Full Ebook of Tilling and Eco Tilling For Crop Improvement 1St Edition Anjanabha Bhattacharya Vilas Parkhi Bharat Char Online PDF All Chapter

TILLING and Eco TILLING for Crop
Improvement 1st Edition Anjanabha

Bhattacharya Vilas Parkhi Bharat Char
Visit to download the full and correct content document:
https://ebookmeta.com/product/tilling-and-eco-tilling-for-crop-improvement-1st-edition
-anjanabha-bhattacharya-vilas-parkhi-bharat-char/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...
TILLING and Eco TILLING for Crop Improvement 1st

Edition Anjanabha Bhattacharya Vilas Parkhi Bharat Char
https://ebookmeta.com/product/tilling-and-eco-tilling-for-crop-
improvement-1st-edition-anjanabha-bhattacharya-vilas-parkhi-
bharat-char/
Complexity in Tsunamis Volcanoes and their Hazards 2nd

edition Robert I. Tilling
https://ebookmeta.com/product/complexity-in-tsunamis-volcanoes-
and-their-hazards-2nd-edition-robert-i-tilling/
Genome Editing Current Technology Advances and

Applications for Crop Improvement 1st Edition Shabir
Hussain Wani
https://ebookmeta.com/product/genome-editing-current-technology-
advances-and-applications-for-crop-improvement-1st-edition-
shabir-hussain-wani/
Silicon and Nano-silicon in Environmental Stress

Management and Crop Quality Improvement: Progress and
Prospects 1st Edition Hassan Etesami (Editor)
https://ebookmeta.com/product/silicon-and-nano-silicon-in-
environmental-stress-management-and-crop-quality-improvement-
progress-and-prospects-1st-edition-hassan-etesami-editor/
I Survived 1st Edition Bruno Vilas Boas
https://ebookmeta.com/product/i-survived-1st-edition-bruno-vilas-
boas/
Bravehearts of Bharat Vikram Sampath
https://ebookmeta.com/product/bravehearts-of-bharat-vikram-
sampath/
Room for Improvement First Edition Jessica Gregory
https://ebookmeta.com/product/room-for-improvement-first-edition-
jessica-gregory/
Cybersecurity and Identity Access Management Bharat S.

Rawal
https://ebookmeta.com/product/cybersecurity-and-identity-access-
management-bharat-s-rawal/
Digital Disruption : implications and opportunities for

economies, society, policy makers and business leaders.
1st Edition Bharat Vagadia
https://ebookmeta.com/product/digital-disruption-implications-
and-opportunities-for-economies-society-policy-makers-and-
business-leaders-1st-edition-bharat-vagadia/
Anjanabha Bhattacharya
Vilas Parkhi
Bharat Char Editors
TILLING and
Eco-TILLING
for Crop
Improvement
TILLING and Eco-TILLING for Crop
Improvement
Anjanabha Bhattacharya • Vilas Parkhi •
Bharat Char
Editors
TILLING and Eco-TILLING

for Crop Improvement
Editors
Anjanabha Bhattacharya Vilas Parkhi
Mahyco Research Centre Mahyco Research Centre
Mahyco Private Limited Mahyco Private Limited
Jalna, India Jalna, India
Bharat Char
Mahyco Research Centre
Mahyco Private Limited
Jalna, India
ISBN 978-981-99-2721-0 ISBN 978-981-99-2722-7 (eBook)

https://doi.org/10.1007/978-981-99-2722-7
# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and
transmission or information storage and retrieval, electronic adaptation, computer software, or by
similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
Preface
Food and nutritional security for feeding the global population remains a constant
challenge in the years to come. The human population is expected to reach 9.7
billion by 2050. Climate change-associated events like heat waves, high
temperatures, drought as well as flooding will constrain food production. Along
with newly emerging anti-globalization sentiments, the supply chain crisis fuels
spiraling food prices, and inflationary pressure is adding fuel to the fire in the
affordability of food for all. Elimination of hunger is envisioned in the UN 2030
agenda as an immediate sustainability goal. Agriculture today needs a technology-
aided solution. The rate of genetic gain in food crops has remained stagnant in the
past decade. The diversity of germplasm remains a key element in enhancing the
breeder’s capacity to breed new crops. The spontaneous mutation also happens in
nature at a very low frequency. In a sense nature is also facilitating mutagenesis all
the time. Thus, intervention by mankind to facilitate the process of creating
variability to breed new crop varieties as per the need of the marketplace. Thus
random mutagenesis approaches have a major role to play by increasing the number
of permutations and combinations among available germplasms which can translate
to designer crops. New biotechnology tools are being discovered at a rapid pace but
the regulatory environment remains hostile. Licensing issues also add multiple
constraints to the commercial use of the recent genome editing technologies and
thus thwart innovation. The first generation of the Green Revolution thus needs to be
repeated using a newer basket of technologies. Mutation breeding has played a
significant role in the development of improved varieties led by agencies like the
Food and Agriculture Organisation (FAO) and Bhabha Atomic Research Centre
(BARC), India, among many others. Thus, by their (FAO & BARC) pioneering
efforts, plant breeding efforts focused on mutation breeding came to the forefront.
Readers are directed to FAO/IEAE mutant database available online at https://mvd.
iaea.org/, which lists more than 3365 mutant released varieties across 214 crop
species. The advent of next-generation sequencing tools has further aided in the
mutation discovery efforts, particularly recessive alleles which are hard to detect by
phenotypic means alone. Given the importance of mutagenesis and technological
advances in creating and identifying useful alleles, this book will appraise the target
audience, viz., students, researchers, policymakers, consumers, and advocacy
groups. This will facilitate further developments in this field of mutagenesis. Since
v
vi Preface
mutagenesis and TILLING have already been accepted by consumers for more than
a century, it is hoped that the relevance and efforts provided by all the stakeholders
including the crop breeding community, seed industry, Government agencies, and
policymakers will remain upbeat even if many advanced technologies start playing
out. This is primarily due to the popularity of mutagenesis being free from any
controversy, regulatory hurdles, licensing required, clean patentscape, and the mass
participation of start-ups, institutes, and industry in developing new products. Public
outreach is one of the objectives of this book in light of precise genome editing,
particularly CRISPR/Cas9.
We hope a bit of illustration on the contents will enhance the book’s appeal to the
target audience. Firstly, the major objective of this book is to introduce the topic of
mutagenesis and mutant screening technique and the relevance of these technologies
in the era of precise genome editing. We have tried our best to include the latest
information and technological advances in this book compilation. Every day new
findings emerge and therefore it is near impossible to claim completeness in the
topics covered in this publication. Inadvertently errors also might have crept in and
we would like our readers to reach to us and we will incorporate them in future
editions. Our mutagenesis and TILLING journey begins with Chap. 1, which is
comprehensively written by the editors themselves and primarily focuses on
introducing the topic of mutagenesis, progress in screening techniques, and compar-
ison with the recently developed genome editing tools. Comprehensive deliberation
is conducted for the social acceptability aspect, patentscape including plant varietal
protection (PVP). Mutagenesis efforts in various crops are described at length for
crop improvement efforts. Chapter 2 is written by Dr. Tyagi and their team, focusing
on induced mutagenesis beginning with the application of various mutagens, both
chemical and physical agents, and their application in vegetative tissue. This chapter
ends with a mention of CRISPR/Cas mutagenesis. Chapter 3 focuses on bioinfor-
matic tools used in the identification of genes of interest, mining genes for functional
SNPs, tools used for the analysis of TILLING by sequencing data, and In Silico
TILLING. Chapter 4 focuses on next-generation mutation detection methods which
are vital in speeding up allele discovery compared to gel-based methods. Chapter 5
focuses entirely on TILLING by sequencing. Chapter 6 is dedicated to natural
TILLING and provides examples of cultivated crops where natural variation can
be exploited to speed up crop improvement efforts. Chapter 7 is on mutagenesis in
somatic tissues and a team led by Dr. Debasis Chakraborty, NBRI, takes the reader
through the various somatic mutagenesis approaches and mutation detection.
Chapter 8 focuses on phenomics and trait selection. Chapter 9 focuses on the
development of climate-resilient crops and Chap. 10 deliberates on the future of
TILLING while the last chapter of the book (Chap. 11) focuses on the perception of
food crops developed by mutagenesis compared to other approaches like
GM. Stakeholder perception will ultimately pave the way for acceptance of food
products and thus the last chapter of the book discusses various topics related to the
use of biotechnological tools in food production.
Preface vii
Our reader may find at times a bit of overlap in contents between chapters which
are kept deliberately as these cannot be seen in isolation and multiple perspectives
are needed to understand various viewpoints.
Thus, with the availability of advanced sequencing systems, bioinformatics tools
can help in the efficient utilization of mutagenesis and help the breeding community
in unleashing a second green revolution. The crops of the future are expected to
withstand the vagaries of climate change and have a positive predictable outcome on
the global food supply chain.
We hope this book will provide a holistic view of the mutagenesis and TILLING
approach toward crop improvement and keep its relevance intact in advancing
modern agriculture.
We at Mahyco live by the vision to feed the world by empowering smallholder
farmers, and we fulfill our mission by providing smallholder farmers with scientifi-
cally advanced genetics, in annual crops, to improve their income sustainably. We
thank our Managing Director, Mr. Shirish Barwale, Mahyco, for letting us undertake
this project. Special thanks to all our contributors spanning several countries and
geographies for undertaking this project despite their busy schedules. Thank you,
team Springer, for wholeheartedly accepting our proposal to edit a book project on
an important topic of mutagenesis and TILLING.
We sincerely hope this book will facilitate further use of mutagenesis and
screening techniques in bringing designer crops into the world which will help to
improve the livelihood of millions of small farmers in India and the World.
We wish everyone a happy read!
Jalna, India Anjanabha Bhattacharya

Jalna, India Vilas Parkhi
Jalna, India Bharat Char
Aims and Scope
The recent pandemic led to severe food supply chain disruption, marked changes in
policy from global to single market, hostile trade barriers, wars, increasing inflation-
ary pressure, and climate change. Self-reliance in food production now is a long-term
strategy for every nation on this planet. For more than a hundred years mutation
breeding has resulted in the release of thousands of crop varieties and thus played a
role in alleviating hunger. The efforts by the Food and Agriculture Organisation
(FAO)-International Atomic Energy Agency (IAEA), Bhabha Atomic Research
Centre (BARC)-Department of Nuclear Agriculture, India, are praiseworthy in the
field of mutation breeding. The list of released mutants is available online at mvid.
iaea.org. The most commonly used mutagen has been physical irradiation and later
on chemicals were used to induce single base pair change. Vegetative parts like
tubers are also used. Consumer distrust remains with other biotechnology-assisted
technologies in several marketplaces. The already widespread acceptability of
mutagenesis-derived products along with multiple CRISPR patents, and licensing
difficulties associated with precise genome editing means the fruits of precise
breeding will still take a few years to fructify. Meanwhile, mutagenesis and
TILLING which are free to practice will remain relevant without any restrictions.
Further advancements in mutation detection led by TILLING by sequencing (TbyS)
will make allele discovery more appealing, economical, and fast. Therefore, this
book aims to focus on mutagenesis and associated allele discovery tools and
platforms. Given the importance of mutagenesis and technological advances in
creating and identifying useful alleles, this book will appraise the target audience,
viz., students, researchers, policymakers, consumers, and advocacy groups. We
sincerely hope this book will provide readers with a holistic view of mutagenesis
and TILLING in the era of precise genome editing like CRISPR/Cas and yet show its
relevance in the modern era of biotechnology. We hope both TILLING and
CRISPR/Cas precise genome editing tools will co-exist in the years to come and
help in creating designer crops for the betterment of humankind.
Jalna, India Anjanabha Bhattacharya

Jalna, India Vilas Parkhi
Jalna, India Bharat Char
ix
Contents
1 Mutagenesis and TILLING in the Era of Precise

Genome Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Anjanabha Bhattacharya, Vilas Parkhi, Bhavesh Palan,
and Bharat Char
2 Induced Mutagenesis for Crop Improvement . . . . . . . . . . . . . . . . . 35
Anshika Tyagi, Rekha Joshi, Nisha Singh, Priyanka Jain,
Kumar Durgesh, and Indumathi Padmanaban
3 Bioinformatics and Candidate Gene Mining for TILLING . . . . . . . 61
Raghavendra Gunnaiah and Mahantesha B. N. Naika
4 Next Generation Mutation Detection Techniques in Crops . . . . . . . 75
Shalu Choudhary, Jayendra Padiya, Abhijit Ubale, Preeti Lohani,
and Venugopal Mikilineni
5 TILLING by Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Swapnil B. Kadam and Vitthal T. Barvkar
6 Advances in Eco-TILLING: In Search of Superior
Natural Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Vaishali Khanale
7 Mutagenesis in Somatic Cell and Tissue . . . . . . . . . . . . . . . . . . . . . 137
Puja Singh, Mrinalini Bhaduri, Monica Kumari,
and Debasis Chakrabarty
8 Forward Genetics: Phenomics and Trait Selection . . . . . . . . . . . . . 151
P. Magudeeswari, A. Loyanganba Meitei, M. James, Wricha Tyagi,
and Mayank Rai
9 Towards the Development of Climate-Resilient Crops . . . . . . . . . . . 175
Elangovan Mani
xi
xii Contents
10 Future of TILLING in Plant Breeding . . . . . . . . . . . . . . . . . . . . . . 185

Vinod Kumar, Muhammad Hafizur Rahman, Sabah AlMomin,
and Anisha Shajan
11 Perception of Food Crops Developed by Mutagenesis
Among Various Stakeholders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Seema Pradhan and Ajay Parida
Editors and Contributors
About the Editors
Anjanabha Bhattacharya is working as a Group leader (Tech) with Mahyco since

2012. He is also an elected member of the prestigious PTCA-I (Plant Tissue Culture
Association of India). His areas of research expertise include TILLING,
Eco-TILLING, crop improvement through physical and chemical mutagenesis,
laboratory stewardship, and tissue culture-assisted technologies including double
haploids for about two decades. He is also an active business excellence practitioner
and worked with an agri-start-up for two years. He obtained his PhD from the
University of Nottingham, England, and undertook postdoctoral training from the
University of Georgia, Tifton, USA.
Vilas Parkhi is a seasoned plant biotechnologist, who has exposure to various

fields of plant biotechnology research on the platform of academic and industrial
setup. He is an alumnus of the International Rice Research Institute, Philippines;
Texas A&M University, TX, USA; and Pune University, India. Currently, he is
engaged in the research area of genome editing of crops and transgenic trait
development at Mahyco Pvt Ltd, Jalna India.
Bharat Char is the Chief Science Officer (CSO) at Mahyco. He obtained his PhD
in Biochemistry and Molecular Biology, University of Southern California Medical
School, Los Angeles, and undertook postdoctoral work at the University of
California, Berkeley, in plant molecular genetics. He has served as a member of
the International Seed Federation Working Group on GM Vegetables.
Contributors
Sabah AlMomin Biotechnology Program, Environment and Life Sciences

Research, Center, Kuwait Institute for Scientific Research, Safat, Kuwait
Vitthal T. Barvkar Department of Botany, Savitribai Phule Pune University,
Pune, India
xiii
xiv Editors and Contributors
Mrinalini Bhaduri Molecular Biology and Biotechnology Division, CSIR-

National Botanical Research Institute, Lucknow, India
Anjanabha Bhattacharya Mahyco Research Centre, Mahyco Private Limited,
Jalna, Maharashtra, India
Debasis Chakrabarty Molecular Biology and Biotechnology Division, CSIR-
National Botanical Research Institute, Lucknow, India
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
Bharat Char Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
Shalu Choudhary Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
Raghavendra Gunnaiah Dept. of Biotechnology and Crop Improvement, College
of Horticulture, Bagalkot, University of Horticultural Sciences, Bagalkot,
Karnataka, India
Priyanka Jain National Institute of Plant Genome Research, New Delhi, India
M. James College of Post Graduate Studies in Agricultural Sciences, Central
Agricultural University (Imphal), Umiam, Meghalaya, India
Rekha Joshi Division of Genetics, IARI, New Delhi, India
Swapnil Kadam Department of Botany, Savitribai Phule Pune University, Pune,
India
Vaishali Khanale Mahyco, Jalna, Jalna, Maharashtra, India
Vinod Kumar Biotechnology Program, Environment and Life Sciences Research,
Center, Kuwait Institute for Scientific Research, Safat, Kuwait
Monica Kumari Molecular Biology and Biotechnology Division, CSIR-National
Botanical Research Institute, Lucknow, India
Preeti Lohani Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
P. Magudeeswari College of Post Graduate Studies in Agricultural Sciences,
Central Agricultural University (Imphal), Umiam, Meghalaya, India
Elangovan Mani Advanta Enterprises Limited, Hyderabad, Telangana, India
Loyanganba Meitei College of Post Graduate Studies in Agricultural Sciences,
Central Agricultural University (Imphal), Umiam, Meghalaya, India
Venugopal Mikilineni Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
Editors and Contributors xv
Mahantesha B. N. Naika Dept. of Biotechnology and Crop Improvement, KRC

