Genetically Modified Crops: Current Status, Prospects and Challenges Volume 2 [1st ed.] 9789811559310, 9789811559327

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P. B. Kavi Kishor Manchikatla Venkat Rajam T. Pullaiah  Editors

Genetically Modified Crops Current Status, Prospects and Challenges Volume 2

Genetically Modified Crops

P. B. Kavi Kishor • Manchikatla Venkat Rajam • T. Pullaiah Editors

Genetically Modified Crops Current Status, Prospects and Challenges Volume 2

Editors P. B. Kavi Kishor Department of Biotechnology Vignan’s Foundation for Science, Technology & Research Guntur, Andhra Pradesh, India

Manchikatla Venkat Rajam Department of Genetics University of Delhi South Campus New Delhi, India

T. Pullaiah Department of Botany Sri Krishnadevaraya University Anantapur, Andhra Pradesh, India

ISBN 978-981-15-5931-0 ISBN 978-981-15-5932-7 https://doi.org/10.1007/978-981-15-5932-7

(eBook)

# Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are reserved 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

Foreword

To get easy access to food and to improve the productivity of crop plants, humans have used methods of domestication and improvement through selective breeding, based on useful phenotypic traits. It was through the work of Gregor Mendel that we learnt about the genetic basis of plant traits. The first hybrid corn was developed in 1922 by an intelligent breeding strategy. Following the discovery of DNA as the genetic material, work of a number of groups led to the concept of gene as the unit of DNA that controls a phenotypic character of an organism. And it was in 1973 that Herbert Boyer and Stanley Cohen developed genetic engineering by inserting DNA from one bacterium to another. Around the same time Jeff Schell and Marc Van Montagu discovered that it is due to the transfer of the plasmid DNA of Agrobacterium tumefaciens that results in tumor formation in plants. This research was a by-product of curiosity-driven science and based on fundamental scientific discovery. Using this information, and developing plant transformation technology, group of Mary-Dell Chilton and R. Fraley and scientists from the Monsanto Company created the first transgenic plant. During the mid-1990s, with the creation of GM tomato, the initial wave of GM plants was set in motion. However, due to certain issues of public acceptability and stringent regulatory laws that were put in place in different countries, the growth of this technology was slowed down. Van Montagu, whom I have had the pleasure of meeting and knowing for a long time, wrote an insightful article in the Annual Review of Plant Biology in 2011 titled “It is long way to GM Agriculture.” Even then this technology has been used in many crops and the global biotech crop area is steadily increasing within many countries which have adopted this technology for crop improvement in their agriculture systems. Unfortunately, due to various social and political issues the adoption of this technology has received resistance. This trend needs to be reversed. In the meanwhile, one has seen the emergence of new technologies like RNAi to silence the expression of genes to understand their role as also to develop novel transgenic plants with useful traits. And since 2015, gene editing technologies have evolved which have become useful and efficient tools to manipulate DNA in plant cells. And now we are moving onwards to the precision genome engineering though prime genome editing which does not involve double-strand breaks and donor DNA templates. Hopefully, these interventions will not be subjected to as much stringent regulatory procedures and will also find better acceptability in the society. v

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An article was published in EMBO Reports by Fagerstrom et al. in 2013, entitled “Stop Worrying Start Growing” with the subtitle, “Risk research on GM crops is a dead parrot, it is time to start reaping the benefits of GM.” This is even more true today. The present volumes by Professors Kavi Kishor, Rajam, and Pullaiah have been compiled to convey the same message by presenting achievements and opportunity of employing different technological tools for genetic improvement of plants. I have known the editors of this volume for a long time. They have themselves made significant contributions in the area of plant biotechnology and are well acquainted with GMOs, in all its perspectives. They are also aware of the views of opponents of this technology. Accordingly, taking these into considerations too, they have broadly outlined the status, prospects, and challenges of different genetic interventions in various plants of economic importance for improving traits like developing resistance to viral, insect, and other diseases and for conferring tolerance to abiotic stresses. With rapid advancements in genome sequencing methodologies and functional genomics tools, it has now been possible to identify the genes which can be deployed in a very precise manner using efficient transformation techniques. These volumes cover, among cereals, a chapter on rice that deals with the use of GM technology to address the problem of food and nutrition security and a chapter each on wheat and finger millet. Legumes, which remained recalcitrant for a long time and an efficient transformation system was not available, have now been tamed. This family of plants have received special attention, and a chapter each on pigeonpea, chickpea, cowpea, and peanut have found a place in this volume. Among vegetables there is a detailed account on the present status on brinjal, tomato, cucurbits, and one chapter each on redpepper and capsicum. Other plants of importance which have been included are sugarcane, cassava, banana, papaya, citrus, mulberry, and jatropha. Work on two oil plants, sunflower and safflower, has been presented in two independent chapters. This approach of illustrating the use of the technology for each species separately, rather than group them on specific trait, I find, provides better perspective to evaluate the importance of GM technology with respect to each plant species. These volumes, I am very sure, will be useful to all students and practitioners of biotechnology, be in colleges, universities, and private organizations, as well as for policy makers and regulators in the government agencies. I look forward to the deployment of the safe use of new tools and techniques of genetic manipulation for the improvement of important plants on a large scale in our agriculture and horticulture system. This will help, along with other breeding methodologies, including marker-assisted breeding, to sustain productivity with limited inputs. We hope to see hunger-free world in the years to come. International Centre for Genetic Engineering and Biotechnology New Delhi, India June 06, 2020

Sudhir K. Sopory

Preface

Plants provide us many essential things in life, including food, feed, cloth, wood, paper, medicinal compounds, industrial products, and most importantly the life sustaining molecule oxygen to breath. Plants are also crucial to clean lifesaving water. There are only six crop plants, viz., rice, wheat, corn, potato, sweet potato, and cassava, which provide about 80% calories to humans. There are other important crops like sugarcane, barley, sorghum, bean, soybean, coconut, and banana, which are also being consumed by humans. But crop plants are vulnerable for various biotic factors (pathogens and pests) and extreme environmental conditions or abiotic stresses (e.g., high salinity, drought, heat and cold, heavy metal and submergence) because of their sessile nature. These stresses cause a colossal loss of crop yields and impair nutritional quality. Otherwise, one can realize the potential and harvest 100% agricultural productivity from all crops. In addition, global warming, shrinking water resources, arable land, and population growth are aggravating the problem of food security. In fact, these are key scientific issues in agriculture besides post-harvest losses and impairment in nutritional quality. Then the critical question that arises in our minds is how to harness the full yield potentials of crops without compromising the quality component. The answer lies evidently in the exploitation of diverse technologies, particularly plant breeding and genetic engineering. Between plant breeding and genetic engineering, the former has contributed significantly for more than seven decades to crop improvement and in fact almost all the new and improved varieties were virtually derived through breeding strategies. However, breeding methods suffer from certain limitations like incompatibility barriers or narrow mobilization of useful genes between closely related species. This leads to the problem of using only limited gene pool and there is no way to transfer a single beneficial gene since we generally transfer a cluster of genes/chromosomes during the crosses, thus subject F1 hybrids for 4–5 back-crosses to chuck away the unacceptable. It takes nearly 10–12 years to develop a new variety with desirable traits and may not be cost-effective. In contrast, genetic transformation by Agrobacterium or other gene delivery systems or transgenic technology offers several advantages such as precise gene transfer from any source to crop plants. This means a huge gene pool exists for transfer of desirable traits across species and takes relatively 7–9 years to develop a transgenic line of interest. Consequently, genetic engineering holds great promise for crop improvement and is essential since huge gap exists between vii

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food production and rate of population growth. Today’s world population is about 7.7 billion and expected to reach 9.7 billion by 2050, and further to an estimated 11 billion by 2100. Human hunger and malnutrition are the major problems, especially in Asian countries due to accelerating birth rate. So, it is a challenge for plant biologists and biotechnologists to resolve the problem of human hunger and malnutrition through crop improvement programs. In reality, about 70% increase in food production is required by 2050 to feed the growing masses; otherwise we may face great famines in the near future. Indeed, this suggests that a second green revolution is the need of the hour to bring food security to the world population, and this can only happen if we couple the conventional breeding strategies with genetic engineering technologies. Transgenic technology has already proven to be novel and a potential alternative for crop improvement, and a handful of transgenic varieties like cotton, corn, soybean, and canola have been commercialized globally. This has led to a substantial increase in crop yield and quality and reduced use of harmful pesticides, reduction in CO2 emissions, and a decrease in the cost of crop production, besides improving the economy of marginal farmers. The first transgenic variety, flavr savr—the slow ripening tomato, was commercialized in 1994 in the USA, and since then there is a steady increase in the adoption of the first generation of genetically modified (GM) crops such as corn, cotton, and soybean for insect resistance, herbicide tolerance, and improvement of oil quality. In 2018, about 475 million acres (191.7 million hectares) of land were under the cultivation of various GM crops in 26 countries (21 developing and five developed countries), including five top countries—USA, Argentina, Brazil, Canada, and India (with the adoption of only Bt cotton) with the largest area of GM crops grown, and an additional 44 countries imported these GM crops. To date, about 525 different transgenic events in 32 crops have been approved for cultivation in different parts of the world. Currently, the next generation of transgenic plants displayed potential for the production of bio-ethanol, bio-plastics, and many pharmaceutically important recombinant proteins and compounds. Interestingly, the recent genome engineering or editing technology is quickly gaining importance for maneuvering genes in crop plants using the gene editing tool, the CRISPR-Cas system. This technology is aiding us in the improvement of many agronomically important traits such as yield, stress tolerance, and nutritional quality. Soon, the gene-edited crop plants with new traits, but not having an alien gene, will be commercialized. Such an endeavor will assist us in meeting the increasing food demands and global food security. This technology can be safely exploited since it has minimum or no regulatory issues. GM crops have the most rapid adoption rate in the history in spite of public concerns as compared to the traditional hybrids like corn, which took more than seven decades for global penetration. Transgenic varieties were released only after passing the tests against environmental aggressiveness, toxicity, allergenicity, after fulfilling the stringent regulatory guidelines laid down by the respective countries, and after exhibiting their superiority for field performance vis-à-vis the untransformed or wild-type plants. The present book brought in two volumes has updated information about the current status of GM crops. While the first volume covers genetic modification

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studies in cereals, pulses, and oil-yielding crops, the second one includes information on important vegetable, fruit-yielding, and commercial crops. These volumes on GM crops will be handy to students of life science stream of both undergraduate and postgraduate studies, research scholars, postdocs and researchers working in plant and agricultural biotechnology organizations, faculty members, biotech companies and professionals alike. Lastly, we would like to express our heartfelt gratitude to Springer Nature for kindly consenting to bring out this book in two volumes and for extending support through various phases and for the timely completion of publishing. Our heartfelt thanks are also due to Prof. Sudhir K. Sopory, ICGEB, New Delhi, for writing the foreword. We would like to thank all the authors/coauthors who have contributed the review articles and also for their cooperation and erudition. Hyderabad, Andhra Pradesh, India New Delhi, India Anantapur, Andhra Pradesh, India

P. B. Kavi Kishor M. V. Rajam T. Pullaiah

Contents

Transgenic Tomatoes for Abiotic Stress Tolerance and Fruit Traits: A Review of Progress and a Preview of Potential . . . . . . . . . . . . . . . . . . P. Hima Kumari, S. Anil Kumar, G. Rajasheker, D. Madhavi, N. Jalaja, K. Kavya Shridhar, K. P. Scinthia, D. Divya, M. Swathi Sri, Ch. Akhila, E. Sujatha, P. Rathnagiri, and P. B. Kavi Kishor

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Genetically Modified Brinjal (Solanum melongena L.) and Beyond . . . . C. Kiranmai, T. Pullaiah, and M. V. Rajam

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Biotechnology of Red Pepper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. V. Rajam, S. Nandy, and R. Pandey

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Non-host Armor Against Insect: Characterization and Application of Capsicum annuum Protease Inhibitors in Developing Insect Tolerant Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. S. Tanpure, K. R. Kondhare, V. Venkatesh, V. S. Gupta, R. S. Joshi, and A. P. Giri

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Transgenic Banana: Current Status, Opportunities and Challenges . . . . 111 T. R. Ganapathi, Sanjana Negi, Himanshu Tak, and V. A. Bapat Transgenic Papaya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Melaine Randle and Paula Tennant Genetically Modified Citrus: Current Status, Prospects, and Future Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Sameena E. Tanwir, Juliana M. Soares, Stacy Welker, Jude W. Grosser, and Manjul Dutt Modified Cassava: The Last Hope That Could Help to Feed the World—Recent Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Charles Oluwaseun Adetunji, Muhammad Akram, Areeba Imtiaz, Ehis-Eriakha Chioma Bertha, Adrish Sohail, Oluwaseyi Paul Olaniyan, Rabia Zahid, Juliana Bunmi Adetunji, Goddidit Esiro Enoyoze, and Neera Bhalla Sarin

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Contents

Transgenics for Targeted Trait Manipulation: The Current Status of Genetically Engineered Mulberry Crop . . . . . . . . . . . . . . . . . . . . . . . 221 K. H. Dhanyalakshmi, H. V. Chaithra, R. S. Sajeevan, and K. N. Nataraja Genetically Engineered Jatropha: A New Bioenergy Crop . . . . . . . . . . . 237 G. Raja Krishna Kumar, Nalini Eswaran, and T. Sudhakar Johnson GM Crops for Plant Virus Resistance: A Review . . . . . . . . . . . . . . . . . . 257 A. M. Anthony Johnson, D. V. R. Sai Gopal, and Chinta Sudhakar

About the Editors

P. B. Kavi Kishor holds a PhD in Botany from Maharaja Sayaji Rao University of Baroda, Vadodara, Gujarat. He was a Visiting Professor at the Biotechnology Center, Ohio State University, Columbus, Ohio, USA, under the Rockefeller Foundation program; Emory University, Atlanta, Georgia, USA; Linkoping University, Sweden; and a Visiting Scientist at the Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany. He has published 255 papers and edited or written five books. He is a Fellow of the National Academy of Sciences (FNASc) and the National Academy of Agricultural Sciences (FNAAS), and he holds one patent. Manchikatla Venkat Rajam is a Professor and UGC BSR Faculty Fellow in the Department of Genetics at the University of Delhi South Campus, New Delhi, and has also served as head of the department. He holds a PhD in Botany from Kakatiya University, Warangal, India, and was a Postdoctoral Fellow at Yale University, New Haven, USA. He also worked as a Visiting Research Associate at Boyce Thompson Institute (BTI), Cornell University, Ithaca, USA. He is a Fellow of the Indian National Science Academy (FNA), National Academy of Sciences, India (FNASc), and National Academy of Agricultural Sciences (FNAAS). He has published 144 papers and is a co-editor of a two-volume book on plant biology and biotechnology, published in 2015 by Springer India. T. Pullaiah is a former Professor in the Department of Botany at Sri Krishnadevaraya University in Andhra Pradesh, India. He has held several positions at the university and was President of the Indian Botanical Society and of the Indian Association for Angiosperm Taxonomy. He holds a PhD from Andhra University, India, and was a Postdoctoral Fellow at Moscow State University, Russia. He was awarded the Panchanan Maheshwari Gold Medal, the Dr. G. Panigrahi Memorial Lecture award of the Indian Botanical Society, and the Prof. Y.D. Tyagi Gold Medal of the Indian Association for Angiosperm Taxonomy. He has authored 51 books, edited 19 books, and published over 330 research papers. He was a member of the Species Survival Commission of the International Union for Conservation of Nature (IUCN).

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Transgenic Tomatoes for Abiotic Stress Tolerance and Fruit Traits: A Review of Progress and a Preview of Potential P. Hima Kumari, S. Anil Kumar, G. Rajasheker, D. Madhavi, N. Jalaja, K. Kavya Shridhar, K. P. Scinthia, D. Divya, M. Swathi Sri, Ch. Akhila, E. Sujatha, P. Rathnagiri, and P. B. Kavi Kishor

Abstract

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop of the world. It is rich in nutrition with zero cholesterol, but highly sensitive to abiotic stresses, especially salt, drought, and high temperatures. Development of transgenic tomatoes that are climate resilient coupled with high nutritional value and improved shelf-life of fruit is the need of the hour. Utilization of conventional plant breeding methods and genetic engineering technologies must therefore be vital to achieve these goals. Tomatoes overexpressing transgenes and transcription factors conferred tolerance against different abiotic stresses with increased fruit production in comparison with wild-type (WT) plants. Similarly, delayed fruit ripening and nutritional quality of the fruit have been achieved in tomato. The present review describes the current status of the development of transgenic tomatoes that are tolerant to diverse abiotic stresses alongside delayed fruit ripening and other quality attributes and projects the potential areas for future research.

P. H. Kumari · G. Rajasheker · D. Madhavi · K. K. Shridhar · K. P. Scinthia · D. Divya · M. S. Sri · C. Akhila Department of Genetics, Osmania University, Hyderabad, India S. A. Kumar · N. Jalaja · P. B. Kavi Kishor (*) Department of Biotechnology, Vignan’s Foundation for Science, Technology & Research, Guntur, Andhra Pradesh, India E. Sujatha Department of Botany, Osmania University, Hyderabad, India P. Rathnagiri Department of Genetics, Osmania University, Hyderabad, India Genomix CARL Pvt. Ltd., Pulivendula, Kadapa, Andhra Pradesh, India # Springer Nature Singapore Pte Ltd. 2021 P. B. K. Kishor et al. (eds.), Genetically Modified Crops, https://doi.org/10.1007/978-981-15-5932-7_1

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Keywords

Lycopersicon esculentum · Fruit quality · Transgenic tomato · Abiotic stresses · Polyamines

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Introduction

Plants always encounter various biotic and abiotic stresses which can trigger a series of biological, morphological, and physiological changes leading to metabolic derailment of cellular activities, resulting in reduced growth as well as yield (Rodríguez et al. 2005; Zhou et al. 2011). Biotic and abiotic stresses bring severe metabolic alterations and affect up to 50% of crop productivity every year (Boyer 1982; Wang et al. 2003; Oerke 2006). Plants respond to these stresses by a cascade of interactions which are complex and integrative (Atkinson and Urwin 2012). It is crucial to produce 50% more food by 2050 in order to meet the demands of the growing population (Godfray et al. 2010). This is further compounded, because of the limitations in the availability of water, land, and other natural resources. Abiotic stresses, especially salinity, drought, and high temperature, are responsible for reduced crop growth and cause economic losses in agricultural production. Further, demands are also raising worldwide for more nutritious fruits. Tomato (Lycopersicon esculentum Mill.) is the second most produced and consumed vegetable crop originated from South America and spread throughout the world after Spanish colonization. It is a diploid (2n ¼ 24) crop that belongs to the genus Lycopersicon of Solanaceae family (Weese and Bohs 2007). The word “tomato” is derived from Spanish “tomate”. Tomato was renamed from Solanum lycopersicum L. to Lycopersicon esculentum based on molecular and phylogenetic data (Foolad 2007; Peralta and Spooner 2007). Botanically tomato is a fruit berry, but due to its vast usage for culinary purposes, it is treated as vegetable. Due to the presence of 16 elements and important nutrients, it is often regarded as “poor man’s orange”. Tomato has become a model organism due to its smaller genome, short life span, absence of gene duplication, ability to grow in a wide variety of climatic conditions, and ease of controlled pollination and hybridization (Ranjan et al. 2012; Bergougnoux 2014; Schwarz et al. 2014). Currently, total world production of tomatoes is about 182 million tonnes. India ranks second with 20 million tonnes after China with 60 million tonnes in its production (FAOSTAT 2017). Asia dominates the global tomato production (Fig. 1).

