlD

Salinity and drought stress Drought stress

MuNAC4 AtDREB2A, AtHB7 and AtABF3 AtDREB1A SbpPAX SbASR-1 AtDREB1A AtHDG11 SbNHXLP AVP1 Agrobacteriummediated (in planta)

Trait Salinity and drought stress Drought stress

Salinity and drought stress Salinity and drought stress Salinity stress Salinity and drought stress Drought stress Salinity and drought stress Salinity stress

Cry1AcF

Salinity and drought stress Insect resistance

PDH45

Drought stress

AtNAC2 (ANAC092) Alfin1, PgHSF4, and PDH45 BjNPR1 and Tfgd

Salinity and drought stress Drought stress

Fungal resistance (Aspergillus flavus and Cercospora arachidicola) Drought stress

References Asif et al. (2011) Qin et al. (2011) Banjara et al. (2012) Vadez et al. (2013) BhatnagarMathur et al. (2014) Bhauso et al. (2014) Pandurangaiah et al. (2014) Pruthvi et al. (2014) Sarkar et al. (2014) Singh et al. (2014) Tiwari et al. (2015) Sarkar et al. (2016) Banavath et al. (2018) Kandula et al. (2019) Qin et al.(2013) Keshavareddy et al. (2013) Manjulatha et al. 2014 Patil et al. (2014) Ramu et al. (2016) Sundaresha et al. (2016)

(continued)

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Table 13.2 (continued) Improvement technique

Mode of transformation

Biolistic

Intragenic

Cisgenic

Transgene MuNAC4, MuWARKY3 and MuMYB96 Barley oxalate oxidase

Trait

Whiskermediated

EN4+RCG 3

Agrobacteriummediated (in planta) Constitutive silencing Agrobacteriummediated (in planta) NA

SGT1

AhKCS1

Field evaluation of transgenic lines for Sclerotinia minor resistance Chitinase gene enhancing against leaf spot disease Induces cell death and enhanced disease resistance Reduced allergen content Drought stress

AhWRKY75

Salinity stress

NA

NA

NA

Ara h 2

References Kiranmai et al. (2018); Venkatesh et al. (2022) PartridgeTelenko et al. 2011 Akram et al. (2016) Kumar and Kirti (2015) Dodo et al. (2005) Lokesh et al. (2019) Zhu et al. (2021) NA

that is, genetic manipulation of the desired trait using a complete copy of natural genes with their regulatory elements that belong exclusively to sexually compatible plants (Mohapatra and Sahoo 2020).

13.2.4 Genome Editing Despite many advantages of genetic engineering, there are many stumbling blocks such as the need for long tissue culture protocol for recovery of transgenic plants, less possibility of obtaining stable transformed plants, the restrictions on certain Agrobacterium species’ host range, and most importantly the compulsion of reporter gene for the identification of phenotype. The major drawback of traditional breeding methods is the lack of precision, and, in the case of transgenesis, random insertion transgene into host genome leads to adverse effects. To overcome the abovementioned limitation of transgenic plants, genome editing has been proven to be a state-of-the-art method for crop improvement. The genome-editing technique is precise in which DNA is manipulated (inserted, deleted, modified, or replaced) in the genome of the host organism. This genetic manipulation comprises the use of programmable nucleases that recognize the target on genomic loci and then repair of the double-strand breaks via homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

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Table 13.3 Genome editing-mediated peanut improvement Improvement technique Meganuclease ZFN TALEN CRISPR

Mode of transformation NA NA Agrobacteriummediated Polyethylene glycol (PEG) mediated Agrobacteriummediated (hairy root)

Transgene NA NA AhFAD2 Ara h 2

FAD2 FAD2 Cis-Regulatory motifs AhFAD2 FAD2 AhNFR1, AhNFR5

Trait NA NA High oleic acid peanut Efficiency check (disruption of peanut allergen) Efficiency check (of FAD2 gene) Increase in oleic acid level

References NA NA Wen et al. (2018) Biswas et al. (2022) Yuan et al. (2019) Neelakandan et al. (2022b)

Efficiency check for nCas9 Efficiency check (of FAD2 gene) Functional validation (role in nodulation)

Neelakandan et al. (2022a) Neelakandan et al. (2022c) Shu et al. (2020)

The site-directed nucleases (SDNs) cause breakages in double-stranded DNA. The major SDNs used in the plant genomic editing are zinc-finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), and CRISPR. ZFNs are targetable DNA cleavage reagents that have been adopted as gene-targeting tools (Carroll 2011). A ZFN consists of a designed zinc finger protein (ZFP) fused to the FokI restriction enzyme. A ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specificities (Urnov et al. 2010). Meganucleases are also known as molecular DNA scissors. Sometimes they are naturally found in the genome of organisms and are made up of large basepair structures having the potential to excise large pieces of DNA sequences (Khan 2019). No literature is available on the applications of both ZFNs and meganucleases in peanut improvement (Table 13.3). Numerous organisms have been genetically altered using transcription activatorlike effector nucleases (TALENs), which contain two functional protein domains that recognize a specific sequence and make double-stranded breaks to initiate mutation. The coding region of fatty acid desaturase 2 gene of peanut was altered through TALEN-mediated mutagenesis and sequencing results revealed the genetic stability of AhFAD2 mutations in up to 9.52 and 4.11%, respectively, of the regeneration plants at two different targeted sites (Wen et al. 2018). No further literature is available on the use of TALENs in peanut. The possible reason(s) for the limited use of TALENs were effective for only mutation, and mutation has mosaicism. The clustered regularly interspaced short palindromic repeat (CRISPR)-mediated mutagenesis took over TALENs because it has many advantages such as

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multiplexing of gRNAs and higher efficiency of transfection, and can be delivered with Agrobacterium-dependent and -independent transformation as well. Genomeediting efficiency in peanut can be enhanced through sequence-specific nucleases, including ZFNs and CRISPR. CRISPR-associated protein 9 nuclease (Cas9) system emerged as a genome-editing tool in 2012 (Jinek et al. 2012) because of its simplicity, ease, and high efficiency (Wada et al. 2020). In peanut, CRISPR was employed to induce mutations in FAD2, which catalyzes the conversion of oleic to linoleic acid. Efficiencies in CRISPR systems were checked on FAD2 by Yuan et al. (2019) and Neelakandan et al. (Neelakandan et al. 2022a;b;c), wherein only Neelakandan et al. (Neelakandan et al. 2022a, b, c) substantiated their claim with phenotypic data on oleic to linoleic ratio. Shu et al. (2020) functionally validated CRISPR system for inducing mutations in AhNFR1 and AhNFR5 governing root nodulation by Rhizobium sp. in peanut. Both PEG- and Agrobacterium-mediated (hairy root) transformations worked well with the CRISPR system. However, most of the work focused on checking the efficiency of the CRISPR system in peanut and requires further phenotypic validation. Storage protein in peanut is the major food allergen for a small, but significant, portion of consumers. RNA interference is used for partial or complete silencing of the expression of genes responsible for allergen production in peanut seeds (Chu et al. 2008;Dodo et al. 2008;Chandran et al. 2015), reduction in the growth of A. flavus, a major producer of aflatoxin (Dodo et al. 2008), and identification of key components of nodule/nitrogen-fixing. Recently, Biswas et al. (2022) tested a CRISPR system to disrupt the peanut allergen production (Table 13.3).

13.3

Impacts of Policy Decisions and Regulations on Genetic Engineering of Peanut

Research and development in the field of genetic engineering of crop plants was at a slower pace in the past three decades due to the policies and regulations of the Government of India. The first genetically engineered crop that was released for commercial cultivation was Bt cotton in 2002. Genetic engineering of around 20 crop plants was taken; however, only 13 crops were identified by a statutory panel of Genetic Engineering Appraisal Committee (GEAC) for confined field trials in India. However, the final approval for the same was not granted by the Government of India for the want of long-term effects of genetically engineered crops on soil. In October 2022, the Government of India approved the environmental release of Dhara Mustard Hybrid (DMH-11), genetically modified herbicide-tolerant mustard with two alien genes, Barnase and Barstar genes. However, its release was challenged in the Honorable Supreme Court of India. Various transgenic lines developed in groundnut for tolerances to drought (mtlD), salinity (Dreb1a), insect (Cry 1Ac), and aflatoxin (Defensin) are in cold storage, awaiting approvals for field trials. In May 2022, the Government of India green-lighted the confined field trials of genome-edited plants developed by using genome-editing techniques employing SDNs such as ZFNs, TALENs, CRISPR, and other nucleases with similar functions

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(Annonymous2022). Researchers concentrating on these new genome-editing techniques for peanut improvement received a major fillip with these policy changes. Rajyaguru and Tomar (2020) have successfully employed CRISPR/Cas9 for inducing mutations in AhFAD2B for improving the shelf life of peanut oil. More such research in genome editing of peanut can be done, evaluated in confined field trials, and possibly recommended for cultivation in the near future.

13.4

Future Prospects and Conclusions

Peanut improvement programs globally have moved from being solely traditional breeding to more sophisticated techniques by employing genetic transformation, genome editing, and molecular tools. Identification and confirmation of gene (s) responsible for a biochemical pathway/trait may help in better introgression of gene or point mutations at the target gene to knock out or enhance the enzyme activities and trait expressions. Recent policy changes have brought fresh impetus to the utilization of site-directed nucleases for crop improvement. With such groundbreaking technologies only varietal improvement can be done at faster rate, with higher precision and economically. Of late, quality parameters of peanut are being considered, especially improving the protein, vitamins, minerals, resveratrol, and sugar content but reducing the phytic acid content in the kernel. Tapping into the diverse market requirements, size, shape, and blanchability of kernels is gaining importance. Genome-editing tools will have a greater role in these areas of research to identify and functionally validate the role of genes governing these quality traits.

References Akram Z, Ali S, Ali GM et al (2016) Whisker-mediated transformation of peanut with chitinase gene enhances resistance to leaf spot disease. Crop Breed Appl Biotechnol 16:108–114 Annonymous (2022). Guidelines for the safety assessment of genome edited plants 2022. https:// dbtindia.gov.in/sites/default/files/Final_%2011052022_Annexure-I%2C%20Genome_Edited_ Plants_2022_Hyperlink.pdf(Accessed 01 December 2022) Anuradha TS, Jami SK, Datla RS et al (2006) Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterlessgus:: nptII fusion gene based vector. J Biosci 31:235–246 Asif MA, Zafar Y, Iqbal J et al (2011) Enhanced expression of AtNHX1, in transgenic groundnut (Arachis hypogaea L.) improves salt and drought tolerance. Mol Biotechnol 49:250–256 Athmaram TN, Bali G, Devaiah KM (2006) Integration and expression of Bluetongue VP2 gene in somatic embryos of peanut through particle bombardment method. Vaccine 24:2994–3000 Badigannavar AM, Mondal S (2007) Mutation experiments and recent accomplishments in Trombay groundnuts. IANCAS Bull 6:308–318 Banavath JN, Chakradhar T, Pandit V et al (2018) Stress inducible overexpression of AtHDG11 leads to improved drought and salt stress tolerance in peanut (Arachis hypogaea L.). Front Chem 6:34 Banjara M, Zhu L, Shen G et al (2012) Expression of an Arabidopsis sodium/proton antiporter gene (AtNHX1) in peanut to improve salt tolerance. Plant Biotech Rep 6:59–67

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Baskaran RKM, Rajavel DS (2013) Yield loss by major insect pests in Groundnut. Ann Plant Protec Sci 21:189–190 Bhatnagar-Mathur P, Devi MJ, Reddy DS et al (2007) Stress-inducible expression of AtDREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water limiting conditions. Plant Cell Rep 26:2071–2082 Bhatnagar-Mathur P, Rao JS, Vadez V et al (2014) Transgenic peanut overexpressing the DREB1A transcription factor has higher yields under drought stress. Mol Breed 33:327–340. https://doi. org/10.1007/s11032-013-9952-7 Bhauso TD, Thankappan R, Kumar A et al (2014) Over-expression of bacterial ‘mtlD’ gene confers enhanced tolerance to salt-stress and water-deficit stress in transgenic peanut (Arachis hypogaea) through accumulation of mannitol. Aust J Crop Sci 8:413–421 Biswas S, Wahl NJ, Thomson MJ et al (2022) Optimization of protoplast isolation and transformation for a pilot study of genome editing in peanut by targeting the allergen gene Ara h 2. Int J Mol Sci 23:837 Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188:773–782 Chandran M, Chu Y, Maleki SJ et al (2015) Stability of transgene expression in reduced allergen peanut (Arachis hypogaea L.) across multiple generations and at different soil sulfur levels. J Agric Food Chem 63:1788–1797 Chu Y, Faustinelli P, Ramos ML et al (2008) Reduction of IgE binding and nonpromotion of Aspergillus flavus fungal growth by simultaneously silencing Ara h 2 and Ara h 6 in peanut. J Agric Food Chem 56:11225–11233 Craufurd PQ, Prasad PV, Kakani GV et al (2003) Heat tolerance in groundnut. Field Crops Res 80: 63–77. https://doi.org/10.1016/S0378-4290(02)00155-7 Dodo H, Konan K, Viquez O (2005) A genetic engineering strategy to eliminate peanut allergy. Curr Allergy Asthma Rep 5:67–73 Dodo H, Konan K, Chen F (2008) Alleviating peanut allegy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant Biotechnol J 6:135–145 Entoori K, Sreevathsa R, Arthikala MK et al (2008) A chimeric Cry1X gene imparts resistance to Spodoptera litura and Helicoverpa armigera in the transgenic groundnut. EurAsia J Biosci 2: 53–65 Feng S, Wang X, Zhang X et al (2012) Peanut (Arachis hypogaea L) expressed sequence tag project: progress and application. Comp Funct Genomics. https://doi.org/10.1155/2012/373768 Gantait S, Mondal S (2018) Transgenic approaches for genetic improvement in groundnut (Arachis hypogaea L.) against major biotic and abiotic stress factors. J Genet Eng Biotechnol 16:537–544 Garcia GM, Stalker HT, Kochert G (1995) Introgression analysis of an interspecific hybrid population in peanuts (Arachis hypogaea L.) using RFLP and RAPD markers. Genome 38: 166–176 Garcia GM, Tallury SP, Stalker HT (2006) Molecular analysis of Arachis inter-specific hybrids. Theor Appl Genet 112:1342–1348 Ghewande MP, Desai S, Basu MS (2002) Diagnosis and management of major diseases of groundnut. National Research Center for Groundnut, Junagadh Gowda MVC, Motagi BN, Naidu GK et al (2002) GPBD 4: A Spanish bunch groundnut genotype resistant to rust and late leaf spot. Int Arachis Newslett 22:29–32 Harish G, Nataraja MV, Holajjer P et al (2014) Efficacy and insecticidal properties of some essential oils against Caryedon serratus (Oliver)—a storage pest of groundnut. J Food Sci Technol 51: 3505–3509 Janila P, Rao TN, Kumar AA (1999) Germination and early seedling growth of groundnut (Arachis hypogaea L.) varieties under salt stress. Ann Agric Res 20:180–182 Janila P, Aruna R, Kumar JE et al (2012) Variation in blanchability in Virginia groundnuts (Arachis hypogaea L.). Indian J Oilseeds Res 29:116–120 Janila P, Nigam SN, Pandey MK et al (2013) Groundnut improvement: use of genetic and genomic tools. Front Plant Sci 4. https://doi.org/10.3389/fpls.2013.00023

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Transgenic Technologies for Fusarium Wilt Management in Banana

14

R. Deepa Sankari, S. Varanavasiappan, L. Arul, K. Eraivan Arutkani Aiyanathan, E. Kokiladevi, and K. K. Kumar

14.1

Introduction

Banana (Musa spp.) is one of the most important fruit crops and a staple food crop for millions of people in developing countries. Most edible bananas are triploid, formed by inter-specific hybridization between two diploid progenitors, namely Musa acuminata (AA genome) and Musa balbisiana (BB genome). Many fungal, bacterial, and viral diseases have a significant impact on the global production of bananas. The most common fungal diseases are Panama disease (Fusarium oxysporum f. sp. cubense (Foc)), black Sigatoka (Mycosphaerella fijiensis), and yellow Sigatoka (Mycosphaerella musicola). Panama disease, also known as Fusarium wilt, is a major threat to banana production. F. oxysporum f. sp. cubense (Foc), belonging to the Ascomycotina class of fungus, is the causal agent of banana (Musa spp.) Fusarium wilt disease (also known as “Panama disease”). The Fusarium wilt pathogen was first discovered in an infected banana var. Sugar (Silk AA) in 1874 at an eagle farm near Brisbane, Queensland, Australia (Bancroft 1876). Foc invades banana root system and colonizes the corm tissue that clogs the water-conducting vessels, thereby causing wilting of the aerial parts of the infested banana plant. The infected plant shows typical discoloration of the corm and pseudostem, yellowing of the foliage, followed by complete wilting. Four races of Foc have been identified so far: race 1 (Foc1) causes disease in Gros Michel (AAA) and cultivars with the AAB genome; race 2 infects race 1-susceptible cultivars and cooking cultivars with ABB genome; race 3 affects Heliconia species; and race 4 has a broad host range, including “Dwarf R. D. Sankari · K. E. A. Aiyanathan Department of Plant Pathology, Tamil Nadu Agricultural University, Madurai, Tamil Nadu, India S. Varanavasiappan · L. Arul · E. Kokiladevi · K. K. Kumar (✉) Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_14

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Cavendish” (AAA genome) as well as all race 1 and race 2 susceptible cultivars. The isolates within race 4 are further divided into “tropical race 4” (TR4) and “subtropical race 4” (STR4) groups (Stover 1990; Ploetz 2005; Ordonez et al. 2015). Foc has spread throughout the world due to the informal exchange of planting material and the movement of spore-bearing soil. Foc isolates were separated into “vegetative compatibility groups,” which are genetically different populations. Some VCG isolates were compatible with isolates from other VCG, resulting in VCG complexes. There have been 24 Foc VCGs discovered so far around the world. However, the use of VCG as a means to classify Foc is also considered to be incomplete as one race could comprise more than one VCG or one VCG occurs in multiple races (Thangavelu et al. 2020). Various agronomic practices such as crop rotations, addition of organic amendments, and flood fallowing have proved to be ineffective in controlling this disease (Bakry et al. 2009). Once the fungal spores get established in the field, they persist in soil for almost three decades. Moreover, the disease is difficult to manage using chemical pesticides (Thangavelu and Mustaffa 2012). One of the sustainable solutions to manage this disease is to develop resistant cultivars by either conventional breeding or genetic engineering approaches.

14.1.1 Fusarium Wilt: International and National Scenarios In banana, Fusarium wilt originated from Southeast Asia and coevolved in conjunction with the Musaceae in its center of origin. The disease is reported in all the banana-producing regions of the world (Fig. 14.1). The first outbreak of this disease in the 1950s caused an estimated economic loss in Gros Michel by Foc race 1 of around US$23 billion. Due to Fusarium disease, banana industries depended on Cavendish cultivars, which were resistant to Foc race 1 instead of susceptible Gros Michel variety. Later in the year 1992, a new strain of Foc was discovered and the strain alone accounted for economic losses of about US $400 million to the banana industries (Ploetz and Pegg 1997; Ploetz 2015). Cavendish was thought to be highly resistant to the disease, but in 1953, Fusarium wilt was found to affect a small number of plants of the Cavendish cultivar “Williams” in Southern Queensland (Purss 1953). A “Williams” isolate was pathogenic to both Cavendish and “Lady Finger,” but a “Lady Finger” isolate did not have impact on Cavendish. This was possibly Australia’s first case of Fusarium wilt caused by TR4 (tropical race 4). This strain was also found to be present, often with race 1 strains, in “Lady Finger” plantations. A race 1 strain of the pathogen was involved and the disease outbreak was exacerbated by adverse environmental conditions (flooding and drought). Before 1930, this strain of Foc was thought to have been introduced with windbreak banana plants (an edible diploid of Musa acuminata) imported directly from Java and Singapore (Pegg et al. 1995;Shivas et al. 1995). A devastating strain of Foc was discovered in Cavendish plantations in Indonesia and Malaysia in 1990. This strain, called Foc TR4, spread to northern Australia in

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Fig. 14.1 Global widespread of Fusarium oxysporum f. sp. cubense (Foc) races. (Source: CABI/ EPPO, EPPO, and PROMUSA)

1997 and was thereafter reported from Taiwan, the Philippines, and mainland China. After being confined to Asia for almost two decades, Foc TR4 has spread to the Middle East and Mozambique. Foc TR4 affects banana cultivars of the “Cavendish” group in Australia and the tropical regions of the southeast (Conde and Pitkethley 2001;Garcia et al. 2014;O’Neill et al.2016) (Fig. 14.1). In India, the disease was first recorded in West Bengal in 1911 (Stover 1962), and the disease is now widespread and destructive in almost all the banana-growing states in India, causing a disease incidence of up to 30% in the main crop and up to 85% in the ratoon crop. The cultivars “Rasthali” (syn. “Malbhog,” “Nanjangod Rasabale”), “Amrithapani,” “Martaman,” AAB, Silk), “Karpuravalli” (syn. “Kanthali,” ABB, Pisang Awak), “Monthan” (ABB) and “Virupakshi” (syn. “Hill Banana,” AAB, Pome) are severely affected by Fusarium wilt (Thangavelu et al. 2001). Tropical race 4 (TR4), a severe disease that first occurred in the Cavendish group of bananas in regions of Bihar (2018), is now spreading to Uttar Pradesh, Madhya Pradesh, and even Gujarat, jeopardizing the country’s 50,000 crore (USD 7.52 billion) banana industry (Dita et al. 2018;Thangavelu et al. 2020).