College of Horticulture, Arabhavi 591218, University of Horticultural Sciences,
Bagalkot, Karnataka, India
Jayendra Padiya Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
Indumathi Padmanaban Pondicherry University, Tirupur, Tamil Nadu, India
Bhavesh Palan Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
Ajay Parida Plant and Microbial Genomics Laboratory, Institute of Life Sciences,
NALCO square, Bhubaneswar, India
Vilas Parkhi Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
Seema Pradhan Plant and Microbial Genomics Laboratory, Institute of Life
Sciences, NALCO square, Bhubaneswar, India
Muhammad Hafizur Rahman Biotechnology Program, Environment and Life
Sciences Research, Center, Kuwait Institute for Scientific Research, Safat, Kuwait
Mayank Rai College of Post Graduate Studies in Agricultural Sciences, Central
Anisha Shajan Biotechnology Program, Environment and Life Sciences Research,
Center, Kuwait Institute for Scientific Research, Safat, Kuwait
Nisha Singh ICAR-National Institute for Plant Biotechnology, IARI, New Delhi,
India
Puja Singh Molecular Biology and Biotechnology Division, CSIR-National
Botanical Research Institute, Lucknow, India
Durgesh Kumar Tripathi Division of Genetics, IARI, New Delhi, India
Anshika Tyagi Department of Biotechnology, Yeungnam University, Gyeongsan,
South Korea
Wricha Tyagi College of Post Graduate Studies in Agricultural Sciences, Central
Abhijit Ubale Mahyco Research Centre, Mahyco Private Limited, Jalna,
Maharashtra, India
Mutagenesis and TILLING in the Era
of Precise Genome Editing 1
Anjanabha Bhattacharya, Vilas Parkhi, Bhavesh Palan,
and Bharat Char
Abstract
The human population is burgeoning and land resource to grow crops is decreas-
ing day by day impacted by climate change. Feeding mankind remains a chal-
lenge. Nutritional security and the development of crops resilient to biotic and
abiotic stress are the need of the hour. For more than a 100 years mutation
breeding has resulted in the release of thousands of crop varieties and thus played
a role in alleviating hunger. The efforts by the Food and Agriculture Organisation
(FAO)-International Atomic Energy Agency (IAEA), Bhabha Atomic Research
Centre (BARC)-Department of Nuclear Agriculture, India is laudable in the field
of mutation breeding. The most commonly used mutagen has been physical
irradiation and later on chemicals were used to induce single base pair change.
Usually, the seed is the primary material for conducting mutagenesis. Vegetative
parts like tubers are also used. Consumer distrust remains with other
biotechnology-assisted technologies in several marketplaces. The already wide-
spread acceptability of mutagenesis-derived products along with patent, and
licensing difficulties associated with the new generation of genome editing
technologies like CRISPR/Cas9 can translate into a favorable shift toward muta-
genesis and TILLING which are free to practice. Plant breeders are in a race
against time to develop the most promising lines to meet the market demand and
thus may not be interested in retrieving traits directly from wild species and fine-
tuning the desirable character. Thus crop breeders may continue to focus on
adapting existing varieties with the much-needed desired traits by mutation
breeding. Combined with the approved use of mutants in organic farming,
A. Bhattacharya · V. Parkhi · B. Palan · B. Char (✉)

Mahyco Research Centre, Mahyco Private Limited, Jalna, Maharashtra, India
e-mail: bharat.char@mahyco.com
# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

Ltd. 2023
A. Bhattacharya et al. (eds.), TILLING and Eco-TILLING for Crop Improvement,
https://doi.org/10.1007/978-981-99-2722-7_1
2 A. Bhattacharya et al.
next-generation sequencing, and improved and rapid detection methods, we feel

the relevance of mutagenesis and TILLING is here to stay.
Keywords
EMS · Mutagenesis · Mutant library · NGS · Allele · TILLING by sequencing
Abbreviations
Acdc1 Adult cyanide deficient class 1

ACS2 Acetyl co-enzyme synthetase 2
BARC Bhabha Atomic Research Centre
C3H Coumarate-3-hydroxylase
CAD Cinnamyl Alcohol Dehydroxygenase
CAGR Compound annual growth rate
CEL1 Endonuclease from Celery
COMT1 Caffeic acid 3-O methyltransferase
CRISPR/Cas9 Clustered regularly interspaced palindromic repeats-CRISPR-
associated proteins
DNA Deoxyribonucleic acid
Eco-TILLING Natural TILLING
emb Embryo specific
EMS Ethyl methanesulfonate
ESC Extended shelf life
FAD Fatty acid desaturase
FAO Food and Agriculture Organisation
GB Giga bases
GBS Genotyping by sequencing
GBSS Granule-bound starch synthase
HCT2 Hydroxycinnamyltransferase-2
IAEA International Atomic Energy Agency
IFOAM International Federation of Organic Movement
INDELS Insertions deletions
KASP Kompetitive allele-specific PCR
MAGIC Multiparent advanced generation intercross
MITE Miniature inverted-repeat transposable elements
NGS Next-generation sequencing
NUE Nitrogen utilization efficiency
PVP Plant varietal protection
QTLs Quantitative trait loci
RMS 3 & 4 RAMOSUS 2 & 4
SAD Stearoyl-acyl carrier protein desaturase
SECO Secoisolariciresnol
SGM Stay green mutants
1 Mutagenesis and TILLING in the Era of Precise Genome Editing 3
SNP Single nucleotide polymorphism

TALEN Transcriptional activator-like effector nucleases
TbyS TILLING by sequencing
Tcd1 Totally cyanide deficient 1
TILLING Targeting Local Lesion in Genome
TRIPS Trade-related aspects of intellectual property rights
ZFNs Zinc Finger Nucleases
1.1 Introduction
The mutation is the source of primary variation in plants (Griffiths et al. 2000) and it
is an abrupt inheritable change in the genetic material of an organism and therefore is
a substantial steering force of evolution in ever-changing climatic regimes. The term
mutation breeding was first coined by Freisleben and Lenin. Mutation-induced
change may or may not result in a noticeable phenotype and can be detected by
visual or molecular means (Brown 2002). Mutation has played a key part in crop
improvement. Hugo De Vries in 1889 while rediscovering Mendel’s law showed
that the inheritance of variation in primrose and snapdragon resulted in traits that do
not follow the typical segregation patterns and at the same time the traits were
heritable (Stamhuis et al. 1999). Hugo De Vries, therefore, is credited with the
discovery of mutation. He described such anomalous plants as mutants (Stoltzfus
and Cable 2014). Later on, Johannsen described these factors as genes, which can
pass from one generation to another (Portin and Wilkins 2017). Mutagenesis has
been in use for a very long time since its initial discovery by Sadler in the nineteenth
century in Agriculture crops for creating random variability (Sikora et al. 2011).
Recently, a paper in Nature by Monroe et al. (2022) showed that mutations in
Arabidopsis thaliana are non-random and follow a typical pattern of occurrence.
They sequenced mutants that otherwise would not have survived in nature and
analyzed over a million mutations. The author was able to decipher that mutation
in genes that are essential for survival, occurs in low frequency than in other regions.
Thus, the probability of finding mutation could be of interest to crop breeders who
are typically interested in one gene for an independent trait that is easy to manage in
plant breeding settings. Rötter et al. (2015) observed that the future crop improve-
ment goals must address food sufficiency by 2050 when climatic events like heat
waves, high temperatures, frequent drought in a few places, and heavy rainfall in
other regions will place new food challenges. Ramirez-Villegas et al. (2015)
described models on genotypes suitable to climate change models, i.e., a type of
ideotype design. The objective was to identify plant processes related to yield, and
growth in face of climate uncertainty. Some reports suggested that Sadler himself
was not so enthusiastic about the technology (Shu et al. 2012). Mutagenesis is also
dubbed as factual due to the oddness of the mutagenesis process and most of the
selection is done visually in the field by the crop breeders. In this chapter, we will
focus on TILLING (Targeting Induced Local Lesions in Genome), the success of

TILLING, and its relevance in the modern era of genome-mediated editing particu-
larly CRISPR/Cas and TALENS to name a few. TILLING came to the limelight in
the year 2000 when Luca Comai first showed mutation detection in the model crop,
Arabidopsis, and soon the range of organisms studied by TILLING method
expanded vastly (refer to reviews by Palan et al. 2021, Shu et al. 2012).
1.2 Understanding Mutation and Mutagenesis
The early days of chemical mutagenesis involved mutagens like mustard gas and
nitrous acid (Povirk and Shuker 1994). Then chemicals like diethyl sulfate, and
alkylating agents like nitroso compounds, acridines, azides, and base analogous
were introduced (Shu et al. 2012). The genetic screen was previously used for the
understanding role of any particular gene. The main purpose of genetic screens is to
phenotype mutants caused by single-gene lesions. Chiefly, mutations are classified
into transition, silent mutation, missense, nonsense, transversions as well as
INDELS, frameshift, and inversion (Website 1 n.d.; Website 2 n.d.). In each of
these cases, the mode of action varies, in almost all cases, it is a case of base
substitution. A silent mutation might cause exon skipping, similarly, a mutation in
the intron may impede self-splicing activity. However, still, such nonsignificant
changes can modify the chaperone activity of the coding protein, modify physiolog-
ical and chemical functions in plants, and are important for improvement efforts. The
green revolution genes of the 1960s were spontaneous mutations (Bhattacharya et al.
2021). Most mutations are silent and only a fraction results in a phenotype. The plant
genome absorbs many rapid mutation-induced changes over a long period without
showing any negative impact. The ability to withstand mutation has been a part of
plant evolution as standing crops endure sunlight along with UV radiation during
their life cycle (Gill et al. 2015). Many of the mutations induced are identified
promptly by the natural cell repair mechanism and can revert to their natural levels
(Alberts et al. 2002). Thus, mutagenized induced breeding is different from natural
mutagenesis as this is a deliberate effort to induce changes in the genome to change
plant phenotype (Jankowicz-Cieslak et al. 2017). Today, physical (gamma rays, fast
neutron mutagenesis), chemical, and biological mutagens like transposon-based
chromosome integration, Agrobacterium-based site-directed, insertional techniques
are used in mutagenesis which has resulted in robust crop breeding, genomic
research (Chaudhary et al. 2019), though is tedious and costly. Even, MITE (minia-
ture inverted-repeat transposable elements) can cause genetic change but, can remain
unstable. Apart from applied Agriculture, TILLING has many applications in basic
sciences like being involved in the identification of genes and pathways related to
plant developmental pathways including flowering, yield, and stress tolerance. In
fact, till recently mutation breeding was only the method available for creating novel
genetic variants in crops, and mostly phenotyping was the sole basis of trait selection
among crop breeders (Muñoz-López and García-Pérez 2010). Few mutations may
also cause epigenetic changes without modifying the DNA molecule and may
remain stable for multiple generations (Osabe et al. 2014). These mutagenesis
methods create SNP, INDEL which is random, takes time, is costly, and mostly
trait discovery is limited to visual inspection (Tadele 2016). Physical mutagenesis
mostly creates knockout or nonfunctional alleles of the gene of interest in the mutant
plants (Alberts et al. 2002). Today, with the common usage of the gamma radiation
facility using Cobalt-60, physical mutagenesis is routinely used in plant breeding.
Cosmic space is another source of inducing mutations that involve cosmic radiation,
weak magnetic fields, and microgravity. Thus, living samples are also experimented
with in space flights (Oladosu et al. 2016). The need for creating variability arose due
to repeatedly using genetically similar germplasm in the breeding program, thus
narrowing the genetic base tremendously. In general, mutation breeding involves
generating mostly recessive mutations with a small fraction of mutations described
as dominant (Loewe and Hill 2010). Mutation in genes can also play an important
role in pleiotropy, which is a term used for a gene influencing a second gene (Paaby
and Rockman 2013). Most of the mutations identified by physical means tend to be
recessive (Holme et al. 2019). Nowadays, both the phenotypic and the genotypic
platforms are automated using an entirely computerized system with artificial intel-
ligence coming to the forefront to predict true mutations from a pool of putative
mutations (Beans 2020). It is possible to link genotype to crop phenotype using high
throughput phenomics platform (Zhao et al. 2019). Many of the previously used
popular mutants like short stature with strong culm which can withstand strong wind
and rain are being now mapped to the specific lesion in target genes (Dockter and
Hansson 2015). Later with the discovery of many other molecular biology tools
detecting subtle mutations was possible thus extending the value proposition, and
drastically decreasing resource requirements including time and labor. Many traits
are hard to detect by visual means alone, for example, quality traits, disease, and pest
resistance to name a few, and require additional interventions by molecular biology
tools (Website 3). In case of resistance to disease tolerance, amino acid changes in
the virulence proteins lead to loss of recognition site (Jia et al. 2000). Thus, the
interaction of protein from the host organism with altered plant protein can lead to
disease tolerance.
1.3 Progress in Sequencing Techniques and Mutation

Detection
Once scarcely available genomic data was used extensively to study gene function in
development, protein–protein interactions, and metabolic pathways by sequencing,
is used today with NGS across organisms (Unamba et al. 2015). Using next-
generation mapping by sequencing approach resulted in the method which does
not depend on any particular reference genome, multiple crosses which are complex
and require further linkage information. Thus, this combined approach is amenable
for species for which no genetic screen exists so far (Schneeberger 2014). Thus, one
of the aims of the vast amount of data generated by various sequencing platforms
across crops is to assign a function to operational genes. Previously, thought of as a
tiresome method to screen for novel traits by visual means over many generations
has gained traction in the recent decade due to the advent of new molecular screening
tools like Mutchrom Seq, Netmap, exome capture sequencing, SNP chips, MutRen
seq, whole-genome sequence by a new generation of sequencing and assembly by
improvised bioinformatic assembly analyzer with in-depth long and short reads
(Unamba et al. 2015; Thao and Tran 2016). A brief overview of various sequencing
techniques is discussed as rapid progress allowed analysis of multiple targets in less
time thus replacing the tedious ways to detect mutations in TILLING with gel-based
methods (Gupta et al. 2017). We aim here to provide a glimpse of the workability of
each system. Sequencing platforms that are used to detect mutations in both
TILLING and modern genome editing are developed by Roche, Illumina,
and Applied biosystems (for more details refer to Dorado et al. 2021, Heather and
Chain 2016). In the primary days of sequencing, two scientists Allan Maxam and
Walter Gilbert used chemical sequencing. This was based on radioactive labeling
and was complex. Thus, the hunt for a reliable, simple method resulted in the chain
termination technique by Sanger and co-workers. Sanger sequencing was further
refined with the inclusion of fluorescent probes, automated platforms, and electro-
phoresis systems (Moorthie et al. 2011). The second generation of sequencing
platforms includes Polony, MPSS (massively parallel signature sequencing),
SOLID, HiSeq, MiSeq from Illumina, and pyrosequencing (Koboldt et al. 2013).
The second-generation pyrosequencing works using streptavidin-coated magnetic
beads with a polymerase (also refer to Pourmand et al. 2002). Then, there is the
Illumina (Solexa) sequencing system which introduced two types of sequencing
platforms, Hi Seq and Mi Seq (refer to Illumina Website 4 n.d.), the other platform is
SOLID sequencing which uses beads containing single copies of a DNA (Website
5 n.d.). MPSS is based on a bead, ligated adapter, which could measure the
expression of genes (Moorthie et al. 2011). 454-pyrosequencing paved the way for
the next generation of sequencing technology, which was mostly dominated by
Sanger sequencing over the last three decades. The new improved sequencing
technology including second and third generations increased precision with high
throughput while decreasing cost immensely. The main difference between the
second (next generation) and third generations is that in the latter a single DNA
molecule is used instead of template DNA. The template DNA reads are prone to
error during the amplification step. Further, parallel sequencing of such smaller
templates increases the chances of error (also refer, Heather and Chain 2016).
Gupta et al. 2017 used MiSeq Illumina sequencing to TILL the tomato mutant
population and analyzed the sequence data by six software namely SNVER,
CRISPR, GENOTYPER, UNIFIED, CAMBA, and VIPR. They found that no single
software was able to predict changes with high accuracy. Tomato samples were
pulled to 128 folds. Only 64 of 75 mutations were verified by Sanger sequencing. In
the latest developed sequencing technology, the nanopore is coming to an age. Here,
each strand of DNA is read without the PCR bias. Therefore, with major strides in
the sequencing platform for the first time mutagenesis could be explored beyond
visual means at the sequence level with certainty. One of the early reports of
TILLING came from McCallum and their research team where the mutation was
detected in chromomethylase genes in a model crop using dHPLC, which was

unsuitable for scaling up discovery activities (McCallum et al. 2000). The detection
activity was further boosted by the discovery of the mismatch enzyme, CEL1 from
celery, Apium graveolens L. (also refer to Comai and Henikoff 2006). A low-cost
detection technique followed by a LICOR-based system (LI-COR4300 DNA ana-
lyzer) came to the forefront to address the bottlenecks to some extent (Taheri et al.
2017). AutoCloner is another program that allows researchers to design primers
suitable for use in TILLING before NGS platforms took over the herculean task of
mining mutations. Wang et al. (2006) advocated the application of TILLING beyond
model crops, thus expanding the scope of the technique to economically important
crops. Before the discovery of the practical application of CRISPR/Cas in
eukaryotes in 2012, TILLING was deemed as a simple, high-efficiency, and sensi-
tive procedure to detect the mutation. Further application of TILLING and
Eco-TILLING can be also used as a haplotyping tool for pinning down allelic
variation in genes attributing specific phenotypes and therefore, creating and
verifying new alleles which otherwise cannot be created by spontaneous mutations
(natural or using tissue culture techniques) thus expanding genetic resources avail-
able with crop breeders for new improved products helping farming community and
consumers.
1.4 Comparison of TILLING and Recent Genome Editing

(GE) Tools
Mutagenesis and TILLING are independent of transformation protocol, and genome

size facilitates scalable mutation and applies to a wide range of crops. The popularity
of TILLING continued for 22 years, i.e., 2000–2022, as is evident from numerous
publications in this field as listed in this chapter and referenced. In the meantime,
CRISPR/Cas (Clustered Regular Interspaced Short Palindromic Repeats/CRISPR
associated proteins) system was discovered for precise gene editing, which was
recognized by the Nobel Prize Committee, and inventors were bestowed with the
coveted Nobel award (Farhud and Zarif-Yeganeh 2020). Previously, other precise
editing systems like homing nucleases (Meganucleases), Zinc finger nucleases
(ZFNs), and TALEN (Transcriptional Activator Like effector nucleases) were used
(Gaj et al. 2016). Due to regulatory and licensing constraints, such tools are not
extensively used in developing a few products (Anonymous 2021; Martin-Laffon
et al. 2019) when compared to random mutagenesis as indicated by the list of
mutants in mvid.iaea.org. Then, CRISPR/Cas due to its simplicity, and scalability
caught the attention and therefore became popular for academic studies while other
genome editing tools require different complex protein rearrangements for each
target gene identified for editing making them less desirable. Such massive develop-
ment along with base and prime editing in the space of precise editing, saw the
decline of popularity with traditional mutagenesis, as evident from the fall of
traditional (chemical and physical) mutagenesis publications compared to a rapid
rise in CRISPR activity (Ghosh, 2020). Further, a vast improvement in massively
parallel sequencing methods provided the much-needed impetus to mutation detec-

tion. Once, a mutagenized population is developed, it can be used multiple times to
screen for new genes. Improved methods of creating a large-scale mutagenized
population in a short time (FAST-TILL, Serrat et al. 2014) and the double mutagen-
esis technique (Gupta et al. 2017) will help to create a high-quality mutant popula-
tion in the shortest possible time. Also, mutagenesis-based methods are free from
genotype recalcitrance which remains a major impediment with non-transformable
systems by genetic transformation. TILLING can be applied to populations created
by vegetative and seed material (Jankowicz-Cieslak and Till 2016). TILLING is a
useful tool to detect allelic series of a target largely composed of SNP, and INDELS
in the gene which are undetectable by visual means. These point mutations in the
genome translate into useful resource development by functional genomic studies.
Mutant phenotype is considered as base germplasm, thus is not directly released as a
variety in most cases. Thus, many a time the original process employed is forgotten
which may be the reason why mutation breeding is not a common theme even among
crop breeding community and industry players. In terms of practical applications,
and products developed and commercialized, the readers are directed to FAO/IEAE
mutant database available online at https://mvd.iaea.org/. Thus, the economic benefit
derived from mutagenesis and TILLING activities stands vindicated by the vast
number of products developed and commercialized. The same website lists more
than 3200 released varieties across 214 crop species. As noted in the IAEA mutant
variety database, the mutation breeding approach has resulted in billions of dollars in
extra income from new and improved varieties. Thus, through the pioneering efforts
of FAO, plant breeding efforts focused on mutation breeding came to the forefront.
Tremendous improvement in mutation detection techniques with terabytes of data
generated from numerous plant genomic sequencing projects has transformed muta-
tion breeding from what was perceived as a random process or empirical task to that
of specific allele identification bringing serious efforts to exploit the mutation
resources. There is an urgent need for new improved varieties related to medicine,
human nutrition, and energy needs, carbon credit which means the process to
achieve the goals should be improved beyond the known methods including inser-
tional and site-directed mutagenesis. Any mutagenesis experiment may have an
outcome that directly relates to the type of source material used, such as vegetative
parts—pollen, tubers, or seed, ploidy level, type, and dose of mutagen. In some way,
mutagen dose is a subtle balance between inducing maximum lesion rate and
eventual recoverable stable lines. Many chimeric lines would eventually get
eliminated in future generations of seed advancements. Consumers still distrust
crops developed by other biotechnology-led interventions (Lucht 2015) party due
to false information. This trend of misinformation continues to this day and has
impeded crop development goals. Thankfully, mutagenesis and TILLING enjoys far
more independence in terms of regulatory hurdle and remains relevant ever after.
1.5 Convergence or Divergence: Mutagenesis and Organic