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Morphological and Geographical Diversity of Tomato

Tomato shows remarkable diversity in morphological/phenotypic characters and geographical distribution. It typically grows up to 1–3 m (3–10 ft) in height and has a weak stem that often sprawls over the ground and vines over other plants.

Transgenic Tomatoes for Abiotic Stress Tolerance and Fruit Traits: A Review of. . .

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Fig. 1 Schematic representation of production share of tomatoes during 2017 by different regions of the world. (Source: FAOSTAT 2017)

Branches are usually sub-opposite with basic cyme inflorescence. It grows as perennial in its native habitat and as an annual in temperate climates. Cultivated tomatoes are generally self-pollinated, though controlled crosses can be made. Tomatoes vary in size, for example, tom berries measure 5 mm, cherry tomatoes 1–2 cm, and wild beefsteak 10 cm or more in diameter. The most widely grown commercial tomatoes vary in the diameter range of 5–6 cm. It also shows variation in fruit weight ranging from 20 g (cherry tomato) to 500 g (beef tomato), and on average, common tomato weighs approximately up to 100 g. Most cultivars produce red fruits, but a number of cultivars produce yellow, orange, pink, purple, green, black, and white also. Multicoloured and striped fruits are quite striking. Different fruit shapes are observed in tomato including round, oblate, pear, torpedo, or bellshaped. More than ten quantitative trait loci (QTLs) are associated with size and shape of cultivated tomatoes (Tanksley 2004). Today, cultivars are seen all over the world, but the wild species are restricted to certain areas (Table 1).

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Nutritional Value of Tomatoes

Tomato (100 g) is one of the low-calorie (18 kcal) vegetables with zero cholesterol levels. The water content of tomato is around 95% and carbohydrates and fibre constitutes the other 5%. It has important nutrients like flavonoids, folic acid, carotenoids, lycopene, and ascorbic acid (AsA), which act as antioxidants. Tomato suppresses cancer cell proliferation, protects from prostate cancers, digestive tract, and cardiovascular disorders (Franceschi et al. 1994; Giovannucci et al. 1995; Levy et al. 1995; Willcox et al. 2003). The consumption of tomato significantly increases the lycopenes, total skin carotenoids, phytofluene, and phytoene levels in human serum which protects the skin against UV-light-induced erythema (Aust et al. 2005). The tomato epicarp contains naringin, which reduces the inflammation, atherosclerosis, cardiovascular disorders, diabetes mellitus and acts as an antioxidant (Bharti

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Table 1 List of cultivated and wild tomatoes and their geographical distribution S. no. 1. 2. 3.

Cultivated/wild Lycopersicon esculentum (Syn.: Solanum lycopersicum) (cultivar) Solanum galapagense (wild) Solanum cheesmaniae (wild)

4. 5. 6. 7. 8. 9.

Solanum pimpinellifolium (wild) Solanum chilense (wild) Solanum chmielewskii (wild) Solanum habrochaites (wild) Solanum pennellii (wild) Solanum eorickii (wild)

10. 11. 12. 13.

Solanum arcanum (wild) Solanum huaylasense (wild) Solanum peruvianum (wild) Solanum corneliomuelleri (wild)

Geographical distribution All over the world Galapagos islands Galapagos islands and Ecuador Ecuador and Chile Peru and Chile Peru and Bolivia Ecuador and Chile Peru and Chile Ecuador, Peru, and Andean valley Peru and Andean valley Peru Peru and Chile Peru

et al. 2014). Tomatoes also play a vital role in bone health with significant increase in the growth of femur and tibia (Choudhary et al. 2016).

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Need for Genetic Engineering of Tomato with Altered Traits

Genetic engineering is as an alternative to conventional plant breeding methods for the development of transgenic crops by introduction of alien genes with better agronomic characters. Conventional methods of breeding takes 5–7 years for the development of new tomato cultivars (Vinocur and Altman 2005; Causse et al. 2007). But through genetic engineering techniques, one can introduce the genes of interest within a short span of time and develop a line with traits of interest. Genetically modified crops for biotic and abiotic stresses are gaining importance all over the world. McCormick et al. (1986) produced the first transformed tomato. The flavr savr (also known as CGN-89564-2 and pronounced as “flavour saver”) is the first commercial genetically modified tomato used for human consumption (Kramer and Redenbaugh 1994). Tomatoes have a short shelf life, but need to be transported for long distances within the country and also to other countries. This necessitates to develop fruits with improved shelf life. Hence, flavr savr tomatoes were marked initially, but withdrawn subsequently. Salt, drought, and other abiotic stress factors show adverse effects on tomato production and fruit quality. We do not have tomato cultivars that can withstand high-salt, severe water-deficit conditions or low and high temperatures. So, to overcome these stresses, one way is to grow tomato plants in saline irrigated lands, and to adopt crop rotation methods. But, this is an ineffective method as most of the soils are saline and availability of fresh water

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is meagre. Another way to overcome these problems is to genetically engineer elite varieties with genes that confer tolerance to salt, drought, cold, and high temperature stresses (Table 2). Therefore, development of tomato and other crops that are tolerant to abiotic stresses is crucial especially in the wake of climate change. Hiwasa-Tanase et al. (2012) reviewed the role of tomato fruits as biofactories for the production of recombinant products and artificial sweeteners. Klee and Giovannoni (2011) elaborately reviewed the process of tomato fruit ripening. In the present study, we reviewed the development of tomato transgenics for abiotic stress tolerance and fruit quality attributes including ripening that have not been covered earlier.

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Development of Transgenic Tomato Plants for Salt Stress Tolerance

Salinity results in the loss of water content and turgor from leaf cells which limits the ability of plant growth and decreases the final productivity. Salinity also affects the closure of stomatal apertures and reduces the photosynthetic rate with an increase in the formation of reactive oxygen species (ROS) resulting in an oxidative stress. The strategies and mechanisms for sodium (Na+) transport include Na+ exclusion from the cell, or its inclusion into vacuole and intracellular compartmentation and acquisition of potassium (K+) to cope with osmotic stress during different developmental stages of plant growth. K+ maintains ion homeostasis of the cell, membrane potential, photosynthesis, and enzyme activation. In many crops, salt tolerance is achieved by Na+ exclusion. This minimizes the damage caused by the accumulation of Na+ ions (Munns 2002; Tester and Davenport 2003). Tomato production has been limited by a high level of salinity in the soil or irrigation water. It is sensitive to moderate levels of salinity like most other crop plants. Seed germination, vegetative growth, and reproductive stages of tomato show high sensitivity to salt stress, and economic yield is drastically reduced under these conditions (Maas 1986; Bolarín et al. 1996). Salinity stress results in the Na+ toxicity which impairs electroneutrality of the cell by altering the metabolic process. Regulations of Na+ transport rate from root to the shoot and tissue tolerance are the critical factors for salinity tolerance (Shabala 2013; Maathuis 2014). Elevated salinity levels for longer period compounds the problem of drought stress also (Munns and Tester 2008). A number of candidate genes associated with salt stress tolerance have been identified and overexpressed for salt stress tolerance in tomatoes which are briefly described below. Transgenic tomato plants overexpressing a vacuolar Na+/H+ antiporter (NHX) accumulated high amount of Na+ in leaves but not in fruits when grown in the presence of 200 mM NaCl (Zhang and Blumwald 2001). Tomatoes expressing Na+/ H+ antiporter-like protein (NHXLP) conferred tolerance to salt stress by low accumulation of Na+. Transgenics displayed higher proline, K+, improved cambial conductivity, and fruit yield in comparison with WT plants (Kumari et al. 2017). Transgenic tomato co-expressing Arabidopsis thaliana vacuolar H+pyrophosphatase (AVP1) and Pennisetum glaucum vacuolar Na+/H+ antiporter

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Table 2 List of tomato transgenics developed for abiotic stress tolerance Genes and their source GRF9 from Arabidopsis SbNHXLP from Sorghum

Improved tolerance to Improved phosphate deficiency Salt stress

Choline oxidase gene (codA) from Arthrobacter globiformis EgCBF3 from Elaeis guineensis

Salt stress

Osmotin-like protein (OLP) and Chitinase (Chi11) from Solanum and rice LeFAD7 from tomato

Salt and drought stresses

FaGalUR from strawberry miR399 from rice ICE1 from Arabidopsis AtHMA4 from Arabidopsis

Abiotic stress

High temperature

AtDREB1A from Arabidopsis BADH from Suaeda liaotungensis

Salt and cold conditions Salt and cold conditions Cold stress Enhanced Zn translocation Reduction in heatinduced photoinhibition Drought stress Salt, drought, and cold stresses Drought and oxidative stresses Drought stress Salt stress

HMA4 (P1B-ATPase) from Arabidopsis halleri

Increased uptake of zinc

MDHAR from tomato SpUSP from tomato

Increased tolerance to salt and osmotic stresses Drought stress

Dehydrin (TAS14) from tomato

Salt and drought stresses

CaRma1H1 from Capsicum annum MhGLB1 from Malus hupehensis mhgai2 from apple

Drought stress Hypoxia stress Salt and drought stresses

MdCIPK6L and MdCIPK6LT175D from apple

Salt, drought, and chilling stress Salt stress

BADH from spinach StAPX from tomato MdoMYB121 from apple ZAT12 from Brassica carinata

Co-expression of AVP1 and PgNHX1 (vacuolar H+ pyrophosphatase from Arabidopsis thaliana and PgNHX1 from Pennisetum glaucum) CaXTH3 xyloglucan endotransglucosylase/ hydrolase from Capsicum annuum

Salt and drought stresses

References Zhang et al. (2018a) Kumari et al. (2017) Wei et al. (2017) Ebrahimi et al. (2016) Kumar et al. (2016) Nakamura et al. (2016) Cai et al. (2015) Gao et al. (2015) Juan et al. (2015) Kendziorek et al. (2014) Li et al. (2014) Sun et al. (2014) Cao et al. (2013) Rai et al. (2012, 2013a) Rai et al. (2013b) Wang et al. (2013) Barabasz et al. (2012) Li et al. (2012) Loukehaich et al. (2012) Muñoz-Mayor et al. (2012) Seo et al. (2012) Shi et al. (2012) Wang et al. (2012a) Wang et al. (2012b) Bhaskaran and Savithramma (2011) Choi et al. (2011) (continued)

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Table 2 (continued) Genes and their source MdVHP1 from apple

Improved tolerance to Salt stress

coda from Arthrobacter globiformis MdSPDS1 (spermidine synthase 1) from apple SlXTH1 (xyloglucan endotransglucosylase/ hydrolase) from tomato HALI from yeast

Salt and drought stresses Salt stress Altered fruit characteristics Salt stress

At-CBF1 (C-repeat binding factor 1) from Arabidopsis CAX1 from Arabidopsis

Cold stress

AtNHX1 from Arabidopsis

Ca2+ accumulation and fruit shelf-life improvement Salt stress

References Dong et al. (2011) Goel et al. (2011) Neily et al. 2011 Ohba et al. (2011) Safdar et al. (2011) Singh et al. (2011) Park et al. (2005)

Zhang and Blumwald (2001)

(PgNHX1) genes showed higher degree of salt tolerance when compared to plants where the genes were expressed individually. With the overexpression of PgNHX1 gene, higher salt stress tolerance was noticed. The tolerance mechanism has been found to be due to the exclusion of Na+ at the root level and also due to sequestration of cytosolic Na+ into the vacuoles. Transgenics also displayed higher proline and chlorophyll content compared to WT plants (Bhaskaran and Savithramma 2011). Genes associated with osmolyte biosynthesis have been deployed for salt stress tolerance with great success. Osmolytes act as osmotic balancing agents and have the ability to quench/scavenge the ROS. Transgenic tomatoes expressing betaine aldehyde dehydrogenase (BADH) gene, driven by CaMV35S promoter, displayed salt stress tolerance. Similarly, overexpression of BADH driven by stress-inducible promoter P5 showed higher tolerance to salt stress than CaMV35S promoter (Wang et al. 2013). Tomato transformed with the choline oxidase gene (codA) from Arthrobacter globiformis accumulated glycine betaine (GB), which was not reported in WT plants. Upon exposure to salt stress, the codA transgenics showed higher photosynthetic rates, antioxidant enzyme activities, and lower accumulation of reactive oxygen species (ROS). The quantitative real-time PCR (qRT-PCR) experiments in codA transgenics revealed higher GB, enhanced expression of K+ transporter, Na+/H+ antiporter, and H+-ATPase genes under salt stress conditions. The codA gene also regulated the ion channel and transporters as evident by high K+ to Na+ ratio in transgenics treated with salt (Wei et al. 2017). Genes associated with antioxidative metabolism have also been tested with over all positive effect. Monodehydroascorbate reductase (MDHAR) catalyses the reduction of monodehydroascorbate (MDHA) to ascorbate (AsA) and the increased AsA alleviates the photoinhibition of PSII. Tomatoes expressing sense MDHAR showed higher mass, height, and increased tolerance to salt and osmotic stress than the

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antisense and WT. The AsA levels were high in sense plants followed by WT and antisense (Li et al. 2012). Tomato containing the gene mhgai2 exhibited more resistance to drought and salt stresses than the WT plants at the seedling stage (Wang et al. 2012a). In addition to transgenes, an array of transcription factors were also employed in genetic transformation of tomato for tolerance to salt, drought, and cold (Liu et al. 1998; Lindemose et al. 2013; Nakashima et al. 2014). Of the various transcription factors, members of the MYB family were widely studied. MdoMYB121 was induced under multiple stress conditions. Compared to WT plants, transgenic tomato expressing MdoMYB121 showed better tolerance to salt stress (Cao et al. 2013). Tomato overexpressing strawberry FaGalUR gene exhibited elevated tolerance to salt stress with twofold increase in AsA content in tomato fruit (Cai et al. 2015). Transgenic tomato overexpressing athmiR399d under the control of rd29A promoter displayed tolerance to salt stress. It also increased the biomass of tomato under low-temperature and phosphate (P) deficiency conditions (Gao et al. 2015). Overexpression of Capsicum annuum gene (CaRma1H1), an endoplasmic reticulum-localized protein, displayed enhanced tolerance to both salt and drought stresses compared to WT tomato plants (Seo et al. 2012). Interestingly, tomato overexpressing osmotin-like protein (OLP) and chitinase (Chi11) double construct exhibited tolerance to salt as well as drought. These plants showed higher proline, K+, total biomass, and relative water content, than the WT under salt and drought stress conditions in the pots (Kumar et al. 2016). Thus, an array of genes isolated from diverse pathways and different species exhibited tolerance to salt stress in tomato upon overexpression, but field level trials were not conducted in majority of the cases mentioned above. However, potential exists for further validating a great deal of transcription factors that have been isolated which can modulate downstream genes and impart salt stress tolerance.

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Transgenic Tomatoes for Water-Deficit Conditions or Drought-Affected Areas

Drought or water deficit is an important abiotic stress which results in reduced growth and final productivity (Jacob 2008). Plants have developed efficient cellular and molecular mechanisms to cope with water-deficit conditions (Kramer and Boyer 1995). Drought stress results in ion imbalance as well as production of ROS (Mittler 2002). To maintain the osmotic potential of the cells, plants accumulate inorganic solutes in the vacuoles (like Na+ and K+) and compatible solutes like proline, GB, and sugars in the cytoplasm and thus favour the uptake of water from the soil. Antioxidative compounds and ascorbate peroxidases (APX) quench the ROS and degrade H2O2 to water. Transgenic tomatoes overexpressing GA3 Della protein (mhgai2) are insensitive to exogenous gibberellins and are more resistant to drought than WT plants, but produced smaller flowers and seeds (Wang et al. 2012a). Similarly, tomatoes overexpressing Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (SlAPX) displayed higher APX activity, with subsequent tolerance to drought (Sun et al. 2014). Transgenics recorded higher yields compared

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to WT plants (Sun et al. 2014). Transcription factors also play crucial roles during water-deficit conditions. Cao et al. (2013) developed transgenics by introducing MdoMYB121 gene, which showed tolerance to water-deprived conditions. Similarly, tomatoes overexpressing AtDREB1A/CBF3 driven by rd29A promoter exhibited drought tolerance with lower levels of superoxide and H2O2 compared to WT plants. A significant increase in the antioxidant activity was noticed in transgenic plants (Rai et al. 2013b). Tomato overexpressing ZAT12, a C2H2 zinc finger, confirmed tolerance against drought and oxidative stresses by accumulating higher proline in comparison with WT plants (Rai et al. 2012, 2013a). The universal stress protein (SpUSP) is induced by salt, drought, abscisic acid (ABA), and oxidative stresses. Loukehaich et al. (2012) demonstrated that transgenic tomato plants expressing SpUSP exhibited increased tolerance to water deficit conditions. SpUSP expression enhanced the accumulation of ABA which in turn regulated water loss by closing the stomata. Besides drought tolerance, SpUSP tomato plants performed well under oxidative stress. It appears that SpUSP gene interacts with annexin and modulates ABA-induced stomatal closure, which prevents water loss, and hence imparts drought tolerance (Loukehaich et al. 2012). Late embryogenesis abundant (LEA) proteins or dehydrins play a prime role during drought stress tolerance. Transgenic tomato expressing dehydrin TAS14 driven by CaMV35S exhibited tolerance to both salt and drought stresses with increased accumulation of K+ and sugars in comparison with WT plants (Muñoz-Mayor et al. 2012).