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Pathogen and Disease Cycle

14.2.1 Symptomatology and Resting Spores of Fusarium Fusarium wilt pathogen belongs to the Ascomycota class. Foc invades banana root system and colonizes the corm tissue and subsequently clogs the water-conducting vessels, causing wilting of the aerial parts of the infected banana plant (Stover 1962). The infected plant shows typical discoloration of the corm and pseudostem and yellowing of the foliage, followed by complete wilting. Generally, the infected plants do not produce fruit, and if produced, fruits are very tiny in size, ripen irregularly, and flesh is pithy and acidic (Thangavelu and Mustaffa 2012;Dale et al. 2017). When the plants are dead, the fungus enters the epidermal cells and intercellular spaces of the banana root and develops numerous microconidia and macroconidia, followed by chlamydospores (Nelson 1991). Macroconidia are sickle-shaped four- to eight-celled with foot-shaped basal cells. Chlamydospores are typically globose in shape and are highly resistant double-membrane propagules that allow the pathogen to survive in the soil for several years in the absence of a host, preventing susceptible cultivars from being replanted in the same soil once affected (Davis et al. 2006).

14.2.2 Life Cycle The Fusarium pathogen penetrates the plant through root tips or natural lesions in the lateral root base, travels through the xylem vessels, and colonizes the rhizome. Internal vascular system discoloration is present at this stage (Robinson 1996). Sieve cells prevent the conidia from spreading further, allowing the spores to germinate, develop, and spread until the entire xylem system is blocked (Jeger et al. 1995). When the plant dies, the fungus produces dormant chlamydospores, which are released back into the soil once the plant has disintegrated (Jones 2000;Pei et al. 2005). The pathogen is mostly disseminated by infected rhizomes used for vegetative propagation. The infection can also spread through soil or running water as spores (Jones 2000). When chlamydospores germinate and infect a new host plant, the disease cycle is restarted (Stover 1962) (Fig. 14.2). Susceptible cultivars cannot be successfully replanted for up to 30 years if the soil has been contaminated with Foc (Ploetz 2006). Studies on the mode of infection and colonization of Foc in banana roots and rhizome region have been carried out by Li et al. (2011) using a green fluorescence protein (GFP)-tagged Foc TR4 isolate. Accordingly, chlamydospores connected to banana roots and root hairs infiltrate through root tips after spontaneous injury. A network of fungal hyphae was observed on the root caps and elongation zone of banana roots at 11 and 15 days post inoculation (dpi) with Foc in cv. Brazilian, respectively. The surrounding tissue became disordered as the rhizome’s vascular space was filled with fungal spores and hyphae.

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Fig. 14.2 Disease cycle of Fusarium oxysporum f. sp. cubense (Foc)

Furthermore, Xiao et al. (2013) provided evidence for the mode of infection by infecting the Cavendish banana cv. B.F. with GFP-tagged Foc race 4. According to the findings, after 3–10 dpi, the conidia and their germ tubes of Foc pierced the epidermis of the young roots. Later, hyphae went into the root xylem, rhizome, and ultimately to the surface of the pseudo-stem xylem. The hyphal population was also found to be higher in the pseudostem than in the roots and rhizomes of the infected plant.

14.2.3 Pathogenicity of Foc A huge proportion of putative virulence-associated genes were discovered in both the Foc race 1 and 4 genomes, including genes involved in root adhesion, cell wall degradation, toxin detoxification, transport, secondary metabolite biosynthesis, and signal transductions. The two-component regulatory system in fungi plays important roles in environmental change detection and adaptation. As a consequence, it was discovered that Foc 1 and Foc 4 could modulate the expression of various histidine kinase (HK) and response regulator (RR) genes during infection, resulting in transcription activation or a mitogen-activated protein kinase cascade (Catlett et al. 2003). Three putative secreted proteins (SPs) encoded by Foc were found to be significantly homologous

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to INF2A, INF2B effectors of P. infestans (Huitema et al. 2005), and PosjNIPw of Phytophthora sojae (Qutob et al. 2002), which are able to elicit hypersensitive response or induce necrosis in host plants, suggesting the involvement of secreted proteins in Foc–banana interaction. To overcome the chemical barrier synthesized in the host, fungi have evolved a diverse array of enzymes, including cytochrome P450s (CYP) of fungal origin, which is essential for secondary metabolite biosynthesis and toxic compound detoxification (Ichinose 2012). Foc 1 encodes 25 peroxidases that break down hydrogen peroxide (H2O2) produced by the host plant during fungal infection, analogous to the catalase peroxidases VlcpeA of Verticillium longisporum (Singh et al. 2012) and CPXB of Magnaporthe oryzae (Tanabe et al. 2011). Li et al. (2013) discovered beauvericin and fusaric acid in all tissues of wilt-infected bananas, which correlated with the virulence of FOC strains. Importantly, in comparison to the Foc race 1 isolate (Foc1), the Foc race 4 isolate (Foc4) has evolved with a few expanded gene families of transporters and transcription factors for toxin and nutrient transport, enabling improved alteration to host environments and contributing to banana pathogenicity. Ma et al. (2010) reported chromosome 14 of Foc1 as a “pathogenicity” chromosome harboring SIX effector genes, namely SIX5, SIX6, and SIX7, and established that the transfer of lineage-specific chromosomes between genetically isolated strains led to the emergence of new pathogenic lineages in F. oxysporum. FraserSmith et al. (2014) used PCR and sequencing to find variation in Foc SIX8 to distinguish Foc race 4 from Foc race 1 and 2 isolates, as well as subtropical and tropical Foc races. Guo et al. (2014) discovered 115 genes encoding putative GPCRs, suggesting that the G protein-mediated signaling pathway is conserved in ascomycete fungi.

14.3

Management of Foc

Management of the Fusarium wilt disease is through on-farm practices that reduce crop loss and prevent pathogen spread. However, there is no means of controlling Fusarium wilt once the plant is attacked since the fungus is found in the soil. Therefore, breeding for resistance is the most preferred method of overcoming the Fusarium wilt of banana. Conventional breeding in banana faces challenges due to various sexual reproduction barriers, such as high sterility, complex genetic background, polypoid, and parthenogenesis (Ghag et al. 2014a), making it extremely difficult to develop new disease-resistant bananas (Czislowski et al. 2018). Therefore, the development of genetically improved Foc-resistant banana cultivars against all races of Foc through genetic engineering is considered a good strategy (Ghag et al. 2014a). Genetic engineering has played a vital role in developing transgenic plant in banana. Different genes were successfully employed for imparting Fusarium wilt resistance in banana (Table 14.1).

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Table 14.1 Transgenic banana developed for Fusarium wilt resistance S. no. 1

Genotype and cultivar AAB cv. Rasthali

2

AAA cv. Williams

3

6

AAA cv. Cavendish AAA cv. Grand naine AAB cv. Sukali Ndizi AAB cv. Rasthali

7

AAB cv. Rasthali

8

AAB cv. Rasthali

9

AAB cv. Rasthali

10

AA cv. Furenzhi

11

AAB cv. Rasthali

12

15

AAB cv. Pisang Nangka AAB cv. Lady Finger AAA cv. Pei Chiao and cv. Gros Michel AAB cv. Rasthali

16

AAA cv. Taijiao

17

AAB cv. Rasthali

4 5

13 14

Race Race 1 TR4

Gene Ace-AMP1 and ca-pflp

TR4

MaLYK1, the sense and antisense fragments ofMaLYK1 ERG6, ERG11

TR4

Ced-9, RGA-2

Race 1 Race 1 Race 1 Race 1 Race 1 TR4

mCed-9 (modified form of ced9)

Race 1 TR4

Ace-AMP1

Race 1 TR4

Bcl-xL, Ced-9, Bcl-23‘UTR

Race 1 TR4 Race 2

MusaDAD1, MusaBAG1, MusaBI1 Defensin (Sm-AMP-D1) Velvet (vel), Fusarium transcription factor1(Ftf1) Petunia floral defensin (PhDef1/PhDef 2) Endochitinase gene (Thchit42)

Thaumatin-like protein (ostlp)

Plant ferridoxin-like protein (pflp), Arabidopsis root-type ferredoxin gene (Atfd3) GmEg Human lysozyme MSI-99 (maganine analog synthetic peptide)

References Sunisha et al. (2020) Zhang et al. (2019) Dou et al. (2019) Dale et al. (2017) Magambo et al. (2016) Ghag et al. (2014c) Ghag et al. (2014b) Ghag et al. (2014a) Ghag et al. (2012) Hu et al. (2013) Mohandas et al. (2013) Mahdavi et al. (2012) Paul et al. (2011) Yip et al. (2011) Maziah et al. (2007) Pei et al. (2005) Chakkrabarti et al. (2003)

14.3.1 Transgenic Approaches for Foc Management Recombinant DNA technology provides opportunity for engineering disease resistance in banana as a sustainable alternative approach (Hu et al. 2013). It can be achieved by means of identifying potential disease resistance gene cloning and introduction of genes into the plants by overexpressing or silencing. Genes such as antiapoptosis-related genes and the defense-related genes that are induced in response to pathogen attack have been used for the development of transgenic plants.

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Similarly, the pathogen genes that play a key role in the invasion, growth, and pathogenesis of plants are selectively silenced through post-transcriptional gene silencing strategies.

14.3.1.1 PR-Related Gene (Defensin Gene) Plant proteins that are produced in response to pathogen invasion are known as pathogenesis-related proteins. At present, 17 families of PR proteins have been identified based on their primary structure homology. Hydrolytic enzymes (chitinase and glucanase), thionins, and defensins are among the most essential PR proteins. With respect to developing transgenic crops resistant to Fusarium wilt of banana, the following are the selected examples involving overexpression of PR genes. Maziah et al. (2007) cloned and expressed the gene encoding β-1-3endoglucanase in banana cv. Rasthali to impart resistance to Foc race 1. The gene was expressed at its highest level, resulting in enhanced resistance against Foc race 1. Wilt-resistant banana plants were developed by Hu et al. (2013) by introducing an endochitinase gene (chit42) into cv. Furenzhi. After 2 months of pathogen inoculation, the majority of transgenic lines (three of seven) expressing chit42 demonstrated a higher level of resistance to Fusarium wilt (Foc race 4), whereas nontransgenic control plants were susceptible. Further, Ghag et al. (2012) transformed two floral defensin genes, phdef1 and phdef2, into banana cv. Rasthali in two independent experiments. The findings of the pot bioassay indicated that transgenic plants expressing the floral defensin gene had less external and internal symptoms than control plants. After 6 weeks of post inoculation with Foc culture, control plants succumbed to wilt disease, but phenotypically normal transgenic plants displayed modest signs and recovered entirely within 3 weeks. These findings showed that the expression of defensin gene in the host plant can enhance resistance to Fusarium wilt. Ghag et al. (2014b) reported that the gene sequence coding for a seed defensin (Sm-AMP-D1) of common chickweed, Stellaria media, protected banana against Fusarium oxysporum with an IC50 value of 0.35. This study demonstrated that overexpression of Sm-AMP-D1 in banana could potentially lead to the development of durable resistance against Foc pathogens. Mahdavi et al. (Mahdavi et al. 2012) introduced the rice thaumatin-like proteins gene (tlp) into the banana Musa sapientum cv. Nangka(AAB). After 30 days of inoculation with Foc race 4 in pot culture, the transgenic plant overexpressing the tlp gene showed increased resistance to Fusarium wilt compared to control plants. 14.3.1.2 Antimicrobial Peptide Gene (Ace-AMP1 Gene) Antimicrobial peptides are small, cationic, and amphipathic peptides enriched with cysteine amino acids and encoded by multigenic families. AMPs have a wide range of activity and are broadly distributed among plant kingdom (Boman 1991;Hancock and Lehrer 1998). These peptides are important in innate immunity and serve as a first line of defense barrier in the plant system against multiple invading phytopathogens.

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Chakkrabarti et al. (2003) cloned MSI-99, a maganin analog, a short alpha-helical peptide of 23 amino acids from the African clawed frog and reported to have broadspectrum action against Gram-positive and Gram-negative bacteria, fungi, and protozoa, as well as antitumorigenic activity. At higher concentrations, MSI-99 peptide, when tested under in vitro conditions, completely inhibited the growth and spore germination of Foc. Banana plants engineered with MSI-99 exhibited resistance to Foc and Mycosphaerella musicola. Pei et al. (2005) developed a transgenic banana plant using a human lysozyme gene. Transgenic plants overexpressing human lysozyme demonstrated the improved resistance to Fusarium wilt disease not only in pots but also in confined fields. Overexpression of the pflp gene in banana cv. Pei Chiao and cv. Gros Michel exhibited a much lower percentage of severity when exposed to Foc race 4 during a 9-week period, showing the potential for Foc resistance (Yip et al. 2011). Mohandas et al. (2013) generated a transgenic banana with the Ace-AMP1 gene and (Sunisha et al. 2020) with pflp gene to evaluate its tolerance to Fusarium wilt disease. The banana cv. Rasthali expressing the Ace-AMP1 and pflp resulted in improved resistance to Fusarium wilt disease. After 180 days of planting, a bioassay against Foc race 1 in pot culture studies revealed improved tolerance in transgenic plants. When compared to untransformed banana cv. Rasthali, two separate transformants revealed a 10–20% vascular discoloration index (96%). The stacked lines revealed higher activity of superoxide dismutase and peroxidase compared to untransformed control that showed higher tolerance to oxidative stress caused by Foc infection.

14.3.1.3 Antiapoptosis-Related Proteins Antiapoptosis genes have been identified as a promising choice for engineering resistance against necrotrophic fungal pathogens that favor dead tissues to induce nutrient leakage in order to survive. Overexpression of mammalian antiapoptotic genes such as Bcl2, Bcl-xl, and Ced9 gene from Caenorhabditis elegans could suppress apoptosis. Paul et al. (2011) transformed banana cultivar Lady Finger with Bcl-xL, Ced-9, and Bcl-23′UTR with a view to prevent necrotrophic death of banana plants due to Foc infection. Transgenic banana plants subjected to a 12-week root challenge with Foc race 1 showed dramatically reduced disease symptoms compared to wild-type. Even after continuous exposure to transgenic plants for 23 weeks, the Bcl23′ UTR expressing transgenic line proved to be highly resistant. Similarly, the popular dessert banana in East Africa, banana cv. SukaliNdizi was engineered with a modified variant of the antiapoptosis gene ced9. When compared to control plants, transgenic plants showed much reduced disease severity at 13 weeks of post inoculation (Magambo 2012). The use of host genes coding for cell death-related proteins to control the Fusarium wilt disease in banana was described by Ghag et al. (2014c). In order to identify cell death-related genes, embryogenic cells were treated with filtrate of Foc culture, Fusarium toxin known as fusaric acid and beauvericin at 50 M and 5 M, respectively. Transgenic banana plants were generated overexpressing the cell death-related genes MusaDAD1, MusaBAG1, and MusaBI1. When compared to

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MusaBI1 and MusaDAD1 overexpressing transgenic banana plants, MusaBAG1 expressing transgenic banana plants demonstrated improved resistance to Fusarium wilt disease. Transgenic banana modified with the antiapoptosis gene, Ced9 from C. elegans, showed enhanced resistance to banana tropical race 4 (TR4) in the field (Dale et al. 2017). Sunisha et al. (2020) showed that the prevention of necrosis is an ideal method to prevent Fusarium infection in banana. Oxidative stress-induced cell necrosis is prevented by the activation of antiapoptotic pathways by an antiapoptotic gene, Ced-9. The transgenic Rasthali banana plants engineered with Ced-9 showed higher level of resistance for more than 6 months.

14.3.1.4 RNAi-Mediated Host-Induced Gene Silencing (HIGS) RNAi has emerged as a new technique for disease management by silencing pathogen-associated gene. Foc is a hemi biotrophic fungus with a short biotrophic phase followed by complete necrotrophy phase inside the host plant (Thaler et al. 2004). Transgenic hairpin RNAs that are targeted against fungal genes have been expressed at high levels in transformed plants in the majority of these studies to confer resistance against various pathogens. The host plant-derived fungal-specific small interfering RNAs (siRNAs) can enter the fungal cell and cause silencing of the target fungal genes, and this mechanism is termed as host-induced post-transcriptional gene silencing (HIGS). Ghag et al. (2014a) employed HIGS in the banana cv. Rasthali to introduce Fusarium wilt resistance. Banana cv. Rasthali plants were transformed with partial coding sequences of velvet family genes (VeA, VelB, and VosA) involved in fungal morphogenesis and Fusarium transcription factor 1 (ftf1) required for fungal colonization and infection. using the Agrobacterium-mediated method. Individual transgenic banana plants expressing siRNAs specific to fungal genes showed significant resistance to Fusarium wilt disease even after 8 months of Foc inoculation in the greenhouse. Zhang et al. (2019) studied the MaLYK1 gene function in Fusarium disease resistance by inoculating the Foc4 in the MaLYK1RNAi-silenced banana lines and overexpressing (MaLYK1-OE) banana lines. Inoculation of MaLYK1-RNAi plants with Foc4 resulted in larger leaf lesions (chlorotic area) while no visible lesions were found in the leaves of inoculated MaLYK1-OE lines. Dou et al. (2020) have shown that the transgenic banana expressing the dsRNA specific to two Foc ergosterol biosynthetic genes, RG6/ERG11, could inhibit the growth and development of Foc tropical race 4. Deepa Sankari et al. (2022) showed that the silencing of two fungal genes, ftf1/velvet, by the host-induced gene silencing (HIGS), could inhibit the growth and development of Foc race 1 in transgenic Rasthali cultivar. Even after 8 months of Foc inoculation in the greenhouse, individual transgenic banana plants expressing siRNAs specific to both fungal genes showed significant resistance to Fusarium wilt disease. Panama wilt pathogen, Foc transformed with RNAi construct to silence the fungal SGE1 (secreted in xylem – gene expression 1) gene showed reduced gene expression (27–47%) in 13 different Foc transformants compared to wild-type strain (Fernandes et al. 2016). The pathogenicity analysis revealed that the transformants were able to reach the rhizomes and pseudostem of the inoculated banana plants.

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However, the transformants induced initial disease symptoms approximately 10 days later than that by the wild-type Foc.

14.3.1.5 Use of Genome-Editing Tool for Disease Management CRISPR, a recently developed precise gene-editing tool, has the potential for the accurate modification of beneficial genes in banana without introducing foreign genes. The CRISPR genome-editing system consists of two essential components: an sgRNA that recognizes the target DNA specifically and a Cas9 endonuclease that precisely cleaves the target DNA (Feng et al. 2013). The CRISPR system has two major advantages: numerous simultaneous mutations and Cas9-free plants (Wang and Chen 2020). . Gene editing has been used to improve banana fruit quality, shelf life, and plant architecture, in addition to developing disease resistance. The first report of banana gene editing was reported in the cultivar “Rasthali” (AAB genome) whose phytoene desaturase (PDS) gene was targeted (Kaur et al. 2018). The researchers used a single sgRNA to create mutations in the PDS gene, resulting in an albino appearance. The mutation rate, however, was only 59%. Furthermore, Naim et al. (2018) have employed a polycistronic tRNA to edit the PDS gene in “Cavendish Williams” (AAA genome) with 100% editing efficacy. Similarly, Ntui et al. (2020) used numerous sgRNAs targeting the PDS gene to achieve 100% mutation efficiency in banana cultivar “Sukali Ndiizi” (AAB genome) and plantain cultivar “Gonja Manjaya” (AAB genome). The CRISPR technology was also used to create a semi-dwarf phenotype using M. acuminata gibberellin 20ox2 (MaGA20ox2) gene, disrupting the gibberellin (GA) pathway of the Gros Michel banana cultivar (Shao et al. 2019). Some progress has been made to build resistance against bacterial pathogens using CRISPR/Cas9-mediated gene editing by knocking out the disease-causing susceptibility (S) genes or activating the expression of the plant defense genes (Tripathi et al. 2022). There has been some success in developing resistance to banana streak virus (BSV) and BXW disease. It was recently shown that editing MusaDMR6 in banana using CRISPR/Cas9-mediated genome editing resulted in increased resistance to BXW disease (Tripathi et al. 2021). Several groups are attempting to enhance TR4 resistance in Cavendish bananas using CRISPR, not only by suppressing the expression of the TR4 susceptible gene but also by expressing dormant TR4 resistance genes (Dale et al. 2017;Maxmen 2019). Banana being an asexually propagated plant, edited plants will have CRISPR/ Cas9 transgenes stably integrated into their genomes. In order to overcome this limitation, DNA-free gene editing can be performed by introducing the preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs) into banana protoplasts. These RNPs cleave target sites soon after transfection and are rapidly degraded by the cell’s endogenous proteases. This allows for specific mutagenesis in Cas-free regenerated plants (Woo et al. 2015).

