Joanna Jankowicz-Cieslak · Thomas H. Tai Jochen Kumlehn · Bradley J. Till Editors Biotechnologies for Plant Mutation Breeding Protocols Biotechnologies for Plant Mutation Breeding Joanna Jankowicz-Cieslak • Thomas H. Tai • Jochen Kumlehn • Bradley J. Till Editors Biotechnologies for Plant Mutation Breeding Protocols Editors Joanna Jankowicz-Cieslak Plant Breeding and Genetics Laboratory Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Vienna, Austria Thomas H. Tai Crops Pathology and Genetics Research Unit USDA-ARS Davis California, USA Jochen Kumlehn Plant Reproductive Biology Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany Bradley J. Till Plant Breeding and Genetics Laboratory Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Vienna, Austria ISBN 978-3-319-45019-3 ISBN 978-3-319-45021-6 (eBook) DOI 10.1007/978-3-319-45021-6 Library of Congress Control Number: 2016959193 © International Atomic Energy Agency 2017. The book is published open access at SpringerLink.com. Open Access This book is distributed under the terms of the Creative Commons Attribution- Noncommercial 2.5 License (http://creativecommons.org/licenses/by-nc/2.5/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. 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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, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG Switzerland In memory of Dr Pierre J.L. Lagoda (1961–2016) for his tireless efforts and endless enthusiasm for plant mutation breeding. Preface Sustainable global food security remains a serious challenge. The compounding factors of a growing population, a changing climate and finite and dwindling natural resources mean that food production needs to increase dramatically in the coming decades. Genetic improvement of crops has historically led to vast gains in yields and subsequently has reduced famine. Variation is the source of all breeding, and inducing mutations is an important and successful approach for generating novel variation and developing new crop varieties that are climate smart and nutritious and increase farmer ’ s incomes. The use of induced mutations dates back to the 1920s, and today there are over 3200 mutant crop varieties registered in a database curated by the FAO/IAEA. The process can be improved through the development, adaptation and transfer of technologies for optimizing the density of induced mutations and increasing the efficiency of phenotypic and genotypic screening of large mutant plant populations. Towards this end, the Joint FAO/IAEA Programme of Nuclear Techniques for Food and Agriculture initiated a collaborative Coordinated Research Project (CRP) titled “Enhancing the Efficiency of Induced Mutagenesis through an Integrated Biotechnology Pipeline“. The project brought together researchers from developing and developed countries with the aim to develop protocols and guidelines to improve the efficiency of the different steps of the plant mutation breeding process. This book provides protocols resulting from this CRP. A range of methods are provided suitable for those new to the field The views expressed in this text do not necessarily reflect those of the IAEA or FAO or governments of their Member States. The mention of names of specific companies or products does not imply an intention to infringe on proprietary rights, nor should it be construed as an endorsement or recommendation on the part of IAEA or FAO. vii as well as to those seeking more advanced phenotypic and genotypic screening techniques. To guarantee high scientific quality, all chapters of this book have been peer reviewed. We are thankful to the participants of the CRP, authors of the chapters and also to all the reviewers (for list, see overleaf) who donated their time to improve the chapters. Vienna, Austria Bradley J. Till viii Preface Acknowledgements We thank the participants of the IAEA Coordinated Research Project (CRP) D24012 for the valuable input into the project which stimulated this book. We also thank many PBG and PBGL staff past and present who supported activities and initiated new thinking about novel biotechnology approaches for increasing effi- ciencies in mutation induction and detection. Specifically, we would like to thank Dr Chikelu Mba for the preparation of the Coordinated Research Programme proposal. Funding for this work was provided by the Food and Agriculture Orga- nization of the United Nations and the International Atomic Energy Agency through their Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture. ix Contents Part I Introduction 1 Mutagenesis for Crop Breeding and Functional Genomics . . . . . . . 3 Joanna Jankowicz-Cieslak, Chikelu Mba, and Bradley J. Till Part II Mutation Induction and Chimera Dissociation 2 Chemical and Physical Mutagenesis in Jatropha curcas . . . . . . . . . 21 Fatemeh Maghuly, Souleymane Bado, Joanna Jankowicz-Cieslak, and Margit Laimer 3 Chemical Mutagenesis and Chimera Dissolution in Vegetatively Propagated Banana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Joanna Jankowicz-Cieslak and Bradley J. Till 4 Mutation Induction Using Gamma Irradiation and Embryogenic Cell Suspensions in Plantain ( Musa spp.) . . . . . . . . . . . . . . . . . . . . 