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High-Temperature Stress-Tolerant Transgenic Tomato

Moderate heat stress inhibits the repair mechanism of photosystem II (PSII) without causing photo-damage directly (Chen and Murata 2011). On the other hand, high temperature induces PSII inhibition and hence affects plant growth and metabolism. Osmolyte GB accumulates rapidly under heat stress and provides tolerance to plants by acting as a compatible solute. Also, it enhances repair of PSII induced by heat stress by lowering the levels of ROS (Allakhverdiev et al. 2007; Yang et al. 2007). Transgenic tomato overexpressing BADH exhibited higher GB accumulation with increase in chlorophyll fluorescence compared to WT plants upon exposure to 42  C. Transgenics revealed an increase in D1 protein content, associated with PSII repair indicating that the osmoprotectant GB plays a pivotal role in temperature stress tolerance. Arguably, exogenous application of GB do not reduce the ROS content directly (Li et al. 2014). Tomato plants transformed with fatty acid desaturase gene (LeFAD7) evinced high-temperature tolerance along with lower amounts of unsaturated fatty acids when compared to WT plants. Nakamura et al. (2016) developed transgenics where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Transgenic lines grew under high-temperature conditions. Further, such a high temperature tolerance was conferred in the nontransgenic tomato scions after grafting onto the silenced root stocks. Such a novel technique may be critical for developing many crop plants with high-temperature tolerance. This can avoid the spread of engineered genes into wild species. However, a comprehensive

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understanding of high-temperature tolerance mechanisms, identification of candidate genes such as heat shock proteins (HSPs) and heat shock factors (HSFs), RNA-binding proteins (chaperones), and their modulation is highly crucial to develop tomato transgenics that can withstand the climate changes in future.

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Low-Temperature or Cold Stress-Tolerant Transgenic Tomatoes

Tomato is highly sensitive to low temperatures or cold stress conditions. Both growth and yield decrease, and plants become non-productive if the temperature drops to 5  C (Lin et al. 2000). For protecting the crop plants from low temperatures, and for generating cold stress-tolerant plants, several antifreeze proteins (AFPs), cold-induced candidate genes, and transcription factors have been identified and isolated (Thomashow 1999). Calcineurin B-like protein (CBL) kinase (CIPK) responds to different abiotic stresses. Tomato overexpressing both MdCIPK6L and MdCIPK6LT175D exhibited enhanced tolerance to multiple stresses like drought, chilling, and salt (Wang et al. 2012b). Experiments conducted by Cao et al. (2013) with MdoMYB121 gene overexpression in tomato revealed tolerance to cold stress conditions. The ICE1 gene [C-repeat/DRE-binding factor, (CBF)], an inducer of CBF expression 1, when overexpressed in tomato displayed tolerance to cold stress with an increase in proline content and catalase activities compared to WT plants. Malondialdehyde (MDA) content remained lower in transgenics than the WT plants (Juan et al. 2015). This indicates that CBF has the ability to regulate antioxidative genes such as catalase and also improve the accumulation of the osmolyte proline. Likewise, transgenic tomatoes expressing strawberry FaGalUR gene displayed tolerance to low temperatures alongside twofold increase in AsA level in tomato fruits (Cai et al. 2015). When compared to the WT plants, enhanced expressions of ethylene biosynthesis-related genes and antifreeze proteins (AFPs) like SlCHI3, SlPR1, SlPR-P2, and SlLAP2 were recorded in transgenic tomatoes with the introduction of oil palm transcription factor EgCBF3. EgCBF3 expression resulted in the delayed leaf senescence and enhanced chlorophyll content suggesting the role of this gene in ethylene biosynthesis-related as well as in AFP genes and, hence, could protect crops from low-temperature stress tolerance (Ebrahimi et al. 2016). However, the mechanistic explanation for the upregulation of ethylene biosynthetic pathway and chlorophyll biosynthetic pathway genes is largely obscure. The nonsymbiotic hemoglobins-1s (nsHb-1s) have very high affinity for oxygen and involved in oxygen transport. Tomato overexpressing the transgene MhGLB1 (nsHb-1s) improved the plant tolerance to hypoxia by the decrease in photosynthetic transpiration and stomatal conductance rates compared to WT plants (Shi et al. 2012).

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11

Transgenic Tomatoes with Heavy Metal and Mineral Stress Tolerance

Among heavy metals, cadmium (Cd) and nickel (Ni) are the most hazardous that cause considerable damage to plant productivity. Exposure of plants to these toxic metals triggers various physiological and metabolic alterations (Villiers et al. 2011). The widespread effects of these metals on plant growth (Sharma and Dubey 2007) include leaf chlorosis, necrosis, root activity, altered photosynthetic, and reduced biosynthetic activities leading to the death of plants eventually (DalCorso et al. 2010). However, plants have developed several tolerance mechanisms which include modulation in physiological and biochemical processes and changes in global gene expressions (DalCorso et al. 2010; Urano et al. 2010). Inhibition of metal uptake or avoidance involves restriction of metal entry to the cell by extracellular precipitation, biosorption to cell walls, reduced uptake, or increased efflux. Further, plants adapt to heavy metals by intracellular metal chelation through the synthesis of organic acids (e.g., malate), glutathione (GSH), or metal binding ligands such as phytochelatins (PCs), and metallothioneins (MTs). Vacuolar compartmentation of metals is a strategy adapted by many plants to avoid the toxic effects of metal stress in the cytoplasm. Further, induction of the antioxidant defence system is crucial to counter the toxic effects caused by metal-induced ROS. Cadmium (Cd) content was quantified in six tomato cultivars, and its effects on the expression of LeNRAMP3, LeFER, LeIRT1, and LeNRAMP1 were evaluated. The six tomato cultivars accumulated high Cd concentrations and were able to transport it to fruits. Among the evaluated genes, the Cd-induced level of LeFER expression appeared to provide evidence regarding the capacity of foliar Cd accumulation in tomato (Hartke et al. 2013). Transgenic tomato was generated by heterologous expression of AhHMA4p1::AhHMA4 from Arabidopsis halleri. This is a Zn export protein implicated in loading of Zn into xylem. AhHMA4 induces uptake of Zn in a Zn-dependent manner and also the activation of Fe-uptake in roots if the Zn ions are taken up in excess. Expression of AhHMA4 gene may also cause cell wall remodelling due to overload of Zn into the apoplast, and thus help in metal homeostasis network in tomato (Barabasz et al. 2012). Though Fe-Zn homeostasis has been studied to some extent in transgenic tomato, heavy metal-tolerant transgenics have not yet been generated in tomato using genetic engineering technologies.

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Biofortification in Transgenic Tomato

Several biofortification strategies have been pursed to improve mineral quality like zinc (Zn) and iron (Fe) in tomato. Zn plays an important role in vegetative growth by regulating root-to-shoot metal translocation through the xylem (Palmgren et al. 2008). Deficiency of Zn results in the reduced crop yields and also Zn malnutrition in humans. Transgenic tomatoes expressing AtHMA4 showed enhanced Zn translocation to shoots, which helps in Zn biofortification. But, strangely, overexpression of

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AtHMA4 resulted in decreased Fe in transgenics compared to WT plants by upregulation of Fe-deficiency marker genes (LeFER, LeFRO1, LeIRT1). Tomatoes transformed with AhHMA4p1::AhHMA4 displayed improved Zn uptake by facilitating root-to-shoot Zn translocation. It also induced the uptake of Fe in the roots. Thus, it appears that AtHMA4 overexpression in tomato alters the crosshomeostasis (Kendziorek et al. 2014). But, no attempts were made till date to identify the number of genes associated with Fe and Zn transport and their tissuespecific expressions under Fe- and Zn-deficient and -sufficient conditions, and also translocation of these metals to the fruits. Such a comprehensive identification and understanding their modulation would help us greatly to generate tomatoes with better accumulation of Fe and Zn contents. Phosphorus (P) plays a pivotal role in plant growth and development, and its deficiency results in decreased productivity and quality of crops. miR399 is essential for phosphate homeostasis, and its overexpression resulted in P toxicity and retarded growth. The athmiR399d showed increased biomass under low temperature and P deficiency conditions (Gao et al. 2015). Thus, it is possible to manipulate P content in tomato. Further work on the efficient uptake of P is necessary by genetic engineering or genome-editing technologies. Overexpression of Arabidopsis General Regulatory Factor 9 (GRF9) improved the tolerance of plants to low phosphate (P) with improvement in root biomass and enhanced P content under hydroponic conditions compared to WT plants. Transgenic tomato also showed higher uptake of P content and transcript levels of LePT1 and LePT2 in both normal and low-P hydroponic solutions. GRF9 overexpression resulted in the exclusion of protons from the roots under low P conditions in transgenic tomato and promotion of fruit production (Zhang et al. 2018a) indicating the importance of the gene GRF9 in fruit yield/final productivity. Calcium (Ca2+) maintains membrane stability and cell wall structure, and its deficiency results in low plant productivity. Exogenous supply of Ca2+ before harvest of fleshy tomato fruits showed improvement in shelf life by maintaining plasma membrane integrity and cell wall firmness (Gerasopoulos et al. 1996). Transgenic tomato expressing Arabidopsis H+/cation exchangers (CAX) displayed increased accumulation of Ca2+ and prolonged shelf life in comparison with the WT plants (Park et al. 2005), indicating that Ca2+ is associated with tomato fruit shelf life. Methylselenocysteine (MeSeCys), a derivative of the amino acid, has anticancer activity in animals. Selenium (Se) hyperaccumulating plants convert Se to MeSeCys by the enzyme selenocysteine methyltransferase (SMT). Overexpression of a cDNA encoding SMT in tomato resulted in high accumulation of MeSeCys in the fruit, but not in the leaves when roots were fed with selenite or selenate (Brummeli et al. 2011). More interestingly, MeSeCys was found heat stable and not destroyed by the processing of the fruit to tomato juice (Brummeli et al. 2011). These results indicate that biofortification of tomato fruit with an anticancer compound methylselenocysteine is possible and can be utilized for controlling cancer. However, more efforts are needed to make such transgenics affordable and acceptable by the consumers.

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13

Modulation of Tomato Fruit Traits Like Shelf Life and Pigments/Carotenoids

Tomato is a fleshy fruit, and several flavour compounds and pigments accumulate during the process of ripening. Such compounds/pigments attract animals/birds that devour them and disseminate the indigestible seeds of tomato at a distant place. This helps them in proper seed dispersal and ensue successful establishment of progeny. Pesaresi et al. (2014) discussed the role of plastid modifications in the tomato fruit maturation and ripening. A large body of information indicates the possible involvement of crosstalk via plastid to nucleus (retrograde) and nucleus to plastid (anterograde) signalling. Nearly 3000 proteins observed in the chloroplast are encoded by nuclear genome, but translated in the cytoplasm. All these proteins are then transported into the cell organelles (Richly and Leister 2004; Li and Chiu 2010). This implies that chloroplast transition to chromoplast involves sizeable exchange of information between the plastids and nucleus. Such an exchange of information between the two organelles is essential to meet the needs of the changing energy and metabolic demands (Chi et al. 2013). Therefore, maturation and ripening of tomato fruit are dynamic and highly complex. A comprehensive understanding of the events are essential to regulate fruit maturation and subsequently improve fruit quality traits including pigments that have antioxidative properties. Carotenoids protect the photosynthetic apparatus from excess light and lycopene β-cyclase (lyc-b) is an essential enzyme for the synthesis of β-carotene via methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. Transgenic tomato fruits expressing Lycb-1 under the control of CaMV35S showed 4.1-fold increases in the production of β-carotene and 30% of total carotenoid content in comparison with WT plants. Expression of Lycb-1 altered the other pathways including fatty acids and flavonoid biosynthesis (Guo et al. 2012). miRNAs also involve in regulating carotenoid content in tomato by targeting the biosynthetic pathways (Koul et al. 2016). Transgenic tomato overexpressing oat arginine decarboxylase displayed improved fruit harvesting attributes (Gupta et al. 2019). Tomato fruit ripening is a complex and genetically regulated process which completes with seed formation. It is a climacteric fruit, where ripening is associated with increased production of ethylene. Ripening of the fruit is regulated by thousands of genes that control fruit softening, accumulation of sugars, volatile compounds, and pigments which increase the palatability. Palma et al. (2019) reviewed the role of ethylene receptors, anthocyanin and carotenoid biosynthesis, auxin signalling, effect of light and vitamin C, and modification of organic acids, and cell wall degrading enzymes involved in the process of fruit ripening. Several studies report the maturation of tomato involving ripeninginhibitor (rin), non-ripening (nor), and colourless non-ripening (cnr) mutants. Different transgenes and transcription factors (TFs) were employed for slowing fruit ripening and improving palatability. The list of such transgenics is shown in Table 3. NON-RIPENING (NOR) NAC transcription factors (TFs) play important roles in fruit ripening, and controls leaf senescence (Ma et al. 2019). In addition to these TFs, it is known that ethylene TFs also play an indispensable role in fruit ripening and shelf life of fruits (Klee and Giovannoni 2011). Miraculin, a glycoprotein, is an

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Table 3 List of tomato transgenics and mutants developed for fruit traits Genes and their source ACS1 from grapes GRF9 from Arabidopsis XTH2 and XTH10 from apple CmLOX18 from Cucumis melo MYB12 from Arabidopsis FaGalUR from strawberry Ornithine decarboxylase from mouse SlSAMS1 from tomato BZR1-1D from Arabidopsis S-adenosylmethionine decarboxylase from human GAD RNAi from tomato RNAi ACO1 DAHPS from bacteria hpRNAi-ACO1 GGP from Actinidia chinensis Lycb-1 from tomato Prosystemin Selenocysteine methyltransferase (SMT) ODO1 from Petunia DHAR from MDHAR Miraculin from Richadella dulcifica Miraculin from Richadella dulcifica Stilbene synthase from grapes AP2a mutant AFSK (an NAK-type protein kinase) from apple gr (green ripe) mutant

Fruit improvement Decreased ethylene production for enhanced shelf life Phosphate deficiency Increased ethylene production

References Ye et al. (2018)

Increased production of C6 volatiles Increased flavonoid content Increased ascorbate accumulation Increased fruit quality

Zhang et al. (2017b)

Increased fruit set and yield and tolerance to alkali stress Increased accumulation of carotenoids Delayed ripening Reduced glutamate accumulation Delayed ripening and increased shelf life Increased aroma Reduced ethylene production and increased shelf life Increased ascorbate accumulation Increased β-carotene and total carotenoid accumulation Increased lycopene content Increased selenium accumulation Increased phenylpropanoid compound levels Increased ascorbate accumulation in fruit but not in leaves Increased miraculin accumulation (a taste-modifying protein) Conversion of sour to sweet taste Induce parthenocarpy Regulates carotenoid and chlorophyll metabolism Higher floral abscission and affection of pollen development Delayed ripening

Zhang et al. (2018a) Zhang et al. (2017a)

Choudhary et al. (2016) Cai et al. (2015) Pandey et al. (2015) Gong et al. (2014) Liu et al. (2014) Madhulatha et al. (2014) Chew and Seymour (2013) Eglous et al. (2013) Tzin et al. (2013) Behboodian et al. (2012) Bulley et al. (2012) Guo et al. (2012) Liu et al. (2012) Brummell et al. (2011) Cin et al. (2011) Haroldsen et al. (2011) Hirai et al. (2011) Hiwasa-Tanase et al. (2011), Kurokawa et al. (2013) Ingrosso et al. (2011) Karlova et al. (2011), Chung et al. (2010) Kim et al. (2011) Barry and Giovannoni (2007) (continued)

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Table 3 (continued) Genes and their source CNR (colourless non-ripe) mutant rin (ripening inhibitor) mutant Nr (never ripe) mutant rin mutant

Fruit improvement Delayed ripening

References Manning et al. (2006)

Delayed ripening

Vrebalov et al. (2002)

Delayed ripening

Yen et al. (1995), Lanahan et al. (1994) Giovannoni et al. (1989)

Degradation of polyuronide but not fruit softening

alternative to more traditional sweeteners. It was discovered in red berries of the miracle fruit of West Africa, which converts sour into sweet tastes (Kurihara and Beidler 1968; Theerasilp and Kurihara 1988). Transgenic tomatoes expressing miraculin driven by E8 tissue-specific promoter and heat shock protein (Hsp) terminator accumulated 30–630 μg miraculin per gram fresh weight of tissue which was four times higher than transgenic tomatoes expressing miraculin driven by the constitutive 35S promoter (Hiwasa-Tanase et al. 2011; Kurokawa et al. 2013). Yet, such fruits have not been marketed, and tomatoes with sweet taste are still a dream to come true to the consumers. In tomato, fruit ripening involves a cascade of physiological and biochemical events like softening, change of fruit pigment, development of flavour components, and more importantly biosynthesis of ethylene. Levels of ethylene content increase which can subsequently trigger multiple physiological changes brought out simultaneously by the expression of several genes. It is known that the MADS-box genes help by way of non-hormonal ripening. Tomato rin mutant has large sepals and loss of inflorescence determinacy. Cloning of the rin locus revealed the presence of two tandem MADS-box genes, namely LeMADS-RIN and LeMADS-MC. While RIN is associated with fruit ripening, MC plays a role in sepal development. The rin mutation alters the expression of LeMADS-RIN and LeMADS-MC genes (Vrebalov et al. 2002). It appears that MADS-box transcription factor RIN is an essential regulator of ripening gene expression network. It interacts with many genes and controls the changes in fruit colour, flavour, texture, and taste during ripening. RIN interacts with the promoters of genes responsible for the overall ripening including the transcriptional regulation, cell wall metabolism, and ethylene and carotenoid biosynthesis. Contrary to this, macrocalyx (MC) has very low expression in tomato fruit, but the function of the fusion gene RIN-MC is not completely clear. It is RIN which acts as a rate-limiting factor in ethylene and carotenoid biosynthesis by interacting with the promoters of several genes. While overexpression of RIN-MC in tomato impaired the process of fruit ripening, downregulation of RIN-MC in the rin mutant stimulated the normal yellow colour of the fruit (Li et al. 2018). The experiments conducted by Li et al. (2018) infer a negative role for RIN-MC fusion gene in fruit ripening, and it encodes a chimeric transcription factor which can regulate many genes associated with ripening. Further, RIN function depends on the normal functioning of ncr gene (Martel et al. 2011). Martel et al. (2011)