System
If we look at EU regulations, crops developed by chemical and physical mutagenesis

are not subjected to GMO directive, 2001/18/EC, vide Article 3, and can be used in
organic agriculture. The International Federation of Organic Movements-IFOAM is
united in the principles of health, sustainability, sustainable livelihood opportunities,
a future for coming generations, and a safe environment. This is more important as
Climate change is real. Sustainable yield increase using increasing fertilizer can
increase carbon footprint which is against climate change agreements. Agriculture is
seen as Carbon negative. While GM crops are discriminated against in the organic
system citing that GM events are not found in nature and therefore man-made, the
scientific understanding itself has evolved and thus Gheysen and Custers in their
2017 paper argue that such bias is unwarranted in today’s situation. Modern-day
cultivated crops which were selected by human intervention differ significantly from
what nature had to offer. A similar dilemma also exists for the newer generation of
CRISPR crops. We believe a small fraction of Agriculture is organic (non-organic is
pegged at 3538336 B USD vs. 200 B for organic) and largely the priority in the
developing and the underdeveloped world is food security and sufficiency. Coming
back to mutagenesis, varieties developed through chemical and physical means
should not be seen outside the organic system which is valued at 200B USD
worldwide and expected growth of CAGR of 9.7% whereas the market in India
though still considered a niche, where buying power of the consumer is still low is
pegged less than a 1B USD and CAGR of 20.5%. Therefore, the opportunity for
varieties developed through mutagenesis in the organic segment exists even though
the contribution to total agricultural produce is abysmal with comparatively higher
margins. Though the same may not be true in all marketplaces. Thus, even with the
discovery of the CRISPR/Cas9 genome editing systems and their improved variants,
in absence of a clear, free regulatory, licensing regime, TILLING and mutagenesis
will continue to strive forward in the twenty-first century. Let us now focus on the
aspects of TILLING having described the relevance of this technology in the
previous sections.
1.6 TILLING Workflow
The first step in a TILLING project is the mutagenized population (Figs. 1.1, 1.2, and
1.3). Mutation sources can be diverse. Physical mutagens such as ultraviolet
radiations, alpha, gamma, and X-rays. Biological agents like virus, bacteria,
retrotransposons, transposons, and artificial T-DNA insertions cause random reloca-
tion and disrupts the function of DNA, however, not used in the crop mutagenesis
process. Commonly used chemical means include nitrous acid (which causes base
pair change from cytosine to uracil), deaminating agents, alkylating agents like
ethylnitrosourea, vinyl chloride, mustard gas, aromatic amines, sodium azide, ben-
zene, and ethidium bromide (causes frameshift mutations). Ethyl methanesulfonate
Mutagenesis of M0explants (seeds, tubers) with mutagens of interest (EMS,

Sodium azide, x rays, Gamma-radiation)
The treated seeds (M0) were grown to obtain M1 Plants
The M1 plants were self pollinated to produce M2 seeds
The M2 plants were used for tissue M2 plants were grown to harvest
collection to extract the DNA which M3 seeds which could be stored
could be stored as DNA Library for as Seed Bank for future
future TILLING experiments requirements
Pooling of DNA samples (2-fold to 8-fold) is done to reduce the efforts for
screening of large samples before PCR amplification of target gene
Enzymatic digestion of hetero-duplexes using dHPLC/endonucleases
Mutants detection with Agarose Gel Electrophoresis/LI-COR DNA

Analyzer/Microchip based Capillary Electrophoresis
Confirmation of SNPs by Sequencing
Fig. 1.1 TILLING workflow
(EMS) is an ethylating agent (C3H8SO3). EMS causes nucleotide substitution A->T,

G->C by guanine alkylation mechanism (point mutation). EMS causes the conver-
sion of guanine to O6-ethylguanine, GC!AT (transition). Other chemical mutagen-
esis agents include methyl methanesulfonate, sodium azide, hydroxylamine,
hydrogen fluoride, and nitrosourea (Viana et al. 2019). Here in this workflow
section, we will focus only on chemical means as these chemicals create uniform
SNPs, and small INDELS in the plant genome increasing plant survival, mutation
acceptance, and stability in the genome. Mutagen is selected depending on the nature
of the desired type of mutation as described in the above paragraph. The commonly
used chemical is EMS. Usually, the seed is the primary material for conducting
Fig. 1.2 NGS platforms for TILLING
mutagenesis. Even vegetative parts like tubers are used. The primary untreated
material is designated as M0 on which EMS treatment is done. After the treatment
seeds are designated M1. Till et al. (2006) reported a protocol for TILLING and
Eco-TILLING in plants using primers targeting particular sequences in DNA, PCR
samples are denatured and annealed. Further, heteroduplexes are cleaved with any
endonucleases, and PCR products are separated by applying denaturing polyacryl-
amide electrophoresis. Several approaches like sample pooling, gel analyzer, and
multi-well can reduce overall cost. Mutagen is selected depending on the nature of
the desired mutation. The primary untreated seeds are termed M0 on which EMS
chemical treatment in phosphate buffer (Hohmann et al. 2005). The treated seeds are
termed M1 and these are planted in soil to get M2 lines. This is our master population.
A master population contains all possible mutations in that line. Thus, master seed
packets contain seeds having related recessive mutations. Many a time growing a
large population (M3 onward) is labor and resource intensive and storing seeds is
also a logistic problem. The population size is also critical. For example, Arabidopsis
requires >100 K population to ensure that each gene is mutated at least once
(Ruegger and Chapple, 2001). Arabidopsis has a genome size of 135 Mb whereas
commercially important crops like corn have a genome size of 2.3 Gb and Cotton is
2.5 Gb (for more information on “The First 50 Plant Genome,” refer Scott and Todd
2013). However, maintaining such a large population in every commercial crop is
not feasible. Also, seeds have a limited viability period. Therefore, storing seeds in
Find or extract the Sequence of the Gene of Interest from the databases such as
GenBank available in different species and then identify the proper homologue
Identify the region within coding sequence with the most potential to generate
deleterious changes with the help of CODDLE (Codons Optimized to Discover
Deleterious LEsions) Software
BLASTN&ClustalW2 softwares to be used for alignment of genomic and amino acid

sequences
PARSESNP (Project Aligned Related Sequences and Evaluate SNPs) analyses the
nucleotide polymorphisms and determines its effect on protein, based on alignment of
related protein with PSSM (Position-Specific Scoring Matrix) and SIFT (Sort
Intolerant From Tolerant) soft wares
Further, to predict the effect of mutation on the secondary structure of proteins before
phenotyping could be done with SAS (Sequence Annotated by Structure) and SOPMA
(Self-OPtimized Method for secondary structure prediction with Alignment) softwares
Fig. 1.3 Bioinformatics workflow for analysis of TILLING data
ambient conditions is essential along with reviving the population from time to time
to preserve the master population. Similarly, DNA libraries prepared from such a
population should also be stored at -80 °C for long-term storage. This DNA library
must be prepared using high-quality DNA kits. Quality of DNA is an important
attribute and therefore, in-house protocols are best avoided. Developing an entire
mutation population is an expensive affair, therefore, care must be taken at each of
the steps for harvesting the entire benefits of novel mutants from such a population.
For practical purposes, a population size of 3000–5000 M2 is preferable. Once, the
population is developed, this can be screened by phenotypes (forward genetics) and
genotypes (reverse genetics). There are different types of mutation. If there is a
mutation in the transcription start site can lead to a loss of function which in most
cases will be a knockout or entire gene inactivation. Similarly, a situation will arise
in the case of mutation in the promoter region. Targeting regulatory sequence and
intronic sequence is desirable only when there is enough evidence, through prior
publication or bioinformatic analysis upstream that such a mutation works consider-
ing the time and resource which is invested in TILLING activities. Whereas any
intronic mutation may affect chaperone activity or make the final protein unstable
due to changes in peptide, hydrophobic, hydrophilic, ionic, disulfide, and hydrogen
bonds. Primer design in diploid species with one copy of the gene is fairly straight-
forward. However, in the case of polyploidy having multiple copies of homeologous
gene sequence, variability between coding, and non-coding regions should be of
focus. The primer must be designed taking into account differences in the 3′ prime
region usually toward the tail sequence thus increasing specificity. Today, KASP
(Kompetitive allele-specific PCR) is increasingly being used for genotyping from
low to high scale. An automated bioinformatic platform like PolyMarker can be used
for homolog-specific assays for polyploidy species. A recent book on TILLING
which also focus on screening mutants by sequencing and utilizing the mutants in
functional genomics can be referred to for further information. In any situation,
reverse genetics is important because traits like quality, phytonutrient, and
bio-fortified with mineral content may not be easily discernable by the naked eye.
Genomic sequence is readily available for the majority of crops. Many individual
lines and cultivars are already resequenced (Hao et al. 2020). Using genomic
information we can detect mutation using traditional TILLING. TILLING involves
amplifying the gene of interest by PCR and digesting the fragment by an endonu-
clease like CEL1, Endo 1 which is found naturally in celery and tomato among many
other sources. Then, these digested products are passed through agarose gel or
LICOR instruments (which contain labeled probes), and only selected samples
showing fragments are sequenced. To reduce the cost involved with molecular
biology aspects, samples are pulled 2–16 folds depending on accuracy and experi-
ence. Once, the mutant and corresponding trait are identified, the lines need to be
segregated further to find a homozygous line (Viana et al. 2019). Further, a few
rounds of backcrosses with non-mutagenized control lines are recommended to
remove background mutation. Line purification and trait stabilization make take
multiple generations, even up to M8 to M10 depending on the stability of the trait
(Das et al. 2014). Increasing the number of seeds will need bulking. This is important
for evaluating agronomic characters.
1.7 Social Study on Consumer Acceptability
Ahloowalia et al. (2004) quantified value addition in various crops by including

mutant varieties throughout the world. Therefore, confirming the wide acceptance of
varieties and hybrids developed through traditional mutagenesis. Several crop
varieties are protected by various laws protecting their cultivation, whereas mutants
are free to use. Mutagenesis continues to play a role in developing improved oil
composition in new genetic material, starch quality, protein content, and resistance
to biotic and abiotic stress tolerance. For example, the early maturity of a crop can
contribute to increasing cropping intensity. In Ahloowalia et al. (2004) paper, the
authors listed the economic impact of mutant varieties of major cereal crops. An
equivalent of sd1 (Dee-geo-woo-gen) was induced by gamma irradiation in rice
cv. Calrose-76. The germplasm (Calrose-76) was used extensively in plant breeding.
A derived line from Calrose-76 is Amaroo which occupies 60–70% of rice area in
Australia. The rice yield rose 2x in Egypt when the dwarf trait was used. No updated
data is available on the mutant varieties in India separately. In an earlier estimation of
productivity increase and value capture, IARI reported a US$1748 value of produce
from 2 PNR (rice) varieties. Further, Ahloowalia et al. (2004) review show value
capture in a wide range of cereal crops showing tremendous acceptance of varieties
developed from random mutagenesis. It seems consumers are not concerned with the
mutation-based approach and it is hoped that the recent plant breeding techniques to
edit endogenous genes similarly will not stir any fresh controversy. The European
Commission had requested an expert panel to address the question of in vitro and
in vivo mutagenesis, i.e., whether there is a difference in the cellular mechanism and
the nature of genetic changes including repair mechanism and the nature of genetic
change in both the processes. The expert panel concluded that such a distinction is
unwarranted and impossible to classify (Mullins et al. 2021). There are other social
studies to find consumer acceptance of the new generation of precise editing of genes
which shows modern-day genetic intervention can make the public skeptical largely
due to misunderstanding perpetuated by vested interest groups. For the time being, it
is safe to conclude that traditional mutagenesis will continue to be used in various
crop improvement efforts in an unhindered manner.
1.8 Patentscape: Plant Patent and Plant Variety Protection

(PVP)
The first criterion for registering any plant variety must be compliance with distinct-
ness, uniformity, and stability (DUS). Any mutant developed by chemical and
physical mutagenesis must fit the description of DUS. Most of the varieties crop
breeders develop are protected under plant variety rights (PVP), the equivalent in
India is ‘The Plant variety protection and Farmer’s rights (PPVFR) Act, 2001 (the
“Act”). Indian PPVFR is aligned to TRIPS (Trade-related aspects of Intellectual
property rights), an agreement that protects varieties as well as essentially derived
varieties. Protection under PPVFR ranges from 15 years (for seeds) to 18 years (for
trees). An inventive step is not essential in PVP the same is a must for patenting. A
plant patent is not allowed in India. However, there are certain advantages to
patenting a plant variety vs. registering the new variety under PVP. A patent prevents
others from developing derived varieties which PVP fails to protect, thus limited in
value (this is not the case with India). Patents are of 20 years duration, thus more than
PVP. However, many countries including India do not allow plant patenting due to
concerns about misuse by the patent holders and throttling further innovation. If we
look at other countries, like European Economic Zone countries, The European
patent office grants patents in exceptional circumstances to plant varieties. Though
there are controversies surrounding the patentability of plant varieties found in
nature, these arise from different interpretations of the “exception” wording used in
the European Patent Act. Plants bred naturally cannot be patented in Europe
(Website 10 n.d.). Plant patenting varies from country to country. For one, plant
varieties already available in the wild and in the cultivated state cannot be patented.
In the USA, under the provision of 35 US C161 which centers on a distinct
phenotype obtained asexually and uncultivated. This includes plant varieties and
mutants that are discovered in cultivation or trial and must have at least one distinct
characteristic not present in nature. The meaning of plant covers fungi and algae and
is limited to asexually reproduced in nature, excluding tubers. The novel plant
variety must satisfy the general requirements of obtaining a patent which will not
be obvious to one generally skilled in the art and is subject to a utility application.
The purpose of asexual reproduction is included to assert that is produced (i.e., A
true genetic copy and stable in nature). A true genetic copy can be maintained by
several horticultural practices like grafting, budding, grafting, and tissue culture
(Website 11 n.d.). Spontaneous mutants are excluded. The botanical classification
of the novel plant to be patented must be included in its entirety, including ones from
breeding trials. The DUS criteria for the UK are listed on Website 12 (n.d.).
1.9 Crop Improvement Using Mutagenesis and TILLING
Here in this section, we will closely look into traits improved by mutagenesis and
TILLING in various crops (also, see Table 1.1 for commercialized mutant products).
Kurowska et al. (2011) found an average of 1 mutation in every 200–500 kb region
screened in various crop species.
1.9.1 Rice
Cooper et al. (2013) proclaimed a strategy for rice TILLING and Eco-TILLING
using a LI-COR DNA analyzer. The developed mutant population can be used to
screen several genes and is offered to the public in a service mode for wider outreach.
Tai (2013) opined that advances in mutation detection techniques have resulted in
renewed interest in the traditional mutagenesis program. Though in the past chemical
mutagenesis has been used extensively for generating useful genetic variation in
breeding programs. From a nutritional viewpoint, the phytic acid complex contains
phosphorous which is not bioavailable. This presents a complex problem as P along
with other micronutrients remains unavailable to humans and livestock as reported
by Kishor et al. 2019. Kim and Tai (2014) identified 4 LPA mutants that showed a
reduction in phytic acid which was up to 46% less than the original line. Similarly,
deploying TILLING in cv. Volcano, Casella et al. (2013) targeted four genes related
to plant architecture height, flowering initiation, aroma, and abiotic stress (drought).
Height in the mutant SD1 (semi-dwarf 1) was reduced by 21%. In this (Casella et al.
2013) mutation density on average was 1/373 kb. Another study in rice by TILL
et al. (2007) used two different mutagens, one was methyl nitrosourea (MNU) and
Table 1.1 Worldwide commercial mutant products (Source: FAO/IAEA Mutant Variety
Database)
Sr. Country of Registration
no. Crop Variety name origin year Improved characters
1 Rice Vikram-TCR India 2021 Dwarf variety with mid
(Trombay to early maturity and
Chhattisgarh high yielding
Rice)
2 Rice CG Jawaphool India 2021 Semi-dwarf stature,
Trombay mid-early maturity,
high yield potential
3 Tomato Girón 50 Cuba 2021 High productive
potential, high content
of total soluble solids,
short cycle, tolerant to
heat and diseases
4 Soybean CUVIN 22 Cuba 2021 High productive
potential, relatively
short cycle, robustness
for disease resistance
5 Lentil Binamasur-12 Bangladesh 2021 Higher yield
6 Rice Kian Iran 2021 Drought-tolerant, early
flowering, dwarf,
optimal grain length
after cooking, and
elongation ratio higher
than parent lines
7 Rice Sinar 1 Indonesia 2020 High yield, higher
aroma level than the
parent, moderately
resistant to brown plant
hopper
8 Rice Sinar 2 Indonesia 2020 Resistant to BLB
diseases, high yield,
higher aroma level than
the parent
9 Barley Madaba1 Jordan 2019 Plant height, biomass,
yield, drought tolerance
10 Banana Pirama 1 Indonesia 2019 Tolerant to fusarium
wilt disease (TR 4),
high vitamin C content
11 Rice Konjikinokaze Japan 2019 Low amylose content
12 Soybean Kemuning 1 Indonesia 2019 High yield, large seed
size, moderately
resistant to leaf rust
disease and
pod-sucking pests,
drought tolerant
(continued)
Table 1.1 (continued)