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Challenges and Future Prospects for the Development of Crop Disease-Resistant Cultivars Using Transgenic Technologies

After rice, wheat, and maize, bananas is the fourth most important crop in developing nations; these crops are vital for food security (Dash and Rai 2016). Banana production can be enhanced in addition to disease resistance by developing bananas with ideal plant architecture. Banana plants with ideal architecture (e.g., dwarfism, strong stems, more upright leaves, and root systems with excellent hydrotropism) would have better light and water utilization efficiency, lodging resistance, yield, and disease resistance. As Fusarium cannot be controlled once it has established itself, sustainable banana production demands the use of Foc-resistant varieties. Genetic modification, which compensates for a lack of traditional breeding options, is an effective method of developing bananas with better agronomic characteristics such as increased disease resistance and yield. Banana breeders face two main challenges in producing genetically modified banana varieties with Foc resistance and no yield penalty. First, genes associated with Foc resistance and other important agronomic traits must be identified, functionally evaluated, and used in target breeding programs. Second, a transformation and regeneration system that is highly efficient and stable must be created. Currently available genetic transformation techniques are variety dependent, and regeneration of entire plants from a single transformed cell is difficult. The differentially expressed genes and putative signaling pathways identified through transcriptomic profiling will ideally accelerate research on banana toward Foc resistance and contribute to a better understanding of the banana defense response to plant pathogens. Functional genomic studies of the M. acuminate and M. balbisiana genomes will aid in elucidating the molecular mechanisms underlying the banana immune response, as well as determining the genomic correlations between disease resistance and banana yield.

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Conclusion

Banana cultivation is seriously affected throughout the world by the devastating Fusarium wilt disease. In most banana-growing countries, wider prevalence of Foc race 4 in the last two decades is able to cause the wilt disease in earlier Foc race 1-resistant banana varieties. Transgenic banana expression PR genes show promise in Fusarium wilt disease control, however, not suitable enough for commercial cultivation. There is a scope for engineering strong Fusarium resistance in banana by employing RNAi and genome editing. The transcriptome data and gene expression profiles of diverse Foc races, as well as the availability of banana wholegenome sequences, will be very useful in the near future for identifying potential target gene for mediating resistance to Fusarium wilt disease. Finally, once the molecular mechanisms are known, banana genetic improvement will have excellent prospects in increasing disease resistance and food security.

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Acknowledgments The authors express their gratitude to the Department of Biotechnology (New Delhi) for the NER banana project grant under the scheme of Development of Fusarium wilt resistant Banana cultivar for NE India.

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Stover RH (1990) Fusarium wilt of banana: some history and current status of the disease. In: Ploetz RC (ed) Fusarium wilt of banana. APS Press, The American Phytopathological Society, St. Paul, MN, pp 1–7 Sunisha C, Sowmya HD, Usharani TR, Umesha M, Gopalkrishna HR, Saxena A (2020) Deployment of stacked antimicrobial genes in banana for stable tolerance against Fusarium oxysporum f. sp. cubense through genetic transformation. Mol Biotechnol 62(1):8–17 Tanabe S, Ishii-Minami N, Saitoh K, Otake Y, Kaku H et al (2011) The role of catalase-peroxidase secreted by Magnaporthe oryzae during early infection of rice cells. Mol Plant Microbe Interact 24:163–171 Thaler JS, Owen B, Higgins VJ (2004) The role of the jasmonate response in plant susceptibility to diverse pathogens with a range of lifestyles. Plant Physiol 135:530–538 Thangavelu R, Mustaffa MM (2012) Current advances in the Fusarium wilt disease management in banana with emphasis on biological control. Plant Pathol:273–298. https://doi.org/10.5772/ 33775 Thangavelu R, Sundaraju P, Sathiamoorthy S, Raguchander T, Velazhahan R, Nakkeeran S, Palaniswami A (2001) Status of Fusarium wilt of banana in India. In: Molina AB, Nikmasdek NH, Liew KW (eds) Banana Fusarium wilt management towards sustainable cultivation. INIBAP-ASPNET, Los Banos, Laguna, pp 58–63 Thangavelu R, Loganathan M, Arthee R, Prabakaran M, Uma S (2020) Fusarium wilt: a threat to banana cultivation and its management. CABI Rev:1–24. https://doi.org/10.1079/ PAVSNNR202015004 Tripathi JN, Ntui VO, Shah T, Tripathi L (2021) CRISPR/Cas9-mediated editing of DMR6 orthologue in banana (Musa spp.) confers enhanced resistance to bacterial disease. Plant Biotechnol J 19(7):1291–1293 Tripathi L, Ntui VO, Tripathi JN (2022) Control of bacterial diseases of banana using CRISPR/Casbased gene editing. Int J Mol Sci 23(7):3619 Wang J, Chen H (2020) A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis. aBIOTECH 1(1):6–14 Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33(11):1162–1164 Xiao RF, Zhu YJ, Li YD, Liu B (2013) Studies on vascular infection of Fusarium oxysporum f. sp. cubense race 4 in banana by field survey and green fluorescent protein reporter. International J Phytopathol 2(1):44–51 Yip MK, Lee SW, Su KC, Lin YH, Chen TY, Feng TY (2011) An easy and efficient protocol in the production of pflp transgenic banana against Fusarium wilt. Plant Biotechnol Rep 5:245–254 Zhang L, Yuan L, Staehelin C, Li Y, Ruan J, Liang Z, Xie Z, Wang W, Xie J, Huang S (2019) The LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE 1 protein of banana is required for perception of pathogenic and symbiotic signals. New Phytol 223(3):1530–1546

Genetic Improvement of Banana

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Soni KB, Anuradha T, Pritam Ramesh Jadhav, and Swapna Alex

15.1

Introduction

Banana (Musa spp.), belonging to the Musaceae family, is a herbaceous, perennial that originated in the tropical region of Southeast Asia. It is the fourth most cultivated crop in the world. Banana possesses nutritional, medicinal, and industrial values and contributes significantly to a major portion of the calorie intake of many populations. Bananas are cultivated in over 140 countries in the tropics and subtropics with a global production of 125 million tons in 2021, with India contributing 35 million tons. Over 1000 varieties of bananas are cultivated in different parts of the world, and the most important commercial variety is Cavendish (Tripathi et al. 2019; Thangavelu et al. 2021), because of their reliability during transport and shelf life. The majority of the existing cultivars of banana and plantain are hybrids produced from the two wild diploid species, namely Musa acuminata Colla (genome A) and M. balbisiana Colla (genome B). The hybrids are of different ploidy levels and genomic combinations, namely AA, AB, AAA, AAB, ABB, AABB, AAAB, and ABBB (Simmonds and Shepherd 1955). In most banana-growing countries, it is produced mainly for local consumption as a staple food or as a dietary supplement. Banana cultivation is seriously affected by many biotic and abiotic stresses (Pillay 2011;Tripathi et al. 2015). Fusarium wilt is the most destructive threat to banana production, which is difficult to control once established. Viruses such as banana bunchy top virus (BBTV), banana bract mosaic virus (BBrMV), cucumber mosaic virus (CMV), and banana streak virus (BSV) cause huge economic losses. Burrowing nematodes and weevils also pose a threat to S. KB (✉) · A. T · P. R. Jadhav · S. Alex Department of Molecular Biology and Biotechnology, College of Agriculture, Thiruvananthapuram, Kerala, India e-mail: [email protected]; [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_15

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banana cultivation. The increasing changes in climate pose a serious impact on yield, specifically when the crop is grown with less or no irrigation. Considering the food and economic security provided by the crop, it is important to take efforts for the genetic improvement of banana. New varieties and hybrids of elite agronomic traits combined with tolerance toward biotic and abiotic stresses are in demand. In the changing climate scenario, resilient varieties are the need of the hour. Other desirable traits include fruit quality, increased shelf life, short stature, and early flowering. During the past years, several strategies have been tried for the genetic improvement of bananas. Many research groups adopted conventional breeding approaches such as diploid breeding and hybridization between triploid cultivars with wild-cultivated diploid parents (Dale et al. 2017). Though there has been success, the higher level of heterozygosity and sterility of the edible varieties due to triploidy hampered many of the breeding processes (Becker et al. 2000;Ortiz and Vuylsteke1995). Other challenges included the lack of desirable traits in the available fertile diploids and the lower seed set in the crosses. Induced mutation using chemical and physical mutagens was another approach that also showed limited success. Molecular marker technology has enabled the characterization of banana germplasm, identifying desirable genes and alleles linked to the elite agronomic traits that can be introduced into popular cultivars by adopting marker-assisted selection or developing transgenics. The advancements in genetic engineering techniques, and the publication of the banana genome, facilitated the genetic improvement programs in banana (D’Hont et al. 2012; Wang et al. 2019; Maxmen2019). During the last two decades, transgenic approaches were tried to introduce desirable traits in bananas. The possibility of selecting desirable genes from noncompatible banana germplasm or from other sources makes this approach suitable for developing cultivars with new traits. Compared to conventional cross-breeding, this approach takes relatively short time without altering the basic genetic traits of the targeted cultivars. Since 1990, methods such as particle bombardment, electroporation, and Agrobacterium-based transformation have been used for genetic transformation in banana (Sági et al. 1995; May et al. 1995). Transgenic approaches are attractive in banana because of the female and male sterility of most edible banana cultivars and the lack of cross-fertile wild relatives in most of the banana-producing areas. Several transgenic banana varieties have been developed for different traits such as Fusarium, bunchy top and bract mosaic virus disease resistance, and qualitative traits. Genome editing using CRISPR/Cas system has become a promising and attractive technology for genetically modifying crops without gene transfer and with a minimum alteration of the genome. Several research groups have tried this technology to alter traits in banana. Editing of the endogenous banana streak virus in the B genome of Musa spp. to overcome the challenge in banana breeding is a promising example (Tripathi et al. 2019).

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Biotechnological Tools for Genetic Improvement of Banana

15.2.1 Somatic Embryogenesis as a Tool for Genetic Manipulation An efficient and reliable in vitro regeneration system is a prerequisite for the success of any genetic improvement method. Due to the high proliferation potential and single-cell origin, somatic embryogenesis is mostly used for genetic manipulation in banana. Genetic instability is also less in this system. In somatic embryogenesis, the somatic cells are differentiated and reprogrammed to follow the embryogenic pathway to produce somatic embryos resembling the zygotic embryos. Somatic embryos are developed either directly or via callus. Among the different explants, the most responsive explant for producing embryogenic cell suspension (ECS) is the immature male inflorescence, which is suitable for transformation studies (Novak et al. 1989; Ganapathi et al. 1999; Morais-Lino et al. 2008; Tripathi et al. 2015; Lekshmi et al. 2016; Nandhakumar et al. 2018). Genotype, age of the explant, and growth regulators are the major factors affecting somatic embryogenesis in bananas. Somatic embryogenic cell suspensions provide an ideal material for in vitro mutagenesis and genetic transformation studies. Figure 15.1 shows different stages of somatic embryo-mediated regeneration in banana.

15.2.2 In Vitro Mutagenesis Mutagenesis is a strategy for developing novel traits in both vegetatively and seedpropagated crops. It is an efficient tool for both forward and reverse genetics approaches (Henikoffet al. 2004). In vegetatively propagated crops such as banana, callus, single-cell suspension, or somatic embryos are preferred for mutagenesis because of their rapid regeneration and ease of recovery of mutants and separation of chimaeras (Van Harten1998). Both chemical and physical mutagens are used for treating the in vitro cultures of banana (Kulkarni et al. 2007). Chemical mutagens include ethyl methane-sulfonate (EMS), sodium azide, and diethyl sulfate). These

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Fig. 15.1 Somatic embryogenesis-mediated regeneration in banana. (a) Immature male flower bud inoculated in semi-solid media. (b) Somatic embryo development. (c) Elongating embryos. (d) Germination of embryos. (e) Maturation. (f) Plant conversion

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chemicals destroy the nuclear DNA and randomly induce mutations during DNA repair process. Gamma radiations are able to produce a broad spectrum of mutations. The important factors determining the success of in vitro mutagenesis include the selection of an effective mutagen for the species, an in vitro plant material having rapid regeneration potential, and an effective mutant screening system. TILLING (targeting-induced local lesions in genomes) is a commonly used method to identify SNPs and indels caused by mutagens.

15.2.3 Genetic Transformation Genetic transformation involves the insertion of foreign DNA into the target cells using direct or vector-mediated methods to develop the desired traits. It is important to know the mechanism of gene action, regulation of expression, and safety of the gene and its product to be utilized. Advancements in genomics and transcriptomics have made the identification and functional analysis of genes easier. Developments in gene transfer techniques also facilitated the process. Genetic transformation is an attractive option for the improvement of banana due to their recalcitrancy to conventional techniques. The genetic modification methods mostly followed in bananas include particle bombardment (Sági et al. 1995), electroporation of the protoplasts (Sági et al. 1994), and Agrobacterium-mediated transformation (May et al. 1995). Agrobacterium-based method is mostly used as it allows stable integration of the insert and limits the number of copies of the gene, thus reducing the possibility of gene silencing. It is important to optimize the density of Agrobacterium for effective transfer of T-DNA, without causing necrosis to the tissues. Banana being a monocot, the addition of phenolic compounds such as acetosyringone in the co-cultivation medium can improve the T-DNA transfer. Washing co-cultured tissue/cells with bacteriostatic agents like cefotaxime and adding it to the selection medium is effective to eliminate Agrobacterium and prevent necrosis of the infected tissue. Culturing the tissue without the selection agent for 1 week can help the tissue recover from infection (Carvalho et al. 2004). The addition of a centrifugation step during the infection process has been shown to improve the efficiency of Agrobacterium-mediated transformation banana (Khanna et al. 2004). Sonication and vacuum infiltration have also been shown to improve Agrobacterium-mediated transformation (Subramanyam et al. 2011).

15.2.3.1 Cisgenic Approach Cisgenesis has been developed as a tool for genetic modification in response to public concerns about safety issues with transgenic crops and to ensure an environmentally acceptable crop improvement technique. While transgenesis is carried out using the genes from a sexually incompatible organism, cisgenesis uses the genes from the organism itself or from a sexually compatible organism. It is a natural variant with its introns, flanking native promoter, and terminator in normal sense orientation inserted as an additional copy to the genome. In contrast to conventional

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breeding, cisgenic crops lack any undesirable genetic components and only have the desired gene or genes. Cisgenesis is advantageous in maintaining the original genetic makeup of the crop and also avoiding the problem of gene flow to wild vegetation. But the inserted cisgene may have an impact on the expression of genes that are already present in the recipient genome. It is impossible to introduce characters outside the sexually compatible gene pool.

15.2.4 RNA Interference (RNAi) It is a natural antiviral defense mechanism found in plants. This mechanism silences the targeted gene in a sequence-specific manner with the help of an RNA-induced silencing complex (RISC) that can identify and cleave the homologous sequence (Hannon 2002). Since the pest or pathogen populations mostly interact with the host plants for nutrition, RNAi constructs targeting pest or pathogen transcripts can be used to develop host resistance. The small interfering RNA (siRNA) produced in the host from the double-stranded RNA will enter the pest or pathogen and cleave the transcripts needed for their growth and disease development. It is also called hostinduced gene silencing (HIGS). Because of the high-sequence specificity, genes can be selectively targeted without affecting any beneficial partners. RNA silencing strategy is an important tool for developing virus resistance in plants since the viral genomes replicate within the host and are amenable to direct silencing (Wang et al. 2012). But in some cases counter defense mechanisms developed by the viruses acting through suppressors of RNA silencing have been identified (Burgyán and Havelda2011;Pumplin and Voinnet2013).

15.2.5 Genome Editing Genome editing is an advanced tool for making precise changes in the DNA sequence of an organism. Gene editing is done using site-directed nucleases that can target a specific DNA sequence. The DNA breaks caused by these nucleases are utilized to introduce desirable changes in the target sites that are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The most popular genome-editing system is CRISPR-Cas9, discovered by Jennifer Doudna, Emmanuelle Charpentier and colleagues in 2012. The CRISPR-Cas9 system comprises a short noncoding guide RNA (gRNA) and a Cas9 nuclease. The gRNA contains a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA). The gRNA unit guides the Cas9 protein to a specific genomic locus, where the Cas9 nuclease induces a double-stranded break at the specific genomic target sequence. Following DNA cleavage using CRISPRCas9, the double-stranded break is repaired by one of the following mechanisms. In the absence of a repair template, the nonhomologous end joining (NHEJ) process results in a heterogeneous population of cells with different insertions or deletions

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(indels) around the gRNA-defined break. This process can be exploited to generate cell lines with random deletions around a specific sequence, producing a functional knockout. Alternatively, if a repair template is provided, a user-defined sequence change can be introduced at a specific locus within the genome via the error-free homology-directed repair (HDR) mechanism. This process can be used to overexpress a novel gene, create disease-relevant cell models, or tag endogenous genes with reportable moieties.

15.3

Genetic Improvement of Banana for Various Traits

15.3.1 Biotic Stress Tolerance The most economically important biotic stresses reducing the productivity of banana worldwide are viral, bacterial, and fungal diseases and nematodes (Fig. 15.2). Genetic engineering strategies have been adopted for the genetic improvement of many cultivars for different traits, including tolerance to biotic and abiotic stresses, quality of fruits, and ideal plant architecture. Some of the studies on the development of biotic stress tolerance in banana are described here.

15.3.2 Virus Resistance Banana bunchy top virus (BBTV), banana streak virus (BSV), banana bract mosaic virus (BBMV), and cucumber mosaic virus are the major viruses that cause economic loss in many banana cultivars. BBTV, a Babuvirus of the family Nanoviridae, is a complex ssDNA virus that multiplies in the phloem tissue of the host plant. Banana aphid (Pentalonia nigronervosa) transmits this virus in a persistent, nonpropagative, circulative manner (Di Mattia et al. 2020;Watanabe et al. 2016). BSV is a Badnavirus of the family Caulimoviridae (Alangar et al. 2016) that is transmitted through several species of mealybugs. The genome of BSV, when it gets integrated into the host genome, is activated to form infectious virions under

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Fig. 15.2 Symptoms of major diseases in banana. (a) Yellowing of leaves due to Fusarium wilt. (b) Chlorotic streaks on leaves infected with BSV. (c) Mosaic symptoms due to CMV infection. (d) Bunch appearance of leaves due to BBTV infection. (e1). Purplish streaks on bracts due to BBrMV. (e2) Traveler’s palm appearance due to BBrMV. (e3) Purplish streaks on pseudostem due to BBrMV

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Table 15.1 List of strategies and target genes used for virus resistance in banana Strategy Transgenic

Target gene Rep

Genotype M. spp. cv. Dwarf Brazilian Grand Naine

Desired trait Tolerance to BBTV

References Borth et al. (2011)

BSV-resistant lines

El-Sayed et al. (2011)

M. spp. cv. Williams M. spp. cv. Rasthali

Tolerance to BBTV Tolerance to BBTV

Ismail et al. (2011) Shekhawat et al. (2012) Krishna et al. 2013

Induced mutation γ rays (20, 40, and 60) Transgenic

BBTV-G-CPb

RNAi

Rep

RNAi

DNA-R, DNA-S, DNA-M, and DNA-C of BBTV genome BBTV rep gene

M. spp. cv. Grand Nain

Tolerance to BBTV

Virupakshi (AAB)

Tolerance to BBTV

RNAi

Replicase gene of BBrMV

Nendran (Musa AAB)

Resistance to BBMV

CRISPR-Cas9

ORF1, ORF2, and ORF3 of BSV

Musa spp. cv. GonjaManjaya

Inactivation of integrated endogenous BSV

RNAi

Elayabalan et al. (2013) Lekshmi et al. (2021) Tripathi et al. 2019

environmental stress. BBMV is a potyvirus and is transmitted by several aphid species in a nonpersistent mode. The small genome size and high genetic mutations occurring due to rapid replication help the plant viruses to evade the defense mechanisms developed by plants in a shorter period. Hence, it is difficult to sustain the disease resistance developed by conventional methods for longer. Advanced crop improvement techniques such as genetic engineering, RNAi, and CRISPR/Cas genome editing can compensate for the lacunas of conventional methods. There are different strategies deployed to control viruses in banana (Table15.1). The target genes for virus resistance included genes encoding antiviral proteins, antiviral signal inducers of systemic nature, and ribozymes specific to the viral genome (Gadani et al. 1990; Mandadi and Scholthof2013). RNAi strategy has been proven effective to develop resistance to Fusarium wilt and BBTD with durable resistance. The transgenic plants of banana cv. Rasthali transformed with the ihpRNA constructs, namely ihpRNA-Rep and ihpRNAProRep, showed complete resistance to BBTD, and no disease symptoms appeared even after 6 months of challenging with viruliferous aphid (Shekhawat et al. 2012). The infected transgenics did not show the transcripts of coat protein, movement protein, and Rep protein, confirming the resistance. Similar strategies of RNA

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silencing have been done in other banana cultivars such as Virupakshi targeting different gene components of BBTV (Krishna et al. 2013; Elayabalan et al. 2017). Though the transgenics were not immune to the virus completely, symptom development was delayed, leading to partial resistance. Lekshmi et al. (2021) developed resistance toward BBMV in cv. Nendran by transforming embryogenic calli with ihpRNA constructs targeting replicase gene of the virus. None of the transgenics were infected when challenged with viruliferous aphids. CRISPR/Cas9 system was used to target multiple ORFs of BSV to inactivate the dsDNA of eBSV integrated into the cultivar “GonjaManjaya” (Tripathi et al. 2019). The mutated viral genome in the regenerated plants was not able to produce the transcripts and infectious viral proteins. The mutated plants were asymptomatic and showed 75% inactivation of viral transcription.