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demonstrated that RIN recruitment to target loci depends on a functioning allele at the ripening-specific transcription factor COLOURLESS NONRIPENING gene locus. Thus, it appears an interaction between the ripening regulators is highly crucial. Biosynthesis of ethylene is regulated by two key enzymes, 1-aminocyclopropane1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) which transform S-adenosyl-L-Met (SAM) into ACC and convert it further into ethylene (Kende 1993). Both ACS and ACO genes show different spatial and temporal expression patterns (Barry et al. 2000; Jiang et al. 2011; Liu et al. 2015; Ye et al. 2017). A total of seven ACO genes have been identified, and LeACO1 and LeACO4 are highly expressed in tomato leaf and flower tissues (Seymour et al. 2013; Chersicola et al. 2017). Overexpression of VvACS1 is the only rachis-specific ACC synthase (ACS) gene in tomato which showed increased activity in rachis without any increase in fruit. Ectopic expression of VvACS1 resulted in decreased ethylene production in flowers, fruits, and leaves of tomato. Its expression does not downregulate the expression of endogenous tomato ACS1 and ACS6 genes. VvACS1 (from grapes) expression in tomato resulted in decreased auxin and increased zeatin contents, suggesting the role of ethylene in auxin transport and distribution during fruit ripening (Ye et al. 2018). Thus, the phytohormone ethylene is at the centre stage of fruit ripening and its regulation is critical for improving shelf life of tomato fruits. Several fruit ripening, single recessive gene mutants such as ripening inhibition (rin) as described above, non-ripening (nor), alcobaca (alc), never ripe (nr), and green ripe (gr) have been known that prolong the shelf life of tomatoes (Chialva et al. 2016; Osei et al. 2017). These gene mutants modify several of the ethylene’s downstream effects, thus inferring a complex fruit ripening gene/ protein network. Therefore, production of ethylene in these gene mutants is limited and hence fruits fail to ripe, thus they contribute to postharvest fruit quality. Further, the discovery of such gene mutants improved our understanding of the molecular mechanisms that help to control fruit ripening. However, in a heterozygotic condition, such mutants exhibit natural fruit colour that is acceptable by consumers, and fruits ripe naturally with enhanced shelf life. Thus, potential exists for the utilization and exploitation of these genes for genetic engineering and for improving the shelf life of tomato fruit with consumer acceptance. Brassinosteroids (BRs) also regulate fruit ripening besides ethylene. Transgenics expressing Arabidopsis BR response transcription factor Brassinazole resistant 1 (BZR1-1D) showed enhanced accumulation of carotenoids, soluble sugars, and ASA during fruit ripening. Transgenics also showed upregulation of SlGLK2 gene involved in chloroplast development. The 2,4-epibrassinolide (EBR)-treated ethylene-insensitive mutant pericarps have never ripen with the accumulation of carotenoids. Thus, EBR and BZR1-1D play vital roles in the accumulation of carotenoids which is attributed to the fruit quality (Liu et al. 2014). Transgenic lines overexpressing BZR1-1D showed 411 differentially expressed proteins with enhanced light reaction pathway during ripening. The increase in 2-oxoglutaratedependent dioxygenase (2-ODD2), a protein involved in gibberellin biosynthesis, was noticed during all the four developmental and ripening stages (Liu et al. 2016).

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Jasmonic acid (JA) is also involved in carotenoid biosynthetic pathway; thereby it can control fruit quality. Both lycopene and ethylene contents decreased significantly in the fruits of JA-deficient mutants (spr2 and def1). This indicates that JA is a crucial player in improving lycopene content in tomatoes. On the other hand, transgenics overexpressing 35S::prosystemin (35S::prosys) displayed increased levels of JA and ethylene. Exogenous application of methyl jasmonate (MeJA) to the mutant fruits spr2 and def1 increased the levels of fruit lycopene. Similarly, exogenous application of MeJA to Never ripe (Nr) and the ET-insensitive mutants increased the lycopene accumulation significantly. Thus, JA appears to promote lycopene biosynthesis independent of ethylene (Liu et al. 2012). But the mechanistic explanations for the roles of these hormones during fruit ripening and accumulation of lycopene are largely not explained properly.

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Transgenic Tomato with Improved Fruit Aroma

Fruits have characteristic aroma volatiles that impart a fruity flavour contributed by ester compounds (Song and Forney 2008; Deflippi et al. 2009). More than 400 different types of volatile compounds have been recorded in tomato, but few of them have an impact on its organoleptic properties (Sitrit et al. 2008). This implies that metabolic engineering of tomato fruits for better taste and flavour qualities is possible. Sitrit et al. (2008) have chosen a terpenoid pathway gene that encodes geraniol synthase (GES) for this purpose. GES produces geraniol, an acyclic monoterpene alcohol, which has a good odour and also acts as a precursor to other scented volatiles like geranial, citronellol, and geranyl acetate. Overexpression of GES gene in tomato under the influence of polygalacturonase promoter (PG) resulted not only in enhanced levels of geraniol, but also aroma and overall flavour of the transgenic fruits (Sitrit et al. 2008). These results indicate that it is feasible to alter aroma and other quality traits through genetic engineering in tomato and other fruit crops. 3-Deoxy-d-arabino-heptulosonate7-phosphate synthase (DAHPS) is the first enzyme of the shikimate pathway which produces the aromatic volatiles. Transgenic tomato expressing bacterial feedback-insensitive AroG gene encoding a 3-deoxy-darabino-heptulosonate7-phosphate synthase (DAHPS) under the influence of a fruit ripening-specific promoter E8 enhanced the levels of metabolites and aroma (Tzin et al. 2013). The synthesis of 2,4,6-carbon chain esters occurs from the degradation of linoleic and linolenic acids. LOX enzymes contribute for the synthesis of ester compounds from alcohols (Baldwin et al. 2000; Contreras and Beaudry 2013) and help improve the flavour quality. When CmLOX18 gene isolated from melon was overexpressed in tomato, it enhanced the biosynthesis of C6 volatiles like hexanal, (Z)-3-hexanal, and (Z)-3-hexen-1-ol, during fruit ripening (Zhang et al. 2017b).

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Transgenic Tomato with Enhanced Vitamin C or Ascorbic Acid

Ascorbic acid (AsA) is an essential component for collagen biosynthesis. Very rich in tomato, it acts as an antioxidant and protects DNA damage from oxidative stress (Raiola et al. 2014). Synthesis of AsA does not occur in humans due to the mutation of L-gulono-1,4-lactone oxidase enzyme; hence, humans obtain it from plant sources (Chatterjee 1973). AsA protects the plants from ROS, but its deficiency activates cell death via redox mechanisms which are independent of natural senescence of the plants (Conklin et al. 1996; Pavet et al. 2005). Biochemical and transcriptomic analyses revealed the relation of AsA content with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME) in Solanum pennellii introgression line (IL12-4-SL). SolyPME, SolyPG, and UGlcAE have been found as candidate genes responsible for an increase in AsA production by affecting the alternative D-galacturonate pathway (Rigano et al. 2018). Overexpression of strawberry FaGalUR gene resulted in twofold increase in AsA levels in tomato fruit with enhanced oxidative and salt stress tolerance in comparison with WT plants. These results suggest that tomato has an alternative D-galacturonate pathway for ascorbate biosynthesis as pointed out by Cai et al. (2015).

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Increased Flavonoid Content

Transgenic tomato fruits expressing AtMYB12 transcription factor displayed significant increase in flavonoid content. Mice fed with such transgenic tomato fruit extracts containing AtMYB12 transcription factor recorded significant increase in femur and tibia bone growth in comparison with WT fruits (Choudhary et al. 2016). This implies that exciting prospects lie to generate transgenic tomato fruits with improved flavonoid content for better bone growth in humans.

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Transgenic Tomato for Expression of Malarial Antigens

It is important to develop transgenics that express animal proteins in high quantities. Kantor et al. (2013) developed transgenic tomatoes expressing the PfCO-2.9 protein which is a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum. Thus, successful transformation of tomato was reported with the expression of malarial antigen (PfCP-2.9). This study holds great promise to express other antigens of commercial importance in tomato.

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Transgenic Tomato Expressing RNA Interference (RNAi)

Double-stranded RNA-mediated interference (RNAi) is an important method of silencing gene expressions in many organisms. RNA is degraded into short RNA molecules which activate ribonucleases to target homologous mRNA. Constructs of ACC oxidase gene1 (RNAi ACO1) displayed low levels of ethylene with increased shelf life of 32-days in comparison with WT plants. The RNAi ACO1 tomatoes showed reduced activity of lipoxygenase (LOX) and MDA content and increased activities of superoxide dismutase, and catalase (Eglous et al. 2013). The delayed ripening by RNAi-mediated silencing of three homologues of ACC gene increased the shelf life to nearly 45 days (Gupta et al. 2013). Ethylene plays an important role in salt tolerance of plants by controlling the entry of Na+ and uptake of K+ for efficient growth of the plants. Transgenic tomatoes expressing hpRNAi-ACO1 displayed lower ethylene production, which increased the shelf life of transgenic tomato to 32 days compared to 10 days in WT fruits. Transgenic fruits also showed reduced activity of pectin methylesterase (PME) and polygalacturonase (PG) activities. Fruit ripening occurs by extensive degradation of pectin catalysed by polygalacturonase (PG). In spite of its expression, no significant change in the levels of β-galactosidase (β-Gal) and ASA was observed in transgenic and WT fruits (Behboodian et al. 2012). In tomato, an expansin (LeExp2) and extension-like protein1 (LeEXT1) genes accumulate during rapid growth of the plant, while endo-1,4-beta-glucanase (EG) accumulates and remains the same during the last stages of fruit growth. The expression of LeExp2, LeEXT1, and cellulase (Cel7) were not noticed during the onset of fruit ripening, which confirms that these proteins play a specific role in loosening the cell wall and implicated in ripening stage only (Catalá et al. 2000). But simultaneous suppression of LePG and LeExp1 genes influenced not only the fruit texture but also the juice viscosity (Powell et al. 2003). These results indicate that transgenes greatly influence both the tomato fruit texture and juice quality. Transgenic overexpression of xyloglucan endotransglucosylase/hydrolase2 and 10 (XTH2 and XTH10) in tomato displayed enhanced expression in the levels of ethylene biosynthesis genes (ACS2, ACO1). Besides ethylene biosynthesis genes, transgenics also displayed enhanced expression of signal transduction (ERF2) and fruit softening [XTHs, PG2A, Cel2 and tomato β-galactosidase4 (TBG4)] genes (Zhang et al. 2017a). Expression of a chimeric PG ripening inhibitor (rin) in tomato blocked the ripening including the activation of PG gene transcription. Expression of rin resulted in the accumulation of active polygalacturonase enzyme and the degradation of cell wall polyuronides in the fruit. But, no change in the fruit softening and ethylene levels were noticed which suggest that PG plays a role in degradation of cell wall polyuronide, but is not sufficient for fruit softening and increased levels of ethylene (Giovannoni et al. 1989). Thus, many efforts were made to generate tomatoes with delayed fruit ripening. But, due to biosafety regulations, such lines were not commercialized. S-adenosyl-L-methionine (SAM), a common precursor for polyamines and ethylene, is synthesized by SAM synthetase from methionine and ATP. A fruit shelf-life regulator (FSR) gene suppression by RNAi resulted in the reduction of cell wall modification-related genes, decreased the activities of PG,

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TBG, CEL, and XYL (β-D-xylosidase), and prolonged fruit shelf life (Zhang et al. 2018b). This points out that SlFSR gene from tomato, a member of the GRAS family, is a potential target for the control of fruit shelf life. Tomato overexpressing SAM synthetase1 (SlSAMS1) exhibited significant increase in tolerance to alkali stress with decreased Na+ absorption and increased fruit set and yield. Transgenics also displayed nutrient and ROS balance, profuse rooting, with improved photosynthetic activity compared to WT plants (Gong et al. 2014), suggesting that SAM synthetase plays a crucial role not only in alkali stress but also in modulating fruit shelf life. Glutamate, responsible for the unique taste sensation termed UMAMI, the fifth taste or brothy taste, is produced from TCA cycle and amino acid metabolism in plants. Tomato cotyledonary explants infected with glutamate decarboxylase RNAi (GAD RNAi) gene using CaMV35S promoter failed to produce transgenics. Expression of GAD RNAi may alter the levels of γ-aminobutyric acid essential for plant survival (Chew and Seymour 2013). Similarly, tomatoes expressing Actinidia chinensis GDP-L-galactose phosphorylase (GGP) under the influence of 35S promoter showed three- to sixfold increase in ascorbate content in the fruits than the WT plants. However, increased accumulation of ascorbate resulted in the loss of seed and the locular jelly (Bulley et al. 2012).

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Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated System for Genome/Gene Editing in Tomato

Genome editing, also known as gene editing, helps us to add, remove, or alter DNA molecule at a specific location in the genome. The CRISPR-Cas9 system is not only accurate but also highly efficient. Tomato is an ideal crop for gene/genome editing using CRISPR/Cas9 (Van Eck et al. 2006). Veillet et al. (2019), using Agrobacterium-mediated delivery of a CRISPR/Cas9, successfully edited the targeted cytidine bases in the gene acetolactate synthase (ALS) and generated tomato plants that are chlorsulfuron-resistant with an efficiency of 71%. Great prospects lie in selecting candidate genes and editing them for generating tomato plants with useful agronomic traits.

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Conclusions

Considerable progress has been made over the past few years in the genetic transformation technique of many tomato varieties. Several candidate genes and transcription factors responsible for the tolerance of tomato to different abiotic stress conditions were validated. Such transgenics displayed not only higher stress tolerance but also better yields by accumulating osmolytes, excluding the Na+ ions, preventing water loss, and with improved antioxidative and photosynthetic capacity in comparison with WT plants. Different regulatory proteins and compounds involved in fruit softening and ripening for enhanced shelf life were also reviewed

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here. However, we are still far away from identifying the candidate genes/transcription factors that help us in regulating tomato fruits with precision and improved abiotic stress tolerance, fruit shelf life and fruit quality including aroma. Further, the generated transgenics were only tested under controlled conditions in the glass house, but not in the field. In the absence of field data under natural conditions, it is difficult to say that these transgenics have higher tolerance in comparison with WT plants. Due to its economic importance world-wide (more than 4 billion US$ per annum), future research should focus mainly on genome editing for developing tomato plants with better tolerance to multiple stresses and improved fruit shelf life, aroma and other qualities, without any decline in the overall productivity. Acknowledgements PBK acknowledges the CSIR, New Delhi, for awarding CSIR Emeritus fellowship.

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Genetically Modified Brinjal (Solanum melongena L.) and Beyond C. Kiranmai, T. Pullaiah, and M. V. Rajam

Abstract

Solanum melongena L., commonly called as brinjal/eggplant, occupies an important position in vegetable rearing across the globe and has been regarded as the poor man’s crop. The estimated production goes over 52,309,119 metric tonnes annually. Traditional plant breeding techniques have played a vital role in developing new cultivars, thereby improving the overall crop production that catered to the needs of the global requirement. However, in the long run, the requirement has risen enormously due to the rapidly growing population. Simultaneously, the reduction in the yield due to various factors including soil quality, environmental vagaries, diseases and pest attacks posed new challenges in the production-consumption landscape. Of all the factors, the threat of the notorious insect pest, Leucinodes orbonalis, commonly known as brinjal shoot and fruit borer (BSFB) which belongs to the phylum Arthropoda and to the order Lepidoptera stood as the greatest challenge to counter as it withstood several broad range insecticides. This situation demanded for BSFB-resistant varieties of brinjal, eventually leading to the development of the genetically modified Bt brinjal. The development of such an insect-resistant variety has been a landmark in brinjal production. The present chapter focuses on transgenic brinjal with improved agronomic traits, particularly insect-resistant Bt varieties, the basic biology of Bt and the major methodologies, the mechanism of action involved in the development of the Bt brinjal.

C. Kiranmai (*) Department of Biotechnology, Vikrama Simhapuri University, Nellore, Andhra Pradesh, India T. Pullaiah Department of Botany, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India M. V. Rajam Department of Genetics, University of Delhi South Campus, New Delhi, India # Springer Nature Singapore Pte Ltd. 2021 P. B. K. Kishor et al. (eds.), Genetically Modified Crops, https://doi.org/10.1007/978-981-15-5932-7_2

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Keywords

Solanum melongena · Brinjal shoot and fruit borer (BSFB) · Bacillus thuringiensis (Bt) · Cry protein · Transgenic brinjal

1

Introduction

Cultivation of vegetables has been the major occupation of several countries including India, representing one of the largest branches of agriculture and had been a great source of income for large number of farming communities over the years. Vegetables are known to be a great source of healthy diet that provides us with high nutrient value containing vitamins, minerals, proteins and carbohydrates. Owing to its high nutritional value, Solanum melongena L., commonly called as eggplant, brinjal, aubergine or baingan (in Hindi), is the most important and widely cultivated vegetable crop in India as well as other tropical and temperate regions across the globe. Brinjal is native to India and has been cultivated in the country for over 4000 years. A total of 1.4 million small family members grow brinjal on 550,000 ha. It is an important crop for poor farmers, who transplant it from nurseries at different times of the year to produce two or three crops. Brinjal provides a steady stream of food for the family, and it also provides a stable income from market sales for most of the year serving as a vehicle for reducing the poverty in rural areas. The estimated total world production for eggplants in 2017 was 52,309,119 metric tonnes. China was by far the largest producer of eggplants, accounting for over 62% of global production. India produces eight to nine million tons, equivalent to one quarter of the global production (http://www.fao.org/family-farming/detail/en/c/ 414901/). Eggplant has been divided into three main types based on the fruit shape. These include egg-shaped (S. melongena var. esculentum), long and slender in shape (S. melongena var. serpentium) and dwarf types (S. melongena var. depressum). Apart from the nutrition point of view, brinjal is believed to possess high medicinal value (Aykroyd 1966; Choudhury 1966; Chandra and Murty 1968; Choudhury and Kalda 1968). Besides being used as an important vegetable, eggplant has been exploited extensively in traditional medicines. For example, tissue extracts have been used for the treatment of asthma, bronchitis, cholera and dysuria; fruits and leaves are beneficial in lowering blood cholesterol. Recent studies have shown that eggplants also possess antimutagenic properties. The medicinal and economic value of eggplant can be found in the Sanskrit literature (Rajam and Kumar 2007). A thicker pericarp with reduced fibre content, longer shelf life with good texture, smoothness and colour are the characteristic features of a good fruit. Since the time of green revolution in India (1960–1970), the scientific advancements in the field of crop improvement have played a great role in enhancing the quality of the vegetable crops in general and brinjal in particular. Previously, a detailed account on eggplant biotechnology and transgenic eggplant has been covered (Collonnier et al. 2001; Kashyap et al. 2003; Rajam and Kumar 2007; Rajam et al. 2008; Saini and Kaushik 2019).