Sr. Country of Registration
no. Crop Variety name origin year Improved characters
13 Barley Haneumamochi Japan 2019 Glutinous endosperm
(low amylose, high
amylopectin)
14 Groundnut BINA Bangladesh 2018 Higher pod number and
Chinabadam- higher yield
10
15 Rice RD77 Thailand 2018 Photoperiod insensitive
the other was EMS. Multiple genes conferring biotic and abiotic stress tolerance
were TILLED. A total of 57 mutations were deciphered. Here, the MNU population
showed an average frequency of 1 in 265 kb, whereas for the EMS population it was
1/294 kb. Ramkumar et al. (2019) developed and screened a mutant population in
rice cv. Nagina 22 and obtained three EMS mutants named stay green mutants
(SGM-1, 2, 3). These mutants were tested for drought tolerance and morphologically
were similar as evident by SNP marker assay involving 72 markers. The stay green
gene results in an increased level of cytokinin. Only SGM-3 displayed delayed
maturation and showed promising results under normal irrigation and water stress
conditions. A set of 15 candidate genes associated with stay green was analyzed and
these lines showed non-synonymous mutations in SGM-1, 2 mutants whereas no
change was found associated with mutant-3. Thus SGM-3 was suggested as a new
mutant with a better harvest index under irrigated and stress conditions.
1.9.2 Wheat
Krasileva et al. (2017) pointed out that genetic resources for polyploidy like wheat
are scarce compared to diploid species. Also, having a high degree of redundancy
enables wheat to tolerate a high degree of mutation. Taking this into consideration
they were able to catalog 10 million mutations translating into 2735 mutants with a
density of 35–40 mutations per kb. Compared to diploid rice, wheat is an
allohexaploid crop with a genome size of 17 GB. TILLING has been reported
extensively in wheat. Traits like high amylase content were reported by Slade
et al. (2012), where SNPs caused altered branching related to starch synthesis
(SBEIIa). This novel allele when used in wheat breeding resulted in elevated starch
content (47–55% amylose) compared with the original line. In another study, Rawat
et al. (2012) developed a mutagenized population of T. monococcum (TA 4342-96),
a spring wheat variety targeting the waxy gene. The mutation frequency achieved
was 1 in 90 kb which is very high. Slade et al. 2005 and Dong et al. 2009 also found a
mutation in the waxy gene in the range of 1 in 17.6–34.4 kb DNA. Rawat et al.
(2012) targeted genes involved in the biosynthesis of lignin, namely, COMT1
(1/86 kb), HCT2 (1/92 kb), and CL1(1/100 kb). They also predicted a 95% proba-
bility, the minimum wheat population required for TILLING at 5520 for obtaining
robust mutation (to generate the sufficient number of knockout—STOP codon/

non-sense mutations for each gene). Here, the thing to be noted is that they used
diploid wheat. Targeting the waxy gene was also done by Dong et al. (2009) in
cv. Ventura variety. They reported a wide range of mutation types including
knockouts, missense, and silent ones. Also, puroindoline genes were targeted thus
a hard grain type was identified. Another study involved the cultivar “Jinmai 47”
which was screened for various genes (Rubisco A/B, Ppd-D1). A total of 31 unique
mutations were reported. There are many online resources for aiding TILLING in
wheat. For example, wheat-TILLING focuses on tetraploid cv. Cedenza. Another
resource, developed by Dr. Debocovsky, from UC Davis focused on “Kronos.”
Further, see Website 6 n.d. and Website 7 n.d. The uniqueness of this project
involves the re-sequencing of the protein-coding regions of the mutant population
using Illumina’s next-generation platform which was then aligned to the Chinese
spring survey sequence. Bovina et al. (2014) developed a 2601 M3 durum wheat
population in cv. Svevo using EMS chemical mutagen was phenotypically
evaluated. The same group obtained a high number of phenotypic mutants (22%)
which considering wheat is significant. Two nonsense mutants in a gene involved in
starch synthesis, SBEIIA and B alleles were obtained which were planned for
stacking. A 2020 study published in BioRxiv by Moehs et al. (later published in
PLoS One) showcased the development of non-transgenic glyphosate-tolerant wheat
by TILLING. The stack mutant carrying T102I and P106S is resistant to commercially
available weed killer, glyphosate. Sharma et al. (2022) identified 44 EMS M4 lines
for variation in grain amylase which is related to grain nutritional quality. An
amylase mutant was found with an overall mutation density of 1 in 65 kb. Genes
that were targeted were alpha-amylase (ZIP), MYB-TF.
1.9.3 Maize or Corn
Corn is an important food, feed, and biofuel crop. The consumption demand for corn
is increasing day by day. Therefore, corn improvement is very critical for high-
yielding traits to meet the gap. Mutagenesis experiments to improve corn traits are
reported in plenty. However, in the history of corn breeding, a unique mutation
transformed wild corn into cultivated one by turning its hard kernel into a soft one
that is easy to consume (Website 7 n.d.). An early study by Freeling (1978) described
the Adh1 gene as a mutagen monitor which was used for assessing hazards from
pollutants thus the indirect utilization of the mutation. We reviewed TILL et al.
(2004) early work where chromomethylase gene was targeted. A total of 17 unique
mutations were found. The mutation rate varied from 0.93 to 2.1/kb in the two
cultivars under study (i.e., B73 and W22). Many of the functional genomic studies in
corn were facilitated by using maize transposons by tagging genes. These mutations
will act as a source of new alleles in functional genomic studies (Walbot 2000). May
et al. (2003) developed a knockout resource in maize by using transposon-
mutagenized seeds. This resource is available to the public and any gene knockout
can be screened. A Robertson’s mutator (MU) pollen parents were used thus this
resource sheds light on the epigenetic and functional genomics of corn. Effective
mutagenesis is a must for establishing the relationship between traits to those of
corresponding sequences (Settles 2020). Chemical mutagenesis can be effectively
used for creating high-density mutation screens. Settles 2020 also reported muta-
genesis using mature pollen. Brunelle et al. (2017) hypothesized the possibility of
using chemical mutagenesis to induce mutation in corn more effectively than
previously reported in Robertson’s mutator germplasm. They screened the EMS
population for embryo-specific (emb) mutations which were found in higher fre-
quency than that of the transposon KO population. Thus the presence of emb loci in
corn which resulted in an abnormal embryo with normal endosperm was validated in
this EMS population. Heuermann et al. (2019) combined next-generation sequenc-
ing with phenotyping to identify induced recessive as well as dominant mutations. A
rapid process to identify corn mutants having dwarf and pale green phenotype which
was obtained through EMS mutagenesis of pollens of M2 family was subjected to
whole-genome sequencing. The state of zygosity was identified in M2 family by
phenotypically analyzing M3. Zygosity was analyzed with sequence data. Therefore,
Mendelian inheritance and individual sequence data passed their zygosity filter for
dwarfism which happened due to insertion in an1 locus and pale green phenotype in
the W2 gene due to non-synonymous amino acid change.
1.9.4 Sorghum
Sorghum has multiple uses for food, feed, and industrial use (bioenergy, remedia-
tion, and use in small industries). Wild relatives have several important traits such as
shattering and disease resistance. Intercrossing wild relatives with traditional lines
and transferring desirable traits will result in improved lines. The other approach
could be mutagenesis in preferred lines. In a recent paper, Hao et al. (2021)
envisioned a paper on sorghum breeding involving NAM (next associated mapping),
MAGIC (multi-parent advanced generation inter-cross), and mutant population.
Going forward the aim is to combine agricultural traits with other industrial traits.
We report the work carried out by Xin et al. (2008) where 1600 mutant lines were
produced using EMS. Mutation frequency was reported at 1 in 526 kb. The mutation
corresponds to the brown midrib (Bmr) phenotype, translating to modified lignin
composition and improved absorption. In another screening study, Nide et al. found
mutants for NAC and Lsi1 (low silicon) using the Illumina HiSeq2500 system. They
generated data of 22 M bytes which were analyzed by ComSeq software. The true
mutation was subsequently validated by phenotyping. Chen et al. (2019) discussed a
plan for the development of a pedigree mutant library using EMS treatment for new
traits. The pedigree population is a powerful resource for identifying mutations
related to agronomy and key biological traits. Combining gene discovery programs
with sequencing will fasten crop breeding. Jiao et al. (2016) stated that sorghum
being a C4 plant has a high-quality sequence available in the BTx623 population.Six
thousand four hundred pedigreed M4 mutants were created from a single seed
descent method. Further, the sequence data clearly showed that variation in the
genome was different from those found in nature. Eight genes attributing to drought
tolerance were identified which had allelic mutations and these showed
co-segregation with phenotype, thus providing a unique platform for functional
validation of genes in Sorghum. Addo-Quaye et al. (2017) advocated that EMS
mutagenesis caused few base pair changes in each line in BTx623. NGS data with 7x
coverage from 586 lines had 5.4 million mutations. The repeat region in the genome
showed poor call reads. The CG-rich patches and overlapping sequences influence
the mutation rate allowing the detection of true mutation. The mutation in a sequence
was linearly correlated with transcript rate and G:C content. Sorghum is indicated as
an emerging bioenergy crop (Ordonio et al. 2016). Present efforts for the characteri-
zation of sorghum mutants are described with the identification of quantitative trait
loci (QTL). Further improvement strategies are discussed in light of genomic and
molecular breeding benchmarks. Sorghum is a stress-tolerant C4 plant and is well-
grown in semiarid and arid zones of the country. Now, sorghum grain is yet again
finding a place in culinary products which are shown as healthy. For the rapid
discovery of genes attributing to food, feed, and energy demands. Screening various
sets of germplasm (Eco-TILLING) and mutants (TILLING) is a useful tool for
breeding (Xin et al. 2021). Striga is a parasite that thrives in sorghum. However, a
low germination stimulant 1 (lgs1) shows low Striga germination or lower induction
(stimulation) for germination to screen 177 accessions. Dhurrin-cyanogenic gluco-
side is a plant defense molecule against insects and pests. Blomstedt et al. (2018)
identified two mutants, tcd1 (totally cyanide deficient 1) and acdc 1 (adult cyanide
deficient class 1). They found the expression levels of dhurrin are more at the initial
growth stages than at the later stage thus a relative trade-off exists between nitrogen
partitioning and dhurrin levels. Another study by Sohail et al. 2020 concluded that
dhurrin production in the initial critical stages particularly seedling emergence gave
plant growth advantages then acyanogenic plants which are typically small in size.
The vegetative growth parameters were not affected in later growth stages.
1.9.5 Barley
Historically first evidence of mutagenesis in Barley was reported by Hermann

Nilson-Ehle and Aka Gustafsson in 1928 (as cited in Lundqvist 2014). Physical
mutagenesis resulted in the development of a mutant named Erectoides. A spectrum
of mutants with a high frequency of lesions in the specific gene was observed which
can be used in gene mapping. The mutant resources can be found in NordGen,
Sweden (Lundqvist 2014). Gao et al. (2018) reported in vitro mutagenesis using
microspore culture to regenerate a double haploid line. EMS and pingyangmycin
were used as mutagens. Mutants were subjected to high and low nitrogen treatment.
A set of five mutant lines having more productive tillers was identified. Results,
however, overall NUE increased but did not entirely depend on the N uptake
parameter only. N translocation improved during grain filling. Three popular barley
mutants in current cultivation include Golden Promise, Diamant, and hybrid
(Ohnoutkova 2019). In the case of barley Talame et al. (2008) reported on a sodium
azide-treated barley population was four genes namely Constans like (Col1), nitrate
reductase (NR), rust resistance protein gene (Rpg1), and eukaryotic factor gene
(eIF4e) were screened (TILLed). Here they had a population size of 3148 M2 in
more background. They obtained a total of 22 mutants with a frequency of 1 in
374 kb. In another study, Bovina et al. (2011) obtained 29 mutations in targeted five
genes (viz. starch metabolism gene, beta-amylase 1, starch synthase I, and starch
synthase II with a mutation density of 1 in 520 kb. Later, Sparla et al. screened lines
for various quality parameters like granule size, which led to the identification of
7 mutant lines with improved quality traits. Again, we refer to another work by
Szurman-Zubrzycka et al. 2018, where they created a HorTILLUS platform by
treating barley on cv. Sebastian. A relatively large population of 9600 M2 plants
was created as a part of the study. A total of 32 genes were screened which played a
role in plant architecture. Dockter and Hansson (2015) reported more than 1000
short-culm mutants from a barley resource. Some of these mutants were linked to
hormonal rhythms. Thus, the entire resource could be used to develop climate-
resilient cultivars.
1.9.6 Oats
In the mutagenesis study of Chawade et al. (2010) in oats where the researchers
developed 2600 mutant resources. To develop new oat varieties, particularly to alter
starch and protein among others, the entire mutation population was screened for
PAL1 (phenylalanine ammonia-lyase gene) and six mutants were identified. Simi-
larly, 10 mutations were identified in the cellulose synthase-like (CslF6), beta-glucan
biosynthesis gene among others. The mutation detection frequency varied from 1 in
20 kb to 38 kb depending on the detection method employed. Hernandez-Hernandez
et al. (2017) created an EMS mutagenesis population. The project aimed to increase
avenanthramide (Arv-A, B, C) which is an antioxidant with health-promoting
properties. Genes involved in avenanthramides are 4-CL, HHT, and CCoADMT
(Caffeoyl-CoA-O-methyltransferase N-hydroxycinnamoyl transferase) {Li et al.
2019}. A total of 17 lines with elevated AVN were obtained with the highest amount
of 227.5 ug/g in flour compared to 78.2 ug/g in control. Thus, the biochemical
selection of mutant lines was sown. Chawade et al. (2010) conducted a study to
develop high beta-glucan oats using 2500 EMS-TILLING populations. They
analyzed 1700 lines biochemically for altered beta-glucan contents. One mutant
line showed a 52% increase in glucan over the non-mutagenesis control.
1.9.7 Flax
In a separate study on Flax by Chantreu et al. (2013) in Linum usitatissimum L. cv.

Diane, a 4894 mutant population was developed. EMS was the preferred source of
mutation. Their main focus was on lignin biosynthesis genes namely C3H and CAD.
Seventy-nine mutations were identified in coumarate-3-hydroxylase (C3H) and
76 mutations in Cinnamyl Alcohol Dehydroxygenase genes. The estimated average

frequency was 1 in 41 kb. The mutant line of CAD showed an orange-brown
phenotype similar to CAD mutant reported in various species. Fofana et al. (2017)
worked on increasing SECO (secoisolariciresinol) diglucoside lignin (SDG) which
helps treat various diseases. This SECO compound is more rapidly accumulated in
the body than in the original plant source. An EMS mutant population of 1996 M2 of
CDC Bethune was created for the study. Mutants showed a range of SDG levels. The
action of UGT74S1 in the biosynthesis of SDG from SECO was proven. Another
study on the fatty acid composition involving 84 cultivars found variation in the
SAD (stearoyl-ACP desaturase) and FAD2 & 3 alleles (Fatty acid desaturase) by
Eco-TILLING. Previously, polymorphisms in SAD and FAD alleles were unknown.
Deep sequencing with 400x coverage with the MiSeq platform was used. The SAD
enzyme converts stearic to oleic acid whereas the FAD2 allele converts oleic to
linoleic and FAD3 linoleic to linolenic acid. The objective of the study was to
evaluate the fatty acid composition and use in the future breeding program. Simi-
larly, earlier Thambugala et al. (2013) reported flax seeds are rich in omega fatty
acids and therefore beneficial for human health. Here, they analyzed 120 accessions
that also contained a few EMS mutant lines. The germplasm was screened for SAD,
FAD2, and FAD3 genes, and isoforms were isolated. The fatty acid profile and oil
content were analyzed with the polymorphism data. Finally, they found six
desaturase varieties from the study. Another study by Burnett et al. (2018) on flax
focused on orbit-wide composition from a core flax collection. Several new variants
of the compound were elucidated in the study. Tombuloglu (2020) reported the role
of MYB-TF in lignin fiber production by analyzing the genome data. Data showed
multiple MYBTFs of which 17 R2R3 MYBs are indicated in lignin biosynthesis.
Upregulation and downregulation of MYB TFs were studied concerning lignin
biosynthesis.
1.9.8 Soybean
Oil quality and content are important traits for fetching higher value in soybeans.
With the same objective to increase Soybean oil production, a JTN-5203, 1820 M1
EMS population was developed by mutagenizing 15,000 seeds. The resultant mutant
population is a resource for a range of genetic diversity. Several lines with an
increase in total protein and total oil were obtained (Espina et al. 2018). A recent
2021 study by Lakhssassi et al. mined new variants in the FAD2 gene, i.e.,
GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E
which grew normally under cold conditions, whereas the earlier alleles did not
grow normally under low temperature. The primary goal of soybean breeders is to
increase the amount of oleic acid and thus bring high oxidative stability in oil and
applied products. In another study, Millas et al. (2019) developed 1820 mutant
populations after treating 15,000 seeds of cv. JTN-5203 and targeted FAD2-1A,
1B genes that are involved in fatty acid biosynthesis. They obtained allelic variants
of the targeted genes. Most of the mutants had GC to AT transition which is a
significant change of EMS wherever the chemical is used. We also looked at another
study by Hoshino et al. (2014) where a very large mutation population consisting of
39,100 lines was developed in five different mutant populations and was analyzed
for several genes. These populations were developed using EMS, and X-rays for
different cultivars. The same authors reported FAD 3-2a mutant which had a reduced
2-linolenic acid profile in soybean oil along with another seven mutants. A triple
combination of mutants had less than 2% linolenic acid profile in oil. In another
study by Cooper et al. (2008), where they subjected soybean seeds to NMU and
EMS-induced mutagenesis in cv. Williams 82 and Forrest. A total of four
mutagenized population was developed. A total of 116 mutations were identified
among seven target genes. For the NMU-induced mutagenized population, a higher
mutation density of 1 in 140 kb was obtained whereas for EMS the range was 1 in
250 and 550 kb, respectively.
1.9.9 Peanut or Groundnut
Guo et al. produced a mutant population in the background of CV724-19-25. Their

main objective was to TILL for two major allergen genes Arah1, Arah2, and fatty
acid desaturase FAD2 (which facilitates oleic to linoleic acid profile). The same
study indicated that various other genes line AhLOX7, and AhPLD were screened.
The mutation density was found to be 1 in 344 kb for single-copy genes whereas
multiple-copy genes showed 1 in 3028 kb. Again, the combined mutation density
was found in the range of 1 in 1066 kb. In another study on peanuts, Knoll et al.
(2011) developed a mutant population in Tifrunner background using two different
doses of EMS viz. 0.4% for 12 h and 1.2% for 270 min. The population size was
3420 M2 individuals, which like Guo et al. 2015 study which is mentioned earlier
also focused on peanut allergens namely, Arah1 and Arah2. The mutation frequency
was 1 in 967 Kb. Knoll et al. (2011) also focused on other seed quality traits like oil
composition. Fatty acids which are largely monosaturated have a longer storage life,
are nutritionally rich, and possess good flavor. Increasing the content of oleic to
linoleic acid is a desirable trait obtained in this study. Fatty acid desaturase (FAD2)
was another target of the study, where a frameshift mutation was identified which
lead to the deactivation of FAD2B protein. In another study, Karaman et al. (2021)
developed a 1541 M2 EMS population. TILLING for the Delta (12) fatty acid
desaturase (FAD2) was conducted for decreasing the conversion of oleic acid to
linolenic acid by creating a variation of the gene. The mutation rate for the popula-
tion was 1 in 2139 kb.
1.9.10 Sunflower
Sabetta et al. (2011) created a mutagenized population in GV 342 background using