15.3.3 Fungal Disease Resistance The fungal diseases seriously affecting the banana production globally are Fusarium wilt (caused by Fusarium oxysporum f. sp. cubense (Foc)) and Sigatoka leaf spot disease (caused by Mycosphaerella fijiensis). Foc, the most potent and soil-borne pathogen, has three pathogenic races called Foc races 1, 2, and 4 (Robinson 1996). Foc race 1 constitutes isolates affecting cultivars such as the Gros Michel, Silk, and Pome. The isolates that initially showed symptoms in Cavendish cultivar were brought under race 4, which was later divided into tropical and race 4 (TR4 and STR4). TR4 strain is the most virulent, and most of the edible banana varieties grown in different regions have succumbed to this strain. The presence of this strain is reported in almost all banana-growing regions. The genetic engineering strategies adopted to control Fusarium wilt include the overexpression of antimicrobial peptides such as defensins and ferredoxin-like proteins, thaumatin-like pathogen-related protein, chitinase, and apoptosis-related genes (Ghag et al. 2012; Sunisha et al. 2020; Hu et al. 2013; Mahdavi et al. 2012, Dale et al. 2017). Other successful methods that showed durable resistance to this fungus include overexpressing proteins, chaperons, and secondary metabolites (Vishnevetsky et al. 2011; Ghag et al. 2014a). Table 15.2 shows some of the research studies conducted for developing Foc resistance in banana cultivars through genetic engineering. Newer control measures such as cisgenesis were also exploited to target endogenous genes in plants. In the cisgenesis, native genes or promoters from related species or from the crop plant itself are introduced for genetic modification. Stacking of resistance genes from the germplasm can also be done for improving the existing varieties. A more attractive linkage-drag-free cisgenic approach utilizes the possibility of stacking resistance genes from the related wild species. A resistance gene analog 2 (RGA2), isolated from a TR4-resistant wild banana (M. acuminata ssp. malaccensis), was introduced to develop Fusarium wilt-resistant lines of Cavendish banana (Dale et al. 2017). The field trial showed >80% survival rate by the transgenic plants.

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Table 15.2 Genetic improvement strategies for Fusarium wilt resistance in banana Strategy Genetic transformation In vitro mutation, γ rays (20Gy) Genetic transformation Genetic transformation Genetic transformation In vitro mutation EMS (300 mM) Genetic transformation

Trait Foc race 2 resistance Foc race 4 resistance

Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation

Foc race 1 resistance TR4 resistance

Genetic transformation

Foc race 1resistance

Foc race 1 resistance Foc race 1 resistance Foc race 1 resistance Five lines resistant to Foc race 4 Foc race 1 resistance

Target gene/ transformed gene MSI-99

Genotype Rasthali (AAB)

–

Dwarf Parfitt (AAA)

GmEg

Rasthali (AAB)

Bcl-xL, Ced-9, Bcl-23’ UTR PhDef1, PhDef2

Lady Finger (AAB) Rasthali (AAB)

References Chakrabarti et al. (2003) Smith et al. (2006) Maziah et al. (2007) Paul et al. (2011) Ghag et al. (2012) Chen et al. (2013)

–

Brazil banana (AAA) Rasthali (AAB)

Ghag et al. 2014b

Rasthali (AAB)

TR4 resistance

VELsen-IntVELas, FTF1sen-IntFTF1as Ace-AMP1 + Ca-pflp Human lysozyme Atfd3, Ca-pflp

TR4 resistance

OsTLP

TR4 resistance

ThChit42

Pei Chiao (AAA) or Gros Michel (AAA) Pisang Nangka (AAB) Furenzhi (AA)

TR4 resistance

MaLYK1

Williams (AAA)

TR4 resistance

RGA2, Ced-9

Grand Nain (AAA)

TR4 resistance

ERG6, ERG11

Cavendish (AAA)

Foc race 1 resistance Foc race 1resistance

Endo β-1-3glucanase Antimicrobial peptide (Ace-AMP1) mCED9a synthetic peptide

M. spp. cv. Rasthali

Sunisha et al. (2020) Pei et al. (2005) Yip et al. (2011) Mahdavi et al. (2012) Hu et al. 2013 Zhang et al. (2019) Dale et al. (2017) Dou et al. (2020) Maziah et al. (2007) Mohandas et al. (2013)

Taijiao (AAA)

M. spp. cv. Rasthali

M. spp. cv. SukaliNdiizi

Magambo et al. (2016)

Host-induced gene silencing (HIGS) is the most effective technology to generate crops resistant to different pathogens by silencing the genes specific to pathogens (Qi et al. 2018;Zhang et al. 2016). Selection of an appropriate target gene is

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important for the success of this technique. The constructs designed for siRNA to target ftf1 (Fusarium transcription factor 1) and the Foc velvet genes developed increased resistance in banana plants to Foc race 1 (Ghag et al. 2014b). Similarly, silencing of genes involved in ergosterol biosynthesis (ERG6 and ERG11) enhanced the resistance to Foc race 4 in Cavendish banana (Dou et al. 2020). CRISPR/Cas 9 technology is a promising technology that can be exploited for Fusarium wilt resistance. Potential targets for editing to get silencing or overexpression of the targeted gene need to be analyzed and confirmed. Some of the potential candidate genes that can be edited to generate Fusarium wilt-resistant banana are listed in Table 15.2. Black Sigatoka causes reduced green canopy and premature ripening and defects in fruits. Control using chemical pesticides is likely to develop disease resistance and environmental and safety issues in the banana-growing regions (Avenot and Michailides2010). Transgenic approaches have been adopted to develop resistance toward Black Sigatoka. Transgenic lines were developed by expressing endochitinase gene (ThEn-42) from Trichoderma and stilbene synthase (StSy) gene from grapes. To the gene cassette, superoxide dismutase gene (Cu,Zn-SOD) from tomato was also added to improve ROS scavenging activity (Vishnevetsky et al. (2011). Four years of field trial generated several transgenic banana lines with improved tolerance to Sigatoka.

15.3.4 Bacterial Disease Resistance The major bacterial diseases affecting banana include banana Xanthomonas wilt (BXW, caused by Xanthomonas campestris pathovar (pv). Musacearum), Moko disease (caused by Ralstonia solanacearum), and blood disease (caused by Ralstonia syzygii subspecies Celebesensis). BXW is the most economically crucial bacterial disease affecting banana production that is mainly transmitted by infected planting materials, insects, and contaminated tools (Shimwela et al. 2017). In severe cases, huge yield loss occurs due to complete wilting of the plant and rotting of fruits. Though it can be contained by following proper sanitary practices and using pathogen-free planting materials, sanitary techniques are labor intensive. Since no naturally resistant source has been identified in banana germplasm, genetic engineering provides an alternative technique to develop BXW-resistant banana varieties. It is mostly done by expressing resistance (R) genes, antimicrobial genes, or defense genes to enhance the host resistance. Some of the selected genes include the defense genes such as hypersensitive-response-assisting protein (Hrap) and plant ferredoxin-like protein (Pflp) genes from sweet pepper to enhance the hypersensitive response (Lin et al. 1997; Chen et al. 2000). Transgenic lines of “SukaliNdiizi” (AAB cv.), and “Nakinyika” (AAA-EAHB cv.) expressing Hrap or Pflp gene showed increased resistance to BXW (Namukwaya et al. 2012; Tripathi et al. 2010). In a confined field trial, out of 65 transgenic events (40 Hrap gene lines and 25 Pflp gene lines), 11 transgenic events showed complete BXW resistance. Stacking of these genes (Hrap-Pflp) did not show any difference in the resistance

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Table 15.3 Summary of potential target genes that could be manipulated in bananas to develop resistance against bacterial pathogens S. no. 1

Mode of action Susceptibility genes

2

Hypersensitivity response

3

4

Pathogen recognition receptors-induced immunity Resistance proteins

5

Defense signaling

Target gene MusaDMR6 SlDMR6, OsSWEET14 OsSWEET11, OsSWEET13 Hrap Pflp Stacked Hrap and Pflp Xa 21

RPM1

NPR 1 and PR 1

Type of manipulation Gene knock out

References Tripathi et al. (2021); Thomazella et al. (2021); Li et al. (2012)

Overexpression

Tripathi et al. (2014)

Overexpression

Tripathi et al. (2014)

Overexpression, CRIPSR activation Overexpression, CRIPSR activation

Tripathi et al. (2019)

Xu et al. (2017)

when compared with individual genes. It is expected that stacking of these genes can provide durable resistance. Expression of rice Xa21 gene in the transgenic lines showed resistance to BXW disease (Tripathi et al. 2014). Mildew resistance 6 (DMR6) ortholog in bananas when knocked down using CRISPR/Cas9 system increased the resistance to BXW (Tripathi et al. 2021). Table 15.3 summarizes the potential target genes to manipulate for resistance to bacterial diseases.

15.3.5 Nematode Resistance The important species of nematodes that cause a serious infestation in the banana fields are Radophilus similis, Meloidogyne spp., and Helicotylenchus. These nematodes are associated with the roots and cause severe yield reduction in banana and can cause loss of up to 50% and increase susceptibility to other diseases and pests. As the usual control measures involve toxic chemicals, genetic engineering will be a better alternative considering the safety of environment and banana growers (Table 15.4). Host-induced gene silencing had been successful against nematode resistance by targeting nematode-specific genes required for growth and development (Fairbairn et al. 2007). Successful nematode resistance was obtained in transgenic banana plants expressing the maize or rice cystatin (Atkinson et al. 2004;Roderick et al. 2012) gene that inhibits the activity of cysteine proteinases, thereby suppressing

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Table 15.4 List of strategies adopted to control banana nematodes System Transgenic Transgenic

RNAi RNAi

Resistance to Radopholus similis Radopholus similis

Target gene/ transformed gene Cystatin (OC-lΔD86) Cystatin (CCII)

Radopholus similis All nematodes

Cathepsin B Proteasomal alpha subunit 4 and actin-4

Genotype M. spp. cv. Grand Naine M. spp. cv. GonjaManjaya

References Atkinson et al. (2004) Roderick et al. (2012); Tripathi et al. (2015) Li et al. (2015) Roderick et al. (2018)

nematode growth and reproduction. Expressing these inhibitors in transgenic banana plants offered resistance to nematodes under confined field conditions (Tripathi et al. 2015). In another approach, the nematodes were targeted using the proteasomal alpha subunit 4 and actin4. Here the plants will secrete a peptide that disrupts chemosensory function in nematodes by interfering with enzymatic cleavage of the neurotransmitter nicotinic acetylcholine receptors (Wang et al. 2011). Based on these studies, banana cv. GonjaManjaya (Musa AAB) was transformed with maize kernel cystatin or chemoreception-disrupting peptide (nAChRbp). Ten transgenic lines challenged with nematode (single species and mixed population) showed significant resistance (Roderick et al. 2012). RNAi is also deployed to get nematode resistance by silencing the cathepsin B (Rs-cb-1) in the nematode that produced reduced hatching and pathogenicity (Li et al. 2015).

15.4

Genetic Improvement for Abiotic Stress Tolerance

One of the crucial challenges that current agriculture faces worldwide is the change in the climate. The drastic change in rainfall, heat, and cold may seriously affect plant growth, yield, and quality of the output. In this challenging scenario, crops such as banana have significant importance as they are crops of food security and can grow in marginal nutrient and climatic conditions. Mostly yield is reduced by abiotic stress. Conventional breeding technology is not successful in bananas for developing abiotic stress-tolerant varieties due to parthenocarpy and polyploidy. Successful results were obtained by using biotechnological tools such as in vitro techniques, in vitro mutations, and transgenic and genome editing.

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15.4.1 Cold Stress Temperatures below 12 °C are critical for banana, and the damages caused by temperatures below 8 °C are irreparable (Santos et al. 2009). Cold induces alterations in membrane shape and fluidity, and also protein denaturation and formation of ROS. Plants sense changes in the environment by signal transduction, and mitogenactivated protein kinases (MAPKs) play an essential role (Hamel et al. 2006) in responding to abiotic stress in plants. Transgenic approaches were tried to develop cold-tolerant banana lines by introducing MAPK coding genes. Transgenic lines overexpressing MusaMPK5 performed better at temperatures of 4 °C and 8 °C. The tolerance was supported by the increased level of proline and reduced level of MDA (Taket al. 2020). When MaMAPK3 in Musa spp. “Dajiao” (ABB Group) was silenced via RNAi, plants exhibited wilting and severe necrotic symptoms in leaves. The expression of the cold-responsive gene MaICE1 was significantly decreased. In Cavendish banana, overexpression of MaICE1 increased cold resistance significantly in transgenic plants (Gao et al. 2021). Aquaporins (AQPs) are important in conferring abiotic stress responses in plants. They increase the permeability of the membranes to small molecules. The banana plants that overexpressed the aquaporin gene MaPIP2-7 showed better resistance to salt, cold, and drought. Under stress and recovery circumstances, these transgenics showed reduced levels of malondialdehyde and ion leakage compared to wild-type plants and increased quantities of abscisic acid, chlorophyll, proline, and soluble sugar (Xu et al. 2014).

15.4.2 Heat, Drought, and Salinity Bananas often face heat and drought stress or sometimes a combined stress during growth as they have to pass through the different seasons due to long duration. Rainfall below 1100 mm per annum can lead to around 65% yield loss (Van Asten et al. 2011). Temperature stress leads to poor plant growth and also affects the quality and postharvest life of the fruit. B genomes contribute to abiotic stress, but most of the cultivated and preferred ones have A genome. In vitro selection method has been adopted by many research groups for inducing drought stress tolerance by treating banana cultures with trehalose and also by screening the improved mutated lines (using chemical mutagens) using PEG (Said et al. 2015, Bidabadi et al. 2012). Drought tolerance is a multigenic trait that makes the genetic improvement programs difficult. Long breeding cycles and co-inheritance of undesirable traits make the conventional breeding less attractive. Transgenic approaches have been found useful in developing drought tolerance in banana. It has been possible due to the discovery of many candidate genes conferring resistance to drought in many crops. Structural genes for the biosynthesis of detoxifying enzymes and osmolytes and regulatory genes encoding transcription factors are important. Banana plants tolerant toward drought and salinity stress have

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been developed by overexpressing WRKY, NAC, and MYB transcription factors, late embryogenesis abundant (LEA) proteins, aquaporin PR proteins, etc. (Sreedharan et al. 2015; Shekhawat and Ganapathi 2013; Rustagi et al. 2015; Dou et al. 2016). ABA levels increase under water stress and result in stomatal closure, thereby reducing water loss through transpiration from leaves. The transcription factors DREB1 and DREB2 are important in the ABA-independent drought tolerance. DREB genes are important targets for genetic engineering because of their functional conservation. MaDREB1F overexpression enhanced resistance against drought in banana by activating ethylene and jasmonate synthesis (Xu et al. 2023). Overexpression of MusaSNAC1 showed higher level of stomatal closure in banana by increasing H2O2 content in guard cells, leading to drought tolerance (Negi et al. 2018). Similarly, overexpression of MusaNAC042 in cv. Rasthali increased drought and salinity stress (Tak et al. 2017). Transgenic plants (banana cv. Matti (AA)) overexpressing salinity-induced pathogenesis-related class 10 protein gene from Arachis hypogaea showed tolerance toward NaCl and mannitol with less membrane damage and also exhibited better photosynthetic efficiency (Rustagi et al. 2015). microRNAs play significant roles in stress responses in plants. These noncoding RNAs are involved in the post-transcriptional regulation of gene expression. Only a few miRNAs have been identified in banana that have a prospective role in stress tolerance (Bhakta et al. 2021). Further studies can develop miRNA-mediated approaches to improving banana. Table 15.5 summarizes the strategies developed for abiotic resistance in banana.

15.4.3 Fruit Quality and Shelf Life Banana is a staple food in several developing countries. Though it contains carbohydrates, nutrients, and minerals, it is deficient in vitamin A, protein, and iron. So, people relying on banana as staple food show deficiency symptoms of these nutrients. Biofortification of bananas is important in this respect. Considering the vitamin A and iron deficiency in the children and young population in African countries, several projects have been initiated for the biofortification of bananas to increase the content of iron and vitamin A. Biofortification is a cost-effective and sustainable method to deliver nutrient-rich food to the population to tackle the nutrient deficiencies. Many research groups have initiated projects on the biofortification of banana, and the first report of successful result was that of Kumar et al. (2011). They introduced soybean ferritin gene into cv. Rasthali (AAB) that resulted in increased levels of iron (6.32-fold) and zinc (4.58-fold). Yadav et al. (2017) used MusaFer1 gene to increase the iron content in cv. Rasthali and proposed it as a candidate gene for the biofortification in banana. Banana-21 project was initiated in 2005 for the biofortification of banana for vitamin A by transgenic approach by overexpressing pro-vit A genes and gene

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Table 15.5 Summary of the strategies for abiotic resistance in Musa species System Genetic transformation Tissue culture Tissue culture Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation Genetic transformation

Trait Drought tolerance Drought Water stress Drought tolerance Oxidative and salinity tolerance Drought tolerance Drought tolerance Drought tolerance Drought tolerance Drought tolerance Cold tolerance Cold tolerance Salt Salt Drought and salinity

Genotype Rasthali (AAB) Gran Nain Berangan (AAA) Rasthali (AAB) Rasthali (AAB) Rasthali (AAB) Rasthali (AAB) Rasthali (AAB) Rasthali (AAB) Gongjiao (AA) Dajiao (ABB) Musa spp. (AAA) Gran Nain Grand Nain Matti (AA)

Gene/ reagents MaDHN-1

MaSNAC1

References Shekhawat et al. (2011) Said et al.(2015) Mahmood et al. (2012) Sreedharan et al. (2012) Shekhawat and Ganapathi (2013) Negi et al. (2018)

MaNAC042

Tak et al. (2017)

MaPIP1;2

Sreedharan et al. (2013) Sreedharan et al. (2015) Xu et al. (2020)

PEG Methyl jasmonate MaSAP1 MaWRKY71

MaPIP2;6 MaPIP2;7 RNAi of MaMAPK3 MaICE1

Gao et al. (2021)

Tps-tpp P5CS

Santamaría et al. (2009) Ismail et al. (2005)

PR10

Rustagiet al. (2015)

Gao et al. (2021)

stacking (Paul et al. 2017, 2018). Cavendish banana (Paul et al. 2017) inserted with the Asupina-derived banana phytoene synthase gene (MtPsy2a) showed enhanced pro-vit A content in field trials in Australia. Transgenic lines with fruit pVAC produced up to 55.0 μg/g two times more than the target level. East African Highland banana cultivar was also genetically modified for vitamin A enrichment (Paul et al. 2018). Recently, using CRISPR/Cas9 technology, the carotenoid cleavage dioxygenase4 (CCD4) gene was found to regulate carotenoid accumulation in banana by using protoplasts transfection (Awasthi et al. 2022). Banana fruits are usually harvested before fully mature for consumption. The fruits are easily deteriorated during storage due to the quick ripening process (Marriott and Palmer 1980;Golding and Shearer 1998). Profit for the banana growers depends on the conservation of the fruit after harvest. It is only possible by

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manipulating the normal postharvest physiology of the fruit and controlling the postharvest disease. The roles of different genes in ethylene biosynthesis and climacteric respiration in fruits have been studied. Transgenic banana plants repressing MaMADS1 and MaMADS2 genes using RNAi showed reduced ethylene synthesis and delayed ripening (Elitzur et al. 2016). They also showed delayed color development and softening, the characteristics contributing to extended shelf life. The fruits of the repression lines responded to exogenous ethylene application and ripened normally. Using the CRISPR/Cas9 system, MaACO1 gene responsible for ethylene production was disrupted in banana plants to improve shelf life. Under natural ripening conditions, the mutant fruits showed a longer shelf life and reduced ethylene synthesis. MaACO1-disrupted fruits were susceptible to ethephon and ripened normally when treated with ethephon (Hu et al. 2021).

15.4.4 Genetic Alterations to Improve Plant Architecture The ideal plant architecture of desirable banana varieties for high-density planting should prevent lodging, facilitate optimal photosynthesis, and efficient water absorption (Dash and Rai 2016). Increased secondary wall deposition can reduce lodging (Velasquez et al. 2010). Secondary wall deposition is tightly controlled by the transcription factor NAC, and overexpression of proteins containing NAC domain (MaVND1, MaVND2, and MaVND3) showed transdifferentiation of different cells into xylem vessel components and deposition of lignin (Negi et al., 2015). Genes involved in the lignin and cellulose biosynthesis pathway were overexpressed in transgenic banana. Functional characterization of the NAC family genes may reveal new ways to develop improved lodging resistance in banana (Negi et al., 2015, 2016). Giberrilins control plant height and mutations in the genes involved in GA biosynthesis and signaling pathway lead to dwarf phenotypes. By using EMS-induced mutagenesis in FJ cultivar, Wang et al. (2021) developed a stably inherited mutant called “ReFen1” (RF1), a semi-dwarf phenotype. These mutant plants showed improved agronomic traits during 5-year multilocation trials in China. The RF1 plants showed significantly enhanced cold tolerance and Sigatoka disease resistance, mainly due to a substantially increased soluble content of sugar and greater starch accumulation, along with reduced cellulose deposition. Semi-dwarf mutants in “Gros Michel” were successfully produced after editing the MaGA20ox2 genes using the CRISPR/Cas9 system (Shao et al. 2020).