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Plant Regeneration in Brinjal

For Agrobacterium-mediated genetic transformation to be successful, a highly valid and reliable protocol for regeneration of a plant material is an important prerequisite. In vitro plant regeneration of a wild species of eggplant (Solanum sisymbriifolium Lam.) was reported for the first time by Fassuliotis (1975). In the subsequent years, several studies were conducted that resulted in various protocols on the regeneration of brinjal through organ as well as callus cultures (Kantharajah and Golegaonkar 2004; Litz 1993) or somatic embryogenesis (SE) (Ammirato 1983). The abovementioned methods could be conveniently exploited for somaclonal variations, haploid production and also somatic hybridization (Collonnier et al. 2001). SE helps in deriving an embryo from any single somatic cell. There are two types of SE namely the direct and indirect SE. The first method does not involve any callus formation and hence the embryos are directly produced from the explants. On the other hand, indirect method of SE involves the formation of a callus prior to the development of somatic embryos (Horstman et al. 2017). Employing the SE protocol, the first ever brinjal was regenerated from immature seeds on Murashige and Skoog’s (MS) medium (Murashige and Skoog 1962) supplemented with the plant hormone indole-3-acetic acid (IAA) (Yamada et al. 1967). Although various parameters such as the type of medium used, the genetic constitution (genotype), type of the explants and their age have been shown to play a critical role in the successful induction and development of somatic embryos (Kantharajah and Golegaonkar 2004). The diamine putrescine caused the promotion of SE, suggesting the intricate regulatory role of polyamines (PAs) in differentiation, specifically putrescine, in SE in eggplant (Yadav 1998; Yadav and Rajam 1997). They have also demonstrated that the explants from different regions of the leaf exhibited significant differences for SE, and discs from the apical region of leaves having higher titres of PAs yielded more somatic embryos than those from the basal region with lower level of PAs. Sharma (1994) and Sharma and Rajam (1995b) reported that high levels of conjugated spermidine along with high levels of total PAs (primarily free fraction) could be correlated with the formation of somatic embryos in terminal segments. Temporal changes in endogenous levels of free, conjugated and bound putrescine, spermidine and spermine were analysed at critical stages of SE from four different hypocotyl segments. Interestingly, the temporal regulation of SE was achieved by adjusting cellular PA content in eggplant (Yadav 1998; Yadav and Rajam 1998). Kinetic studies of the up- and downregulation of SEs revealed that putrescine and difluoromethylarginine (an inhibitor of a key PA biosynthetic enzyme, arginine decarboxylase) pretreatments were most effective before the onset of SE. Similarly, the embryonic potential can vary with the type of explants. Ray et al. (2010) demonstrated the use of stem, leaf and root of the Jhumki cultivar of brinjal as the explants under the combined influence of 6-benzylaminopurine (BAP) and α-naphthalenea cetic acid (NAA) for callus initiation. The study also suggested that the protocol developed could be used for the generation of disease resistant or disease free plantlets (Ray et al. 2010). Similarly, other explants could also be used, which include the immature seed embryos, hypocotyls, cotyledons, roots and leaves

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for efficient SE (Habib et al. 2016). Yesmin et al. (2018) demonstrated successful regeneration of healthy brinjal plants from the cotyledon explants of Bari Begun-4 and Bari Begun-6 cultivars by fortifying the medium with 2 mg/L BAP and 0.1 mg/L IAA. This protocol has resulted in enhancing the multiple shoot generation. The plants produced by this method were looking normal like that of seed raised plants and could be acclimatized to the natural conditions. With the incorporation of optimal nitrates, ammonium and NAA in the medium, Gleddie et al. (1983) could produce somatic embryos from the leaves of the brinjal cultivar Imperial Black Beauty. Similarly, SE was achieved from leaves, hypocotyls, epicotyls and cotyledons of F100 variety. In this case the proembryo formation was initiated within 48 h of culturing (Tarré et al. 2004). Organogenesis (OG) is another method of plant regeneration that can be obtained from various explants such as leaf, shoot tip, root tip and flower bud. In OG, a number of meristematic zones are originated from various parts of the source material. However, the efficiency of regeneration varies with the type of explants used and plant growth regulators used in the nutrient medium (Sharma 1994; Sharma and Rajam 1995a; Mir et al. 2008; Sidhu et al. 2014; Zhang et al. 2014). Sharma (1994) and Sharma and Rajam (1995a) reported that among the explants, hypocotyls yielded the maximum number of adventitious shoots followed by cotyledons and leaves. The embryogenic response of leaves and cotyledons was, however, significantly higher than the hypocotyl explants. Significant differences for morphogenetic potential were also observed within a single explant (hypocotyl). There was a basipetal gradient for organogenesis while the terminal hypocotyl segments showed better embryogenic potential than the median segments. In addition to the explants, the age of the explants also dictates the success of organogenesis. While the explants isolated from younger plants show increased vigour and result in enhanced response, older explants tend to show decreased response (Franklin et al. 2004; Prakash et al. 2015). Using brinjal hypocotyls as a source of explants, Matsuoka and Hinata (1979) showed cultivar variations for in vitro responses. Treatment with NAA resulted only in callus formation, while supplementation with BAP significantly enhanced the shoot differentiation (Matsuoka and Hinata 1979). Sarkar et al. (2006) demonstrated high frequency of regeneration from hypocotyls and cotyledons of Singhnath and Kazla cultivars of brinjal and also obtained viable seeds from the healthy regenerated plants through direct OG. Cotyledons of the eggplant were also used and cultured on medium fortified with a combination of zeatin (Zn) and IAA, eventually resulted in bud differentiation (Xing et al. 2010). Saiki et al. (2009) observed increased rates in plant regeneration from microspore cultures of eggplant. The various stages of SE and organogenesis have been shown in Figs. 1 and 2 respectively.

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Fig. 1 Regeneration of somatic embryos from leaf explants of brinjal (a). Induction of somatic embryos of different stages in callus cultures; (b–d) Scanning electron microscopy photographs of the various stages of somatic embryogenesis—globular (b), heart-shape (c) and torpedo (d) stage embryos from the brinjal culture. (Source: Yadav 1997; Yadav and Rajam 1997)

3

Transformation Protocols

Agrobacterium-mediated genetic transformation is commonly employed for engineering eggplant with new traits (Kumar 2003; Rajam and Kumar 2007). Following the successful demonstration of microprojectile bombardment technique in tobacco, similar studies were carried out on eggplants, and useful genes were transferred, including the Cry genes for insect resistance. Various types of explants were utilized for the transformation of eggplant for disease and insect resistance by using the biolistic approach, but cotyledons displayed high efficiency and potential in terms of regeneration (Christou 1992; Twyman and Christou 2004).

3.1

Agrobacterium tumefaciens-Mediated Gene Transfer

Agrobacterium tumefaciens has been widely used in the field of genetic engineering for the transfer of genes since it can infect a broad variety of host plants by transferring single gene copy number (Nester 2015). The use of gene transfer by the A. tumefaciens has been demonstrated to be quite successful with high efficiency in several members of Solanaceae, including the eggplant (Van Eck 2018). Guri and Sink (1988) were the first to demonstrate the genetic transformation in eggplant by Agrobacterium-mediated transformation. Kanamycin-resistant plants of S. melongena L. cv. Picentia were obtained following the co-cultivation of leaf explants with A. tumefaciens. Since then, several studies on eggplant transformation have been reported (Fillippone and Lurquin 1989; Komari 1989; Rotino and Gleddie 1990; Fári et al. 1995; Iannacone et al. 1995; Billings et al. 1997; Franklin and Lakshmi Sita 2003; Kumar 2003; Rajam and Kumar 2007). Although the protocol for eggplant transformation has been relatively well developed, production of transgenic plants expressing genes for agronomically important traits has been limited. Binary vector system containing the neomycin phosphotransferase (npt-II) gene as the selectable marker and chloramphenicol acetyltransferase (cat) as a reporter

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Fig. 2 Regeneration of brinjal (var. Pusa Purple Long) via organogenesis. (a–c) Induction of several adventitious shoot buds; (d) Elongated shoot cultured on rooting medium; (e) In vitro rooted shoot; (f) Regenerated plantlets; (g) Regenerated plant established in pot condition. (Source: Sharma 1994; Sharma and Rajam 1995a)

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gene was employed to develop transgenic brinjal for resistance against Colorado potato beetle (Leptinotarsa decemlineata), another major insect pest of eggplants that developed resistance against the insecticides (Rotino and Gleddie 1990). Several reporter genes such as gus (encoding β-glucuronidase; Rotino et al. 1992; Sunseri et al. 1993; Chen et al. 1995; Fári et al. 1995; Billings et al. 1997; Jelenkovic et al. 1998), cat (encoding chloramphenicol acetyl transferase; Rotino and Gleddie 1990) and luc (encoding luciferase; Komari 1989) have also been used for eggplant transformation. Kumar (2003) and Rajam and Kumar (2007) used a chimeric gfp: gus reporter gene to monitor transgene expression in an attempt to develop an efficient transformation system for eggplant. Small phenolic molecules, released by wounded plant tissue have been shown to induce the virulence (vir) genes of Agrobacterium tumefaciens, which is a prerequisite for T-DNA transfer. It has been shown that the transformation efficiency is dependent on the vir gene induction by the host plant tissue. To exploit this, and to improve transformation efficiency, new strategies have been applied to use compounds so as to have an increased vir gene induction. Kumar (2003) and Kumar and Rajam (2005a) reported an improved protocol for Agrobacteriummediated transformation of eggplant by modulation of vir gene induction using a low molecular phenolic compound, acetosyringone during infection and co-culture. It is noteworthy to mention that PAs—putrescine and spermidine enhances vir gene induction when Agrobacterium cells are treated prior to acetosyringone addition, and this was confirmed by plant transformation experiments which have shown that modulation of PA levels in Agrobacterium results in the enhanced T-DNA transfer (Kumar 2003; Kumar and Rajam 2005b). They suggested that these findings may be useful in obtaining a high transformation frequency in those plant species, which show minimal vir gene induction. Agrobacterium-mediated genetic transformation of brinjal using gfp + gus fused gene construct is shown in Fig. 3.

4

Need for Brinjal Transgenics

Eggplant, as said earlier, is an important vegetable, and poor people are highly dependent on this vegetable as a source of nutrition. The traditional plant breeding techniques have played a vital role in increasing the crop yield and also the quality of the fruits. These techniques have helped to generate better breeds, which are responsible for the market demand. Most of the cultivated varieties are highly susceptible to diseases caused by various pathogens (fungi, bacteria and viruses) as well as other stresses, and such stresses result in significant loss of crop yield and quality. Eggplant wilt diseases mainly caused Fusarium oxysporum, Verticillium dahliae and Rhizoctonia solani cause considerable loss in crop yield annually. The wild species are a source of resistance against the bacterial and fungal diseases and therefore can be used as useful germplasm to develop resistant varieties of eggplant cultivars against the pathogens through inter-specific hybridization. However, the inter-specific hybrids

3

2

hpt

35 S CaMVP Lac Z alpha

4

NcoI 35SCaMVP

BglII

mgfp

SpeI

gusA

His Nos RB Poly A tag

Fig. 3 Agrobacterium-mediated genetic transformation of brinjal. (1) gfp + gus fusion gene construct, pCAMBIA 1304; (2) (A) Induction of adventitious shoots from co-cultured leaf explants; (B, C) Elongated shoots; (D) Rooted in vitro shoot; (E) Regenerated plant established in pot condition; (3) (A–D) GUS activity in transformed tissues; (E–G) gfp expression in transformed tissues ; (40 PCR analysis for the presence of marker gene, hpt (upper panel) and Southern analysis for transgene integration and copy number (bottom panel). (Source: Kumar 2003; Kumar and Rajam 2005a)

LB Poly A

1

38 C. Kiranmai et al.

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between wild species and cultivated varieties have been successful in only a few cases. Therefore, the introgression of desired traits from the wild relatives into the cultivated varieties is not very successful, although there are some successful stories (Plazas et al. 2016). Even though success rate is limited, nearly 25 wild species were crossed to impart resistance (Rotino et al. 2014). Plants resistant to fungi and bacteria were developed by interspecific hybridization (Rotino et al. 1997a). Behera and Singh (2002) successfully transferred the desirable agronomic characters by crossing S. melongena and other related Solanum species. However, conventional breeding approaches for developing insect resistance for brinjal is limited due to sexual incompatibilities, prevalence of sterility in the progeny and lack of natural resistance sources (Magioli and Mansur 2005; Shivaraj and Rao 2010). Thus, there is an urgent need to adopt the transgenic strategies to engineer eggplant for resistance against wilt diseases (Singh et al. 2014) and other traits like insect resistance. In fact, the development of a genetically modified (GM) brinjal is indispensable, and this had led to the development of the Bt brinjal. Among the biotic stresses associated with brinjal yield losses, insect pests have been the major threat in brinjal production. To combat this, farmers resort to the application of costly pesticides. Therefore, the pests have become resistant to several of the agrochemicals and cause more damage to the productivity of brinjal. The biggest damage is usually caused by the pest Leucinodes orbonalis, commonly called as BSFB. The life cycle of BSFB includes four important stages of development. They are egg, larva, pupa and adult stages. Normally, 200–250 eggs are laid by a mature female moth on the surface of the tender and early developing stem and also on the newly forming brinjal fruits. Upon hatching of the eggs, a slightly pinkish coloured, transparent, slender, caterpillar that measures about 20 mm is developed. Within 2 weeks of time, the larva turns into a pupa with a cocoon and further develops into an adult moth (Talekar 2002). BSFB is the intensely attacking pest on brinjal breeds, and it survives by feeding on brinjal. This insect pest is known to be a monophagous that attacks only brinjal. Its distribution is global ranging from India, Bangladesh, Sri Lanka, Malaysia, China, South America and South Africa (Choudhary and Gaur 2009). This pest does not reside on the outer surface of the host. BSFB is characterized as a borer and hence enters into the tender shoot or fruit and takes its hostage inside and completes the life cycle resulting in the wilting of the shoot and inhibiting the growth of the plant (Rattan et al. 2016). Similarly, when the hatched larva bores into a fruit, it attenuates the healthy and proper development of the brinjal fruit. Since it is not exposed to the pesticides applied, it escapes the death and reproduces inside the fruit or shoot. In addition to the invisibility of the BSFB, it is also well known for its high reproductive capacity and viability. The under developed and damaged brinjal fruits are prone to rejections in the commercial market and are not fit for a healthy diet, thereby causing heavy loss to the farmers. Further, owing to the holes caused by the pest on the fruit by boring, the aesthetic value is also reduced and doubles the loss incurred (Puranik et al. 2002). It is estimated that a single larva can infest and damage up to 5–6 fruits. Nearly 75–90% damage has been reported by the fruit borers in brinjal (Atwal and Verma 1972; Gangwar and Sachan 1981; Peswani and Lal 1964). The stem borer infestation

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was found to be 78.66% (Singh et al. 2000) during the vegetative stage, while it was 67% in the fruiting stage (Naik et al. 2009; Kumar and Singh 2013). Breeding efforts could not result in reducing the crop damage and hence were considered quite unsuccessful (Kashyap et al. 2003; Choudhary and Gaur 2009). Further the innate resistance in the germplasm was gradually lost against this deadly pest. Since traditional plant breeding methodologies were futile, there was a need to develop BSFB resistant varieties of brinjal across the world by using transgenic approaches. Although genetic transformation of eggplant was demonstrated long time ago, the application of this technology for genetic improvement is still in its infancy. To date, only a few traits of agronomical importance have been introduced into cultivated eggplants. These traits include insect resistance, parthenocarpic fruit production and tolerance to salinity and drought stress (Table 1). Therefore, the development of eggplant transgenics with various new traits is need of the hour.