0.7% EMS for 6 h. A total of 3651M2s were obtained. Screening of the mutant
population was restricted to 1152 lines and limited to four genes. The target genes
involved conferred resistance to downy mildew resistance and others involved in

fatty acid biosynthesis (Kas II, Kas III, and FAD 2-1). The mutation frequency was
limited to 1/475 kb and nine mutants were identified. In another study, Kumar et al.
(2013) created a mutant population of 5000 mutagenized individuals. The same
population was screened for two fatty acid biosynthesis genes, FAD A and SAD. A
total of 26 mutations were identified with a total mutation density of 1 in 480 kb.
1.9.11 Brassica
The Brassicaceae family consists of economically important species like B. napus,

B. rapa among others. In one study by Stephenson et al. (2010), where they
developed 9216 M2 lines and screened six genes which included orthologues of
Arabidopsis REPLUMLESS (RLL1), METHYLTRANSFERASE1 (MET1). The
mutation density was found to be 1 in 60 kb. A canola cv. DH12075 mutant
population was developed by Gilchrist et al. 2013. They developed 3158
EMS-mutagenized lines. A total of 432 mutations in 26 target genes were identified
which included multiple alleles for each gene. The mutation types were classified as
missense (42%) and mutation frequency was calculated at 1 in 109 kb. In another
study, Harloff et al. (2012) developed a mutagenized population in two yellow-
seeded rapeseed lines, Spring variety YN01-429 and winter type cultivar, Express
617 with a population of 5361 and 3488 plants, respectively. Here, they tested BnaX.
SGT and BnaX.REF1, having 229 and 341 mutations. The goal of the project was to
develop a new resource for breeding low-sinapine oilseed rape. The mutation
frequency varied from 1 in 12 to 1 in 22 kb for the first population, Express
617, and 1 in 27–60 kb for the YN01-429 population. While Guo et al. 2014 also
studied Express 671, a rapeseed population consisting of a 3488 M2 EMS popula-
tion, targeting BnC6F1 paralogs. This gene (BnC6F1) is involved in flowering time
pathways. Mutation in the target gene resulted in altered flowering habits with yield.
The hybrid involving mutants showed positive heterosis in terms of increased seed
number per pod and per plant. In another study by Himelblau et al. 2009, a mutant
population consisting of 960 M2 in cv. T01000 background was screened for
15 different genes mainly related to abiotic stress. A total of 25 mutations with
1/447 kb density were observed. Altered plant architecture was observed in the Bna.
AP1.A02 K domain having STOP mutation. Other phenotypes like dwarfism were
observed in 18 mutants with reduced internode elongation and delayed flowering
when compared with controls. Again, Shah et al. (2018) used the Express 617 culti-
var to characterize AP1 orthologs in rapeseeds. A STOP codon in AP1 showed an
increase in seed number per plant. The mutation rate varied from 1 in 17.4–26 kb in
A02/C02 analogs in the study by Shah et al. (2018). The mutant showed a 200%
average seed yield over the control. In another study, a CAX1 gene was TILLED and
the mutant showed lower total chlorophyll which was linked to lower photosynthesis
and higher ROS levels. The mutant also showed a higher accumulation of Fe along
with Ca and Mg.
1.9.12 Potato
In the case of potatoes, GBSS (Granule Bound Starch Synthase) gene was screened
in a mutagenized potato population. The mutant contained no amylase and only
amylopectin starch, which is most useful in the industrial use of paper, textiles, and
food. The mutant line was named “Super potato” and this became the first product
developed by TILLING in Germany with market readiness (ISAAA 2009).
1.9.13 Tomato
Tomato finds a wide range of applications in the food and processing industries. The
genome size is fairly placed at 950 Mb and this crop has been sequenced and many
cultivars have been resequenced multiple times. Thus tomato is used as a model crop
to understand gene functions (Okabe et al. 2013). Tomato finds its place in Indian
cuisine. Seasonal supply and continuously growing demand result in drastic price
movement. Moreover, postharvest damage is in the range of 20–35% adding to the
problem of scarcity and price fluctuations (Fig. 1.4). Thus, extended shelf life (ESC)
is an important trait of interest. TILLING has been used extensively in tomatoes with
a focus on improving fruit quality and shelf life (Okabe et al. 2011; Minoia et al.
2010; Piron et al. 2010). A micro-tom population of 3052 lines was developed by
Okabe et al. 2011 to elucidate functional genomics studies. The population was
developed using EMS treatment at 0.5% and 1%. The mutation frequency varied
from 1 in 1710 kb for 0.5%EMS and 1 in 737 kb for 1% EMS population in
10 distinct genes which are involved in fruit quality and neutraceutical application,
gamma-aminobutyric acid (GABA) metabolism with 1 in 1737 kb mutation fre-
quency. Two mutants showing altered/reduced ethylene response were identified in
the same mutant population. Minoia et al. 2010 reported a mutagenized population
of tomato cultivar cv. Red Setter by treating seeds with EMS at 0.7 and 1.0% dose.
Primarily, the study focused on fruit quality traits. The target genes included RIN
(ripening inhibitor), Green ripe (Gr) involved in the maturation of the fruit, Rab
GTPase, expansin 1 involved in cell wall ripening or maturation, and genes involved
in carotenoid biosynthesis. In this study, a total of 9.5 kb region of tomato genome
was investigated and 66 mutations were identified in the two mutant populations
(EMS 0.7%, 1%). A broad spectrum of mutations was identified 37.6% silent, and
62.4% missense mutation with STOP codon. The mutation density was in the range
of 1 in 322–574 kb. In another study, Silletti et al. (2013) screened the tomato “Red
Setter” population. The cyc-B locus was targeted with 8 identified new alleles. The
phenotypic analysis resulted in the identification of a missense mutation with higher
lycopene accumulation with effecting plant traits including fruit quality. Jones et al.
(2012) developed a mutant population intending to focus on nutritional quality. A
point mutation in DE-ETIOLATED 1 resulted in high pigment fruits with increased
carotenoid, and phenylpropanoid phytonutrients, and better fruit types. In India, a lot
of commercial interest is seen by private players. Chief among them is the collabo-
ration of Shriram Bio seeds and Arcadia BioSciences for developing extended shelf
bhavesh.palan@mahyco.com anjanabha.bhattacharya@mahyco.com vilas.parkhi@mahyco.com bharat.char@mahyco.com
ABSTRACT RESULTS
Tomato finds a wide range of applications in food and processing industries.
The genome size is well-established at 950Mb and the species has been
extensively studied at the genomic level with related information available in
abundance. Tomato is therefore used as a model crop to understand gene
function. In India, seasonal supply and continuously growing demand for a
tomato results in drastic fluctuations in pricing. Moreover, post harvest
damage of fruits is in the range of 20-35%, further adding to the problem of
scarcity and price instability. Thus, extended shelf life (ESC) is an important
trait of national interest. One of the solutions for developing ESC could be
with the use of mutagenesis and TILLING (Targeting Induced Local Lesion
Fig.1 Germination % in different
in Genome). Mutation is the source of primary variation in plants. EMS doses
Mutagenesis induced breeding is different from natural breeding as this is a
deliberate effort to induce changes in the genome with the objective to
change the plant phenotype. The economic benefit derived from mutagenesis
Fig.2 Tomato mutants
and TILLING activities stands vindicated by the vast number of products
(a) Seedless (b) shelf life traits
developed and commercialized as reported in mvd.iaea.org. Here we report a
tomato mutagenesis population, developed by using a proprietary cultivar,
which was treated with ethyl methyl sulfonate (EMS) and the optimum dose
standardized for the development of a robust TILLING population. The
tomato seeds were treated with six different concentrations of EMS (0.5%,
0.75%, 1%, 1.5% and 2%) including control for 8 hrs. Further, a standard
kill curve was established for the cultivar. The tomato mutants were grown
till maturity to study the morphological changes occurring in the fruit
development stages. A range of phenotypes like seedless, extended shelf life,
different fruit shapes and sizes were observed at maturity among the
developed mutants. This mutant population is continuously being TILLED
for shelf life, quality and yield traits.
Key words: Tomato, Non-GM, Mutagenesis, TILLING, Shelf life, Yield
INTRODUCTION
Considering the wide application of tomato in food processing industry
and post harvest damage, there is a need of producing tomato cultivars
with extended shelf life with increased yield to fulfill the demand. This can Fig.3 Tomato mutant plants in Greenhouse Fig.4 Variation in Fruit shapes and sizes
be achieved with one of the many available crop improvement methods
including mutagenesis and TILLING. According to FAO/IAEA Mutant CONCLUSION
Variety Database (MVD), 3365 mutant varieties were released worldwide 1. Induced mutagenesis with EMS showed a wide range of phenotypic
till date. India shared 4 out of 25 mutant varieties of tomato listed in MVD. variability in a Mahyco proprietary tomato cultivar.
We report a similar strategy to TILL for tomato mutants with desirable 2. It was observed that germination percentage decreased with increase in
traits like shelf life, seedless among others. Thus, EMS is used as a concentration of EMS in general.
mutagen for the development of TILLING population due to its wider
3. The population will be evaluated for a wide range of phenotypes like
acceptability (Palan et al. 2021). Further, exonic mutations increases
chances of developing new traits. Different concentrations of EMS were seedless, extended shelf life, different fruit shapes and sizes.
used for induction of mutations and kill curve was developed. The mutant 4. The mutant population will also be utilized to TILL genes of interest
tomato population could be utilized for screening of several traits of specifically linked to important commercial traits like yield.
interests in future.
REFERENCES
MATERIALS & METHODS 1. Joint FAO/IAEA Programme’s Mutant Variety Database
1. The seeds of a proprietary tomato cultivar were received from https://mvd.iaea.org/#!Home (accessed on December 30, 2021).
Breeding team for mutagenesis experiment. 2. Palan B, Bhattacharya A, Char B. (2021) TILLING in the era of precise
2. The seeds were soaked in proprietary buffer for an hour before genome editing. Indian J. Biotechnol. 20 (1): 9-16.
adding EMS. 3. Laskar RA, Chaudhary C, Khan S, Chandra A. (2018) Induction of
3. Six different concentrations of EMS (Zero, 0.5%, 0.75%, 1%, 1.5% mutagenized tomato populations for investigation on agronomic traits
and mutant phenotyping. J Saudi Soc. Agril. Sci. 17: 51-60.
and 2% for 8 hrs) were used for optimization of dose.
4. Minoia S, Petrozza A, D’Onofrio O, Piron F, Mosca G etal, (2010) A
4. Later, the seeds were thoroughly washed with running tap water for
new mutant genetic resource for tomato crop improvement by
removing the traces of EMS. TILLING technology. BMC Res. Notes 3: 1-8.
5. The treated seeds were sown in pro-trays for germination. 5. LycoTILL: Tomato mutant DB http://www.agrobios.it/tilling/
6. The germination percentage was noted down across the treatments. (accessed on December 30, 2021).
7. The germinated seedlings were transplanted in the field at
Greenhouse located in campus. Presented at 43rd Annual Meeting of Plant Tissue Culture Association-
India (PTCA-I) and International Symposium on Advances in Plant
8. The tomato plants were grown till maturity to study the
Biotechnology and Nutritional Security- APBNS 2022 organized by
morphological changes occurring in the fruit development stages and ICAR- National Institute for Plant Biotechnology, New Delhi during
mutant seed library was developed. February17-19, 2022.
Fig. 1.4 Example of development of a Tomato TILLING population
life (ESL) tomatoes using TILLING. The gene selected was the non-ripening (nor)
gene. For, this technology Arcadia holds a patent, US 9392759B2. In another study,
4759 M3 mutant lines were developed in the background of M82 as reported by
Piron et al. (2010). A total of 19 genes were screened including eIF4E and 4G
(Eukaryotic initiation factors) and 256 SNPs were identified. The average mutation
density in this study was 1 in 574 kb.
1.9.14 Cucumber
A mutant population of 768 lines in “Poinsett 76,” using 1.5–2% EMS was devel-
oped and screened for six genes which included phytoene desaturase-3, a gene
involved in carotene biosynthesis, female (f) gene that is involved in the formation
of female flowers and at the same time suppression of male flowers. The other genes
involved in the study were Ramosus 3 and 4, which play a role in apical dominance,
a self-pruning gene (sp) that confers determinate growth, and CUM-1, a MAD box
gene. In this project, mutation density was found in the frequency of 1 in 1030kbp. In
another study, Boualem et al. (2014) developed a mutagenized population of
3331 M2 by EMS treatment (0.5% and 0.75%) in the “Beit Alpha” background.
Here, they screened the mutant population for five genes (CsACS2, ACSIG, WIP
1, RMS 3, and 4). In this study, a total of 26 mutants with a frequency of 1 in 1147 kb
were found and confirmed by sequencing. Predominantly, the type of mutation
reported was mainly G/C to A/T transitions.
1.9.15 Melon
In Melon, a TILLING population in the background of CharMono, Charentais

cultivar was created using an EMS dose of 1–3%. The focus of the screening
study was enhanced shelf life. Population size was 4023 M2s and 11 different
genes which attributed to ethylene biosynthesis, fruit ripening, cell growth, and
flower type. Mutation frequency was estimated to be about 1 in 573 kb. One of the
identified mutants had late ripening along with improved shelf life. In another study,
a mutant population of M62–113, and other commercial cultivars were created. The
traits which were screened had disease resistance, fruit quality, and virus resistance.
The mutation frequency was estimated to be about 1 in 1.5 Mb. A total of
33 mutations were identified, the majority of which were of missense type. Some
of the mutants showed Mendelian segregation (Dahmani-Mardas et al. 2010).
Gonazalez et al. (2011) reported the development of a TILLING population in
melons cv. Inodorus Piel de Sapo andromonocious. A range of mutant phenotypes
was observed. The mutant density of the population was 1 mutation for every 1.5 Mb
observed. The list of genes that were TILLEd is Cm-ACO1, Cm-NOR, Cm-DET1,
Cm-DHS, Cm-eIF4E, Cm-eIFI(iso)4E, and Cm-PDS (which particularly showed the
correlation between phenotype and genotype).
1.10 Conclusion
A large number of peer-reviewed articles have been published in the area of