15.5

Biofarming

Genetic transformation of banana plants for molecular pharming has been attempted in recent years (Tak et al. 2016). Molecular farming is the production of pharmaceutically important products such as antibodies, hormones, and vaccines

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using plants (Schillberg and Finnern 2021). Banana is an ideal host for the production of recombinant proteins because of several factors. It is available year-round and is easily digestible and palatable by infants. It is cultivated in many developing countries, and genetic transformation protocols are available. Due to their vegetative propagation, transgene containment is possible (Kumar et al. 2004;Ganapathi et al. 2001). The embryogenic cells of banana were successfully transformed with hepatitis B surface antigen (HBsAg) for producing edible vaccines against hepatitis B (Kumar et al. 2005). Expression of HBsAg was higher under in vitro conditions with EFE promoter (38 ng/g F.W.) and under greenhouse conditions (19.92 ng/g F.W.) with ubq3 promoter (Kumar et al. 2005). The HBsAg extracted from the leaves were almost similar to the HBsAg from human serum. Higher level of recombinant protein in fruit tissue can be achieved by employing promoter of abundant fruitspecific proteins. Banana can provide a suitable platform for the large-scale production of a range of pharmaceuticals. A limitation of biopharming using bananas is the low protein content in the fruits that demands advanced research.

15.6

Biosafety Aspects of Genetically Modified Banana

Genetic modification of crops is always a subject of debate globally. The biosafety regulations differ among countries ranging from prohibitions and moratorium on GM crops to regulations that treat genetically modified crops similar to the conventional plants. India follows a stringent three-tier system of regulation of genetically modified organisms. Because of the resistance from the public and environmental activists, many of the transgenics developed so far are still in pipeline. In the recent amendments, the genome-edited plants belonging to the categories SDN1 and SDN2 are exempted from the regulations. Countries such as Argentina, Australia, Brazil, Canada, Chile, Colombia, Japan, Israel, and the United States also follow the same regulations (Schmidt et al. 2020;Tripathi et al. 2020). But if any T-DNA portions are inserted into their genome, they have to undergo the normal biosafety regulations as it can be segregated out by crossing. The European Union and New Zealand consider genome-edited crops as other genetically modified crops and are under the existing biosafety regulations (Schmidt et al. 2020).

15.7

Challenges and Future Prospects

The genetic improvement in banana through techniques such as in vitro mutation, genetic engineering, and genome editing helped us develop many potential varieties with beneficial agronomic traits. These programs also elucidated valuable information on molecular mechanisms behind biotic and abiotic stress resistance. Despite these efforts, global banana cultivation and production are still facing challenges such as changes in climate, slow commercial approval of transgenic bananas due to

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regulatory issues, and new races of bacterial and viral strains that can break the resistance of already developed varieties. The majority of the transgenic developed for different improved traits are greenhouse restricted and have not undergone field trials. Hence, immediate attention should be given to these challenges. Among the currently available tools, genome editing is the most promising for developing commercial banana varieties having biotic and abiotic stress resistance, improved shelf life, and biofortified nutrient content. Since this tool enables precise editing of the genome with no footprints and off-targets, the edited lines can evade regulatory constraints. But targeting multiple alleles and gene copies in mostly cultivated triploid banana by genome editing is challenging. Use of multiple gRNAs in editing can be a solution to solve this problem (Ansari et al. 2020). Mostly Agrobacterium-mediated CRISPR/Cas editing is done in bananas, which will leave T-DNA in the mutants. Hence, they need to undergo the biosafety regulations, and to overcome this problem, there is a need for transgene-free genome-editing system in banana. Direct delivery of preassembled RNPs into regenerative cells through bombardment can be adopted in banana (Tripathi et al. 2020). Currently to screen the edited lines for mutations, PCR and sequencing-based methods are used, which are costly. Cost-effective screening methods need to be developed (Awasthi et al. 2022;Ntui et al. 2020). Damages caused by viruses, fungi, and bacteria still pose major problems in banana cultivation. Hence, to sustain banana cultivation in the future, the development of new elite banana varieties is required. Newer genomics approaches may be utilized for identifying and validating genes to facilitate genome-editing techniques. Attention is to be given to designing climate-resilient crops for the future.

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Sunisha C, Sowmya HD, Usharani TR, Umesha M, Gopalkrishna HR, Saxena A (2020) Deployment of stacked antimicrobial genes in banana for stable tolerance against Fusarium oxysporum f. sp. cubense through genetic transformation. Mol Biotechnol 62:8–17. https://doi.org/10.1007/ s12033-019-00219-w Tak H, Negi S, Ganapathi TR, Bapat VA (2016) Molecular farming: prospects and limitation. In: Mohandas S, Ravishankar KV (eds) Banana: genomics and transgenic approaches for genetic improvement. Springer, Cham, pp 261–275 Tak H, Negi S, Ganapathi TR (2017) Banana NAC transcription factor MusaNAC042 is positively associated with drought and salinity tolerance. Protoplasma 254:803–816 Tak H, Negi S, Rajpurohit YS, Misra HS, Ganapathi TR (2020) MusaMPK5, a mitogen activated protein kinase is involved in regulation of cold tolerance in banana. Plant PhysiolBiochem 146: 112–123 Thangavelu R, Gopi M, Pushpakanth P, Loganathan M, Edwin Raj E, Marimuthu N, Prabakaran M, Uma S (2021) First Report of Fusarium oxysporum f. sp. cubense VCG 0125 and VCG 01220 of Race 1 Infecting Cavendish Bananas (Musa sp. AAA) in India. Plant Dis 105:1215. https:// doi.org/10.1094/PDIS-09-20-2052-PDN Thomazella DP, Seong K, Mackelprang R, Dahlbeck D, Geng Y, Gill US, Qi T, Pham J, Giuseppe P, Lee CY, Ortega A (2021) Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance. Proc Natl Acad Sci 118(27):e2026152118. https://doi.org/ 10.1073/pnas.2026152118 Tripathi L, Mwaka H, Tripathi JN, Tushemereirwe WK (2010) Expression of sweet pepper Hrap gene in banana enhances resistance to Xanthomonas campestris pv. musacearum. Mol. Plant Pathol 11(6):721–731 Tripathi JN, Lorenzen J, Bahar O, Ronald P, Tripathi L (2014) Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum. Plant Biotechnol J 12:663–673 Tripathi L, Babirye A, Roderick H, Tripathi JN, Changa C, Urwin PE, Tushemereirwe WK, Coyne D, Atkinson HJ (2015) Field resistance of transgenic plantain to nematodes has potential for future African food security. Sci Rep 5:8127 Tripathi JN, Ntui VO, Ron M et al (2019) CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding. Commun Biol 2:46. https://doi.org/10.1038/s42003-019-0288-7 Tripathi L, Ntui VO, Tripathi JN (2020) CRISPR/Cas9-based genome editing of banana for disease resistance. Curr Opin Plant Biol 56:118–126 Tripathi JN, Ntui VO, Shah T, Tripathi L (2021) CRISPR/Cas9-mediated editing of DMR6 orthologue in banana (Musa spp.) confers enhanced resistance to bacterial disease. Plant Biotechnol J 19(7):1291–2193 Van Asten PJ, Fermont AM, Taulya G (2011) Drought is a major yield loss factor for rainfed East African highland banana. Agric Water Manage 98:541–552 Van Harten AM (1998) Mutation breeding theory and practical applications. Cambridge University Press, Cambridge, p 353 Velasquez AHI, Ruiz CAA, Junior SDO (2010) Ethanol production process from banana fruit and its lignocellulosic residues: energy analysis. Energy 35:3081–3087 Vishnevetsky J, White TL, Palmateer AJ, Flaishman M, Cohen Y, Elad Y, Velcheva M, Hanania U, Sahar N, Dgani O, Perl A (2011) Improved tolerance toward fungal diseases in transgenic Cavendish banana (Musa spp. AAA group) cv. Grand Nain. Transgenic Res 20:61–72. https:// doi.org/10.1007/s11248-010-9392-7 Wang D, Jones LM, Urwin PE, Atkinson HJ (2011) A synthetic peptide shows retro-and anterograde neuronal transport before disrupting the chemo sensation of plant-pathogenic nematodes. PLoS One 6:e17475. https://doi.org/10.1371/journal.pone.0017475 Wang Z, Zhang J, Jia C, Liu J, Li Y, Yin X, Xu B, Jin Z (2012) De novo characterization of the banana root transcriptome and analysis of gene expression under Fusarium oxysporum f. sp.

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Genetic Improvement of Mustard

16

Khadija Mika Dawud, Chongtham Allaylay Devi, and Ashutosh K. Pandey

16.1

Introduction

Oilseed crops come from a variety of species in the kingdom Plantae. Soybean and groundnut are legumes from the Fabaceae (Leguminosae) species that were cultivated in the United States for centuries (Nikolaou et al. 2022). Behind the United States, Brazil, and China, India scores the fourth largest oilseed production. India is the fourth largest edible oil economy in the world after the United States, Brazil, and China. India occupies a distinct position not only in terms of area under oilseed crops but also in terms of diversity in cultivated oilseeds. India accounts for 19% to feed over 16.1 of the world’s population. India has 19% of land, and its production is 9% of the total production worldwide (Velasco et al. 2021). Nine oilseed crops, viz., groundnut, rapeseed–mustard, soybean, sesame, linseed, castor, safflower, sunflower, and Niger, are cultivated in India and comprise the second largest commodity after cereal, sharing 14% of the gross cropped area, accounting for 5% of the gross national product and 10% of the value of the agricultural products (Akinyele and Shokunbi 2015). Besides oilseeds, crops play a major role in relieving the malnutrition and calorie nutritional problem of human beings and livestock due to high protein and mineral content. In Asia, oleiferous brassicas (including rapeseed and mustard) are major food crops (Sneha et al. 2018). Oilseeds are India’s second largest agricultural product, with oleiferous brassicas providing the most edible oil to a substantial section of the population among the nine annual oilseed crops farmed (Long et al. 2007). In K. M. Dawud · A. K. Pandey (✉) School of Agricultural Science, Sharda University, Greater Noida, Uttar Pradesh, India e-mail: [email protected] C. A. Devi School of Agriculture, Galgotias University, Greater Noida, Uttar Pradesh, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_16

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addition, India accounts for roughly 30% of global acreage and 20% of global production of oilseed brassicas, respectively. It is one of the best edible oils available, with the lowest level of saturated fats compared to certain other vegetable oils, and supplies both vital fatty acids and animal feed in the form of an oil-free meal rich in protein with a well-balanced amino gram (Chahal et al. 2020). Brassica species produced as oilseeds include rapeseed and mustard. For ages, this has been one of the most important sources of food (Wu et al. 2021). Under climate change scenarios, increased frequency of severe occurrences and, consequently, more harmful effects on the yield of oleiferous brassica are projected (Khatoon 2017). Agricultural sustainability has a significant problem in improving crop output and nutritional value while preserving soil quality, particularly the soil carbon pool. The physical, chemical, and biological qualities of soil are declining worldwide as a result of intensive farming techniques based on high agricultural inputs (widespread use of synthetic pesticides and fertilizers). According to estimates, leaching, drainage, and volatilization remove about 75% of phosphatic and nitrogen fertilizers from the soil (Diepenbrock 2000). Further inefficient fertilizers usages combined with incorrect method of application have led to extensive pollution problems in air, water, and soil (Jangir et al. 2018). The transition to sustainable agriculture must thus be made as soon as possible, while minimizing tradeoffs, by adopting appropriate techniques such as appropriate soil amendments, vermicomposting, green manure, farmyard manure, and rice straw for increasing crop yield and soil fertility (Jangir et al. 2018). Mustard in India is mostly grown in northern Indian states, including Rajasthan, Uttar Pradesh, Madhya Pradesh, Gujarat, and Haryana. Conventional breeding techniques have not been very successful in transferring polygenic characteristics for coping with abiotic stressors to date (Nanjundan et al. 2020). The output of crops must rise proportionally to the daily rise in world population. Brassica crops have been exposed to a wide range of stresses such as any other agricultural crop. The plant may adapt to abiotic stressors by raising levels of photoprotective enzymes and stress metabolites, as well as by changing the shape, size, and thickness of its leaves, the density and distribution of its stomata, and the structure and function of its chloroplasts. Depending on the nature of the plant, these sorts of adaptations may occur within a few days or a week. Around the world, illnesses and insect infestation cause a 25% reduction in crop productivity. Abiotic stress, which reduces average yields for the majority of major crop plants by more than 50%, is the main cause of crop loss globally (add reference). More specifically, by the year 2050, severe salinization of more than 50% of all arable lands has been reported from the devastating effects of drought and salinity in many parts of the world (Rizwan et al. 2018). In recent years, research has focused mostly on how plants react to biotic and abiotic stresses. In the field condition, plants have to face more than one stress simultaneously, and the response cannot be predicted based on the plant’s response to the single stress. According to the ambient environment and the stage of the plant’s growth, plants can exhibit various levels of sensitivity (Niwas and Khichar 2016). Whether biotic and abiotic pressures are more antagonistic, synergistic, or

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additive, increasing or decreasing vulnerability to a particular type of stress is unclear. Most of the time, being exposed to various stresses can also cause the plants to react in an antagonistic way. For instance, when drought stress is present, common beans that have been infected with Macrophomina phaseolina exhibit more symptoms, and applying abscisic acid (ABA) to tomato leaves makes them more susceptible to Botrytis cinerea (Masaka et al. 2021). For agricultural crops, there are two major strategies to deal with environmental stress: management and biotechnological methods. The consequences of abiotic stress can be controlled by agronomic techniques, including irrigation with clean water, soil improvement, and adjusting sowing times.

16.2

Effects of Abiotic Stresses on Vegetables Crops

It is anticipated that the concept toward global warming will continue and that variations in temperature, precipitation, and carbon dioxide will have an impact on how plants grow and develop, how pests and diseases spread, and how the climate affects the production of vegetable crops. Abiotic stressors cause a greater than 50% reduction in vegetable crop output on a global scale. The degree of abiotic stress placed on vegetable crops will vary depending on climate changes (Sneha et al. 2018). There are numerous commercially significant crops, including B. rapa, B. oleracea, B. napus, and others, among the important and diversified vegetable crops known as brassicas. About 85% of the water in brassica veggies makes them soft and succulent. As a result, both inadequate and excessive water use has a substantial impact on the production and quality of these crops. Consequently, we require a long-term remedy to address the stressors that affect vegetable production. Plants have an in-built mechanism for the growth with variable stress tolerance. Plants under different biotic and abiotic stresses upregulate their defensive mechanisms by differential expression of genes to adopt to stresses (Yadav et al. 2014). However, when plants are exposed to stressors, only specific number of genes becomes active. Researchers have exploited these genes to create crop types that can withstand stress throughout their life cycle, Brassica crops encounter many pressures, and they attempt to resist those challenges by adapting to or creating various mechanisms to combat. For Brassica vegetable crops; cold stress is one of the most damaging stress. For example,when plants are exposed to cold stress, the water in their bodies freezes and bursts their cells, resulting in large yield losses and serious dangers to the future of the world food supply (Lal et al. 2019). When it is administered during the seedling and reproductive phases, cold stress is significantly more damaging. The osmotic movement of water out of plant cells is brought on by drought stress, which raises the concentration of solutes in the soil. Crop output will be significantly impacted by severe drought stress conditions (Yahaya et al. 2018). Most vegetable crops, especially during blooming and seed development stages, are vulnerable to drought stress. The problem of salinity for vegetable crops is comparable to that of drought. According to studies, rising salinity will have an impact on 17% and 30%,

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respectively, of the world’s cultivated fields and irrigated agricultural land (MartínHernández et al. 2008). As a result of the high rates of evapotranspiration and hot, dry climates in tropical regions, significant amounts of water are lost, producing salty deposits around the roots of the plants that prevent them from absorbing water. Under salt stress, a plant’s symptoms include loss of turgor, reduced fertility, wilting, leaf curling, leaf abscission, altered respiration, loss of cellular integrity, tissue necrosis, and perhaps death may occur. During the seedling and early developing phases, most vegetable crops, especially Brassica crops, are vulnerable to salt stress (Khattab et al. 2017). As a result, it is widely known that the duration and intensity of the stress, as well as the stage at which the plant is growing, all influence how plants respond to abiotic stressors.

16.3

Types of Abiotic Stresses and Strategies for Reducing Their Impact

16.3.1 Soil pH The acidity and alkalinity of soils are gauged by the pH of the soil. The pH scale goes from 0 to 14, with 7 representing neutrality, less than 7 indicating acidity, and more than 7 indicating alkalinity (Mourato et al. 2015). Though several plants may grow at pH levels that are outside the recommended range of 5.5–7.0, this range is where the majority of them do best. The physical, chemical, and biological characteristics and functions of the soil, as well as plant development, are all impacted by the pH of the soil. It is crucial to maintain the right pH level to maximize a plant’s yield potential since pH levels regulate various chemical reactions that occur in the soil, notably, the availability of plant nutrients (Kaur and Sardana 2018). Depending on the kind of soil, different additions are used to maintain the pH level that the plant requires.

16.3.2 The Acidity of Soil One of the biggest variables affecting crop output is the acidity of the soil. In theory, soil hydrogen (H+) and aluminum (Al3+) concentrations may be used to measure how acidic soil is. Toxicities and inadequacies of minerals and elements, poor activity of helpful microbes, and decreased plant root development that reduces nutrient and water uptake are just a few of the many reasons that make soil acidity a problematic issue for crop productivity. Acid soils are also susceptible to compaction and water erosion and have a limited capacity to retain water (Arifullah 2011). When hydrogen ions replace basic elements retained by soil colloids, such as calcium, magnesium, sodium, and potassium, soils turn acidic. When compared to soils created under more dry circumstances, soils created under conditions of significant annual rainfall are more acidic. As a result, compared to soils in the Midwest and the Far West, the majority of southern soils are naturally more acidic. Because nutrient transformations and nitrogen fixation are restricted in acidic soils due to decreased

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activity of the microorganisms responsible for carrying out these processes, plantavailable forms of N, S, and P are reduced in acidic soils, as well as in symbiotic N fixation by leguminous crops (Wang et al. 2012). The availability of the trace element molybdenum as well as the key plant nutrients nitrogen, phosphorus, potassium, sulfur, calcium, and magnesium is decreased in acidic soils. These soils’ pH can be raised by using lime, which keeps these nutrients as readily available as possible.

16.4

Factors Affecting Soil Acidity

16.4.1 NPK Fertilizers The release of organic acids during the breakdown of crop residues and the addition of organic wastes lower the pH of the soil, which causes soil acidity. Long-term use of ammonium fertilizers, removal of cations in the harvested portion of crops, leaching, and release of organic acids also contribute (Ayerza and Coates 2011). For increased agricultural yields throughout all ecosystems, it is essential to use nitrogen fertilizer in sufficient quantities. The two main nitrogen carriers utilized for crop production worldwide are urea and ammonium sulfate. Nitrosomonas and Nitrobacter are the two most significant autotrophic bacterial genera (Rai et al. 2016). Legumes may make the soil more acidic when grown constantly or in a rotation with other crops.

16.4.2 Amendments to Reduce Soil Acidity Liming, because the Fe and Al soil components (sesquioxides) fix a significant amount of P, soil acidity has a detrimental impact on crops mostly through P unavailability from P fixing in soils (Johnston et al. 2002). As indicated by the suppression of root extension and generally slowed crop development, excess Al3+ ions from acidic soil tend to collect in plant roots and obstruct P, Mo, and other ions from moving from the roots to the tops. Al3+ cations have a more pronounced negative impact than H+ ions, although an excess of H+ ions in acid soils reduces the permeability of plant root membranes, which prevents ions from moving through the soil. To promote the translocation of P, Cu, Mn, and other nutritional ions, lime is applied to the soil, which raises the pH, while also reducing the exchangeable Al and accessible Fe and Zn ions (Johnson et al. 2013). Despite the pH profile of the soil being comparable to the unlimed control, the increase in Brassica crops was documented utilizing a 1-tonne/ha lime treatment. Since it was found that the unlimed treatment had the highest number of weeds, the 1-tonne/ha lime treatment may reduce weed competition for water and nutrients.

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16.4.3 Soil Alkalinity The term “alkaline soil” refers to soils with a pH of more than 7. Alkaline soils are occasionally described as sweet. Alkaline soils, also known as alkali soils, are clay soils with a high pH (>8.5), weak soil structure, and little ability for infiltration (Hossain et al. 2019). At a depth of 0.5–1 m, they frequently contain a hard calcareous layer. The overwhelming presence of sodium carbonate, which makes the soil expand and difficult to settle, is the primary reason for the negative physical and chemical characteristics of alkali soils. Sodium is a member of the family of elements known as alkali metals and may cause basicity (Bansil 1997).

16.4.4 Causes of Soil Alkalinity Both natural and man-made factors contribute to soil alkalinity. Since soil minerals produce sodium carbonate when they weather, this natural development is caused by their existence (Chahal et al. 2020). The use of irrigation water surface or groundwater that contains a disproportionately high amount of sodium bicarbonates and the repetitive addition of lime to the soil without a need is the cause of the man-made development.