5

Generation of Bt Brinjal

Bacillus thuringiensis (Bt) is a member of the Bacillus cereus group that also includes B. cereus, B. anthracis and B. mycoides (Helgason et al. 2000). The feature that distinguishes Bt from the other members of the Bacillus cereus group is its entomopathogenic properties. B. thuringiensis is a Gram-positive bacterium (Salamitou et al. 2000; Bartoszewicz et al. 2009). This is a naturally soil inhabiting bacterium that has the ability to form spores upon reaching its steady or stationary phase of the microbial growth. The spores synthesized by the bacterium contain specific crystals called as “cry” proteins (δ-endotoxin proteins). These proteins exhibit toxicity against the insects (Aronson et al. 1986; Whiteley and Schnepf 1986). Several different strains of B. thuringiensis with different insect host spectra have been identified (Burgers 1981; Roh et al. 2007). All these strains were well categorized into different serotypes or subspecies based on the immunogenic properties elicited by the antigenic proteins present in the flagella (Iqbal et al. 2000; Bishop 2002). The distribution of this bacterium is cosmopolitan in nature across the globe irrespective of the climate, type of habitat, be it sandy, clay, rocky or any other kind. B. thuringiensis exhibits a broad diversity with different types of strains coming under its category cumulatively responsible for synthesizing more than 150 types of “cry” protein crystals (Bravo 1997; de Maagd et al. 2001). Majority of these proteinaceous agents produced by various strains are proven to be quite effective against larvae of certain members of the Lepidoptera. However, few cases of its toxicity against Diptera and Coleoptera species have also been reported (Bravo et al. 2013; Federici et al. 1990; Krieg et al. 1983). No lethal effects are shown on the insect caterpillar pests on direct contact or exposure to the toxin; however, the toxin was able to kill the pest when the Bt toxinsprayed leaves were eaten. Although different insects produce varied toxin products with minor variations, these toxins show profound adverse actions on a broad spectrum of insect gut system and few other lower invertebrate species. However,

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Table 1 Transgenic eggplants with improved agronomic traits Transgene Bt (Cry IIIB) Bt (Cry IIIB)

Bt (Cry IIIB) Bt (Cry IIIA) DefH9-iaaM Bt (Cry III) syn.Bt (Cry IAb) syn.Bt (Cry IIIA) Bt (Cry IIIB)

Δ-9 Desaturase DefH9-iaaM(j) Yeast Δ-9 desaturase mtlD nptII Gfp:gus, hpt(b) VirE:LacZ Mi-1.2, Mi-1, Cry1Ab and cystatin Oryza cystatin

Remarks Resistance against insects Modified Bt gene confers resistance to Colorado potato beetle (CPB) Insect resistance Resistance to fruit borer Parthenocarpy in transgenic plants Resistance against insect pests in S. integrifolium and S. melongena Resistance against fruit borer (Leucinodes orbonalis) Resistance to neonate larvae and adult CPB in T0 and F1 population Insect resistance; studied ecological impact assessment of transgenics Increased 16:1, 18:1 and 16:3 fatty acids Parthenocarpic transgenic plants Resistance to Verticillium wilt Salinity, drought and chill tolerance Efficient and stable transformation Increased transformation rate and vir genes Expression by adding 100 μM acetosyringone Resistance to root knot nematode

Resistance to aphids

Reference(s) Chen et al. (1995) Arpaia et al. (1997)

Billings et al. (1997) Hamilton et al. (1997) Rotino et al. (1997b), Donzella et al. (2000) Iannacone et al. (1997) Kumar et al. (1998) Jelenkovic et al. (1998)

Acciarri et al. (2000)

Rotino et al. (1997b), Donzella et al. (2000), Acciarri et al. (2002) Xing and Chin (2000) Prabhavathi et al. (2002) Franklin and Sita (2003) Kumar (2003), Kumar and Rajam (2005a, b) Goggin et al. (2006); Frijters et al. (2000); Papolu et al. (2016); Phap et al. (2010) Ribeiro et al. (2006)

it does not exhibit any toxic effects on humans. In order to understand the mechanism of Bt proteins, Mattes (1927) isolated the pure strain of Bt bacterium whose potential was demonstrated by Husz (1930). Bravo et al. (2007) studied the mode of action of Cry and Cyt toxins of B. thuringiensis and shown their potential for insect control. These results proved to be beneficial and important for developing a Bt insecticide in the year 1938. Subsequently, the genes which encode for different cry proteins were deployed in various crops for insect resistance, hence the crop yield

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can be enhanced due to the protection from insect pests (De Maagd et al. 2001; Pigott and Ellar 2007). Using the Agrobacterium-mediated transformation, Narendran (2006) has designed a protocol for the cotrasformation of two genes simultaneously to generate marker-free insect-resistant plants. In this study, two plasmids containing cry2Ab gene in one plasmid and gus gene in the other were transferred to produce transgenic insect-resistant plants (Narendran 2006). Cry3B codes for another toxin that has been shown to be highly effective against insects. This toxin is coded by the mutagenized Bt gene called berl. Three transgenic lines of eggplants, namely 3-2, 6-1, 9-8, were developed by introducing berl gene coding for Cry3B toxin. While the 3-2 and 9-8 lines were found to be highly insect resistant, they also resulted in enhancing the yield of the crop. On the other hand, the line 6-1 exhibited only moderate levels of insect resistance and was not much different from the controls (Rotino et al. 1992). The modified Bt gene coding for Cry3B gene was also transformed in different explants through tissue culture. Transformation with Cry3B in cotyledons (Fári et al. 1995), hypocotyl (Chen et al. 1995) and leaf segments (Jelenkovic 1998) of different lines of eggplant showed resistance against the various Coleopterans. Pal et al. (2009) developed transgenic brinjal resistant to shoot and fruit borer by introducing Cry1Ac gene.

5.1

Choosing the Right Bt Strain

As said earlier, there exists a variety of strains in the Bt species. Due to the wide diversity, each of the strains is highly specific to a particular receptor only. Such specificity helps in identifying the compatible receptor for its binding. Hence, the damage is highly specific and not random. So, it becomes important for us to choose the particular Bt strain that is capable of targeting the pest causing the damage. Else, there is a danger of eradicating the farmer friendly insect pests as well. Hence, a word of caution needs to be followed in choosing the Bt strain (De Maggad et al. 2001; Roh et al. 2007). The major achievement in eggplant biotechnology is the development of insectresistant transgenic plants by overexpressing the Bt genes that encode the crystal protein endotoxin. Bt CryIIIb was used to develop plants resistant to Colorado potato beetle (CPB; Rotino et al. 1992). Field trials showed that insect-resistant transgenic lines had a significantly higher yield (Arpaia et al. 1997; Acciarri et al. 2000). Resistance to BSFB, a lepidopteran insect that causes extensive damages to fruits and shoots, was obtained in transgenic plants produced through transformation of cotyledon explants with the strain EHA105 harbouring a synthetic cry1Ab gene modified for rice codon usage and carrying a castorbean catalase intron (Kumar et al. 1998).

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Bt Brinjal Status in India and Bangladesh

After the success of Bt cotton that was deployed in 2002 in India, the focus shifted towards developing the Bt brinjal to target the devastating pest BSFB. MAHYCO, the Maharashtra-based company, was the pioneer and took the lead in developing a new DNA construct whose gene sequence contained an entomopathogenic protein CRY1AC. It was designed such that the insecticidal protein will be expressed in the whole brinjal plant. The Cry1AC was incorporated into the host DNA in association with two other selectable marker genes, namely npt-II and aad (aminoglycoside N60 -acetyltransferase). These genes have been shown to function in combination that helps in the synthesis of a novel protein that has been proved lethal against BSFB. The CRY1AC gene inserted into the host construct was isolated from HD73, a strain classified under the subspecies kurstaki, of B. thuringiensis. This CRY1AC construct with few modifications resulted in the production of active gut targeting insecticidal protein which was inserted into the host plant. In the year 2002, Bt brinjal was developed in India, and all official permissions from Indian government were procured (Kameswara Rao 2010). The potency, bio-safety and field accomplishment were performed by 12 public and private institutions, and a Review Committee for Genetic Manipulations (RCGM) reviewed the results obtained from field testing. Genetic Engineering Appraisal/Approval Committee (GEAC) has been appointed with two expert committees for verification of data. The report committee had approved the release of Bt brinjal termed as EE-1into the market in 2009. Following the approval, Bt EE1 was introgressed into local cultivars in Karnataka and Tamil Nadu which include Bt Malapur local (S), Bt Manjari Gota, Bt Udupi Gulla, Bt Rabkavi local, Kudachi local and Bt Go-112. These cultivars were evaluated along with their non-Bt plants. Ever since it got its clearance for release, Bt brinjal became a topic of controversy. Although the firm claimed enhanced yield due to pest resistance, there were concerns on its safety from various corners. This controversy led to a strong nationwide protest as a result of which the approval for the Bt brinjal was recalled indefinitely by the Ministry of Environment, Government of India. However, the same Bt brinjal that was supplied to Bangladesh was approved by the Government of Bangladesh. Subsequently Bangladesh developed few other varieties, namely Manjarigota, Ruchira, Poona selection and Krishna Kathi (Jadhav et al. 2015), and such Bt brinjal hybrids are performing very well with-reduced pesticide use, yet with enhanced yields.

6

Genetically Modified Brinjal for Biotic Stresses

Apart from insects, the other predominant biotic factors that cause reduced production of brinjal are bacterial and fungal wilt diseases. The wilt diseases caused by Verticillium dahlia and Fusarium oxysporum are the major diseases of eggplant. Although host resistance has been the frequently used method to control these diseases, the resistance developed by the pathogenic strains results in the failure of the crop. The use of excess fungicides has been on the rise and has reported to be

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toxic to the health of the consumer and further, it has been reported that it can cause degradation of the surrounding environment. Hence, in order to protect the loss due to the microbial pathogens, genes that are effectively induced by the plants following the attack of fungus, and other microbes have been identified and isolated from various sources. Chitinase is one such class of proteins that has been understood to be induced in host plants as a defence mechanism to protect against a broad range of microbial pathogens, fungus in particular. Genetic engineering of disease resistance by transfer of plant defence-related genes into crops is valuable in terms of cost, efficacy and reduction of pesticide usage. The variation in the degree of fungal resistance in different transgenic lines could be due to the position effect-mediated variation in transgene expression (Prabhavathi and Rajam 2007a). Overexpression of a yeast Δ9-desaturase gene in eggplant has been attempted with the objective of developing disease-resistant plants. Transgenic plants were shown to contain higher concentrations of 16:1, 18:1 and 16:3 fatty acids and exhibited increased resistance to Verticillium wilt (Xing and Chin 2000). Transgenic plants challenged by Verticillium could also result in a marked increase in the content of 16:1 and 16:3 fatty acids. Results also showed that cis-Δ916:1 fatty acid was inhibitory to Verticillium growth. A gene encoding Oryza cystatin was introduced in eggplant and the effect on Myzus persicae and Macrosiphum euphorbiae was examined (Ribeiro et al. 2006). The transgenic eggplant reduced the net reproductive rate, the instantaneous rate of population increase, and the finite rate of population increase of both aphid species compared with a control eggplant line. Age-specific mortality rates of M. persicae and M. euphorbiae were higher on transgenic plants. These results indicate that expression of oryza cystatin in eggplant has a negative impact on population growth and mortality rates of M. persicae and M. euphorbiae and could be a source of plant resistance for pest management of these aphids. The class I chitinases are the vacuolar proteins capable of degrading the cell walls of invading phytopathogenic fungi, and they have been implicated in the defence of plants against fungal pathogens. Studies of Singh et al. (2015) have revealed that the transgenic lines of brinjal that were overexpressed with chitinase gene (chi) derived from rice plant conferred high resistance against the fungal pathogens, V. dahlia and F. oxysporum. In this study, class I rice endochitinase gene was introduced into eggplant under the control of a constitutive CaMV 35S promoter by Agrobacteriummediated transformation. Molecular analysis of putative transformants revealed the presence of transgene and its expression in several transformants of eggplant. The transgenic lines also showed higher chitinase activity as compared to the untransformed controls. Fungal resistance assays of transgenic lines against the wilt causing fungi, V. dahliae and F. oxysporum, exhibited the delay in the onset of disease by 5–7 days as well as enhanced resistance against wilt diseases (Singh et al. 2015). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed by Singh et al. (2014) to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using npt-II gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by PCR analysis and Southern blot analysis and transgene

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expression by western blot analysis and glucanase activity. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wiltcausing fungi with a delay of 5–7 days in the disease development as compared to wild-type plants. Prabhavathi and Rajam (2007a) have shown that transgenics expressing mtlD gene with mannitol accumulation exhibit increased resistance against three fungal wilts caused by F. oxysporum, V. dahliae and Rhizoctonia solani under both in vitro and in vivo growth conditions. Mannitol levels could not be detected in the wild-type plants, but the presence of mannitol in the transgenics could be positively correlated with the disease resistance. Similarly, Wasabi defensin is another gene that has been isolated from Wasabia japonica, a Japanese horseradish that has been shown to be good source of antimicrobial proteins. This gene has been identified to be highly inducible against fungal attacks. Marker-free fungal-resistant transgenic brinjal plants were developed against Alternaria solani employing site-specific method of recombination to clone Wasabi defensin gene (Darwish et al. 2014). In this study the Wasabi defensin gene was isolated and cloned from the binary vector pEKH-WD, to an ipt-type MultiAuto-Transformation (MAT) vector system, pMAT21, and then transferred to A. tumefaciens strain EHA 105. Transgenic brinjal plants have also been developed to confer protection against viral diseases such as cucumber mosaic virus and tomato chlorotic spot virus. A. tumefaciens strain LBA4404 carrying the coat protein gene of cucumber mosaic virus (CMV-CP) was used for genetic transformation of the cotyledonary explants obtained from S. melongena cv. Pusa Purple Long variety of eggplant. Such transformed lines exhibited increased resistance to the CMV virus (Pratap et al. 2011). Similarly, eggplants that were transformed with SW-5 gene were found to be effective against tomato chlorotic virus and were regenerated through organogenesis and somatic embryogenesis conferred resistance to the tospovirus (Picoli et al. 2006). Expression of Mi-1.2 gene isolated from tomato in eggplant cv. HP 83 conferred resistance to root knot nematode Meloidogyne incognita (Goggin et al. 2006). Previously, Mi-1 gene was used to engineer eggplant for resistance to M. incognita (Frijters et al. 2000). Nematode (M. incognita) resistant brinjal transgenic plants were also developed by using cry1Ab gene from B. thuringiensis (Phap et al. 2010). Recently, the expression of a cystatin transgene has exhibited resistance against M. incognita in eggplant (Papolu et al. 2016).

7

Genetically Modified Brinjal for Abiotic Stresses

Eggplants are also affected by various abiotic stresses. The compatible osmolytes such as carbohydrates, sugar alcohols, proline and glycine betaine, and the increased levels of such osmolytes help in eliciting better response to various abiotic stress conditions. Efforts were made to develop such plants that produce increased osmolytes to counter the stress. Transgenic lines of brinjal were developed in

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order to improve the tolerance against abiotic stresses. Introduction of bacterial gene mannitol-1-phosphodehydrogenase (mtlD) into the eggplant through A. tumefaciensmediated transformation resulted in transgenic lines, which exhibited tolerance against osmotic stress induced by drought, salinity and cold (Prabhavathi et al. 2002). Incorporation of a binary vector pCAMBIA2300 containing rd29A-DREB1A gene into eggplant through A. tumefaciens-mediated transformation resulted in increased tolerance against moisture-induced stress (Sagare and Mohanty 2012). Transgenic lines in brinjal were also developed by the overexpression of a foreign gene HAL1 obtained from yeast exhibited increased tolerance to high concentration of salts in soils (Sugumaran et al. 2014). Polyamines (putrescine, spermidine and spermine) have been shown to be important for abiotic stress tolerance. Prabhavathi and Rajam (2007b) investigated the stress tolerance in eggplant by the introduction of a key polyamine biosynthetic gene arginine decarboxylase (adc) under the control of a constitutive promoter, CaMV35S through Agrobacterium-mediated transformation. The developed transgenic lines have shown an enhanced level of polyamines due to the increase in ADC enzyme activity. The diamine oxidase (an enzyme involved in putrescine and spermidine degradation) activity was also increased in these transgenic plants. Polyamine-accumulating transgenic plants exhibited an increased tolerance levels to multiple abiotic stresses such as salinity, drought, low and high temperature and heavy-metal and resistance against fungal wilt disease caused by F. oxysporum.

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Transgenic Brinjal with Other Traits

A significant achievement in brinjal biotechnology is the development of transgenic brinjal with parthenocarpic fruits by manipulating the concentrations of endogenous auxin during fruit development. This was demonstrated by the production of transgenic brinjal expressing a chimeric iaaM gene from Pseudomonas syringae, which encodes tryptophan mono-oxygenase, under the control of an ovule-specific DefH9 promoter from Antirrhinum majus (Rotino et al. 1997b). In the absence of pollination, transgenic eggplants developed seedless parthenocarpic fruits in the absence of exogenous phytohormones, even at low temperatures which normally prohibit fruit production in untransformed lines (Rotino et al. 1997b). When pollinated, the parthenocarpic plants produced seeded fruits. Trials in an unheated glasshouse showed significantly higher yields in transgenic plants than in untransformed controls and a commercial hybrid (Donzella et al. 2000).

9

Conclusions and Future Prospects

Although genetic engineering has helped a great deal in the process of crop development, the status of genetically modified crops is still in its early phases confining to very few countries. The development of Bt brinjal has received mixed response

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from different committees. Being a developing country, India is facing problems with the rapidly rising population. With increasing demand for food, Bt crops are gaining importance and hence could be a possible solution to the problem. Apart from increasing the farmer’s income, Bt also helps arrests the draining of the chemicals into the water bodies, thereby reducing the aquatic contaminations. Such steps also help in increasing the diversity. Although there are certain questions relating to the safety and associated problems with Bt brinjal, Bt brinjal is being marketed in Bangladesh. The product cannot be just ignored citing these concerns. Further research is warranted to improve the safety and nutritional aspects. Unfortunately, no significant progress has been made in determining the safety of the Bt brinjal since the time of recall of Bt brinjal in India. Therefore, it is safer to wait until any further work evaluates and confirms that the consumption of Bt brinjal is safe before it can be considered for commercialization. Also, we need to look for safer and improved alternatives in GM technology, where in the Bt gene can be confined to the specific tissue only rather than being present in multiple tissues. Also, gene editing tools, particularly CRISPR-Cas9 system can be adopted for the editing of those genes which is associated with eggplant improvement.

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Biotechnology of Red Pepper M. V. Rajam, S. Nandy, and R. Pandey

Abstract

Red pepper (Capsicum spp.) is an indispensable spice used as a basic ingredient in a great variety of cuisines all over the world. It is a recalcitrant for in vitro plant regeneration as well as genetic transformation as compared to other members of the Solanaceae family like tobacco, tomato, brinjal and potato. Considerable efforts have been made during the last two decades on the establishment of optimum conditions for plant regeneration from various seedling explants, zygotic embryos, anthers, cells and protoplasts. Micropropagation protocols have been developed by culturing shoot tips, meristems and nodal bud segments. Likewise, genetic transformation procedures have been established via Agrobacterium-mediated transformation and gene gun methods. These procedures have been used to develop transgenic red peppers with new traits, including biotic and abiotic stress tolerance and post-harvest characteristics. The biotechnological developments are of enormous value for the improvement of red peppers. In this chapter, we have discussed the developments, limitations and applications of red pepper biotechnology. Keywords

Capsicum spp. · Micropropagation · Plant regeneration · Genetic transformation · Transgenic red pepper

M. V. Rajam (*) · S. Nandy · R. Pandey Department of Genetics, University of Delhi South Campus, New Delhi, India # Springer Nature Singapore Pte Ltd. 2021 P. B. K. Kishor et al. (eds.), Genetically Modified Crops, https://doi.org/10.1007/978-981-15-5932-7_3

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Introduction

All chilli peppers belong to the genus Capsicum of Solanaceae family. Pepper, chili, chile, chilli, aji, paprika and Capsicum are used interchangeably to describe the plants and fruits of the genus Capsicum. Capsicum (chilli peppers) is a New World genus from the nightshade family originated and domesticated in the American tropics. Capsicum is derived from the Greek word ‘Kapsimo’ meaning to bite (Basu and De 2003). The first varieties of chilli pepper originated in the remote geologic past in an area bordered by the mountains of southern Brazil to the east, by Bolovia to the west and by Paraguay and northern Argentina to the south (DeWitt and Bosland 1993). This location is called a nuclear area and has the greatest concentrations of wild species of chilli pepper in the world. Chilli is an indispensable spice used as a basic ingredient in a great variety of cuisines all over the world. It is also used as flavourant and colourant and adds tang and taste to the otherwise insipid food. The nutritive value of Capsicum is high and an excellent source of vitamins C (ascorbic acid), A, B-complex and E along with minerals like molybdenum, manganese, folate, potassium and thiamine. Vulnerability of the chilli pepper genotypes to a multitude of abiotic and biotic stresses has restricted their potential yield. Capsicum members have shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it difficult or inefficient to apply to recombinant DNA technologies via genetic transformation aimed at improvement. In this chapter, the advances, limitations and applications of red pepper biotechnology are discussed.