mutagenesis and TILLING, many examples have been listed in this chapter. Muta-
genesis and the associated screening technique, TILLING, has inherent limitations
involving randomness, huge effort in terms of labor and time involved, and technical
complexity which is equally negated by the absence of multiple licensing, regu-
latory foresight, complex genotype independent transformation systems across crop
species, quick time to market, and clarity on the return on investment by investors
and businesses. The same mutant population once developed can be screened any
number of times for numerous targets and no need to start from scratch for new
targets. TILLING can be applied to any crop regardless of its genome size, and
propagation type (Kurowska et al. 2011). Then next-generation sequencing when
coupled with techniques like MutMap can make the mutant screening process more
focused and scalable. We believe mutagenesis and TILLING are here to stay till the
bottlenecks of regulations in the different marketplaces and licensing of a new
generation of genome editing tools are sorted, which at present seems confusing.
As noted in the IAEA mutant variety database, the mutation breeding approach has
resulted in billions of dollars in extra income from improved varieties across species.
Mutagenesis will continue to play its role in developing climate-resilient crops and
remain popular with the breeding community in years to come.
References
Addo-Quaye C, Buescher E, Best N, Chaikam V, Baxter I, Dilkes BP (2017) Forward genetics by
sequencing EMS variation-induced inbred lines. G3 (Bethesda) 7:413–425
Ahloowalia BS, Maluszynski M, Nichterlein K (2004) Global impact of mutation derived varieties.
Euphytica 135:187–204
Alberts B, Johnson A, Lewis J et al (2002) Molecular biology of the cell, 4th edn. Garland Science.
DNA Repair, New York
Anonymous (2021) License CRISPR patents for free to share gene editing globally. Nature 597:152
Beans C (2020) Inner workings: crop researchers harness artificial intelligence to breed crops for the
changing climate. PNAC 44:27066–27069
Bhattacharya A, Parkhi V, Char B (2021) Genome editing for crop improvement: a perspective
from India. In Vitro Cell Dev Biol Plant 26:1–9
Blomstedt CK, Rosati VC, Lindberg Møller B, Gleadow R (2018) Counting the costs: nitrogen
partitioning in Sorghum mutants. Funct Plant Biol 45:705–718
Boualem A, Fleurier S, Troadec C, Audigier P, Kumar APK, Chatterjee M, Alsadon AA (2014)
Development of a Cucumis sativus TILLinG platform for forward and reverse genetics. PLoS
One 9:e97963
Bovina R, Brunazzi A, Gasparini G, Sestili F, Palombieri S, Botticella E, Lafiandra D, Mantovani P,
Massi A (2014) Development of a TILLING resource in durum wheat for reverse- and forward-
genetic analyses. Crop Pasture Sci 65:112–124
Bovina R, Talame V, Silvio S, Sanguineti MC, Trost P, Sparla F, Tuberosa R (2011) Starch
metabolism mutants in barley: a TILLING approach. Plant Genet Res: Characterization and
Utilization 9:170–173
Brown TA (2002) Genomes, 2nd edn. Wiley-Liss, Oxford. Chapter 14, Mutation, Repair and
Recombination
Brunelle DC, Clark JK, Sheridan WF (2017) Genetic screening for EMS-induced maize embryo-
specific mutants altered in embryo morphogenesis. G3 (Bethesda) 7:3559–3570
Burnett PG, Young LW, Olivia CM, Jadhav PD, Okinyo-Owiti DP, Reaney MJT (2018) Novel flax
orbitide derived from genetic deletion. BMC Plant Biol 18:90
Casella L, Greco R, Bruschi G, Wozniak B, Dreni L, Kater M, Cavigiolo S, Lupotto E, Piffanelli P
(2013) TILLING in European Rice: hunting mutations for crop improvement. Crop Sci 53:
2550–2562
Chantreu M, Grec S, Gutierrez L, Dalmais M, Pineau C, Demailly H, Paysant-Leroux C (2013)
PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.)
mutant population and TILLinG platform for forward and reverse genetics. BMC Plant Biol 13:
159
Chaudhary J, Deshmukh R, Sonah H (2019) Mutagenesis approaches and their role in crop
improvement. Plants (Basel, Switzerland) 8:467
Chawade A, Sikora P, Brautigam M, Larsson M, Vivekanand V, Nakash MA, Chen T, Olsson O
(2010) Development and characterization of an oat TILLING-population and identification of
mutations in lignin and β-glucan biosynthesis genes. BMC Plant Biol 10:86
Chen J, Zou G, Xin Z (2019) Development of a pedigreed sorghum mutant library. Methods Mol
Biol 1931:61–73
Comai L, Henikoff S (2006) TILLING: practical single-nucleotide mutation discovery. Plant J 45:
684–694
Cooper JL, Henikoff S, Comai L, Till BJ (2013) TILLING and eco-TILLING for rice. Methods Mol
Biol 956:39–56
Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, El-Mellouki T et al (2008)
TILLING to detect induced mutations in soybean. BMC Plant Biol 8:9
Dahmani-Mardas F, Troadec C, Boualem A, Leveque S, Alsadon AA, Aldoss AA, Dogimont C,
Bendahmane A (2010) Engineering melon plants with improved fruit shelf life using the
TILLING approach. PLoS One 5:e15776
Das P, Mishra M, Lakra N, Singla-Pareek SL, Pareek A (2014) Mutation breeding: a powerful
approach for obtaining abiotic stress-tolerant crops and upgrading food security for human
nutrition. In: Tomlekova N, Kozgar I, Wani R (eds) Mutagenesis: exploring novel genes and
pathways. Wageningen Academic Publishers, The Netherlands
Dockter C, Hansson M (2015) Improving barley culm robustness for secured crop yield in a
changing climate. J Exp Bot 66:3499–3509
Dong C, Dalton-Morgan J, Vincent K, Sharp P (2009) A modified TILLING method for wheat
breeding. Plant Genome 2:39–47
Dorado G, Gálvez S, Rosales TE, Vásquez VF, Hernández P (2021) Analyzing modern
biomolecules: the revolution of nucleic-acid sequencing—review. Biomol Ther 11:1111
Espina MJ, Ahmed CMS, Bernardini A (2018) Development and phenotypic screening of an ethyl
methane sulfonate mutant population in soybean. Front Plant Sci 9:394
Farhud DD, Zarif-Yeganeh M (2020) CRISPR pioneers win 2020 Nobel prize for chemistry. Iran J
Public Health 49:2235–2239
Fofana B, Ghose K, Somalraju A, McCallum J, Main D, Deyholos MK, Rowland GG, Cloutier S
(2017) Induced mutagenesis in UGT74S1 gene leads to stable new flax lines with altered
secoisolariciresinol diglucoside (SDG) profiles. Front Plant Sci 8:1638
Freeling M (1978) Maize Adh1 as a monitor of environmental mutagens. Environ Health Perspect
27:91–97
Gaj T, Sirk SJ, Shui SL, Liu J (2016) Genome-editing technologies: principles and applications.
Cold Spring Harb Perspect Biol 8:a023754
Gao R, Guo G, Fang C, Huang S, Chen J, Lu R, Huang J, Fan X, Liu C (2018) Rapid generation of
barley mutant lines with high nitrogen uptake efficiency by microspore mutagenesis and field
screening. Front Plant Sci 9:450
Ghosh P (2020) Patent landscape of CRISPR/Cas. In: Bhattacharya A, Parkhi V, Char B (eds)
CRISPR/Cas genome editing. Concepts and strategies in plant sciences. Springer, Cham. https://
doi.org/10.1007/978-3-030-42022-2_11
Gilchrist EJ, Sidebottom CHD, Koh CS, Maclnnes T, Sharpe AG, Haughn GW (2013) A mutant
Brassica napus (canola) population for the identification of new genetic diversity via TILLING
and next generation sequencing. PLoS One 8:e84303
Gill SS, Anjum NA, Gill R, Jha M, Tuteja N (2015) DNA damage and repair in plants under
ultraviolet and ionizing radiations. Sci World J
Gonazalez M, Xu M, Esteras C, Roig C, Monforte AJ, Troadec C, Pujol M et al (2011) Towards a
TILLING platform for functional genomics in Piel de Sapo melons. BMC Res Notes 4:289
Griffiths AJF, Miller JH, Suzuki DT (2000) An introduction to genetic analysis, 7th edn. W. H.
Freeman. Sources of variation, New York
Guo Y, Abernathy B, Zeng Y, Ozias-Akins P (2015) TILLING by sequencing to identify induced
mutations in stress resistance genes of peanut (Arachis hypogaea). BMC Genomics 16:157
Guo Y, Hans H, Christian J, Molina C (2014) Mutations in single FT- and TFL1- paralogs of
rapeseed (Brassica napus L.) and their impact on flowering time and yield components. Front.
Plant Sci 5:282
Gupta P, Reddaiah B, Salava H, Upadhyaya P, Tyagi K, Sarma S, Datta S, Malhotra B, Thomas S,
Sunkum A, Devulapalli S, Till BJ, Sreelakshmi Y, Sharma R (2017) Next-generation sequenc-
ing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum
lycopersicum) population. Plant J 92:495–508
Hao C, Jiao C, Hou J, Li T, Liu H, Wang Y, Zheng J, Liu H, Bi Z, Xu F, Zhao J, Ma L, Wang Y,
Majeed U, Liu X, Appels R, Maccaferri M, Tuberosa R, Lu H, Zhang X (2020) Resequencing of
145 landmark cultivars reveals asymmetric sub-genome selection and strong founder genotype
effects on wheat breeding in China. Mol Plant 13:1733–1751
Hao H, Li Z, Leng C, Lu C, Luo H, Liu Y, Wu X, Liu Z, Shang L, Jing HC (2021) Sorghum
breeding in the genomic era: opportunities and challenges. Theor Appl Genet 134:1899–1924
Harloff HJ, Mittasch LJ, Frolov A, Wu JG, Dreyer F, Leckband G, Jung C (2012) A mutation
screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis
mutants. Theor App Genet 124:957–969
Heather JM, Chain B (2016) The sequence of sequencers: the history of sequencing DNA.
Genomics 107:1–8
Hernandez-Hernandez O, Pereira-Caro G, Borges G, Crozier A, Olsson O (2017) Characterization
and antioxidant activity of avenanthramides from selected oat lines developed by mutagenesis
technique. Food Chem 343:128408
Heuermann MC, Rosso MG, Mascher M, Brandt R, Tschiersch H, Altschmied L, Altmann T (2019)
Combining next-generation sequencing and progeny testing for rapid identification of induced
recessive and dominant mutations in maize M2 individuals. Plant J 100:851–862
Himelblau E, Gilchrist EJ, Buono K, Bizzell C, Mentzer L, Vogelzang R, Osborn T, Amasino RM,
Parkin IAP, Haughn GW (2009) Forward and reverse genetics of rapid-cycling Brassica
oleracea. Theor App Genet 118:953–961
Hohmann U, Jacobs G, Jung C (2005) An EMS mutagenesis protocol for sugar beet and isolation of
non-bolting mutants. Plant Breed 124:317–321
Holme IB, Gregersen PL, Brinch-Pedersen H (2019) Induced genetic variation in crop plants by
random or targeted mutagenesis: convergence and differences. Front Plant sci 10:1468
Hoshino T, Watanabe S, Takagi Y, Anai T (2014) A novel GmFAD3-2a mutant allele developed
through TILLING reduces α-linolenic acid content in soybean seed oil. Breed Sci 64:371–377
ISAAA Issue (2009) Amylopectin Potatoes by Precision Breeding. Crop Biotech Update December
11, 2009
Jankowicz-Cieslak J, Mba C, Till BJ (2017) Mutagenesis for crop breeding and functional
genomics. In: Jankowicz-Cieslak J, Tai T, Kumlehn J, Till B (eds) Biotechnologies for plant
mutation breeding. Springer, Cham. https://doi.org/10.1007/978-3-319-45021-6_1
Jankowicz-Cieslak J, Till BJ (2016) Chemical mutagenesis of seed and vegetatively propagated

plants using EMS. Curr Protoc Plant Biol 1:617–635
Jia Y, McAdams SA, Bryan GT, Hershey HP, Valent B (2000) Direct interaction of resistance gene
and avirulence gene products confers rice blast resistance. EMBO J 19:4004–4014
Jiao Y, Burke J, Chopra R, Burow G, Chen J, Wang B, Hayes C, Emendack Y, Ware D, Xin Z
(2016) A sorghum mutant resource as an efficient platform for gene discovery in grasses. Plant
Cell 28:1551–1562
Jones MO, Piron-Prunier F, Marcel F, Piednoir-Barbeau E, Alsadon AA, Wahb-Allah MA, Al-Doss
A et al (2012) Characterisation of alleles of tomato light signalling genes generated by
TILLING. Phytochemistry 79:78–86
Karaman K, Kizil S, Başak M, Uzun B, Yol E (2021) Development of EMS-induced mutagenized
groundnut population and discovery of point mutations in the ahFAD2 and Ara h 1 genes by
TILLING. J Oleo Sci 20:1631–1640
Kim S, Tai TH (2014) Identification of novel rice low phytic acid mutations via TILLING by
sequencing. Mol Breed 34:1717–1729
Kishor DS, Lee C, Lee D, Venkatesh J, Seo J, Chin JH, Jin Z, Hong SK, Ham JK, Koh HJ (2019)
Novel allelic variant of Lpa1 gene associated with a significant reduction in seed phytic acid
content in rice (Oryza sativa L.). PLoS One 14:e0209636
Knoll JE, Ramos ML, Zeng Y, Holbrook CC, Chow M, Chen S, Maleki S, Bhattacharya A, Ozias-
Akins P (2011) TILLING for allergen reduction and improvement of quality traits in peanut
(Arachis hypogaea L.). BMC Plant Biol 11:81
Koboldt DC, Steinberg KM, Larson DE, Wilson RK, Mardis ER (2013) The next-generation
sequencing revolution and its impact on genomics. Cell 155:27–38
Krasileva KV, Vasquez-Gross HA, Howell T, Bailey P, Paraiso F, Clissold L, Simmonds J,
Ramirez-Gonzalez RH, Wang X, Borrill P, Fosker C, Ayling S, Phillips AL, Uauy C,
Dubcovsky J (2017) Uncovering hidden variation in polyploid wheat. Proc Natl Acad Sci U S
A 114:E913–E921
Kumar APK, Boualem A, Bhattacharya A, Parikh S, Desai N, Zambelli A, Leon C, Bendahmane A
(2013) SMART- sunflower mutant population and reverse genetic tool for crop improvement.
BMC Plant Biol 13:38
Kurowska M, Daszkowska-Golec A, Gruszka D, Marzec M, Szurman M, Szarejko I, Maluszynski
M (2011) TILLING: a shortcut in functional genomics. J Appl Genet 52:371–390
Lakhssassi N, Lopes-Caitar VS, Knizia D, Cullen MA, Badad O, El Baze A, Zhou Z, Embaby MG,
Meksem J, Lakhssassi A, Chen P, AbuGhazaleh A, Vuong TD, Nguyen HT, Hewezi T,
Meksem K (2021) TILLING-by-sequencing+ reveals the role of novel fatty acid desaturases
(GmFAD2-2s) in increasing soybean seed oleic acid content. Cell 10:1245
Li Z, Yi C, Dauenpen M, Xiao Q (2019) The biosynthetic pathway of major avenanthramides in oat.
Meta 9:163. https://doi.org/10.3390/metabo9080163
Loewe L, Hill WG (2010) The population genetics of mutations: good, bad and indifferent. Philos
Trans R Soc Lond Series B, Biol Sci 365:1153–1167
Lucht JM (2015) Public acceptance of plant biotechnology and GM crops. Viruses 7:4254–4281
Lundqvist U (2014) Scandinavian mutation research in barley–a historical review. Hereditas
151:123–131
Martin-Laffon J, Kuntz M, Ricroch AE (2019) Worldwide CRISPR patent landscape shows strong
geographical biases. Nat Biotechnol 37:613–620
May BP, Liu H, Vollbrecht E, Senior L, Rabinowicz PD, Roh D, Pan X, Stein L, Freeling M,
Alexander D, Martienssen R (2003) Maize-targeted mutagenesis: a knockout resource for
maize. Proc Natl Acad Sci U S A 100:11541–11546
McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeted screening for induced mutations.
Nat Biotechnol 218:455–457
Millas R, Espina M, Ahmed CMS, Bernardini A, Adeleke E, Yadegari Z, Dumenyo K, Taheri A
(2019) Detection of novel allelic variations in soybean mutant population using Tilling by
Sequencing. bioRxiv:711440. https://doi.org/10.1101/711440
Minoia S, Petrozza A, D’Onofrio O, Piron F, Mosca G, Sozio G, Cellini F, Bendahmane A, Carriero