16.4.5 Treatments to Reduce Soil Alkalinity Utilizing grass cultures to ensure the inclusion of mulch as an acidifying organic material, alkali/sodic soils with solid CaCO3 can be recovered. The calcareous subsoil is deeply plowed and mixed with the topsoil (Shekhawat et al. 2012). As gypsum combines with sodium carbonate to produce sodium sulfate, a neutral salt that does not contribute to high pH is formed. Farmers can use urea to primarily lower the soil’s alkalinity and salinity when it is inexpensively accessible to them (Bhandari et al. 2002). The strong cation Na is released from the soil structure into the water by the weak cation NH4+ (ammonium), which is found in urea. In contrast to other soils, alkali soils therefore absorb and use more urea. As the top layer is most vulnerable to structural degradation, most efforts are consequently focused on improving the top layer alone (the first 10 cm of the soil).

16.4.6 Soil Salinity The majority of the elements required for plant developments are provided by soluble salts, but too much of them can be harmful. In this case, the soil is referred to as saline when soluble salts are present in excess that can adversely affect plants (Kannan et al. 2019). Salinity-related substances such as chlorides, sulfates, carbonate, sodium, calcium, and magnesium bicarbonates are frequently found in saline soils. Depending on the source of the salts, the proportions may vary based on the

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location. In general where yearly precipitation is inadequate to fulfill the evapotranspiration demand of plants, salt-affected soils are frequently seen. Salts do not seep down, and as a result, staying in the soil layer where they are harmful to plant growth (Nanjundan et al. 2020). Saline soils offer the potential for production if amendments are made in a timely and effective manner because they contain an extensive nutrient store due to reduced loss by plants and yearly fertilizer input.

16.4.7 Impacts of Soil Salinity Friable structure is seen in saline soils. The soil has a limited ability to vaporize and absorb water. Plants are impacted by salinity in a variety of ways, including osmotic impacts, specific ion toxicity, and/or nutritional problems, though it speeds up plant maturity and salt stress slowed down leaf growth (Kjellstrom 1993). When salt stress was removed at the late leaf primordial stage or double ridge stage, or when it was postponed until after the terminal spike development, grain yields were maximized. Additionally, wheat growth and development are adversely affected by brief bouts of salt stress during organogenesis. Stress from salt triggers the process of reproduction, but it hurts the growth of wheat spikes and lowers the potential yield (Zhu et al. 2020).

16.4.8 Measures to Reduce Soil Salinity In the short term, plant development is improved by mechanically scraping out salts, but this only lasts so long since the salts keep building up.

16.4.8.1 Leaching The only feasible method for removing excessive salts from the soil is by leaching utilizing irrigation or rainwater. When the soil is salty and has a sound internal drainage system, leaching is effective. Applying up to 48 acre-inches of water is required to flush heavily salinized soil. 16.4.8.2 Moisture Stress In addition to dry and drought-prone areas, places with ample rainfall are also experiencing a shortage of water. When the water supply to a plant’s roots becomes insufficient or when its transpiration rate increases, a plant experiences water stress. A water deficiency, such as a drought or excessive soil salinity, is what largely causes water stress. Water stress is mostly caused by a water shortage, such as a drought or severe soil salinity (Rashid et al. 2015). From germination through physiological maturity, Brassica development is impacted by moisture stress. Depending on the level of stress, pace, length of exposure, and stage of plant growth, Brassica and other crops respond to stress differently. In places of heavy rainfall and undulating terrain, the water runoff is reduced by tillage choices, including zero tillage with or without residue retention on the bed. As a result, there is a decrease in

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the loss of the most fertile topsoil, and the preservation of residue causes a buildup of organic matter in the soil (Bello et al. 2021). This eventually leads to sustainable production over time. Crop production will be impacted during this time because crops are vulnerable to water stress throughout the reproductive development phases. Limiting irrigation during the development phases that are least vulnerable to water stress while preserving water for the crucial growth stages might be a useful method to maximize yield return from irrigation when growers have limited water sources but control over when they can irrigate.

16.4.8.3 Water Logging Insufficient oxygen in the soil pore space prevents plant roots from breathing, which leads to water logging. As a result, the root zone accumulates root-harming chemicals, including carbon dioxide and ethylene, which hinders plant growth and development. In wet soils, ethylene is generated by both microbes and roots (Sharma and Sardana 2016). To prevent water from percolating and generate flooding conditions for mustard farming, soils are often puddled. Grains are planted after wheat but with less-than-ideal soil physical conditions because of soil puddling (Meers et al. 2008). When excessive irrigation or rainfall occurs, the soil pan that was purposefully produced for mustard growth may block water from moving, resulting in waterlogging.

16.4.9 Stress Caused by Temperature 16.4.9.1 High Temperature High temperatures shorten the tillering period, which in turn speeds up crop growth by causing it to enter the jointing stage too early. This causes the number of tillers to decline, which in turn lowers the overall crop production. The time of grain filling is shortened by high temperatures throughout the flowering and grain-filling stages, which leads to early maturity and lowers crop output (Aina et al. 2018). The duration of each stage of the crop’s development is shortened by the presence of high temperatures, which lowers the amount of photosynthesis that must accumulate for the crop to produce its maximum yield. Seedlings are protected from temperature stress by the use of mulch and zero tillage. Shielding the soil from incoming solar radiation and retaining moisture helps to keep soil temperature lower during the day (Ashfaque and Inam 2019). Mulch helps plants overcome terminal heat stress by reducing soil cooling at night, increasing transpiration as soil temperature drops, and lowering the canopy’s temperature as a result. Due to the crop’s delayed maturity, its growth characteristics increase, which ultimately increases the crop output, and when compared to conventional tillage, the zero tillage option improved yield parameters such as plant height, effective tillers per m-1, and grains (Khan et al. 2019).

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16.4.9.2 Drip Irrigation/Use of Sprinkler This lowers canopy/soil temperature by assisting with keeping enough soil moisture. Drip irrigation increased grain output by 24% compared to full irrigation and by 59% compared to the standard rule treatment. 16.4.9.3 Low Temperature Frost damage in cereal crops affects the number of kernels per spike during blooming and the early stages of grain growth (Chauhan et al. 2011). Each time there is a frost, a narrower band on the spikes forms, the awns curl, and the spikes are erect as they approach maturity due to the lower grain weight. Reduced germination of pollen grains in both aestivum and durum wheat can be attributed to low temperatures during the anthesis stage, resulting in increased pollen sterility (Zhai et al. 2020). Because of the extremely cold temperatures, Brassica species whose anthesis occurred in December and January produce more sterile pollens and, as a result, produce less Brassica crop yield is adversely affected. 16.4.9.4 Nutrient Deficiency Essential nutrients are necessary for plants to operate normally and flourish. The range of nutrients required to satisfy the demands of a plant and promote growth is known as its sufficiency range. Various plant species and a specific nutrient will determine this range’s width (Begum et al. 2019). Due to either a nutrient deficit or toxicity, nutrient levels outside of a plant’s sufficient range reduce crop development and health overall. When a nutrient is presently more than a plant’s requirements, it becomes toxic and stunts the development or quality of the plant. The following are the three fundamental methods for determining nutritional deficits and toxicities: (1) plant analysis, (2) visual field observations, and (3) soil testing. Brassica yield has decreased as a result of farmers applying nutrients in an unbalanced and insufficient manner. The nutrients that the crop removes from the field on an annual basis are replenished to keep the fertility state of the soil under high-intensity crop production (Jat et al. 2017). Nutrient management strategies, such as INM, site-specific nutrient management, remote sensing, etc., should be used to address this issue of nutrient deficiency/toxicology. Fertilizers should be used by crop growth requirements and agro-climatic conditions. Additionally, it is important to reduce the detrimental effects of outside influences. Overusing fertilizers does not considerably increase crop nutrient uptake or yields because it is neither economical for farmers in industrialized nations nor is it effective (Rathore et al. 2019). Instead, their excessive usage wastes money and may have negative environmental effects. On the other hand, the improper application slows down plant development and lowers crop output. Inorganic and organic fertilizers are more effectively absorbed by plants when they are applied in a better and more sufficient manner, which also helps to save soil nutrients.

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16.4.9.5 Reduced Water Quality A significant barrier to agricultural development in the country’s dry and semi-arid regions is the lack of suitable irrigation water. While irrigated agriculture has significantly enhanced agricultural yield, improper and wasteful irrigation water usage has destroyed productivity and changed the ecosystem of large tracts of land while also contaminating surface and groundwater. In many locations, surface and groundwater have been poisoned by the indiscriminate use of pesticides, herbicides, and fertilizers. Of the 135 billion cubic meters of groundwater extracted yearly in India, 32 billion cubic meters are thought to be salty (Anjum et al. 2012). As water flows between the soil profile and the groundwater layer, processes take place that lead to saline groundwater. Saline groundwater irrigation can have a negative longterm effect on crop development and yields by deteriorating the quality of the soil. The amount of fresh and saltwater that may be combined limits the amount of water that might potentially be used (Chin et al. 2021). It is necessary to develop and use innovative methods to address the issue of saltwater consumption in agriculture. Better strategies must be devised to more effectively execute current techniques to prevent excessive water usage and conserve scarce water supplies. The irrigation water management system must include the reuse of wastewater, including drainage water and shallow saline groundwater, for crop production. To maintain irrigated agriculture and stop contamination of related water resources, effective salinity management strategies must be created and put into practice (Culver and Precious 2018). Effective salinity control techniques must be developed and implemented to preserve irrigated agriculture and prevent the polluting of associated water resources. 1. Choosing plants or plant kinds that can survive in salty or acidic environments. 2. Certain planting techniques should be used to reduce salt buildup around the seed. 3. Regular salt leaching from the soil and irrigation keep the soil at a high level of moisture. 4. Applying techniques for soil preparation to promote infiltration, leaching, and salt removal while ensuring that water is distributed uniformly. 5. Maintaining soil permeability and tilth requires special techniques such as tillage, the use of chemical amendments, organic matter, and the cultivation of green manure crops. To better regulate salt and water distributions, micro-irrigation devices such as drip and sprinklers are used. This increases the saline water’s usage efficiency, particularly for high-value crops (Nikolaou et al. 2022). Reduced soluble salt concentrations in the seedbed during germination and improved crop establishment are the result of pre-emergence sprinkler applications of saline water. When the crops can withstand greater salt, we may then move to low-quality water. Although salts build up on the edges of the wetted region, drip irrigation is suggested for use with saline irrigation water because it lowers salinity and matric stressors in the root zone. To remove the salt that has been collected during earlier irrigations, more water must be supplied (Akinyele and Shokunbi 2015). Irrigation techniques as well as the crop resistance to salt are the key factors that affect the leaching needs (Figs. 16.1 and 16.2).

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Fig.16.1 Abiotic stressors affecting agriculture

Fig. 16.2 Biotic stressors affecting agriculture

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Function of Genes in Reducing Abiotic Stresses

Plants evolve several defensive mechanisms through the activation of a complicated signaling cascade under different stress conditions. Reactive oxygen species (ROS) and phytohormones such as salicylic acid, jasmonic acid, and abscisic acid (ABA) are produced when a plant is subjected to biotic and abiotic challenges, which encourage the activation of protein kinase cascades and particular ion channels (Thakur et al. 2020). Different mechanisms of plant responses to abiotic stressors, together with those corresponding, have been described in a straightforward model to facilitate comprehension. The transcriptional regulatory networks of abiotic stress signals and gene expression have been investigated in light of our current knowledge of stress signaling pathways. Sensors or receptors for plant cells that detect stress stimuli are found in the cell wall or membrane (Zanetti et al. 2013). There are two types of abscisic aciddependent and abscisic acid-independent pathways used for abiotic stress signal transduction. ABRE serves as the major abscisic acid-responsive component in the abscisic acid-dependent pathway, activating the genes involved in stress response. Dehydration-responsive elements (DRE), on the other hand, play a role in the regulation of genes that are responsive to salt, cold, and drought stress in the abscisic acid-independent pathway. Cells attempt to respond to the signals by creating reaction oxygen species (ROS), calcium ions, inositol phosphate, etc., inside the cells (Shukla et al. 2019). These signals are received by cell membrane sensors that pass from plants when they encountered abiotic challenges. The lipid, protein, and DNA may all be damaged by excessive levels of reactive oxygen species (ROS) in the intracellular environment. Additionally, protein kinase-mediated phosphorylation aids in the activation or suppression of a variety of transcription factors (TFs), which bind specifically to cis-acting elements in the promoters of genes that respond to stress (Long et al. 2007). This regulates the transcriptional level of these genes to counteract abiotic stresses. Meanwhile, additional upstream elements are regulated by transcription-level TFs and are prone to various levels of post-transcriptional modifications, such as ubiquitination and sumoylation, to govern the expression of stress-responsive genes. The function of these genes is to detoxify ROS among different Brassica species (Chahal et al. 2020).

16.6

Factors Affecting Success of Genetic Transformation in B. juncea

The genotype used and age of the explants, the preculture, the concentration and length of infection with Agrobacterium, efficacy of antibiotics used to kill the excess Agrobacterium cells, and the selection medium used to select the transformed cells are the major factors affecting the success of any genetic transformation protocol. In vitro plant regeneration methodology varies from genotype to genotype, and both factors are important for successful plant regeneration (Qamar et al. 2020). In an investigation, three genotypes of B. juncea were used and cotyledonary explants

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were used to determine the regeneration frequencies; while the explants mostly removed from 4–6-day-old in vitro grown seedlings in B. juncea exhibit improved shoot growth due to its superior ability to withstand the shock of transformation compared to other types of explants, the hypocotyl is frequently used as an excellent explant in investigations on the genetic transformation of the Indian mustard (Thakur et al. 2020). Before combining the explants with the Agrobacterium strain, it is advised to give them 48-h, preculture incubation. Given that plant cells are better prepared to withstand the impact of transformation at this time, it has been proposed that this phase is ideal for obtaining higher transformation frequencies. Plant cells begin to regenerate after 2 days, making them ineligible for transformation and decreasing the frequency of transformations (Chauhan et al. 2011). Another key element affecting the transformation frequency is Agrobacterium concentration. It was observed that infection duration of 30 min improved the outcomes for the transformation of B. juncea utilizing cotyledonary petiole explants. The time for infection of explants with the Agrobacterium strain ranges from 1 to 30 min. After co-cultivation, it is necessary to control the expansion of agrobacterial cells since they might consume the entire explant and cause the experiment to fail (Khan et al. 2019). The most often used antibiotics to stop the growth of agrobacteria are cefotaxime, carbenicillin, and timentin at a concentration of 100–500 mg/l. The transformed explants are put on selective media containing a selection agent after co-cultivation to isolate the transformed cells and tissues. The type of antibiotics employed and the concentration utilized are both important factors in the efficient selection of transformed cells. The type of marker gene utilized in the gene construct for genetic transformation determines which antibiotic should be used in the media for tissue culture (Bailey 1896). When the NPT-II gene was included in the gene construct, kanamycin antibiotic was utilized as a selection agent. To select altered cells and tissues, the culture media is also treated with many different selection agents, including hygromycin, 2,4-D, and phosphinothricin, among others.

16.7

Different Genes Expressed in Brassica Under Abiotic Stresses

Due to the rapidly changing global climate, abiotic stressors are becoming a serious problem for the development of Brassica crops everywhere as depicted in Fig. 16.3. In response to these kinds of stressors, B. rapa and B. oleracea exhibit distinct gene expressions according to earlier investigations (Kayum et al. 2016). In response to several abiotic stressors, B. rapa WRKY and Alfin-like transcription factors have been widely studied. When exposed to cold stress, Chiifu displayed about 32-, 42-, 17-, and 54-fold increased expressions of the BrWRKY22, BrWRKY70, WRKY, BrWRKY72, and BrWRKY44 (Meena et al. 2016). Therefore, to create coldtolerant lines, the BrWRKY22 and BrWRKY44 genes might be introduced or overexpressed in B. rapa. In addition, under salt stress conditions, BrWRKY17, 58, and 57 showed about 5-, 2-, and 3-fold higher expression, respectively, and BrWRKY, 65, 104, 51, 98, and 98 showed their highest expression after drought

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Fig. 16.3 Oxidative stress factors and impacts on mustard plants

stress treatment and were approximately 15-, 38-, 85-, and 18-fold higher than the control, respectively (Kayum et al. 2016). However, in response to a combination of abiotic stimuli (cold, salt, and drought), BrAL 3, 9, 13, 14, 15, 2, and 7 displayed greater expression. The B. rapa longevity assurance gene and several copies of the BZR transcription factor responded to ABA, cold, and drought stressors. BoAL 4, 6, 8, 12 1.9, 7, and 5 are Alfine-like transcription factors that have been shown to respond to abiotic stressors such as cold, salt, drought, and ABA in B. oleracea. Among the MYB genes, ABA treatment considerably upregulated six genes (BrMYB210 BrMYB172 BrMYB208, BrMYB140 BrMYB137, and BrMYB229), whereas auxin treatment significantly downregulated the same genes (Nanjundan et al. 2020). Leaves treated with auxin revealed downregulation of BrMYB210 (an ortholog of AtMYB96).

16.8

Different Genes Expressed in Brassica Under Biotic Stresses

Plants and vegetable crops are extremely vulnerable to the catastrophic effects of biotic stress. Figure 16.4 illustrates the sequence of genetic factors in mustard plant alongside the effect of the oxidative stressor that lead to the wilting or death of the plant. Many genes showing differential expression in response to various biotic stressors have been well documented in previous studies (Kannan et al. 2019). After P. carotovorum sub sp. carovorum infection, BrWRKY141 displayed an approximately 180-fold increase in expression. BrAL 3, 9, 14, 2, 15, 7, and 13 were expressed in response to both biotic and abiotic stressors after infection with

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Fig.16.4 A hierarchical graphical representation of genetic consequences of mustard plants

F. oxysporum f. sp. conglutinant, and they exhibited many folds greater expression after infection (Velasco et al. 2021). However, upon infection with P. carotovorum sub sp. carotovorum in B. oleracea, two Alfin-like genes (BoAL8 and BoAL12) were activated. Following P. carotovorum sub sp. carotovorum infection in cabbage plants, 3 of the 12 thaumatin-like proteins showed differential expression. Furthermore, Table 16.1 depicts some examples of current yield-associated traits involving CRISPR/Cas9 gene in mustard.

16.9

Marker Genes

Marker genes can either be measured so that their expression can be observed visually or they can be selectable so that any selective agents added to the medium, such as antibiotics or herbicides, can be detoxified before they can be used to identify transformed cells among the background of nontransformed cells (Nanjundan et al. 2020). In B. juncea transformation, the GUS and NPT-II genes are the most often utilized selectable and extract valuable markers. It was achieved by using an Agrobacterium-mediated gene transfer approach to genetically alter B. juncea cv. Pusa Jaikisan with the GUS gene. Explants from cotyledonary petioles were used to optimize several variables, including explant age, preculture duration, bacterial density, and AgNO3 concentrations (Long et al. 2007). The integration and overexpression of the osmotin and ferritin transgenes in B. juncea were then carried out using the standardized technique, and this was further supported by PCR and southern and western blot studies of the T0 and T1 generations. Choudhary et al. (2015) used hypocotyl explants to refine a genetic transformation technique for B. juncea that included the GUS and NPT-II genes. Using the same procedure, it was also possible to create transgenic Indian mustard plants that had desired genes for fungus and insect resistance. However, there was no noticeable resistance to these biotic stress factors in transgenic plants (Wu et al. 2021).

BnJAG.AO2, BnJAG.CO2, BNJAG.CO6, BnJAG.AO7, BnJAG.AO8 BnaMAX1

BnaSVP

BnaAO3.BP

BnaEOD3

BnAO4.CLV3, BnCO4. CLV3, BnCO2. CLV3

3

5

6

7

8

4

BnSHP1/ BnSHP2homeologs

Target genes BnD14

2

S no. 1

Formation of several siliquae

Height and branch angles of the plant Amount of seeds in each siliquae

Height of the plant, number of major branches, and siliquae count Blooming period

Plant height was reduced by 35%, the number of main branches was increased by three times, and the overall number of siliquae rose by 65% Reduced time till blossoming by 40–50%

Branch angle was decreased from 84 to 14, and plant height was lowered by 16% The number of seeds per siliquae increased by 42% despite shorter siliquae length and smaller seeds Growth in seed weight per siliquae by 74%

Resistance to pod breaking

Resistance to pod breaking

Trait Number of branches, plant height, and internode length

Two times more durable against pod breaking

Phenotype 37% increase in the number of blooms per plant, a 34% decrease in plant height, and 200% more branches Ten times more durable against pod breaking

Table 16.1 Mustard plants improvement through CRISPR/Cas9 gene

CLAVATA3(CLV3)

BREVIPEDICELLUS (BP) ENHANCER3 OF DA1(EOD3)

Short vegetative phase (SVP)

More Axillary Growth (MAX)

JAGGED(JAG)

SHATTERPROOFF1/ 2

Homolog DWARF14(D14)

Yang et al. (2018)

Zheng et al. (2020) Ahmar et al. (2022) Fan et al. (2021) Khan et al. (2020)

References Stanic et al. (2021) Zaman et al. (2021) Zaman et al. (2019)

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16.10 The Use of Molecular Markers to Screen Elite Germplasm Breeding and creating drought-tolerant cultivars is the greatest strategy to address all the issues in agriculture. Analysis of plant methods for drought resistance has been a big difficulty for plant breeders due to the issues with drought as a stress signal (Rana et al. 2020). The genetic improvement of drought tolerance in rapeseed has, nevertheless, been the subject of several investigations. In response to bulk selection for yield, 1000-seed weight, harvest index, and seeds per pod in a drought setting, Brassica campestris was assessed for the enhancement of seed yield. The individual selection was 20% less successful than bulk selection for yield solely during drought (Chahal et al. 2020). Individual plant selection for harvest index or for blooming time was preferable to individual selection for yield in the Brassica napus for achieving genetic improvement. Nevertheless, a 16% increase in output was obtained by bulk selection for yield and flowering period. Moricandia arvensis and Salix alba are two crop wild relatives (CWR) of Brassica that can withstand drought stress better. To introduce these beneficial features into Brassica crops, intergeneric crosses between Mentha arvensis and Brassica oleracea were created. In the BC2 population, a few monosomic lines of B. oleracea were established that included a chromosome from Mentha arvensis (Khattab et al. 2017). These lines might be useful for future genetic and breeding studies. Along with the 18 chromosomes of Brassica oleracea, these BC1 plants also carried one, five, or six extra Salix alba chromosomes.