2

Plant Regeneration

In vitro plant regeneration from cells, tissues and organ cultures is a fundamental process for the application of plant biotechnology to plant propagation, plant breeding and genetic improvement. While many members of the family Solanaceae are facile with regard to cell culture and regeneration, red pepper and sweet pepper (Capsicum annuum L.) are considered to be recalcitrant, and for the same reason, reports on chilli pepper plant regeneration from established callus lines, cell suspensions and protoplasts are currently very scarce. Its regeneration has been achieved mainly through organogenesis, but recent reports have increased the information on somatic embryogenesis. In vitro plant regeneration is the interplay of many factors, and in the case of Capsicum these factors are explant source, culture medium, concentrations of plant growth regulators and culture conditions. Many researchers have effectively demonstrated this. Considerable efforts have been made in recent years to optimize various factors and establish efficient regeneration methods (Ochoa-Alejo and Ramirez-Malagon 2001).

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Organogenesis

Organogenesis is a complex phenomenon involving de novo formation of organs (shoots or roots). Shoots can be derived either through differentiation of non-meristematic tissues known as adventitious shoot formation or through pre-existing meristematic tissues known as axillary shoot formation. Gunay and Rao, for the first time, made attempts for in vitro plant regeneration from hypocotyls and cotyledon explants (Gunay and Rao 1978). They chose three cultivars, and in all friable callus formation was induced but not root and shoot bud formation. Fari and Cźako (1981) studied the relationship between the position and morphogenetic responses of hypocotyl explants. They observed shoot regeneration in the apical segments, while the middle and basal sections produced only roots and callus, respectively. Some of the regenerated shoots developed into whole plants after rooting on culture medium lacking growth regulators. Differentiation of multiple shoot buds and plantlets in cultured embryos was reported by Agrawal and Chandra (1983). Philips and Hubstenberger, in 1985, reported the importance of light regime, growth regulators and carbon source on shoot organogenesis. Glucose was found to be a superior carbon source in comparison with sucrose, and shoot elongation was shown to be the limiting factor in plant regeneration. In a series of reports, Sripichitt et al. (1987, 1988a, b) investigated the in vitro shoot-forming capacity of cotyledonary explants, the effect of exposure dose and dose radiation. They employed this protocol to induce and recover mutant plants from in vitro cultures. Benzyladenine (BA) was found to be more effective than kinetin (Kn) in inducing shoot formation in cotyledon explants cultured on Murashige and Skoog’s (MS) medium (Murashige and Skoog 1962). Agrawal et al. (1989) used segments of roots, hypocotyls, cotyledons, stem internodes, stem nodes, leaves and shoot tips as explants and studied their response on MS medium supplemented with BA or Kn alone or in combination with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthalenacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). It was observed that with increasing concentrations of BA, shoot buds were formed directly from both hypocotyl and cotyledonary explants, while stem internode and leaf segments showed scanty callus formation. Further increase in the concentrations of BA increased the shoot bud induction directly in all explants. Cotyledon, leaf and stem explants exhibited higher bud formation than hypocotyls. Cultured hypocotyl tissues of chilli pepper derived from aseptically germinated seedlings were evaluated on the basis of their morphogenic responses to combinations of various hormones (Ochoa-Alejo and GarćiaBautista 1990). The differential in vitro response in hypocotyl explants of red pepper was reported (Christopher et al. 1991). They have also used hydroxylamine-treated tissue cultures for shoot regeneration in red pepper (Christopher et al. 1990). Induction of multiple shoots was achieved by gamma rays from cotyledon cultures of red pepper (Subhash and Prolaram 1987). The influence of chilli pepper cultivar on the potential of hypocotyl tissues to form adventitious shoots was tested, and explants from 16 cultivars were tested on six culture media (Ochoa-Alejo and Ireta-Moreno 1990). Cultivar differences were

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observed with regard to their ability for in vitro differentiation of shoots. Optimal shoot regeneration medium varied with the cultivar. They extensively studied the effect of explant source (cotyledons, hypocotyls and zygotic embryos), diverse components of the nutrient medium (culture medium, vitamins and gelling agents), growth regulators, incubation conditions and culture container on organogenesis. The results showed that the highest percentage of organogenesis was achieved in the presence of BA or zeatin (Zn). Thidiazuron (TDZ) alone or in combination with IAA or with either BA or Zn did not significantly improve the production of buds, leafy structures or shoots. Significant differences in organogenesis were demonstrated using different gelling agents, Difco Agar being the most effective. A favourable effect of continuous light as compared to photoperiod and dark conditions on organogenesis was also demonstrated. In vitro plant regeneration from cotyledon and hypocotyl explants in two bell cultivars was reported by Arroyo and Revilla (1991). Hypocotyls differentiated into shoot buds and rosettes. Subsequently, the plantlets were successfully transferred to pots. Many researchers observed the formation of ill-defined buds or leafy or shoot-like structures in cotyledon, leaf or hypocotyl explants. Other approaches were tried to overcome the difficulties of shoot bud elongation. In one strategy, rooted hypocotyls from three cultivars were cultured upside down on shoot inducing MS medium with different combinations of plant growth regulators and were supplemented with silver nitrate to prevent ethylene action (Valera-Montero and Ochoa-Alejo 1992). Hypocotyls bearing buds, after culture in the induction medium, were placed in the normal polarity on fresh MS medium lacking growth regulators to promote shoot elongation and development. Ebida and Hu (1993) reported that morphogenetic responses of seedling explants and plantlets were established successfully. Mature seeds have also been used as explants for in vitro chilli pepper plant regeneration through organogenesis (Ezura et al. 1993; Dabauza and Pena 2001). Shoot formation was achieved from excised explants consisting of proximal part of hypocotyls and radicle of mature seeds cultured in MS medium without growth regulators. This plant regeneration approach is simple as it involved medium without growth regulators, and is also rapid with minimal detectable variations among the regenerants. Binzel et al. (1996a) modified this method for in vitro regeneration of a mild and a pungent chilli pepper. Half-seed explants were cultured on MS medium with or without cytokinins. Cytokinins in the culture medium dramatically increased both the percentage of explants forming buds and the number of buds per explant and also hastened the rate of bud production. The elongation of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins. Investigations on plant regeneration using the most beneficial methods and culture media were described earlier (Gunay and Rao 1978; Fari and Cźako 1981; ValeraMontero and Ochoa-Alejo 1992), and culture media supplemented with TDZ were carried out by Szász et al. (1995). With the incorporation of TDZ in the medium, direct shoot induction was achieved. Hyde and Philips (1996) observed plant regeneration via bud inductions, bud enlargement, shoot elongation and rooting of shoots. The effect of silver nitrate on bud proliferation and shoot elongation was also

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investigated. Treatment with silver nitrate was necessary for multiple shoot production and elongation. The relative importance of genotype, explant, medium and their interactions in two wild species and five cultivars of C. annuum on three regeneration media was reported by Christopher and Rajam (1996). Shoots were induced from hypocotyls, cotyledons and leaf explants. The most significant effect on shoot regeneration was due to the type of explant used. Leaf explants consistently generated more shoots than hypocotyls or cotyledons in all genotypes. Determination and competence of chilli pepper hypocotyl cultures during shoot formation seemed to be dependent on BA and sucrose (Ramage and Leung 1996). Simultaneous presence of these two components has been found to be obligatory for high-frequency shoot formation. BA and sucrose seem to act independently on different aspects of the competence of explants to respond to the components of induction medium during shoot initiation. Ramírez-Malagón and Ochoa-Alejo (1996) investigated five factors that influence shoot regeneration, i.e. age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture medium and types of cultivars. It has been observed that growth regulators are needed in this method to induce bud and shoot elongation, and their efficiency is higher than in previously reported systems (Valera-Montero and Ochoa-Alejo 1992; Ezura et al. 1993). Other growth regulators have been tested to overcome the problems faced with shoot bud elongation. A plant steroid lactone (24-epi-brassinolide: EBR) was employed for the elongation of shoot buds (Franck-Duchenne et al. 1998). The presence of EBR induced stem elongation along with leaf elongation. It appears that EBR acts indirectly on stem elongation as an elicitor or enhancer of elongation in combination with endogenous or exogenously added growth regulators. Phenylacetic acid (PAA) has also been found to improve chilli pepper shoot bud elongation (Husain et al. 1999). Hyperhydricity in chilli pepper plants regenerated in vitro was studied by Fontes et al. (1999). Plants regenerated by the system were investigated by ultrastructural analysis, SDS-PAGE and immunoblot analysis. Direct and indirect in vitro plant regeneration was reported by Berljak (1999). He reported that an important step for shoot development from chilli pepper was the transfer of callus after 2 weeks on to the regenerative medium. Plants regenerated from callus cultures grown ex vitro showed differences in their morphological and physiological traits. This is the first preliminary report of a regenerable callus system for Capsicum species. Mihalka et al. (2000) reported an efficient in vitro regeneration system that uses the basal part of young cotyledons for shoot induction. Though the application of this protocol resulted in whole plant regeneration in a wide range of genotypes, but considerable efficiency and reproducibility could only be obtained in a particular genotype only. Enhanced plant regeneration of pepper seedlings in four cultivars consisting of the radicle, hypocotyls and one cotyledon were obtained after removing the primary and axillary meristems (Pozueta-Romero et al. 2001). Until now there has been no efficient in vitro culture system for chilli pepper varieties of Tunisia. Published regeneration protocols for pepper have not been suitable for regeneration on Tunisian varieties due to the genotype effect. This

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difficulty has been noted by many authors (Szász et al. 1995; Franck-Duchenne et al. 1998; Mihalka et al. 2000). Arous et al. (2001) optimized an adapted protocol of in vitro plant regeneration for Tunisian pepper. Dabauza and Pena (2001) intensively studied the effect of various explant types on bud regeneration and subsequent elongation in eight varieties of sweet pepper. Efficacy of different seedling explants (cotyledons, leaves and cotyledonary node shoot tip) and embryonic explants (embryonic cotyledon, embryonic hypocotyls and wounded seedlings) was evaluated for plant regeneration response. High regeneration frequency was obtained from all varieties, when explants were cultured on 2 mg/L TDZ. TomaszewskaSowa et al. (2002) studied the effect of cytokinins on in vitro morphogenesis and ploidy level in pepper. Their experiments resulted in regeneration of pepper plants, which flowered in vitro, and also showed that shoot regeneration did not involve callus, which decreased the probability of somaclonal variation. Amzad et al. (2003) used cotyledonary node explants of two chilli pepper varieties for plant regeneration, and the R1 regenerants have shown variations in plant growth habit, stem colour, flower and fruit colours and the expression of anthocyan in unripe fruits. The R1 regenerated lines have also exhibited differences as compared to their parents, and these variations are early flowering and increase in yield components. They have suggested that these somaclones would be important for chilli pepper improvement. Cytokinins present in the initial medium do not disturb mitosis and changes in ploidy in regenerants, which suggests that the protocol optimized is justified in micropropagation of valuable sweet pepper genotypes. Venkataiah et al. (2003) established a promising system for direct regeneration in cotyledon and leaf explants of red pepper, induced by TDZ over the traditional use of BA in combination with IAA (Szász et al. 1995; Venkataiah and Subhash 2001). Kumar et al. (2005) reported direct multiple shoot formation from seedling explants. They were able to induce regeneration by inverting the explant. The MS medium used was supplemented with ethanesulphonic acid (MES) buffer along with BA, IAA and silver nitrate and could get profuse bud induction. Li et al. (2001) reported the effects of cytokinins on shoot organogenesis from cotyledonary explants of pepper. Qin et al. (2005) reported the effects of the exogenous plant growth regulators on in vitro regeneration of cotyledonary explants in pepper. Ahmad et al. (2006) used nodal explants of C. annuum (cv. Pusa Jwala) for the induction of multiple shoots, and the rate of multiple shoot induction was high on MS medium fortified with 1.0 μM TDZ. The regenerated plants were morphologically and cytologically normal. Siddique and Anis (2006) reported TDZ-induced high-frequency shoot bud formation and plant regeneration from cotyledonary node explants of C. annuum. Golegaonkar and Kantharajah (2006) investigated the shoot-forming capacity of leaf and cotyledon explants from five Indian chilli pepper cultivars (Gujarat-1, Gujarat-2, Guntur-4, Selection49 and Jwala), and they found that regeneration frequency was highly influenced by the explant (49.7% of the accounted total variation), culture media (29.2%) and cultivar (14.2%). The young leaf explants of all the cultivars consistently gave higher regeneration of adventitious shoots than cotyledon explants. A highly efficient procedure for shoot multiplication and plant regeneration of capsicum was developed by Venkataiah et al. (2006). Various cytokinins, viz. BA, kinetin, zeatin

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and TDZ, were tested for plant regeneration from shoot tip explants. TDZ-induced maximum number (4.2–22.4) of shoots in all the Capsicum species tested. Multiple shoot elongation occurred upon transfer to BA (0.22 μM) + IAA (0.48 μM). Rooting of regenerated shoots was achieved on medium supplemented with 5.71 μM IAA. Rooting was observed in 72–94% of shoots obtained from TDZ-containing medium followed by elongation treatment in contrast to 8–22% of shoots obtained without an elongation treatment. Plantlets obtained on TDZ-containing media were normal diploids (2n ¼ 24) and could readily be established in the soil under greenhouse conditions with a survival frequency of 68–84%. Regenerated plants developed into morphologically normal, fertile plants and able to set viable seeds. Joshi and Kothari (2007) emphasized the importance of higher copper levels in the medium for enhanced shoot bud differentiation and elongation from cultured cotyledons of C. annuum cv. X-235. Shoot buds were induced from cotyledons on BA (22.2 μM) and PAA (14.7 μM) supplemented medium and subsequently elongated on BA (13.3 μM) along with GA3 (0.58 μM); both shoot bud differentiation and subsequent elongation media were supplemented with different levels of CuSO4 (0–5 μM). The highest number of shoot buds per explant was obtained when the level of CuSO4 was increased 30 times the normal MS concentration. Shoot bud induction frequency together with elongation in terms of percentage response and length of shoots was better than that on control. Sharma et al. (2008) investigated the influence of silver nitrate (AgNO3) and cobalt chloride (CoCl2) on shoot multiplication and in vitro flowering in C. frutescens Mill. The exogenous administration of AgNO3 and CoCl2 at a concentration of 30 μM resulted in the maximum tissue response in terms of shoot length and number of shoots after 45 days of culturing on MS medium. Both silver nitrate (40 μM) and cobalt chloride (30 μM) influenced in vitro flowering after 25 and 45 days, respectively. This is the first report on in vitro flowering in C. frutescens. Kumar et al. (2007) in a novel approach employed aseptically grown seedling explants of C. frutescens devoid of roots, apical meristems and cotyledons in inverted position on various media. Profuse shoot buds were obtained per explant under continuous light. Sanatombi and Sharma (2008) studied the effect of different explants in six cultivars belonging to three species of Capsicum (C. annuum, C. frutescens and C. chinense). In agreement with previous reports, they also found leaf and cotyledons to be the most responsive explants in comparison to hypocotyls. Valadez-Bustos et al. (2009) reported in vitro chilli pepper plant regeneration using three different explants and three different media. With embryos and hypocotyls as explants, development of morphologically normal adventitious shoot bud formation was reported, while cultured cotyledons resulted in non-elongating rosette-shaped shoot buds. In another study, hypocotyl, cotyledon and shoot tip explants of C. annuum were cultured to determine their regeneration potential. Callus induction and shoot initiation were found to be higher in hypocotyls than cotyledons. Shoot tips regenerated plantlets with sporadic small amount of callus at the base. Shoot elongation was achieved using additional supplementation of GA3 and AgNO3 (Ashrafuzzaman et al. 2009). Song et al. (2010) developed a simple and efficient protocol for in vitro propagation of two miniature paprika cultivars

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(C. annum cv. Hivita Red and Hivita Yellow). Their seeds were decontaminated and placed in a Petri dish containing half-strength MS medium and then incubated in dark for 7–10 days for germination. Leaf explants were excised from 1-month-old aseptic seedlings and cultured on MS medium supplemented with TDZ at different concentrations or in a combination with NAA for 4 weeks. MS medium containing BAP (4.0 mg/L) and IAA (0.5 mg/L) was found to be the best for in vitro shoot bud differentiation from hypocotyl explants of chilli peppers (C. annuum) (Aniel Kumar and Subba Tata 2010). Similarly, an efficient procedure for in vitro plant regeneration through direct shoot bud induction was observed for different explants of C. annuum by Otroshy et al. (2011). The best performance was recorded on MS medium containing BAP (6.0 mg/L) and IAA (1 mg/L). Kumari et al. (2012) have established a high regeneration potential in sweet pepper (C. annuum cv. Yolo Wonder and California Wonder). In vitro plant regeneration was reported from hypocotyl explants, which were collected from aseptically raised seedlings of popular Capsicum F1 hybrids Bharat and Indra by Hegde et al. (2017). They observed varied responses to in vitro morphogenesis with different genotypes and combinations of growth regulators used. Plant regeneration procedure was developed for Naga King Chilli (C. chinense Jacq.) using nodal segment explants by Jamir et al. (2019). They recorded maximum response (88.88%) for shoot regeneration on medium containing 8 mg L1 BAP + 0.5 mg L1 IAA, and the highest number of functional roots (14.55 per explant) on rooting medium fortified with 1 mg L1 IBA. The plantlet survival was 70% during hardening.