F (2010) A new mutant genetic resource for tomato crop improvement by TILLING technology.
BMC Res Notes 3:69
Monroe JG, Srikant T, Carbonell-Bejerano P, Becker C, Lensink M, Exposito-Alonso M, Klein M,
Hildebrandt J, Neumann M, Kliebenstein D, Weng ML, Imbert E, Ågren J, Rutter MT, Fenster
CB, Weigel D (2022) Mutation bias reflects natural selection in Arabidopsis thaliana. Nature.
https://doi.org/10.1038/s41586-021-04269-6
Moorthie S, Mattocks CJ, Wright CF (2011) Review of massively parallel DNA sequencing
technologies. HUGO J 5:1–12
Mullins E, Bresson JL, Dalmay T, Dewhurst IC, Epstein MM, Firbank LG, Guerche P, Hejatko J,
Moreno FJ, Naegeli H, Nogué F, Sánchez Serrano JJ, Savoini G, Veromann E, Veronesi F,
Casacuberta J, Lenzi P, Munoz Guajardo I, Raffaello T, Rostoks N (2021) EFSA panel on
genetically modified organisms (GMO), in vivo and in vitro random mutagenesis techniques in
plants. EFSA J 19:e06611
Muñoz-López M, García-Pérez JL (2010) DNA transposons: nature and applications in genomics.
Curr Genomics 11:115–128
Ohnoutkova L (2019) Mutation breeding in barley: historical overview. Methods Mol Biol 1900:7–
19
Okabe Y, Ariizumi T, Ezura H (2013) Updating the micro-tom TILLING platform. Breed Sci 63:
42–48
Okabe Y, Asamizu E, Saito T, Matsukura C, Ariizumi T, Bres C, Rothan C, Mizoguchi T, Ezura H
(2011) Tomato TILLING technology: development of a reverse genetics tool for the efficient
isolation of mutants from micro-tom mutant libraries. Plant Cell Physiol:1994–2005
Oladosu Y, Rafii M, Abdullah N, Hussin G, Ramli A, Rahim HA, Miah G, Ugman M (2016)
Principle and application of plant mutagenesis in crop improvement: a review. Biotechnol
Biotechno Equipment 30:1–6
Ordonio R, Ito Y, Morinaka Y, Sazuka T, Matsuoka M (2016) Molecular breeding of Sorghum
bicolor, a novel energy crop. Int Rev Cell Mol Biol 321:221–257
Osabe K, Clement JD, Bedon F, Pettolino FA, Ziolkowski L, Llewellyn DJ, Finnegan EJ, Wilson
IW (2014) Genetic and DNA methylation changes in cotton (Gossypium) genotypes and tissues.
PLoS One 9:e86049
Paaby AB, Rockman MV (2013) The many faces of pleiotropy. TIG 29:66–73
Palan B, Bhattacharya A, Char B (2021) TILLING in the era of precise genome editing. Indian J
Biotechnol 20:9–16
Piron F, Nicolai M, Minoia S, Piednoir E, Moretti A, Salgues A, Zamir D, Caranta C, Bendahmane
A (2010) An induced mutation in tomato eIF4E leads to immunity to two potyviruses. PLoS
One 5(6):e11313
Portin P, Wilkins A (2017) The evolving definition of the term "gene". Genetics 205:1353–1364
Pourmand N, Elahi E, Davis RW, Ronaghi M (2002) Multiplex pyrosequencing. Nucleic Acids Res
30:e31
Povirk LF, Shuker DE (1994) DNA damage and mutagenesis induced by nitrogen mustards. Mutat
Res 318:205–226
Ramirez-Villegas J, Watson J, Challinor AJ (2015) Identifying traits for genotypic adaptation using
crop models. J Exp Bot 66:3451–3462
Ramkumar MK, Kumar SS, Gaikwad K, Chinnusamy V, Singh NK, Singh AK, Mohapatra T,
Savanthi AM (2019) A novel stay-green mutant of rice with delayed leaf senescence and better
harvest index (HI) confers drought tolerance. Plan Theory 8:375
Rawat N, Sehgal SK, Joshi A, Rothe N, Wilson DL, McGraw N, Vadlani PV, Li W, Gill BS (2012)
A diploid wheat TILLING resource for wheat functional genomics. BMC Plant Biol 12:205
Rötter RP, Tao F, Höhn JG, Palosuo T (2015) Use of crop simulation modelling to aid ideotype
design of future cereal cultivars. J Exp Bot 66:3463–3476
Ruegger M, Chapple C (2001) Mutations that reduce sinapoylmalate accumulation in Arabidopsis
thaliana define loci with diverse roles in phenylpropanoid metabolism. Genetics 159:1741–1749
Another random document with
no related content on Scribd:
In this manner the court proceeded through Lahore and the plains
of the Pundjâb towards Cashmere; but as their motions were slow,
they were overtaken in those burning hollows which condensed and
reflected back the rays of the sun like a vast burning-glass, by the
heats of summer, which are there little less intense than on the
shores of the Persian Gulf. No sooner had the sun appeared above
the horizon than the heat became insupportable. Not a cloud stained
the firmament; not a breath of air stood upon the earth. Every herb
was scorched to cinders; and throughout the wide horizon nothing
appeared but an interminable plain of dust below, and above a
brazen or coppery sky, glowing like the mouth of a furnace. The
horses, languid and worn out, could scarcely drag their limbs along;
the very Hindoos themselves, who seem designed to revel in
sunshine, began to droop, and our traveller, who had braved the
climate of Egypt and the Arabian deserts, writing from the camp, on
the tenth day of their march from Lahore, exclaims, “My whole face,
hands, and feet are flayed, and my whole body is covered with small
red pustules which prick like needles. Yesterday, one of our
horsemen, who happened to have no tent, was found dead at the
foot of a tree, which he had grasped in his last agonies. I doubt
whether I shall be able to hold out till night. All my hopes rest upon a
little curds which I steep in water, and on a little sugar, with four or
five lemons. The very ink is dried up at the point of my pen, and the
pen itself drops from my hand. Adieu.”
His frame, however, was much tougher than he imagined; and he
continued to proceed with the rest, till having crossed the Chenâb,
one of the five rivers, they ascended Mount Bember, and found
themselves in Cashmere, the Tempé of Hindostan. The traditions of
the Hindoos respecting the formation of this beautiful valley greatly
resemble those which prevailed among the Greeks about that of
Thessaly, both being said to have been originally a lake enclosed by
lofty mountains, which having, been rent by the agency of
earthquakes, or bored by human industry, suffered the waters to
escape. Whatever was its origin, the Indian Tempé, though vaunted
by less renowned poets, is no way inferior in fertility or beauty to the
Thessalian. Fields clothed with eternal green, and sprinkled thick
with violets, roses, narcissuses, and other delicate or fragrant
flowers, which here grow wild, meet the eye on all sides; while, to
divide or diversify them, a number of small streams of crystal purity,
and several lakes of various dimensions, glide or sparkle in the
foreground of the landscape. On all sides round arise a range of low
green hills, dotted with trees, and affording a delicious herbage to
the gazelle and other graminivorous animals; while the pinnacles of
the Himalaya, pointed, jagged, and broken into a thousand fantastic
forms, rear their snowy heads behind, and pierce beyond the clouds.
From these unscaleable heights, amid which the imagination of the
Hindoo has placed his heaven, ever bright and luminous,
innumerable small rivulets descend to the valley; and after rushing in
slender cataracts over projecting rocks, and peopling the upland with
noise and foam, submit to the direction of the husbandman, and
spread themselves in artificial inundations over the fields and
gardens below. These numerous mountain-torrents, which unite into
one stream before they issue from the valley, may be regarded as
the sources of the Jylum or Hydaspes, one of the mightiest rivers of
Hindostan.
The beauty and fertility of Cashmere are equalled by the mildness
and salubrity of the climate. Here the southern slopes of the hills are
clothed with the fruits and flowers of Hindostan; but pass the summit,
and you find upon the opposite side the productions of the temperate
zone, and the features of a European landscape. The fancy of
Bernier, escaping from the curb of his philosophy, ran riot among
these hills, which, with their cows, their goats, their gazelles, and
their innumerable bees, might, like the promised land, be said to flow
with milk and honey.
The inhabitants of this terrestrial paradise, who were as beautiful
as their climate, possessed the reputation of being superior in genius
and industry to the rest of the Hindoos. The arts and sciences
flourished among them; and their manufactures of palanquins,
bedsteads, coffers, cabinets, spoons, and inlaid work, were
renowned throughout the East. But the fabric which tended most
powerfully to diffuse their reputation for ingenuity were their shawls,
those soft and exquisite articles of dress which, from that day to this,
have enjoyed the patronage of the fair throughout the world. In the
days of Bernier these shawls were comparatively little known in
Europe; yet his account of them, though highly accurate as far as it
goes, is brief and rather unsatisfactory.
During the three or four months which he spent in this beautiful
country he made several excursions to the surrounding mountains,
where, amid the wildest and most majestic scenery, he beheld with
wonder, he tells us, the natural succession of generation and decay.
At the bottom of many precipitous abysses, where man’s foot had
never descended, he saw hundreds of enormous trunks, hurled
down by time, and heaped upon each other in decay; while at their
foot, or between their crumbling branches, young ones were
shooting up and flourishing. Some of the trees were scorched and
burnt, either blasted by the thunderbolt, or, according to the traditions
of the peasantry, set on fire in the heat of summer by rubbing against
each other, when agitated by fierce burning winds.
The court, having visited Cashmere from motives of pleasure,
were determined to taste every species of it which the country could
supply; the wild and sublime, which must be sought with toil and
difficulty, as well as those more ordinary ones which lay strewed like
flowers upon the earth. The emperor accordingly, or at least his
harem, ascended the lower range of hills, to enjoy the prospect of
abyss and precipice, impending woods, dusky and horrible, and
streams rushing forth from their dark wombs, and leaping with
thundering and impetuous fury over cliffs of prodigious elevation.
One of these small cataracts appeared to Bernier the most perfect
thing of the kind in the world; and Jehangheer, who passed many
years in Cashmere, had caused a neighbouring rock, from which it
could be contemplated to most advantage, to be levelled, in order to
behold it at his ease. Here a kind of theatre was raised by
Aurungzebe, for the accommodation of his court; and there they sat,
viewing with wondering delight this sublime work of Nature,
surpassing in grandeur, and by the emotions to which it gave birth,
all the wonders of man’s hand. In this instance the stream was
beheld at a considerable distance rolling along its weight of waters
down the slope of the mountain, through a sombre channel overhung
with trees. Arriving at the edge of a rock, the whole stream projected
itself forward, and curving round, like the neck of a war-horse, in its
descent plunged into the gulf below with deafening and incessant
thunder.
An accident which occurred during these imperial excursions
threw a damp over their merriment. In ascending the Peer Punjal,
the loftiest mountain of the southern chain, from whose summit the
eye commands an extensive prospect of Cashmere, one of the
foremost elephants was seized with terror, occasioned, according to
the Hindoos, by the length and steepness of the acclivity. This beast
was one of those upon which the ladies of the harem were mounted;
and fifteen others, employed in the same service, followed. The
moment his courage failed him he began to reel backwards; and
striking against the animal which immediately succeeded, forced him
also to retreat. Thus the shock, communicated from the first to the
second, and from the second to the third, in an instant threw back
the whole fifteen; and being upon the giddy edge of a precipice, no
exertion of their drivers or of the bystanders could check their fall;
and down they rolled over the rocks into the abyss, with the ladies
upon their backs. This accident threw the whole army into
consternation. A general halt took place. The most adventurous
immediately crept down the cliffs, and were followed by the rest, to
aid such as should have escaped with life, and remove the bodies of
the dead. Here, to their great astonishment, they found that, by the
mercy of Providence, only three or four of the ladies had been killed;
but the elephants, which, when they sink under their prodigious
burdens even on a smooth road, never rise again, had all been
mortally wounded by the fall, and could by no means be lifted from
the spot. Even two days afterward, however, when Bernier again
visited the place, he observed some of the poor animals moving their
trunks.
On returning to Delhi from Cashmere, our traveller appears to
have remained quiet for some time, pursuing his researches amid
the mazes of the atomical philosophy; for he was a disciple of
Democritus, and enjoying those “noctes cœnæque deorum” which
seem to have constituted one of the principal pleasures of his friend
Danekmend Khan. His influence with this chief he exerted for the
benefit of others no less than for his own. Numerous were the
individuals who owed to his interference or recommendation their
admission into the service of the khan, or the speedy termination of
their affairs at court, where Danekmend, who possessed the
especial favour of the emperor, could almost always procure an
audience, or give success to a petition. These kind offices were
uniformly repaid with abundant flattery, if not with gratitude; and the
skilful practitioners invariably discharged a portion of the debt
beforehand. Putting on a grave face—a possession of infinite value
in the East—every person who had need of his services assured him
at the outset of the affair that he was the Aristotalis, the Bocrate, and
the Abousina Ulzaman (that is, the Aristotle, the Hippocrates, and
the Avicenna) of the age. It was in vain that he disavowed all claim to
such immediate honours; they persisted in their assertions; argued
down his modesty; and eternally renewing the charge, compelled
him to acquiesce, and consent to allow all the glorious attributes of
those illustrious men to be centred in his own person. A Brahmin
whom he recommended to the khan outdid them all; for, upon his
first introduction to his master, after having compared him to the
greatest kings and conquerors that ever reigned, he concluded by
gravely observing, “My lord, whenever you put your foot in the
stirrup, and ride abroad accompanied by your cavalry, the earth
trembles beneath your feet, the eight elephants which support it not
being able to endure so great an exertion!” Upon this, Bernier, who
could no longer restrain his disposition to laugh, remarked to the
khan, that since this was the case, it was advisable that he should
ride as seldom as possible on horseback, in order to prevent those
earthquakes, which might, perhaps, occasion much mischief. “You
are perfectly right,” replied Danekmend, with a smile, “and it is for
that very reason that I generally go abroad in a palanquin!”
In the year 1666, while Bernier was still at Delhi, there happened
an eclipse of the sun, which was attended by so many curious
circumstances that, should he have lived for ages, he declares it
never could have been obliterated from his memory. A little before
the obscuration commenced, he ascended to the roof of his house,
which, standing on the margin of the Jumna, commanded a full view
of the stream, and of the surrounding plain. Both sides of the river for
nearly a league were covered with Hindoos of both sexes, standing
up to the waist in the water, anxiously awaiting for the
commencement of the phenomenon, in order to plunge into the river
and bathe their bodies at the auspicious moment. The children, both
male and female, were as naked as at the moment of their birth—the
women wore a single covering of muslin—the men a slight girdle, or
cummerbund, about the waist. The rajahs, nobles, and rich
merchants, however, who, for the most part, had crossed the river
with their families, had fixed up certain screens in the water, which
enabled them to bathe unseen. Presently the dusky body of the
moon began to obscure a portion of the burning disk of the superior
planet, and in a moment a tremendous shout arose from the
multitude, who then plunged several times into the stream, muttering
during the intervals an abundance of prayers, raising their eyes and
their hands towards the sun, sprinkling water in the air, bowing the
head, and practising a thousand gesticulations. These ceremonies
continued to the end of the eclipse, when, throwing pieces of money
far into the stream, putting on new garments, some leaving the old
ones, besides the gifts which in common with all others they
bestowed, for the Brahmins, others retaining them, the whole
multitude dispersed.
The Hindoos, however, were not singular in the superstitious
feelings with which they regarded eclipses of the sun. Twelve years
previous Bernier had witnessed the effects which one of these
phenomena produced in his own country, where the madness
exhibited itself in the guise of fear. Astrologers, possessing the
confidence of the Fates, had predicted that the end of the world, that
unfailing bugbear of the middle age, was now to take place, and the
terrified rabble of all ranks, conscious of guilt, or oppressed by
gloomy fanaticism, immediately crept, like rats, into their cellars, or
dark closets, as if God could not have beheld them there; or else
rushed headlong to the churches, with a piety begotten by
apprehension. Others, who only anticipated some malignant and
perilous influence, swallowed drugs, which were vaunted by their
inventors as sovereign remedies against the eclipse disease! Thus it
appears that the superstition of the Hindoos was the less despicable
of the two.
During his long residence in India our traveller twice visited
Bengal. Of his first journey into that province the date is unknown,
but his second visit took place in 1667, the year in which he finally
quitted the country. He seems, on this occasion, to have approached
the place by sea, for we first find him coasting along the Sunderbund
in a small native bark, with seven rowers, in which he ascended by
one of the western branches of the Ganges to the town of Hoogly.
The beauty of this immense delta, divided into innumerable islands
by the various arms of the stream, and covered by a vegetation
luxuriant even to rankness, delighted him exceedingly. Even then,
however, many of these romantic isles had been deserted, owing
principally to the dread of the pirates who infested the coast; and as
in India the spots which cultivation abandons quickly become the
abode of pestilential miasmata, which thenceforward forbid the
residence of man, no one now ventured to disturb the tigers and their
prey, which had taken possession of the soil. It was here that for the
third time in his life he enjoyed the sight of that rare phenomenon, a
lunar rainbow. He had caused his boat to be fastened to the branch
of a tree, as far as possible from the shore, through dread of the
tigers, and was himself keeping watch. The moon, then near its full,
was shining serenely in the western sky, when, turning his eyes
towards the opposite quarter, he beheld a pale, bright arch, spanning
the earth, and looking like a phantom of the glorious bow which,
impregnated with the rich light of the sun, gladdens the eye with its
brilliant colours by day. Next night the phenomenon was repeated;
and on the fourth evening another spectacle, now familiar to most
readers by description, delighted our traveller and his boat’s crew.
The woods on both sides of the stream seemed suddenly to be
illuminated by a shower of fire, and glowed as if they had been
clothed with leaves of moving flames. There was not a breath of
wind stirring, and the heat was intense. This added to the effect of
the scene; for as the countless little fires streamed hither and thither
in columns, or separated, and fell like drops of rain, or rose thick like
the sparks of a furnace, the two Portuguese pilots whom our traveller
had taken on board, imagined they were so many demons. To add to
the effect of this exhibition of fireflies, for, as the reader will have
foreseen, it was they who were the actors, the swampy soil sent up a
number of those earthly meteors which often glide over large
morasses, some in the form of globes, which rose and fell slowly, like
enormous rockets, while others assumed the shape of a tree of fire.
From Bengal our traveller proceeded along the Coromandel coast
to Masulipatam, and having visited the kingdoms of Golconda and
Bejapore, quitted Hindostan, after a residence of twelve years, and
returned by way of Persia and Mesopotamia to Europe. The exact
date of his arrival in France I have not been able to discover, but it
must have been somewhere in the latter end of the year 1669, or in
the beginning of 1670; for the first two volumes of his “History of the
Revolutions of the Mogul Empire,” which would require some time to
prepare them for the press, were published in the course of that
year. The third and fourth volumes appeared in 1671, and so great
was the reputation they acquired, that they obtained for our traveller
the surname of “The Mogul.” These works, which have frequently
been reprinted under the title of “The Travels of M. François Bernier,
containing the Description of the Mogul Empire, of Hindostan, of the
Kingdom of Cashmere, &c.,” were immediately translated into
English, and appear to have been the means of introducing their
author to the most distinguished individuals of his time. Among those
most distinguished by his friendship were Ninon de l’Enclos,
Madame de la Sabliere, St. Evremont, and Chapelle, whose Eloge
he composed. To many of these his speculative opinions, which
were any thing but orthodox, may have rendered him agreeable; but
to Ninon, his handsome person, easy manners, and fascinating
conversation, which he knew how to enliven with a thousand
interesting anecdotes, must have proved by far his greatest
recommendation. By St. Evremont he was called “the handsome
philosopher;” and in a letter to Ninon, this same writer observes,
“Speaking of the mortification of the senses one day, to M. Bernier,
he replied, ‘I will tell you a secret which I would not willingly reveal to
Madame de la Sabliere or to Ninon, though it contains an important
truth; it is this—the abstaining from pleasure is itself a crime.’ I was
surprised,” adds St. Evremont, “by the novelty of the system.” Upon
this M. Walkenaer shrewdly observes, that this system could have
possessed but very little novelty for Mademoiselle de l’Enclos; and
he might have added that the surprise of the writer of the letter must
either have been affected, or else betrayed a very slight
acquaintance with the history of philosophy. The other works of
Bernier, which have been suffered to sink into much greater neglect
than they perhaps deserve, are,—1. “An Abridgment of the
Philosophy of Gassendi:” in which, according to Buhl, the acute and
learned historian of Modern Philosophy, he not only exhibited the
talents of an able and intelligent abbreviator, but, moreover, afforded
numerous proofs of a capacity to philosophize for himself. On
several important points he differed from his friend, with whom,
previous to his travels, he had lived during many years on terms of
the strictest intimacy, and who died shortly after his departure from
France. 2. “A Memoir upon the Quietism of India,” which appeared in
the “Histoire des Ouvrages des Savans,” for September, 1668. 3.
“Extract of various Pieces sent as Presents to Madame de la
Sabliere.” 4. “Eloge of Chapelle.” 5. “Decree of the Grand Council of
Parnassus for the Support of the Philosophy of Aristotle.” 6.
“Illustration of the Work of Father Valois, on the Philosophy of
Descartes,” published by Boyle. 7. “A Treatise on Free Will.”
The travels of Bernier, which enjoy a vast reputation among the
learned, have never, perhaps, been popular, and can never become
so, unless the various letters and treatises of which the work is
composed be properly arranged, and the whole illustrated with
copious notes. As an acute observer of manners, however, he has
seldom been surpassed. His history of the revolutions of the Mogul
empire entitles him to a high rank among the historians of India; and
his description of Cashmere, though brief, is perhaps the best which
has hitherto been given of that beautiful country. In his private
character he appears to have been generous, humane, and amiable,
constant in his friendship, and capable, as may be inferred from the
singular affection entertained for him by Gassendi and Danekmend
Khan, of inspiring a lasting and powerful attachment. Still, his
inclination for the dull, unimaginative, unspiritual philosophy of
Epicurus bespeaks but little enthusiasm or poetical fervour of mind;
and this feature in his intellectual character may account for the
inferior degree of romance with which we contemplate his
adventures.
SIR JOHN CHARDIN.
Born 1643.—Died 1713.
Sir John Chardin was born at Paris on the 16th of November,