16.11 Biotic Stress Tolerance 16.11.1 Resistance to Aphids, Insects, and Pests The mustard aphid, one of several insect pests, significantly reduces the Indian mustard’s productivity. Lipaphis erysimum (L). Mustard aphid, the aphid species known as Kaltenbach, which is a member of the order Hemiptera, feed on phloem sap. In India, compared to other mustard-growing nations, it results in considerable yield losses for the Indian mustard (Chahal et al. 2020). The mustard plant’s leaves, stalks, twigs, and flower buds are all covered by it. The nymph and adult stages of this aphid during blooming and seed setting phases are responsible for the majority of yield losses to the Indian mustard. Due to the lack of a successful resistant Brassica cultivar or genetic line, breeding efforts to develop aphid resistance have not been successful to date. There have been attempts to introduce the aphid resistance trait into cultivated B. juncea types using some wild species of Brassica, such as B. fruticulosa, but no reports of success have been reported yet. Plant lectins, which are proteins that attach to carbohydrates, are very efficient in preventing sap-sucking pests like aphids attributed to their agglutination characteristics (Anjum et al. 2012). Aphid resistance has been discovered to be conferred by wheat germ agglutinin, the lectin protein from wheat germ that binds to chitin. Aphids’ midgut glycoprotein binds to water germ agglutinin, which prevents food

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absorption and results in insects starving to death. Southern blotting was used to confirm transgene integration, and northern and western blotting was used to show that the transgene was expressed (Wang et al. 2012). A direct deterrent impact on aphid colonization was discovered in the EF gene isolated from Mentha arvensis. In transgenic plants that express the E-F gene, a comparably large decrease in the seasonal mean population of aphids was observed. To prevent the target protease from separating the peptide bonds of the essential proteins, protease inhibitors directly attach to the active sites of proteases inside the cell (Rizwan et al. 2018). It prevents the target protease from absorbing and using the proteins for the aphids’ growth and development.

16.11.2 Disease Resistance Numerous fungi, such as Alternaria brassicas, which causes black spot disease, Albugo candida, which causes white rust, Sclerotinia sclerotiorum, which causes stem rot, Erysiphe cruciferarum, which causes powdery mildew, and Hyaloperonospora parasistica, which causes Downey mildew, seriously impair the productivity of the Indian mustard crop. By developing black patches with concentric ring structures on leaves and pods, this fungus spreads on mustard plants. The pathogen white rust may infect almost all kinds of Indian mustard that are now grown in India (Saharan et al. 2014). One of the main biotic stressors limiting the yield of mustard is Alternaria brassicae. The growth of stag heads, which eventually do not carry seeds, is caused by the systemic transmission of the fungal pathogen from the early vegetative stage to the inflorescence. This infection induces hypertrophy of blooming tissues. In the past 4–5 years, stem rot has emerged as a serious hazard to the Indian mustard. Except for the white rust disease, which may be used in breeding programs for resistant plants, no sources of resistance to these biotic stressors have been identified too far. Additionally because of the pathotypes’ propensity for constant change, chemical fungicides are completely ineffectual at exerting any control. To attack such terrifying infections, these circumstances made it necessary to create transgenic techniques. The production of pathogenesis-related (PR) proteins, which are typically found in very little levels in plants, can be increased as one method of enhancing the plant’s defense response against invading plant diseases (Hossain et al. 2019). In transgenic plants compared to control plants, the onset of disease signs was postponed by a period of 10–15 days. Lectins are common carbohydrate-binding proteins that are present in plants as secondary metabolites. In addition to defending plants from aphids, they also help to give tolerance against variety of biotic and abiotic stressors. As determined by the number of lesions and the length of time it takes for leaf necrosis to occur, Varuna imparts resistance against A. brassicae in transgenic lines, with the protection varied in the range of 36–60% in various transgenic crops compared to the nontransformed plants (Kumar et al. 2020). Both water stresses brought on by mannitol and salt stress (NaCl) were also tolerated by the transgenic

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plants. Similarly by introducing the MPK3 gene into B. juncea, Tasleemet al. (2017) developed the plants and investigated how it contributed to A. brassicae resistance. To maintain homeostasis inside the plant cell, it has been discovered that the majority of abiotic stressors cause stress genes to express themselves in overlapping patterns and interact with different signaling pathways (Rathore et al. 2019). To counteract the effects of abiotic stresses, transgenic plants have been created that express genes for the biosynthesis of a variety of substances, such as mannitol, metal ion transporters, glycine betaine, late embryogenesis abundant (LEA) proteins, and PR proteins with specific role in ion homeostasis, membrane stability, and osmolyte accumulation. Plants produce glycine betaine during the abiotic stress phase, and this compound functions as an osmoprotectant to help the plants fend off numerous abiotic stressors. In comparison to control plants, transgenic mustard lines showed a greater germination rate when subjected to saline stress. Due to cracking and breakage brought on by high-temperature stress at the terminal stage, the Indian mustard suffers significant yield losses. March is when the Indian mustard matures and is harvested in the majority of India’s mustard-growing regions, mainly in northern India, and it is also a period when the atmosphere is still very hot (Long et al. 2007). Early harvesting can be done to prevent terminal heat losses since early blooming in Indian mustard results in an early seed set and maturity. Additionally, the intervention of early blooming in the Indian mustard might be advantageous for such crop’s compatibility for cultivation in multicropping systems and its extension into less conventional locations, such as the northeast or southern regions of India. By utilizing hypocotyl explants to transfer the leafy gene from Arabidopsis thaliana, Sahni et al. (2013) aimed to promote early blooming in the Indian mustard. If cultured under in vitro conditions, transgenic mustard plants begin to blossom after 12 weeks, but nontransgenic plants do not begin to flower until 16 weeks had passed.

16.11.3 Resistance to Herbicides There has been a reported movement toward employing new genes, such as herbicide-tolerant genes, as selection agents in genetic transformation studies due to the health risks caused by the use of antibiotic-resistant marker genes. In addition, several weedy species are infesting Indian mustard fields that compete with the mustard plants for nutrients, reducing productivity. In mustard farms, broad-leaved herbicides are commonly engaged to prevent weeds while also damaging mustard plants (Yadav et al. 2014). Therefore, the introduction of transgenic Indian mustard with a herbicide-resistant characteristic gives a chance for the management of weeds, especially invasive weeds such as Orobanche aegyptiaca. The transgenic mustard plants will not be harmed by the herbicide since the transgene expressing the herbicide-tolerant trait prevents the weedy plants from being killed by the herbicide spray. The pollen-mediated genetic transformation has been proposed as a more suitable and effective approach to developing transgenic plants as this route helps to circumvent the problems associated with tissue culture-based methods of gene

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transfer. This may often lead to somaclonal variations due to callusing phase, resulting in morphological abnormalities in plants.

16.12 Conclusion Millions of people still experience malnutrition despite attempts to reduce food insecurity. Global climate change and exponential increase in the global population both possess a serious threat to food and nutritional security by adversely affecting the crop production and crop quality. Application of recent genetic engineering approaches has accelerated the crop improvement programs in order to minimize the gap between the realized and potential yield of Brassica spp. Therefore, toward optimizing the maximum yield potential and quality improvement for the crops selected, gene-editing protocols can play an important role, such as genes related to the low erucic content, high–low glucosinate content, high fatty acid content, and essential oil that is of primary target.

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Genetic Improvement of Mustard for Food and Health Security

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Gohar Taj, Sandhya Upadhyay, and Anjali Sharma

17.1

Introduction

Mustard is a well-known crucifer and the earliest crop domesticated and cultivated around the world for its green leaves, oil (cooking and biodiesel), and fodder. It can be grown with few requirements. Mustard is recognized in several areas such as in ayurveda, food industries (taste enhancer and preservative), cosmetics and daily healthcare products, and a constituent in many other formulations; thus, it has a good weightage in the progress of economy. Mustard oil contributes to about 28.6% in the Indian economy, and the total production value was estimated at 293 billion in 2020, while in the United States the production was 81.8 million pounds at a value of $22.1 million. According to the Central Organization for Oil Industry and Trade, the production of mustard is still rising due to its taste, usability, and additional benefits to human health. By 2025, the global market for mustard is expected to reach around US$7.442 billion. However, numerous factors were identified that have been affecting the productivity of mustard, which presents a challenge to the breeders and researchers to introduce such cultivars that are able to tolerate stress and sustain themselves. A report from the Indian Ministry of Agriculture & Farmer Welfare in 2019–2020 had confirmed the issue, which states that the production of mustard decreased by about 1.40 lakh tons compared to the previous year. Mustards are known to have a reasonable number of fatty acids (erucic, oleic, linolenic, stearate, and etc.), sugars, ascorbic acids, antioxidants (carotenoids, tocopherols, hydroxybenzoic acid, ferulic and sinapic acid), vitamins, minerals, and numerous other bioactive compounds comprising multiple functions, and are thus considered a superfood (Lenzi et al. 2022; Grygier 2022). For example, the bioactive compound glucosinolate (GSL) is responsible for mustard’s pungent flavor G. Taj (✉) · S. Upadhyay · A. Sharma Molecular Biology & Genetic Eng., G.B.Pant University, Pantnagar, Uttrakhand, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_17

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and is a good source for human health (Sun et al. 2019). GSLs were also reported to play a role in plant defense such as repelling pests and inhibiting microbial growth (Peng et al. 2014) as well as suppressing unwanted cell growth (Wang et al. 2021) when converted into isothiocyanates. Another bioactive compound is proapoptotic lipopeptide, which is reported to be involved in cancer suppression (Mondal et al. 2022). Besides the above-described properties, mustard varieties were also reported to accumulate heavy metals that disseminate around metallurgy. Screened Brassica juncea cultivars with increased accumulation of zinc and cadmium. Furthermore, to enhance accumulation, some other components were also added. Mustard was first domesticated around 3000 BC and to date is continuously grown and harvested. Due to some phenomena such as gene flow, genetic mutations, and gene duplication within the starting varieties of mustard, it has now become a huge diversified family. These species are not only diverse in genotype but also in morphology and physiology (Appel and Al-Shehbaz2003). Many of the species of the mustard family are known to develop resistance or tolerance over various factors while some are known to diversify to produce different degrees and types of fatty acids; in addition, they are continuously co-evolving with respect to the various stresses. This diversification of mustard species is very helpful in enhancing the germplasm of cultivated mustard varieties via breeding strategies that gear up in assembling the desired characters. Hence, the first step taken toward genetic improvement was identification, selection, and evaluation of mustard varieties. For protection and development of plant varieties, various centers and organizations were developed around the world to provide beneficiary products to breeders, farmers, and growers as follows: 1. 2. 3. 4.

Directorate of Rapeseed-Mustard Research U.S. Department of Agriculture and Agricultural Research Service European Cooperative Programme for Plant Genetic Resources Gene Bank Committee of the European Association for Research on Plant Breeding 5. International Board for Plant Genetic Resources 6. International Union for the Protection of New Varieties of Plants Now the question arises, what are the desired traits and why are we concerned with developing other lines? The answer lies in the demands of the rising population; insufficient fertile lands, food insecurity, health deprivation, and malnutrition. Furthermore, the continuous exploitation of fossil fuel is desolating the boreholes and its uneven combustion adds up the detrimental compounds in the environment, leading to an increase in Earth’s temperature as well as the concentration of pollutants. The consequences of changing climates and interaction of variable factors over plants are narrowing the harvest. The rising temperature not only affects the plant’s physiology, phenology, morphology, and biochemistry but also the microbiotas associated with it. The production under variable environmental conditions can be predicted through crop modeling technology (Ray et al.2019).

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Approaches Required to Improve Mustard Genome

To grade up and restore the plant functionality, several steps have been taken; especially at the initial level, fertilizers and pesticides were used for their threshold that leach within the ecosystem. Some symbiotic microbes were also incorporated within the crop fields so that they increase the uptake of water added with minerals and ensure a better product. But somehow the results were not satisfactory and unsustainable. Thus, to enhance the mustard production under challenging conditions with escalating harvest, we must strengthen and improve its genomic content. For such purpose, the approaches used are mentioned in Fig. 17.1 and described within the subtopics. The diversified family of mustard is a boon for developing various breeding populations (Wang et al. 2016). In the past, to exploit the potential benefits of mustard, breeding was performed among numerous varieties of mustards. The procedure includes the selection of parents, which was highly dependent on morphological and biochemical markers. However, due to some events within the genome such as dominance, crossing over, gene masking, and intergenic gene interaction, as well as under the changing environment, traceability of traits with respect to morphological and biochemical marker get affected. Moreover, the traditional breeding strategies take much time, are tedious to perform, and are unable to differentiate multiple allelic forms. The crop evolution through conventional breeding strategies undoubtedly narrowed the genome of elite cultivars. Besides, a tool called mutagenesis was introduced to generate novel variability in plants. In this technique, plants were intensively introduced with either physical (UV, X, and gamma rays) or chemical (ethyl methane sulfonate) mutagenic agents that recast the genome architecture (Sikora et al. 2011). Evidently this approach is based on the hit-and-trail mechanism, followed by the screening of phenotype that resembles the desired product and then its incorporation into the breeding program. The mutation cause changes within the Fig. 17.1 Approaches used to strengthen the desirable crop production

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nucleotides of genome (regulatory sequences and ORFs); hence, the resultant plant trait/product varies. Furthermore, the variability can also be generated through the in vitro pressure selection technique called somaclonal variation. This technique relies on the alteration of media composition with each and every subculture (Rai 2021). RNA interference (RNAi) technology has been applied for performing expressional change within the mustard genome. This is a post-transcriptional gene-silencing mechanism that depends on the site-specific degradation of doublestranded RNA strands and associated gene function (Fire et al. 1998). The unrequired expression of any mRNA expression will be downregulated or blocked via this system. Several examples of modification through RNAi are available in mustard varieties such as to get rid of pest herbivory and improve seed meal and fatty acid accumulation, which are explained later. Another technology, the next-generation sequencing (NGS), which is composed of various molecular tools, gave birth to a new field called molecular breeding. It makes it easier to identify the allelic variation, genetic variability, and quantitative loci traits; thus, it is helpful in developing and characterizing genomic maps, markerassisted selection (MAS), genome-wide association study (GWAS), as well as association maps (Tandayu et al. 2022). The detection is based on the polymorphism of DNA, which is unaffected toward the changing environment. Markers such as RFLP (Cheung et al. 1997), SSR (Rajpoot et al. 2020), and AFLP and RFLP (Pradhan et al. 2003) are often used in mapping. Moreover, these markers are also utilized for the analysis of diversity, phylogeny, and breeding program prototypes (Singh et al. 2022). Thus, the implication of molecular-based approach in breeding and genetic recovers the bridge between necessity and fabrication of mustard genome. A transgenic approach is much different from gene editing. One involves the introduction of genes from different genus, family, or kingdom, and the other involves editing/modification of own genome. These strategies were applied when the desired agronomical traits were not sufficient or unable to provide appropriate results. Transgenic approaches are precise, fast, and reliable; and shorten the release time compared to conventional breeding. The transgenes are generally genes from different organisms and are expressed to produce a specific protein that can be used for a defective or unexpressed pathway and can be introduced through numerous transformation techniques such as vector-less (biolistic, PEG, microinjections, and electroporation) and vector-mediated (co-integrated or binary agrobacterium vector) (Dutta et al. 2008). The transgene was transferred within the tissue culture lines (somatic embryo or protoplast) so that the gene remains in every plant cell and no chimeras are formed. Several traits were transferred through this approach, as listed in Table 17.1. Although the transgenic approaches were known to be surrounded by some controversies such as health concerns, ethical issues, and percentage of consumers, gene escape is a hectic and expensive regulatory process (Watanabe et al. 2005). The drawbacks of transgenic plants were overcome by gene-editing technology, which involves the modification of plant genome as per the requirements. It mainly

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depends on the activity of programmable nucleases that make desirable changes to the nucleotide sequences through indel or frameshift. These nucleases are zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/CAS9 system (Manghwar et al. 2019). They are directed to the specific regions within the genome to produce double-stranded breaks (DSB) in the DNA stretch. Whenever breaks are introduced, two types of repair mechanisms play a role: non-homologous end joining (NHEJ) and homologous direct repair (HDR). Inducing indel or substitution with the region of break may generate modifications in nucleotide sequences or a nonfunctional product. Compared to the others, the most preferable programmable nuclease is the CRISPR/CAS system, which has been reported in mitigating stress (Miglani2017).

17.3

Augmentation of Desired Fatty Acids in Seeds

Among the oilseed crops, mustard is listed in the third position in oil production. Several cuisines utilize mustard oil to elevate the texture and flavor of the meal. Mustard oil is loaded with numerous saturated and unsaturated fatty acids. Fatty acids are essential for humans as they are important for metabolic activities and brain development. Fatty acids such as stearate and oleic acid reduce cholesterol and have a sloth rancidification (Kaushik and Agnihotri 2000), whereas some fatty acids are undesirable for human health. A majority of saturated as well as transfatty acids are associated with coronary disease and diabetes mellitus. Other components such as erucic acid and bisphenol F are toxic to animal and human health; however, they are favorable for industrial purposes (Lietzow2021). The mustard family contributes differential constituents and compositions of fatty acids in their seeds, and thus could be a better option to use as a cooking oil or biofuel. Hence, exploitation of the valuable fatty acid composition either for edible or nonedible material-specific changes within the genetic, metabolic, and regulatory pathways should be carried out. To improve seed meal quality, the level of glucosinolate is reduced via RNA interference technology. In 2007, Sinha et al. used hairpin-RNA for silencing the fatty acid elongase gene in B. juncea that downregulates erucic acid production (Yusuf and Sarin 2007). To enhance the oil quantity in the mustard seeds, a transgene expressing WRI1 transcriptional factor, a member of the APETALA2/ethylene-responsive element binding (AP2/EREBP) proteins, was introduced in the site-specific region (seed). WRI1 is known to regulate genes that are involved in carbon allocation into triacylglycerols in plants (Cernac and Benning 2004) and is also responsible for the regulation of genes involved in late glycolysis and fatty acid biosynthesis. Another change within mustard genome was performed; the functional downregulation of ADP-glucose pyrophosphorylase (AGPase) enzyme required in the initial step of starch synthesis pathway. Thus, due to the lack of AGPase, the photosynthetic carbon flux is channelized toward fatty acid biosynthesis and hence the seed filling is acquired with increased oil quantity (Bhattacharya et al. 2016).

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To enhance the synthesis of stearate with the downregulation of erucic acid, a gene MlFatB that encodes fatty acyl-ACP thioesterase (Fat)B from Madhuca longifolia (latifolia) was expressed in mustard seed. The (Fat)B releases the free fatty acids from the fatty acyl-ACP molecules through hydrolysis of thioester bond, which further terminates the acyl chain elongation. The terminated free fatty acids then translocate toward cytoplasm as acyl-CoA, where they are further modified and incorporated into triacylglycerol (TAG) for oil synthesis (Bhattacharya et al. 2015). Mustard oil is a better substitute for fossil fuel because of the reasonable qualities such as a high amount of erucic acid, reduced flash points and lubricator (Premi et al. 2013), combustion with no residue, no toxicity, keeping the environment neat and tidy, as well as the seed meal being consumable as fodder having good nutrients. The current scenario of world’s air pollution index is running down with elevated global warming due to the uneven burning of fossil fuels and loss of vegetation. Moreover, the unrestricted exploitation of fossil fuel is also draining the level of the borewell. These are threatening to give rise to the concept of biofuel. Resources such as sugarcane, cereal grains, and mustard seeds were used as biofuel feedstock to produce bioethanol, biobutanol, and biodiesel. According to the findings of the USDA, the crushed carinata seeds can be used as aviation fuel as they reduce 68% of carbon emissions compared with a unit of conventional aviation fuel. However, the difference between edible and fuel oil with upgraded quantity and quality gives rise to several strategies. Generally, seed oil has lengthy fatty acids, causing increased viscosity, and thus are improper for combustion. But some varieties of mustard can produce short-chain fatty acids: Brassica carinata, Brassica nigra, Sinapisalba L., Brassica napus, and Brassica carinata A. Braun. Breeding between the abovementioned varieties has significant results for biodiesel products (Thakur et al. 2019) while others can be utilized by altering the length of fatty acids into shorter chains (Liu et al. 2015). A gene expressing diacylglycerolacetyltransferase (DAcT) from Euonymus alatus is responsible for the unusual acetated short triacylglycerol’s fatty acid production. In 2020, Naeem et al. reported DAcT gene introduction within the genome of B. juncea via callus culture lines with an increased percentage of regeneration.