2.2

Somatic Embryogenesis

Somatic embryogenesis is an alternative morphogenetic route for obtaining in vitro plants. It is a more efficient morphogenic pathway than organogenesis for regenerating and propagating plants with relatively high genetic uniformity. Harini and Lakshmisita (1993) reported for the first time, the regeneration of chilli pepper plants through direct somatic embryogenesis from immature zygotic embryos. Globular- and heart-shaped zygotic embryos failed to survive, whereas cotyledonary stage embryos responded well, but early cotyledon stage produced more somatic embryos than did larger or smaller embryos. MS medium supplemented with 4–18 μM 2,4-D and 3–10% sucrose has been observed to promote somatic embryo formation in chilli pepper explants (Binzel et al. 1996b; Buyukalaca and Mavituna 1996), whereas cytokinins seemed to have no significant effect on somatic embryogenesis in chilli pepper (Kaparakis and Alderson 2008). Jo et al. (1996) reported plant regeneration through somatic embryogenesis from immature zygotic embryo culture in red pepper. Germination of somatic embryos has been induced by GA3 or TDZ, alone or in combination (Binzel et al. 1996b), whereas abscisic acid (ABA) has been used to promote maturation of somatic embryos (Buyukalaca and Mavituna 1996). Mavituna and Buyukalaca (1996) have studied the bioreactor type and

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oxygen uptake for the induction of somatic embryogenesis of pepper in bioreactors. The effect of leaf position, illumination and explant pretreatment with high cytokinin concentrations was studied for the induction of somatic embryogenesis from young, fully expanded leaves of chilli pepper (Kintzios et al. 2000). Kintzios et al. (2001) studied the influence of vitamins and inorganic micronutrients on callus growth and somatic embryogenesis from leaves of chilli pepper. A protocol for the separation of somatic embryos from embryogenic suspension cultures based on a cold treatment has been reported by Buyukalaca et al. (2003). Direct somatic embryogenesis and plant regeneration from stem segments and shoot tips of C. annuum on TDZ supplemented medium have been described by Khan et al. (2006). A system for the induction of direct somatic embryogenesis and plant regeneration was established using immature zygotic embryos (Binzel et al. 1996b). The whole process of induction and maturation was achieved on the same medium. Secondary somatic embryogenesis also occurred directly from the primary somatic embryos. Bodhipadma and Leung (2003) have reported in vitro fruiting and seed set in plantlets regenerated via somatic embryogenesis from immature zygotic embryos of C. annuum (cv. Sweet banana). Buyukalaca and Mavituna (1996) developed a protocol for plant regeneration from embryogenic cell suspensions. Embryogenic callus masses were induced directly on mature zygotic embryo explants and transferred to liquid MS medium to establish repetitive embryogenic cell suspensions. Somatic embryos at late torpedo stage were matured on paper bridges in half-strength liquid MS medium and converted into plants with a high efficiency. Steinitz et al. (2003) reported failure of proper shoot development in regenerants obtained by direct somatic embryogenesis. They observed that regenerants lacked a shoot irrespective of the auxin-type applied and across all responsive genotypes. In conclusion, only a few reports on chilli pepper regeneration through embryogenesis have been published, and it is evident that more investigations on the factors involved in this process are needed to establish efficient regeneration systems.

2.3

Shoot Tip Culture

Apical shoots, axillary or apical buds and meristems are explants extensively employed to obtain genetically identical plants in large numbers through micropropagation. Since chilli peppers do not have a natural ability for vegetative or asexual propagation, in vitro propagation methods are useful alternative techniques for clonal propagation. Plant regeneration, from shoot tips, was examined by Fári (1986). Shoot tip cultures were established using different accessions; however, majority of the shoot tips died after the second or third passage in the media, while in some accessions, the tips grew continuously at the same intensity, even after eighth or tenth subculture. Shoot tip explants were also used for micropropagation of C. annuum (var. Mathania) by Agrawal et al. (1988). Gururaj et al. (2004) have reported in vitro clonal propagation of bird eye chilli (Capsicum frutescens Mill.). A protocol was developed to obtain whole plants from apical shoot

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meristems (Madhuri and Rajam 1993). Multiple shoots were produced and the shoots further developed upon transfer to agar-solidified medium and complete plants were obtained. Shoot tips were sub-cultured every 2–3 weeks to a fresh medium (Christopher and Rajam 1994). Shoot tip explants excised from these cultures were inoculated on culture media containing BA to promote shoot proliferation. Addition of 2,3,5triiodobenzoic acid (TIBA), an inhibitor of polar transport of auxins, and low levels of BA significantly increased shoot number. Interestingly, plantlets obtained from TIBA plus BA treatments were normal diploids, whereas those from BA alone exhibited chromosomal aberrations in root preparations. Regenerants from TIBA plus BA treatments were established in soil, and they flowered with normal meiotic behaviour with 100% pollen viability. Shoot tips also have been utilized by Tisserat and Galletta (1995) to study in vitro floral and fruit development in MS liquid medium without growth regulators. Thirty-day-old shoot tips germinated under sterile conditions were cultured in an automated plant culture system. Shoot tips were allowed to grow, which flowered subsequently. Direct plant regeneration from shoot tips supported by histological details in C. annuum cv. PLR-1 was reported by Sobhakumari and Lalithakumari (2003). Axillary shoot buds were induced from shoot tip explants cultured on MS medium supplemented with 6 mg/L BA and 2 mg/ L IAA. Small leafy shoots were kept on 1.5 mg/L BA and 1 mg/L GA3 for elongation. Direct shoot organogenesis from shoot apices of two C. annuum cvs. Arka Abhir and Arka Lohit were successfully obtained by Kumar et al. (2005). They induced regeneration in these varieties by inverting the aseptically raised seedling explants like decapitated roots, apical meristems and cotyledons. Inclusion of 2 (Nmorpholino) ethanesulphonic acid buffers along with 26.63 μM BA, 2.28 μM IAA and 10 μM silver nitrate induced profuse shoot buds. Shoot buds were induced earlier when the auxin transport inhibitor TIBA was employed in the medium. In vitro regeneration and rapid multiplication have been achieved by employing shoot tip and axillary shoot explants of C. annuum cultivars Meiteimorok and Haomorok (Sanatombi and Sharma 2006). Shoot tip explants excised from in vitro raised seedlings were used for multiple shoot bud induction on MS medium supplemented with BA alone or in combination with IAA. Maximum number of shoot buds was obtained on MS medium containing 22.2 μM BA or 44.4 μM BA in the cultivar Meiteimorok and on medium containing 22.2 μM BA in the cultivar Haomorok after 4 weeks of culture. Rooting and elongation of the regenerated shoot buds were achieved on medium containing 2.46 μM IBA or 4.90 μM IBA in ‘Meiteimorok’ and on 2.85 μM IAA or 5.71 μM IAA and 2.46 μM IBA or 4.90 μM IBA in ‘Haomorok’. Axillary shoots were induced in the rooted plantlets by decapitating the plantlets. About 4–6 axillary shoots were developed in both the cultivars within 2 weeks of decapitation. The regenerated shoots were rooted on medium supplemented with IAA or IBA. The rooted plantlets were further decapitated for mass multiplication. Sanatombi and Sharma (2007a) have developed an efficient micropropagation protocol for the ornamental cultivar Morok Amuba (Capsicum annuum) using axillary and shoot tip explants. Multiple shoot buds were induced from shoot tip explants on MS medium containing 45.6 μM Zn followed by 22.2 μM BA in

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combination with 5.7 μM IAA. A novel micropropagation protocol was established for C. frutescens cv. Uchithi, a pungent chilli cultivar, through induction of axillary shoot proliferation of in vitro raised plantlets by decapitation and using the axillary shoots as explants for multiple shoot bud induction (Sanatombi and Sharma 2007b). About 2–6 axillary shoots were induced within 2 weeks when 4-week-old in vitro raised plantlets were decapitated. The axillary shoot tip explants produced multiple shoot buds when cultured on MS medium containing 8.8–44.4 μM BA or 9.3–46.7 μM Kn alone, or 8.8–44.4 μM BA along with 4.6 μM Kn, or 5.7 and 28.5 μM IAA. Maximum number of shoots (5.6) was induced on medium containing 22.2 μM BA in combination with 4.65 μM Kn. The separated shoots were rooted and elongated on medium containing 2.8 μM IAA or 2.4–4.9 μM IBA. Kehie et al. (2012) developed micropropagation protocol for C. chinense Jacq. using nodal segments and shoot tips from a highly pungent chilli cultivar of northeast India cv. Naga King Chilli. Micropropagation of C. chinense Jacq. (cv. Lota bhot) through indirect organogenesis was reported by Bora et al. (2014). Meena et al. (2014) have generated chilli plants free from leaf curl virus by meristem tip culture. They used MS medium supplemented with BAP (3.0 mg/L) and IAA (3.0 mg/L) for shoot proliferation, and shoots were rooted on half-strength MS medium. Gayathri et al. (2015) have achieved in vitro micropropagation of North Indian cultivar Naga King of C. chinense Jacq. In vitro plant regeneration was obtained for chilli Dalle Khursani (C. annuum), an important cultivar of Sikkim using aseptic cotyledon, shoot tip and hypocotyl explants (Bhutia et al. 2016).

3

Genetic Transformation in Red Pepper

Plant genetic transformation is currently the approach of choice for transfer of specific genes encoding some agronomically important traits. Application of genetic transformation requires the development of efficient techniques for the transfer of foreign genes into plant cells. In general, Agrobacterium tumefaciens has been used as the vector for genetic transformation of diverse dicotyledonous species, but biolistic bombardment is also a useful technique to introduce foreign DNA into plant cells of monocotyledonous and dicotyledonous plants, particularly recalcitrant plants like legumes. In the case of chilli pepper, genetic transformation is certainly an important goal to facilitate genetic improvement. However, the advances in this area have been limited because of low efficiency for in vitro plant regeneration. Also, Capsicum spp. are recalcitrant to genetic transformation by A. tumefaciens. Although several papers have been published on red pepper transformation, the protocols are not routinely applicable (Heidmann and Boutilier 2015). In red pepper, the low frequency of cells that are susceptible for Agrobacterium infection as well as their ability to regenerate is the main bottleneck (Heidmann et al. 2011).

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Establishment of Genetic Transformation Protocols for Red Pepper

Some advances have been made in recent years concerning the establishment of different approaches for generating and selecting red pepper transformants. For example, Heidmann et al. (2011) have developed a protocol for the efficient plant regeneration of transgenic sweet pepper (C. annuum) by inducible activation of the BABY BOOM (BBM) AP2/ERF transcription factor. They have used this strategy for reproducible transformation of different genotypes of C. annuum with the ability to regenerate fertile shoots. Further, a tissue culture-independent system (called in planta transformation) was used to develop transgenic bell pepper plants by A. tumefaciens-mediated procedure (Manoj Kumar et al. 2009). Liu et al. (1990) published the first results on chilli pepper genetic transformation. The explants (hypocotyls, cotyledons and leaves) from in vitro raised seedlings were co-cultured with individual suspensions of the wild tumorigenic strains A281 and C58 of A. tumefaciens, and with a disarmed strain bearing the plasmid pGV3850 containing the neomycin phosphotransferase gene (npt-II) and the β-glucuronidase gene (gus) under the control of 35S promoter from cauliflower mosaic virus (CaMV). Production of kanamycin-resistant cell lines was more effective for cotyledons and leaves than for hypocotyls. Only cotyledons and leaf tissues formed callus, along with leaf-like structures and occasional shoot buds in the presence of kanamycin. Although a number of kanamycin-resistant shoot buds were obtained, no further elongation and plantlet formation occurred. Sections from leaf-like structures and shoot buds showed GUS activity. Several factors were examined for the optimization of a protocol for transient genetic transformation of Habanero pepper (C. chinense Jacq.) by A. tumefaciens (Arcos-Ortega et al. 2010). Delis et al. (2005) have obtained the adventitious shoot buds from the cultured hypocotyl explants of C. annuum L. after co-culture with A. tumefaciens. A patent describing a complex and time-consuming method for genetic transformation and plant regeneration of chilli pepper was registered by Engler et al. (1993). Cotyledons from mature seeds were removed and cut into three or four sections, and submerged in a liquid medium, namely OMSG, having MS salts, B5 vitamins, 1.6% glucose and 600 mg L1 MES. The liquid medium was removed, and the explants were co-cultured with a suspension of the LBA4404/p5T35AD strain of A. tumefaciens. This strain contained the binary vector p5T35AD in which the CaMV 35S promoter drives a double mutant form of the acetolactate synthase (ALS) gene, which confers resistance to the herbicide chlorsulfuron. After the co-cultivation period, the cultures were subjected to a number of culturing regimes and the whole process took around 10 months. But the transformation efficiency was only 0.7%. Further modifications to this technique were introduced to increase the transformation efficiency (Engler et al. 1993). They worked on the protocol and were able to get a putative transformation efficiency of 27%. However, inheritance data and molecular evidences of transgene integration were not provided. Further, they used at least two binary vectors carrying genes encoding kanamycin and hygromycin resistance and β-glucuronidase as the reporter gene for genetic transformation. But

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they could only obtain one shoot in each case. Lee et al. (1993) co-cultured the cotyledons with Agrobacterium strain LBA4404 carrying a binary vector pRok1/105 harbouring the cDNA of cucumber mosaic virus I17N-satellite RNA. PCR and northern analyses showed that the introduced gene was integrated and stably expressed in the regenerated plants. Fertile transgenic sweet pepper plants were generated at a relatively high rate from different explants co-cultured with A. tumefaciens strain GV3111-SE harbouring a plasmid containing the cucumber mosaic virus coat protein gene (CMV-CP) (Zhu et al. 1996). The transformation efficiency was less again, and the regenerated plants were positive for the presence of the introduced gene as revealed by PCR. Manoharan et al. (1998) established a protocol for regeneration and genetic transformation for chilli pepper. Shoot bud regeneration was achieved using cotyledonary explants. A. tumefaciens strain EHA105 carrying a binary vector plasmid (pBI121) was used for genetic transformation. Histochemical staining of GUS, PCR and Southern analysis of npt-II gene confirmed the transgenic nature of the regenerated plants, but again the transformation frequency was less. RamírezMalagón (1997) performed chilli transformation using LBA4404 (pBI121), and the transgenics showed positive GUS activity. Progeny (T1) derived from GUS-positive regenerants (T0) exhibited 3:1 GUS (+)/GUS () ratio, indicating a monogenic Mendelian inheritance of the gus gene. Interestingly, an increase in transformation efficiency was observed when seedlings from the T1 progeny that segregated as negative for GUS activity were infected by the same procedure and the shoots were regenerated. Plants that were found to exhibit GUS activity were further analysed by PCR and Southern blot hybridization to detect the presence of npt-II gene. Mihalka et al. (2000) optimized protocols for efficient plant regeneration and gene transfer in pepper. To enable the comparison of different selection markers in identical vector background (pGPTV), a set of binary vectors containing the marker genes for npt-II (kanamycin and geneticin resistance), hpt (hygromycin resistance), dhfr (methotrexate resistance) and bar (phosphinotricin resistance) with CaMV 35S promoter/enhancer-GUS chimaeric gene was constructed and introduced into four different Agrobacterium (C58C1Rif, LBA4404, EHA101 and A281) host strains, respectively. The response of pepper tissues to different host strains showed essential differences. The disarmed C58C1Rif and EHA101 seem to be more efficient for pepper transformation than LBA4404. Shivegowda et al. (2002) regenerated welldeveloped shoots from cotyledonary explants transformed with Agrobacterium strain C58, containing binary vector pGV1040 harbouring the nptII and gus genes. The presence of the transgene was confirmed through histochemical staining of GUS, PCR and Southern hybridization analysis of npt-II gene. Li et al. (2003) established a highly efficient transformation system for pepper. They transformed chilli with Agrobacterium harbouring pBI121 plasmid, having npt-II and gus genes. They tested four genotypes of pepper, and all presented a high differentiation (81.3% on average), elongation (61.5%) and rooting efficiencies (89.5%). PCR analysis showed that 40.8% of the regenerated plantlets were transgenic. More recently, Mehto et al. (2019) reported a highly efficient Agrobacteriummediated transformation protocol for two elite varieties of red pepper (C. annuum),

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Fig. 1 (a) T-DNA map of the pCAMBIA2301 vector; (b) steps involved in the development of C. annuum var. Pusa Sadabahar (a – g) transformants. (a, b) Hypocotyl and cotyledonary explants on selection shoot regeneration medium (SRM); (c, d ) Regenerated shoots on selection shoot regeneration medium. (e) Elongated shoots on rooting medium; ( f ) Rooted plants on soilrite medium for hardening; (g) Potted plants in green house. The size of the bar represents (a, b: 2.25 cm), (c, d: 0.6 cm), (e: 2 cm) and (g: 7 cm); (c) GUS histochemical assay of genetically transformed C. annuum var. Pusa Sadabahar (PSB) (a – f ). (a, d ) Hypocotyl as explant. (b, e) Cotyledon as explant. (c, f ) Mature leaf. The size of the bar represents 200 μm. (Source: Mehto et al. 2019)

Pusa Sadabahar and Pusa Jwala using hypocotyl and cotyledonary explants and with GUS reporter gene construct (Mehto et al. 2019; Fig. 1). Dabauza and Pena (2003) studied the response of sweet pepper genotypes to Agrobacterium strains (A281, Ach5, C58, 42CNBP and 1102) as a means of selecting proper vectors for genetic transformation. While some pepper varieties show low response to the Agrobacterium infection, others display high frequency (80–100%) of infection. Furthermore, strains C58 and 1102 showed significantly greater virulence and also induced more tumours per wound than the other strains. Lee et al. (2004) reported a new selection method for pepper transformation. The most critical point in the protocol was the selection of shoot growing on calli referred to as callus-mediated shoot formation (indirect shooting) because shoots not regenerated from the callus (direct shooting from the wounded surface) developed into non-transformants.