1643. He was the son of a rich Protestant jeweller, who, as soon as
his education, which appears to have been carefully conducted and
liberal, was completed, intrusted him with the management of a
commercial speculation in the East, and thus at once gratified and
influenced the passion for visiting new and remote regions which had
already taken possession of the mind of our traveller. Leaving Paris
at the age of twenty-two, he visited Hindostan and Persia, where he
remained several years, and was appointed merchant to the king.
His manly but shrewd character, united with extensive knowledge
and great suavity of manners, procured him numerous friends at the
court of Ispahan, some of whom filled important offices in the
government, and were thus enabled to lay open to him the interior
movements of the great political machine which he afterward
described with so much vigour and perspicuity. He accompanied the
shah on his visits to various portions of his dominions, and in this
way was enabled to traverse with pleasure and advantage the wilder
and least accessible districts of Persia, such as Mazenderan, Ghilan,
and the other provinces bordering on the Caspian Sea. Of this
portion of his life, however, he did not judge it necessary to give any
detailed account; perhaps because he had afterward occasion to
visit the same scenes, when his mind was riper, his views more
enlarged, and his powers of observation and description sharpened
and invigorated by experience and habit.
Returning to France in 1670, he remained fifteen months in the
bosom of his family, and employed this period of tranquillity and
leisure in the composition of his “History of the Coronation of
Solyman III., King of Persia;” a small work usually appended to his
account of his travels. The desire of fame and distinction, however,
which in youthful and ardent minds is generally the ruling passion,
urged him once more to quit his native country, where, as he himself
observes, the religion in which he was educated excluded him from
all hope of advancement or honours, in order to revisit those regions
of the East where his faith would be no bar to his ambition, and
where commerce was not thought to degrade even the majesty of
kings.
Having collected together the jewels, gems, and curious clocks
and watches which he had been commissioned to purchase for the
King of Persia, he repaired to Leghorn, where he embarked with his
mercantile companion for Smyrna. Owing to the unskilfulness of the
mariners, the variableness of the winds, and the badness of the
weather, this short voyage was not performed in less than three
months, during which the passengers endured all the privation and
misery which such a voyage could inflict. From Smyrna he
proceeded to Constantinople, where, through the aid of M. de
Nointel, the ambassador of France, he was initiated in all the
mysteries of diplomacy, which he unveils in his travels with infinite
skill and naïveté for the amusement of his readers.
In other respects his connexion with the French ambassador was
rather prejudicial than useful to him; for M. de Nointel having
conducted himself in all his negotiations with the Turks in a puerile
and fluctuating manner, passing by turns from extreme haughtiness
to extreme cringing and servility, the anger of the Porte was roused,
and directed against the whole French nation; and Chardin, when he
became desirous of departing, was denied a passport. From this
difficult and somewhat dangerous position he was delivered by the
ingenuity of a Greek, who contrived to procure him a passage to
Azoph, on the Palus Mæotis, on board of a Turkish vessel then
about to set sail with the new commandant and fresh troops which
the Porte sent every year to that remote fortress. The Black Sea,
which receives its appellation from the gloomy clouds and
tempestuous winds which hover over and vex its waters in almost
every season of the year, was now to be traversed; and considering
the unskilfulness and apathy of Turkish sailors, who creep timidly
along the shore, and have little knowledge of the use of the
compass, our traveller was not without his apprehensions. After a
voyage of eight days, however, they arrived at Caffa, in the Crimea,
where, by the help of the Greek friend who had enabled him to laugh
at the sultan’s beard and embark without a passport, he eluded the
exorbitant demands of the custom-house, and transported his
merchandise on board another vessel bound for Mingrelia.
Setting sail from Caffa, where there was little to be seen but
stinking Tartars and caviare, they arrived in twenty-four hours at
Touzlah, or the Salt Marshes, a vast sweep of low shore, alternately
covered by the waters of the sea, artificially introduced, and a white
saline crust, looking like a sheet of snow from a distance. Here
upwards of two hundred ships are annually freighted with salt; and it
was for the purpose of taking on board a cargo of this useful
merchandise that the vessel in which Chardin and his companion
were embarked now touched at the place. On landing, the village
was found to consist of about ten or twelve houses, with a small
mosque, and a considerable number of felt-covered tents, which
served for stables, kitchens, and dormitories for the slaves. Salt was
by no means the only article of commerce obtained at this place.
Every morning fires were observed lighted along the shore, as
signals that the brigands of the country had laid violent hands upon a
number of their fellow-creatures, and had them conveyed thither,
chained together like cattle, for sale. These fires being observed,
boats were immediately sent on shore; and when they returned,
crowds of women and children, half-naked, or covered with rags and
filth, but resplendent with beauty, were hoisted on board, where their
wretched apparel was exchanged for clean neat garments, and
where, perhaps, for the first time in their lives they tasted bread. The
men and boys were chained two and two every night; the women,
from whom no danger was apprehended, were permitted the free
use of their limbs. These Circassians did not fetch a great price. A
Greek merchant, whose cabin was contiguous to that of Chardin,
purchased for twelve crowns a woman of extraordinary beauty, with
an infant at the breast. What chiefly surprised our traveller in the
circumstances of this affair was, the coolness and serenity with
which these honest people submitted to their fate. Had not the
women, much against their will, been compelled to occupy
themselves with needlework, and the men with such little matters as
they could perform on board, they would have been perfectly happy.
Idleness was their summum bonum; and this the most beautiful
among the women knew they were about to enjoy in the harems of
Turkey.
On arriving at Isgaour, in Mingrelia, the place where the general
market of the country is held, Chardin naturally expected to find
human dwellings, with provisions, and such other necessaries as in
civilized countries are everywhere attainable for money. In this hope
he went on shore, accompanied by the Greek merchant, who had
hitherto been in a manner his guardian angel; but on entering the
place, they indeed found two long rows of huts formed of the
branches of trees, where merchandise and provisions had once
been exposed for sale, but now empty and deserted. In the vicinity of
the place neither house nor habitation appeared as far as the eye
could reach. Two or three peasants, however, who flitted about like
spectres among the deserted huts, engaged to bring on the morrow
a quantity of that species of grain called gom, which is bruised,
boiled, and eaten instead of bread, together with wine and other
provisions. There being no alternative, they were compelled to rely
on the promises of these men, as they were nearly in want of every
necessary of life; but their presents failing them, it became
necessary to dissemble with his servants, who already began to
murmur aloud and curse the persons by whose advice he had taken
the route of the Black Sea, relying for the future upon the bounty of
Providence. The reason why the market of Isgaour was thus
deserted was, that the Abcas, a neighbouring people of savage
character and barbarous manners, having made an irruption into the
country, were now ravaging it with fire and sword, while the
peasantry and their lords were flying before them in dismay, or
plunging for refuge into the deepest recesses of their forests. Ten
days after their arrival these savages passed along the shore in
search of plunder; and finding none in this celebrated market, set the
huts on fire and reduced them to ashes.
In this dilemma, Chardin had much difficulty in determining what
course to take. He had immediately on landing applied for aid to the
Catholic missionaries of Colchis, the chief of whom promised in reply
to be with him by a certain day, but failed in his engagement; and
when after a second application he repaired to the place of
rendezvous, it was less with the design of forwarding our traveller’s
views than of dissuading him from attempting the journey at all.
Perceiving, however, that his advice could not be followed, he
rendered the travellers every service in his power with alacrity, but
without in the least concealing the magnitude of the danger they
were about to incur.
It was now the beginning of October, and Chardin, irritated at the
numerous obstacles and hinderances which had impeded his
progress, was so extremely impatient to be in Persia that no dangers
appeared to him so terrible as delay. He had very soon cause to
repent his impetuosity. The evils he had hitherto endured dwindled to
nothing when compared with those which now rushed upon him like
a torrent, and threatened to swallow up in a moment his wealth, his
ambitious projects, and his life. Nevertheless, with that unshrinking
courage which his total ignorance of the future and the pressure of
present evils bestows upon man, he hastened to put his foot upon
the shores of Mingrelia; and embarking with all his merchandise on
board the felucca in which the monk had arrived, set sail for
Anarghia, where they next day arrived. Here his followers made
themselves ample amends for the scarcity they had endured at
Isgaour; for poultry, wild pigeons, pork, goats’ flesh, wine, and other
provisions were abundant and cheap.
After remaining nine days at Anarghia, they departed on the 14th,
two hours before day, and having sailed about six miles up the river,
disembarked their merchandise and provisions, with which they
loaded eight small vehicles, and proceeded on their journey by land.
The report that a party of Europeans were passing with incalculable
riches through the country was soon spread; and as few rich
travellers ever traversed Mingrelia, this rumour immediately inflamed
to the highest degree the cupidity of the hungry prince and his
feudatories, who forthwith formed the design of appropriating these
treasures to themselves. They arrived, however, on the evening of
the same day at Sipias, the residence of the missionaries, where
they proposed to remain a few days in order to prepare themselves
by a little repose for the fatigues which were to come, as well as to
deliberate with the monks respecting the means of escaping from the
rapacity of the rulers of Mingrelia.
Four days after his arrival, the princess, or queen, as she termed
herself, of Mingrelia, came to Sipias to visit our traveller, attracted by
the rumours of his wealth, as vultures are attracted by the scent of a
carcass. Her majesty was followed by a train of eight women and ten
men, to all of whom a decent suit of clothes and a tolerable beast to
ride on would have been a welcome present, for they were very
badly mounted and meanly clad. In order to ward off, as far as
possible, the dangerous reputation of being rich, which is elsewhere
so much coveted, our travellers endeavoured to pass for Capuchin
friars, and pretended that the baggage with which their vehicles were
loaded consisted entirely of books. The princess believed neither of
these stories. Being informed that Chardin understood Turkish and
Persian, she tormented him, by means of a slave who could speak
the former language, with a thousand questions, of which the greater
number turned upon the subject of love. After pushing these
questions beyond the verge of decency, to the great amusement of
her suite, who appeared to be more delighted in proportion as her
majesty became more obscene, she suddenly turned to a still more
embarrassing topic—demanding to examine the effects of our
traveller, and the stores of the monks. They all now trembled for their
property. Whatever she should have seen would have been lost. To
allay her cupidity, therefore, and at least put off the evil day, the
principal monk humbly informed her that the usual present should be
sent on the morrow, accompanied by another from the travellers.
With this assurance she appeared to be satisfied, and departed.
On the next day our traveller and two of the monks were invited to
dine with the princess, and were of course careful not to present
themselves before her empty-handed, it being a crime in the East for
an inferior to come into the presence of his superior without some
gift, in token of dependence and homage. Her highness of Mingrelia,
who had painted her face and adorned her person to the best of her
ability, in order to appear to advantage in the eyes of the traveller,
seemed to be highly gratified with his present, which, though tasteful
and elegant, was of small value, the better to maintain a show of
poverty. Some ten or twelve ragged but merry-looking wenches, and
a crowd of half-naked ragamuffins, constituted the court of this
princess, her maids of honour having, as she assured the traveller,
taken refuge in a neighbouring fortress on account of the war! The
better to enjoy the pleasure of tormenting M. Chardin, she caused
him to sit near her, and commenced her attack by observing, that it
was her will and pleasure that he should marry one of her friends,
and settle in the country, when she promised to bestow on him
houses, lands, slaves, and subjects. From all he had heard and seen
of the women of Mingrelia, our traveller would have felt less
repugnance to marrying a vampire than one of them, beautiful as
they were; so that the bare possibility of the thing made him shudder.
He was for the present delivered from the discussion of this painful
topic by the appearance of dinner, during which the princess
inflamed her naturally ardent temperament by copious libations of
wine, which stifled whatever remains of shame might have lingered
in her soul, and impelled her to exhibit all the importunity and
effrontery of a courtesan.
The menaces of this princess, who gave them clearly to
understand that she had determined upon visiting the monastery, for
the purpose of examining their treasures, caused them to return
dejected and melancholy from the castle, the monks apprehending
new extortions and vexations, and Chardin the loss of all he
possessed. The remainder of the day was passed in deliberating
upon the present posture of affairs, and it was at length resolved,
that as soon as it was night, pits should be dug, and the most
valuable portion of their merchandise buried in the earth.
Accordingly, the sun had no sooner set behind the mountains, than
they commenced operations, first digging a pit five feet deep in the
apartments of one of the monks, where they buried a large chest
filled with watches and clocks set with jewels. When this had been
done, and the earth smoothed over, and made to appear as before,
they repaired under cover of the darkness to the church, where the
principal monk advised our traveller to open the grave of one of the
brotherhood, who had been interred there some six years before,
and deposite among his ashes a small casket filled with the most
costly gems of the East, designed for the princesses and great ladies
of Persia. A secret presentiment prevented Chardin from following
this advice, who selected in preference an obscure corner of the
church, where accordingly a pit was sunk, and the casket carefully
interred. Other costly articles, as a sabre and poniard set with jewels,
were concealed in the roof of the monastery; and such articles of
great value as were small and portable our travellers retained about
their persons.
Many days had not elapsed before they were convinced that their
fears were not without foundation. It was now Sunday, and Chardin,
in offering up his prayers to God, according to custom, would not
presume, he says, to petition his Maker for freedom, so persuaded
was he that slavery was to be his fate; he merely prayed for a mild
master, and to be delivered from a Mingrelian wife. While the
classical idea of Medea was haunting his imagination, and disturbing
his devotion, a person came running in, exclaiming that two
neighbouring chiefs, with a band of followers, armed to the teeth,
were knocking at the outer gate, and demanding admittance. There
being no alternative, they were allowed to enter, which they had no
sooner done than they seized and bound the travellers, commanded
the monks to retire, and threatened to put to death the first person
who should make the least stir or resistance. The principal friar was
terrified and fled; but the rest stood firmly by their guests, particularly
the lay-brother, whom not even a naked sword pointed at his throat
could induce to abandon them. When the bandits proceeded to bind
their servants, one of the latter, who had a large knife in his hand,
endeavouring to defend himself, was instantaneously struck to the
earth with a lance, bound hand and foot, and fastened to a tree. This
being done, the ruffians informed the travellers that they wished to
examine their effects. Chardin replied that it was within their power;
that they were but poor monks, whose whole wealth consisted in
books, papers, and a few wretched garments, the whole of which, if
they would abstain from violence, should be shown them. Upon this
he was unbound, and commanded to open the door of their
apartment, where their books, papers, and wardrobe were kept.
Chardin’s companion had sewn the most valuable of his jewels in the
collar of his coat; but our traveller himself had made two small
packets of his, which were sealed, and put among his books, not
daring to carry them about him lest he should be assassinated,
stripped, or sold for a slave. In order to gain a moment to withdraw
these packets, he requested his companion and the lay-brother to
hold the chiefs in conversation, by pretending to negotiate with them,
and offering them a small sum of money. The stratagem succeeding
for an instant, he darted upstairs, their apartment being on the first
floor, entered the chamber, and locked the door. His design was
suspected, and the whole band of ruffians rushed up after him; but
the door being somewhat difficult to be broken open, he had time to
take out his packets and conceal them in the roof of the house. His
companion, however, who was in the room below, called out to him
that he ought to be on his guard, for that he was observed through
the cracks in the floor. Upon hearing this, and seeing that the door
was giving way, he became confused, and scarcely knowing what he
did, took down the jewels out of the roof, thrust them into his pocket,

Full Ebook of Tilling and Eco Tilling For Crop Improvement 1St Edition Anjanabha Bhattacharya Vilas Parkhi Bharat Char Online PDF All Chapter

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Full Ebook of Tilling and Eco Tilling For Crop Improvement 1St Edition Anjanabha Bhattacharya Vilas Parkhi Bharat Char Online PDF All Chapter

Uploaded by

Copyright:

Available Formats

TILLING and Eco TILLING for Crop

Improvement 1st Edition Anjanabha

TILLING and Eco TILLING for Crop Improvement 1st

Complexity in Tsunamis Volcanoes and their Hazards 2nd

Genome Editing Current Technology Advances and

Silicon and Nano-silicon in Environmental Stress

Bravehearts of Bharat Vikram Sampath

Room for Improvement First Edition Jessica Gregory

Cybersecurity and Identity Access Management Bharat S.

Digital Disruption : implications and opportunities for

TILLING and Eco-TILLING

ISBN 978-981-99-2721-0 ISBN 978-981-99-2722-7 (eBook)

Jalna, India Anjanabha Bhattacharya

Jalna, India Anjanabha Bhattacharya

1 Mutagenesis and TILLING in the Era of Precise

10 Future of TILLING in Plant Breeding . . . . . . . . . . . . . . . . . . . . . . 185

About the Editors

Anjanabha Bhattacharya is working as a Group leader (Tech) with Mahyco since

Vilas Parkhi is a seasoned plant biotechnologist, who has exposure to various

Sabah AlMomin Biotechnology Program, Environment and Life Sciences

Mrinalini Bhaduri Molecular Biology and Biotechnology Division, CSIR-

Mahantesha B. N. Naika Dept. of Biotechnology and Crop Improvement, KRC

A. Bhattacharya · V. Parkhi · B. Palan · B. Char (✉)

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

next-generation sequencing, and improved and rapid detection methods, we feel

EMS · Mutagenesis · Mutant library · NGS · Allele · TILLING by sequencing

Acdc1 Adult cyanide deﬁcient class 1

SNP Single nucleotide polymorphism

focus on TILLING (Targeting Induced Local Lesions in Genome), the success of

1.2 Understanding Mutation and Mutagenesis

1.3 Progress in Sequencing Techniques and Mutation

detected in chromomethylase genes in a model crop using dHPLC, which was

1.4 Comparison of TILLING and Recent Genome Editing

Mutagenesis and TILLING are independent of transformation protocol, and genome

parallel sequencing methods provided the much-needed impetus to mutation detec-

1.5 Convergence or Divergence: Mutagenesis and Organic

If we look at EU regulations, crops developed by chemical and physical mutagenesis

1.6 TILLING Workflow

Mutagenesis of M0explants (seeds, tubers) with mutagens of interest (EMS,

The treated seeds (M0) were grown to obtain M1 Plants

The M1 plants were self pollinated to produce M2 seeds

Enzymatic digestion of hetero-duplexes using dHPLC/endonucleases

Mutants detection with Agarose Gel Electrophoresis/LI-COR DNA

Confirmation of SNPs by Sequencing

Fig. 1.1 TILLING workﬂow

(EMS) is an ethylating agent (C3H8SO3). EMS causes nucleotide substitution A->T,

Fig. 1.2 NGS platforms for TILLING

BLASTN&ClustalW2 softwares to be used for alignment of genomic and amino acid

Fig. 1.3 Bioinformatics workﬂow for analysis of TILLING data

1.7 Social Study on Consumer Acceptability

Ahloowalia et al. (2004) quantiﬁed value addition in various crops by including

1.8 Patentscape: Plant Patent and Plant Variety Protection

1.9 Crop Improvement Using Mutagenesis and TILLING

Table 1.1 (continued)

robust mutation (to generate the sufﬁcient number of knockout—STOP codon/

1.9.3 Maize or Corn

Historically ﬁrst evidence of mutagenesis in Barley was reported by Hermann

In a separate study on Flax by Chantreu et al. (2013) in Linum usitatissimum L. cv.

76 mutations in Cinnamyl Alcohol Dehydroxygenase genes. The estimated average

1.9.9 Peanut or Groundnut

Guo et al. produced a mutant population in the background of CV724-19-25. Their

Sabetta et al. (2011) created a mutagenized population in GV 342 background using

involved conferred resistance to downy mildew resistance and others involved in