17.4

Intensive Yield Potential

The production of mustard is being largely affected by numerous pathogens causing diseases, which in turn deteriorate the yield of crops by terribly hampering the growth and quality of production. These diseases are the result of host–pathogen interactions, and the severity depends on the characteristics of host, pathogen, and environment. Abiotic stress has a significant impact on production, that is, reduced due to disturbance in their normal growth caused by water deficit, high temperature, and more stress.

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17.4.1 Abiotic Stress The stress due to the unfavorable condition alters plant metabolism, resulting in low production, reduced plant growth processes, or damage to plant organ, thereby inducing changes in molecular biochemical pathways. Cold, heat, and freezing caused by extreme temperatures, drought and flooding due to water availability, and vital abiotic stresses have severe effects on the production and growth of plants globally (Mahajan and Tuteja 2005). By comparing record yields and average yields for different crop species under biotic and abiotic conditions, the estimate is reduced by around 20% of their genetic potential. Therefore, it is evident that there is a critical need to strengthen a plant’s ability to withstand abiotic stress. With higher rate of increase in global population and reduction in food production because of various stress factors, increasing the crop production worldwide has become the topmost priority. The scope of improvements in stress tolerance within crops through conventional breeding programs is limited by the complicated mechanisms. Additionally, the methods used in selecting tolerant plants require a lot of time and are therefore expensive. The production of many varieties with relevance to growth and yield is seriously hampered by salinity and drought. One of the main abiotic stresses that affect 20–33% of the world’s cultivated agricultural land is salinity. The foundation of traditional breeding procedures is the availability of genetic resources in addition to the generation and utilization of unique diversity. Through selection and evaluation in the targeted areas affected by salinity and drought, cultivars with improved performance have been created. Salinity-resistant cultivars, including CS52, CS54, and CS56, have been created and released because of concerted breeding efforts. The use of genomics-type technology is starting to have an effect, improving our comprehension of how plants react to abiotic challenges that obstruct their regular growth and metabolism. Abiotic stressors, including low temperature and high salinity, have a negative impact on B. juncea seedling vigor and fertility, which reduces yield. It has been discovered that most abiotic stresses cause overlaps in the expression patterns of stress genes that interact with different signaling pathways, maintaining homeostasis inside the plant cell. To combat the effects of abiotic stresses, transgenic plants have been developed that express genes for the biosynthesis of many substances, including glycine betaine, metal ion homeostasis, late embryogenesis abundant (LEA) proteins, and PR proteins with fixed roles in membrane stability, and osmolyte accumulation (Saha et al. 2016). CODA gene coding for choline oxidase enzyme was incorporated in mustard for salinity tolerance. The enzyme oxidizes choline to betaine, and this increased accumulation is responsible for salt tolerance. Higher germination was seen in transgenic mustard lines compared to control plants exposed under saline condition (Prasad et al. 2000). Transgenic mustard Pusa Jai kishan was developed by incorporating osmotin gene of tobacco for drought and salinity tolerance (Bansal et al. 2007).

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17.4.2 Biotic Stress Various stresses are causing damage to the overall production of the crop. Fungus is the most important viral pathogen causing damage to the crop. A. brassicae causes major biotic stresses and damages the mustard productivity. A wide range of plant diseases are chronic in nature and significantly lower plant productivity. Plants experience a rapid decline in production, along with increased susceptibility to other harmful environmental conditions. Bruce and Pickett (2007)studied the defense signaling that was induced in plants after biotic stress. There is a highly specific initial signaling that elicits differences in defense of attacking organisms; however, some show similarities to subsequent signaling and gene expression responses to different types of attacks (Taylor et al. 2004). The pathways that play a role in defense responses are SA, JA ET, and abscisic acid or methyl jasmonate (Doughty et al.1995). Both JA and SA pathways are involved in various pathogen and herbivore interactions depending on the species involved (Stout et al. 2006). JA- signaling pathways is activated by chewing insects such as lepidopteran, but SA and JA can be induced by spider mites (Leitner et al. 2005). Although plant defenses are frequently tailored to specific intruders, there is still much to learn about the processes underlying this difference (Kaloshian and Walling 2005). Mustard is attacked by different insect pests, among which aphid causes serious damage and loss of yield. Lipaphis erysimi (L.) Kaltenbach of order Hemiptera is a phloem sap-sucking aphid. This aphid covers whole surface of plants, including floral buds, stems, and leaves (Das et al. 2018). An antisense of sucrase1 (SUC1) is introduced in mustard plant, and when aphids rely on mustard sap the RNAi within the gut disturb the expression of SUC1 that is responsible for osmoregulation of phloem sap inside the gut of aphid. The unavailability of SUC1 ultimately reduces the fecundity in aphids (Dhatwalia et al. 2022). The antisense for AQP1 is also a good source for controlling insect herbivory as it plays an important role in osmoregulation (Jing et al. 2016). There were no effective resistant Brassica/germplasm line that led to a complete failure of breeding efforts. Plant lectins are proteins that specifically bind to carbohydrates and are very effective in preventing sap-sucking pests such as aphids due to their agglutination capabilities. It is also known that wheat germ agglutinin (WGA) confers resistance against aphids. WGA binds to the midgut glycoprotein of aphids, which prevents food absorption and results in starving, thereby causing the death of the insect. WGA gene cDNA was overexpressed in the Indian mustard variety RLM-198 by Kanrar et al. 2002using an Agrobacterium-mediated gene transfer approach. Southern blotting confirmed transgene integration, while northern and western blotting proved its expression. High aphid mortality and lower fecundity were observed during a bioassay with mustard aphid using transgenic plants’ leaf discs, showing the efficacy of WGA protein in reducing aphid population. Colocasia esculenta tuber agglutinin (CEA) gene was incorporated for resistance against mustard aphid, L. erysimi. High aphid mortality was observed in transgenic plants expressing CEA protein (Das et al. 2018). Alternaria brassicae Sacc. causes

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Alternaria blight, which is the topmost disease of mustard causing the highest damage and yield losses. In wet seasons and areas of high rainfall, the pathogen reports the highest disease incidence as it is greatly influenced by the weather. At every stage of growth and development, including seed A. brassicae is able to cause diseases. Characterization of symptoms is done by the formation of spots on leaves, stem, and fruit (siliquae). Meur et al. 2015 studied Brassica juncea for determining the route of progression of diseases in Alternaria brassicicola infection. Reactive oxygen species was produced within a period of 16–24 h in Brassica juncea after it was infected with A. brassicicola. After 2 days, severe necrosis with necrotic DNA was seen in the leaves of B. juncea. It was observed that after post infection with A. brassicicola jasmonic acid carboxyl methyl transferase (JMT) was transcriptionally activated. JMT was differentially expressed in different mustard tissues, and protein was detected in leaves of mustard when treated with jasmonic acid, but in young buds it was always found and to a lesser extent in opened flowers. JMT could be a promising target to achieve resistance toward necrotrophic infections since it has engaged in positive feedback regulation of jasmonate system and its responsive genes. Brassica induces an antifungal compound called brassinin after microbial infection. The functions of the Bdtf1 gene were studied in Alternaria brassicicola for testing the importance of brassinin in defense (Srivastava et al. 2013). In Brassica species, some strains of this gene’s mutants were weakly pathogenic, producing lesions in three Brassica species that were 70% smaller in diameter compared to the wild type. In Arabidopsis thaliana, these mutants were nevertheless just as pathogenic as the wild type. Another finding showed that HSP90 is a potential target for suppression in stressed A. brassicicola and indicates that brassinin has substantial negative effects on the plant. This suggests that the fungus’ detoxification of brassinin lowers an essential layer of the plant’s defense (Pedras and Minic2012). Camalexin is a type of phytoalexin that is an indole derivative and plays a major role in disease tolerance against the pathogenic fungus by getting accumulated in the plants. Gaur et al. 2018 studied MAPK signaling cascade, which leads to camalexin biosynthesis that induces defense responses in B. rapa when it comes into contact with the devastating necrotrophic fungus, Alternaria brassicae. Using prior knowledge of MAPK cascade in Arabidopsis thaliana, the following cascade was discovered in MAPK of Brassica rapa genome using molecular modeling, docking, and protein–protein interaction. When anticipated models were molecularly docked to identify probable partners for MAPKs, it was discovered that MKK1, MKK4, MKK5, MAPK3, and MAPK6 had strong interactions with MKK9. Different genes that express and have a significant role in camalexin production in B. rapa during the defense response to A. brassicae are also shown in the MAPK signaling cascade. It will be easier to build plans to make Brassica crops resistant to disease if we have a better understanding of the MAPK defensive signaling system in B. rapa against the destructive fungal pathogen Alternaria brassicae. In the past few years, Indian mustard has been facing a major threat from stem rot. To date, only rust disease is being deployed in resistant breeding programs among other biotic stresses. Chemical fungicides are not effective because the pathotypes

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have an ever-evolving nature. Candidate R genes against S. sclerotiorum, of CCNBS-LRR class, are found on chromosome A9 in B. napus (Mei et al. 2013). The majority of QTLs for S. sclerotiorum have been identified in C genome (C9 and C6) (Li et al. 2015), indicating that B. oleracea is a good source of R genes for S. sclerotiorum. BjuWRR1 gene, which codes for CC-NB-LRR protein, was isolated and identified from an exotic mustard line, Donskaja-IV, and transferred through Agrobacterium to confirm its role in resistance against A. candida pathogen causing white rust (Arora et al. 2019).

17.5

Phytoremediation Agent

Anthropogenic and geogenic activity leads to the release of hazardous substances that contaminate the environment. Generally, these substances are heavy metals and metalloids. These are nondegradable, toxic, and sometimes mutagenic, and are thus inappropriate for agricultural practices as they change the properties and microbiota of soil that leads to reduced fertility. Moreover, they can cause lethal disorders when they come in contact with any living organism through food, water, and air. Metals such as lead and mercury cause neurological disorders and cadmium coagulates in the kidney. Metalloids such as antimony and arsenic are also hazardous. These metals are often absorbed by plants, causing unsuitable changes in enzymatic activity, DNA synthesis, photosynthetic pigments, etc. Apart from this, it has been observed that many plants are able to sequestrate, degrade, and stabilize heavy metals into nontoxic substances within their tissue (Padmavathiamma and Li 2007). For a long time, mustard plants have been utilized in remediation due to their effectiveness and low-cost cultivation. But somehow when the concentration of heavy metals in the mustard changes from moderate to high, a decrease in the height and biomass is observed. Whereas apart from this, there are some varieties that can tolerate the harmful effects and could be isolated for breeding purposes. For instance, Brassica juncea can extract mercury from fly ash (Raj et al. 2020) while lead and cadmium accumulation from mine tailing (Bassegio et al. 2020). Besides, other efforts were made to enhance the level of accumulation without any unacceptable changes in mustard plants. Certain strategies were introduced in which the plants were grown in association with substances such as plant growth regulators, microbes chelating, and acidifying agents (Sarwar et al. 2017). Some illustrations of these approaches are that mustard, in association with plant growthpromoting bacterial (PGPB) species such as Enterobacter and Serratia, has encouraged phytostabalization and phytoextraction, respectively, with increased biomass and root structure (Mendoza-Hernández et al. 2019). In 2021, Rathika et al. reported the enhanced growth and increased survival of mustard plants with biochar and EDTA with lead uptake. Furthermore, Rahman et al. 2013 confirmed that the administration of nitrogen in soil contaminated with copper and lead assists mustard in extracting more metals. The cadmium uptake is highly toxic as it inhibits the root growth and causes accumulation of hydrogen oxides, which damage cells,

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but in the presence of castasterone, the uptake is increased and the addition of citric acid resolves the oxides (Kaur et al. 2017). Other strategies based on the genetic modification of mustard include the production of cisgenic and transgenic varieties through breeding and genetic engineering, respectively, with improved and asymptomatic hyperaccumulation of heavy metals. Moreover, both reassemble heritable quality. Engineering techniques are based on adding such genes that are required to perform translocation and sequestration, thus providing tolerance toward metal stress (Van Aken et al. 2008), and those that are able to equilibrate the toxicity generated via metal uptake. In response to metal stress such as cadmium, vanadium, copper, and mercury, the concentration of non-protein thiol (NPT) pool, glutathione, and phytochelatin is increased (Hou et al. 2019). These molecules reduce the damage caused by oxidative stress. Thus, keeping in mind the natural mechanism of plants, the transformation is of γ gshI gene from E. coli to the chloroplast of B. juncea as performed by Zhu et al. 1999. This gene is responsible for expressing γ glutamylcysteine synthetase, an enzyme that acts as a precursor of glutathione and plays a role in maintaining the redox state within the cell. Furthermore, AtPCS gene, which encodes phytochelatin synthase that is required to convert glutathione into phytochelatin, is exploited from Arabidopsis thaliana to integrate into mustard genome to exhibit enhanced tolerance toward arsenic and cadmium (Gasic and Korban 2007). Whereas to increase tolerance of selenium and its forms selenite and selenate, the Indian mustard was introduced with genes expressing selenocysteine methyltransferase and ATP sulfurylase from Astragalus bisulcatus, which generally grows in selenium-rich soil. The genes accomplished the increased uptake of selenium with no toxic effects (LeDuc et al. 2006).

17.6

Other Improvements

In addition to the above-discussed topics, several other genetic improvements in mustard were also performed such as biofortification and herbicide tolerance. The fortification concept is proposed due to the concern of pseudo hunger that is caused by micronutrient deficiency. As described earlier, mustard is consumed throughout the world, thus it makes sense to fortify mustard with such microelements to fulfill the needs. Moreover, the fortification assists in the defense from different stress. Just like other dietary seeds, mustards are also a source of γ tocopherol, known to have antioxidant properties and useful in lowering the risk of cardiac disorder, cancer, neuron degeneration, and aging. Whereas humans are only able to ingest and absorb α form of tocopherol through hepatic α tocopherol protein receptor (Bramleyet al.2000). However, when the enzyme γ tocopherol methyl transferase (γTMT) is introduced into mustard, it converts the γ into α form of tocopherol (Yusuf and Sarin 2007). Another example of transgenic mustard is fortified with anticancer compound. Glucosinolate is a bioactive compound present in sufficient amount in mustard species and is supposed to provide anticancerous effects when converted into

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sulforaphane. However, an enzyme called AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK has a substrate in between the conversion pathway of glucosinolates to sulforaphane. Thus, these enzymes inhibit the production of sulforaphane. The mustard varieties were designed to suppress the GSL-ALK enzymes (Augustine and Bisht 2015). Bra j 1, a protein of prolamin superfamily found in mustard seeds, is known to cause allergic reaction in people. The accidental consumption of this allergen may lead to life-threatening diseases. Though several approaches such as mutagenesis, RNAi, and breeding were applied to target the allergen, the potency of CRISPR/CAS9 system was high in comparison. The lines obtained through the introduction of CRISPR/CAS9 have confirmed the mutation in the region of Bra j1 gene, and no allergenicity was detected through immunoblotting (Assou et al. 2022). Some of the genetic modified traits with transgene are listed in Table 17.1. Moreover, these traits are approved for cultivation in numerous countries (ISAAA).

17.7

Conclusion

The versatility of mustard makes it an important seed crop; however, the growth potential can be enhanced through numerous gene modification technologies. It has the ability to break all barriers to develop a desirable mustard genome that can combat any unfavorable condition and provide yield to its highest potential. The Brassica crop, which can tolerate biotic as well as abiotic stress, will require fewer agrochemicals and will increase arable land; as a result, the production will increase and eventually the cost will reduce. It is imperative to combine traditional disease control methods with modern biotechnological tools for developing disease-resistant crops. These techniques create numerous varieties with elevated yield and are eco-friendly.

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Table 17.1 Functional traits introduced from various sources in mustard varieties Modified trait Phytase production

Gene introduced and source phy A from Aspergillus niger

Herbicide tolerance

bxn from Klebsiella pneumoniae

Nitrilase degrades oxynil herbicides

Bar from Streptomyces hygroscopicus

Phosphinothricin N-acetyltransferase (modifies phosphinothricin through acetylation) Modified form of 5-enolpyruvulshikimate3-phosphate synthase (EPSPS) enzyme

MON88302 x MS8 x RF3

Barnase ribonuclease (RNAse) enzyme, interfere with the expressed of RNAs in tapetum cells Inhibit the activity of barnase ribonuclease inhibitor Delta-12-desaturase, converts oleic acid to linoleic acid Delta-15-/omega-3desaturase, converts linoleic acid to a-linolenic acid Delta-6-desaturase, convert a-linolenic acid to stearidonic acid Delta-6-elongase, convert stearidonic acid to eicosatetraenoic acid

PHY14, PHY25, PHY35, and PHY36

Cp4 epsps from Agrobacterium tumefaciens strain CP4

Male sterility

Barnase from Bacillus amyloliquefaciens

Fertility restoration

Barstar from Bacillus amyloliquefaciens Lackl-delta12D from Lachancea kluyveri Picpa-omega-3D from Pichia pastoris

Modified fatty acids

Micpu-delta-6D from Micromonas pusilla Pyrco-delta-6E from Pyramimonas cordata

Function Break down the phytase

Event name MPS961, MPS962, MPS963, MPS964 and MPS965 OXY-235

MON88302

DHA canola

Regulatory approval/year The United States/1999

Australia, Canada, China, Japan, New Zealand, and the United States European Union, Japan, Mexico, South Korea, and Taiwan European Union, China, the Philippines, Singapore, Japan, Mexico, South Korea, and the United States European Union, Japan, Mexico, South Korea, and Taiwan

Australia, Canada, New Zealand, and the United States

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Tinkering with Stevia rebaudiana Genome to Improve Its Sweetening Property and Productivity

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Rinku Mondal, Shreyasi Kundu, and Abhijit Bandyopadhyay

18.1

Introduction

Stevia rebaudiana, a member of the Asteraceae family, is perennial in nature and a bushy shrub of South America. Its leaves are being used for various purposes by indigenous people of South America for the past 200 years (Rai and Han 2022). Among 230 species of Stevia, only Stevia rebaudiana Bertoni is capable of producing a sweet diterpene compound, that is, steviol glycosides (SGs) (Peteliuka et al. 2021). In 1899, Moises Santiago de Bertoni, a Paraguayan scientist, recorded Stevia plant for the first time as Eupatorium rebaudianum (Ashwell 2015). Finally, in 1905, Eupatorium rebaudianum was renamed as Stevia rebaudiana, a name based on the chemist Ovidio Rebaudi (Gerwig et al.2016). Stevia is more popularly known as “candy leaf,” “sweet herb,” and “honey leaf” of Paraguay (Rai and Han 2022). Japan is the first country to make Stevia commercial and popular in 1970. Currently, Stevia is distributed to Southeast Asia, Brazil, Kenya, and the United Kingdom (Sararom et al.1982). One of the most important sweetening compounds, that is, Stevioside, was first isolated from Stevia leaves by Rebaudi and Resenae (Bell 1954;Bridal and Lavielle 1931). Steviol glycosides (SGs), the most predominant active constituent of Stevia, responsible for their sweetening property, are approximately 200–300 times sweeter than normal table sugar or sucrose (Singh and Rao 2005). Predominantly, steviol glycosides are synthesized in leaves but are also present in stem in small amounts (Ceunen and Geuns 2013). Due to the presence of additional quality to control various diseases such as obesity, diabetes, cardiac blockage, and hypertension, along with being a healthy alternative sugar source, Stevia has become a third-generation zero-calorie sweetener (Ashwell 2015). Steviol glycosides are neither metabolized R. Mondal · S. Kundu · A. Bandyopadhyay (✉) Plant Genetics & Biotechnology Section, Department of Botany, University of Burdwan, Burdwan, West Bengal, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 S. Tiwari, B. Koul (eds.), Genetic Engineering of Crop Plants for Food and Health Security, https://doi.org/10.1007/978-981-99-5034-8_18

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nor absorbed in the human body (Magnuson et al.2016). Diterpene SGs are also found in Rubus suavissimus (Uhler and Yang 2018;Ohtani et al. 1992; Tanaka et al.1981), Angelica keiskei (Zhou et al.2012), and Stevia phelophylla (Kinghorn et al. 1984), but in trace amounts (Libik-Konieczny et al.2021). That is why Stevia rebaudiana assumes greater economic importance for higher commercially acceptable SG content. There are more than 60 types of SGs present in Stevia rebaudiana and possess properties such as high sweetness but lower calorific value (Petit et al.2020). Along with having a premium quality of sweetness property, SGs show clinically significant attributes (Ahmad et al.2020). Extracts of Stevia leaves help in reducing the level of blood sugar for patients suffering from diabetes (Ahmad et al.2018). A potent variety of steviol glycosides, steviol possesses powerful anticancerous effects toward human gastrointestinal cancer cells (Chen et al.2018a). It also demonstrates the time-dependent and dose-based activity against the expansion of human bone osteosarcoma cell line (U2OS) (Chen et al.2018b). Apart from SGs, there are many other secondary metabolites present in Stevia rebaudiana, such as flavonoids (19.93 mg/g), tannins (56.7 mg/g), phenol (24.61 mg/g), coumarins, and a few important essential oils (Yadav et al.2011). Among various types of SGs, stevioside and rebaudioside (Reb) are the most plentiful in nature. The concentrations of stevioside, rebaudioside A, and Reb M are (5–10%), (2–4%), and (