55 Jorge Lo ́pez, Ayme ́ Rayas, Arletys Santos, Vı ́ctor Medero, Yoel Beovides, and Milagros Basail 5 Optimisation of Somatic Embryogenesis in Cassava . . . . . . . . . . . . 73 Kenneth E. Danso and Wilfred Elegba 6 Creation of a TILLING Population in Barley After Chemical Mutagenesis with Sodium Azide and MNU . . . . . . . . . . . . . . . . . . . 91 Iwona Szarejko, Miriam Szurman-Zubrzycka, Malgorzata Nawrot, Marek Marzec, Damian Gruszka, Marzena Kurowska, Beata Chmielewska, Justyna Zbieszczyk, Janusz Jelonek, and Miroslaw Maluszynski 7 Site-Directed Mutagenesis in Barley by Expression of TALE Nuclease in Embryogenic Pollen . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Maia Gurushidze, Stefan Hiekel, Ingrid Otto, G € otz Hensel, and Jochen Kumlehn xi 8 Doubled Haploidy as a Tool for Chimaera Dissolution of TALEN-Induced Mutations in Barley . . . . . . . . . . . . . . . . . . . . . 129 Maia Gurushidze, Hannes Trautwein, Petra Hoffmeister, Ingrid Otto, Andrea M € uller, and Jochen Kumlehn Part III Phenotypic Screening 9 Field Evaluation of Mutagenized Rice Material . . . . . . . . . . . . . . . 145 Sydney D. Johnson, Dennis R. Taylor, Thomas H. Tai, Joanna Jankowicz-Cieslak, Bradley J. Till, and Alpha B. Jalloh 10 Root Phenotyping Pipeline for Cereal Plants . . . . . . . . . . . . . . . . . 157 Michal Slota, Miroslaw Maluszynski, and Iwona Szarejko 11 Breeding New Aromatic Rice with High Iron Using Gamma Radiation and Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Phuong Tan Tran and Cua Quang Ho 12 Utilising NIRS for Qualitative and Non-destructive Identification of Seed Mutants in Large Populations . . . . . . . . . . . . . . . . . . . . . . 193 Johann Vollmann and Joanna Jankowicz-Cieslak 13 Proteome Analyses of Jatropha curcas . . . . . . . . . . . . . . . . . . . . . . 203 Fatemeh Maghuly, Gorji Marzban, Ebrahim Razzazi-Fazeli, and Margit Laimer Part IV Genotypic Screening 14 Low-Cost Methods for DNA Extraction and Quantification . . . . . . 227 Owen A. Huynh, Joanna Jankowicz-Cieslak, Banumaty Saraye, Bernhard Hofinger, and Bradley J. Till 15 A Protocol for Benchtop Extraction of Single-Strand-Specific Nucleases for Mutation Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . 241 Bernhard J. Hofinger, Owen A. Huynh, Joanna Jankowicz-Cieslak, and Bradley J. Till 16 A Protocol for Validation of Doubled Haploid Plants by Enzymatic Mismatch Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Bradley J. Till, Bernhard J. Hofinger, Ays ̧e S ̧ en, Owen A. Huynh, Joanna Jankowicz-Cieslak, Likyelesh Gugsa, and Jochen Kumlehn 17 Bioinformatics-Based Assessment of the Relevance of Candidate Genes for Mutation Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Michal Slota, Miroslaw Maluszynski, and Iwona Szarejko 18 Mutation Detection by Analysis of DNA Heteroduplexes in TILLING Populations of Diploid Species . . . . . . . . . . . . . . . . . . 281 Miriam Szurman-Zubrzycka, Beata Chmielewska, Patrycja Gajewska, and Iwona Szarejko xii Contents 19 Determining Mutation Density Using Restriction Enzyme Sequence Comparative Analysis (RESCAN) . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Diana Burkart-Waco, Isabelle M. Henry, Kathie Ngo, Luca Comai, and Thomas H. Tai 20 Next-Generation Sequencing for Targeted Discovery of Rare Mutations in Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Diana Burkart-Waco, Helen Tsai, Kathie Ngo, Isabelle M. Henry, Luca Comai, and Thomas H. Tai Contents xiii Chapter Reviewers Yosvanis Acanda University of Florida, Lake Alfred, FL, USA Carlos Alonso-Blanco Dpt Genetica Molecular de Plantas, Centro Nacional de Biotecnologia, Madrid, Spain Saleha Bakht John Innes Centre, Norwich, UK Babita Dussoruth Food Agricultural Research and Extension Institute, Reduit, Mauritius Anne Edwards John Innes Centre, Norwich, UK Brian P. Forster Biohybrids International Ltd, Earley Reading, UK Ricardo F.H. Giehl Molecular Plant Nutrition, Leibniz Institute of Plant Genetics & Crop Plant Research, Seeland/OT, Gatersleben, Germany Tilo Guse Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria Thomas Halbach Strube Research GmbH & Co. KG, S € ollingen, Germany Isabelle M. Henry Davis Genome Center and Department of Plant Biology, University of California, Davis, CA, USA John S. (Pat) Heslop-Harrison Department of Genetics, University of Leicester, Leicester, UK Inger Holme Department of Molecular Biology and Genetics, Slagelse, Denmark Ivan Ingelbrecht Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria Daniela Isola Laboratorio di Botanica Sistematica e Micologia, Universit a delle Tuscia—DEB, Viterbo, Italy xv Shri Mohan Jain Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland Yulin Jia U.S. Department of Agriculture, Agricultural Research Service, Dale Bumpers National Rice Research Center, Stuttgart, AR, USA Kamila Kozak-Stankiewicz Kutnowska Hodowla Buraka Cukrowego Sp. z o.o., Kłodawa, Poland Jens Leon Institute of Crop Science and Resource Conservation (INRES), Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Fatemeh Maghuly Plant Biotechnology Unit (PBU), Department of Biotechnol- ogy, BOKU–VIBT, University of Natural Resources and Life Sciences, Vienna, Austria Martina Marchetti-Deschmann Institute of Chemical Technologies and Analyt- ics, Vienna University of Technology, Vienna, Austria Chikelu Mba Seeds and Plant Genetic Resources Team, Plant Production and Protection Division, Food and Agriculture Organization of the United Nations, Rome, Italy Heinrich Orsini-Rosenberg Bruker Austria GmbH, Wien, Austria Peggy Ozias-Akins Department of Horticulture, University of Georgia, Tifton, GA, USA Andy Phillips Plant Biology and Crop Science Department, Rothamsted Research, Harpenden, United Kingdom J. Neil Rutger (retired) U.S. Department of Agriculture, Agricultural Research Service, Dale Bumpers National Rice Research Center, Stuttgart, AR, USA Qing-Yao Shu Institute of Crop Science and Centre for Bio-breeding, Zhejiang University, Hangzhou, China Nils Stein Leibniz Institute of Plant Genetics and Crop Plant Research, Seeland/ OT, Gatersleben, Germany Michael J. Thomson Texas A&M AgriLife Research, Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, USA Trevor Wang John Innes Centre, Norwich, UK Hao Wu University of Florida, Lake Alfred, FL, USA Janice Zale University of Florida, Lake Alfred, FL, USA xvi Chapter Reviewers Contributors Souleymane Bado Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria Milagros Basail Instituto de Investigaciones de Viandas Tropicales (INIVIT), Santo Domingo, Villa Clara, Cuba Yoel Beovides Instituto de Investigaciones de Viandas Tropicales (INIVIT), Santo Domingo, Villa Clara, Cuba Diana Burkart-Waco Crops Pathology and Genetics Research Unit, USDA-ARS, Davis, CA, USA Department of Plant Sciences, University of California, Davis, CA, USA Beata Chmielowska Department of Genetics, Faculty of Biology and Environ- mental Protection, University of Silesia, Katowice, Poland Luca Comai Genome Center, University of California, Davis, CA, USA Department of Plant Biology, University of California, Davis, CA, USA Kenneth. Ellis Danso Ghana Atomic Energy Commission, Biotechnology and Nuclear Agriculture Research Institute, Legon-Accra, Ghana Wilfred Elegba Ghana Atomic Energy Commission, Biotechnology and Nuclear Agriculture Research Institute, Legon-Accra, Ghana Patrycja Gajewska Department of Genetics, Faculty of Biology and Environ- mental Protection, University of Silesia, Katowice, Poland Damian Gruszka Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland Likyelesh Gugsa Ethiopian Institute of Agricultural Research, Holetta, Ethiopia xvii Maia Gurushidze Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Seeland/OT, Gatersleben, Germany Isabelle M. Henry Genome Center, University of California, Davis, CA, USA Department of Plant Biology, University of California, Davis, CA, USA G € otz Hensel Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Seeland/OT, Gatersleben, Germany Stefan Hiekel Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Seeland/OT, Gatersleben, Germany Cua Quang Ho SocTrang Department of Agriculture and Rural Development, SocTrang, VietNam Petra Hoffmeister Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Seeland/OT, Gatersleben, Germany Bernhard Hofinger Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria Owen A. Huynh Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria Alpha B. Jalloh Rokupr Agricultural Research Centre (RARC), Sierra Leone Agricultural Research Institute (SLARI), Freetown, Sierra Leone Joanna Jankowicz-Cieslak Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA Lab- oratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria Janusz Jelonek Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland Sydney D. Johnson Rokupr Agricultural Research Centre (RARC), Sierra Leone Agricultural Research Institute (SLARI), Freetown, Sierra Leone Jochen Kumlehn Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Seeland/OT, Gatersleben, Germany Marzena Kurowska Department of Genetics, Faculty of Biology and Environ- mental Protection, University of Silesia, Katowice, Poland Margit Laimer Plant Biotechnology Unit (PBU), Department of Biotechnology, BOKU–VIBT, University of Natural Resources and Life Sciences, Vienna, Austria xviii Contributors Jorge Lo ́pez Instituto de Investigaciones de Viandas Tropicales (INIVIT), Santo Domingo, Villa Clara, Cuba Fatemeh Maghuly Plant Biotechnology Unit (PBU), Department of Biotechnol- ogy, BOKU–VIBT, University of Natural Resources and Life Sciences, Vienna, Austria Miroslaw Maluszynski Department of Genetics, Faculty of Biology and Envi- ronmental Protection, University of Silesia, Katowice, Poland Gorji Marzban Plant Biotechnology Unit (PBU), Department of Biotechnology (DBT), University of Natural Resources and Life Sciences (BOKU), Vienna, Austria Marek Marzec Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland Chikelu Mba Seeds and Plant Genetic Resources Team, Plant Production and Protection Division, Food and Agriculture Organization of the United Nations, Rome, Italy Vı ́ctor Medero Instituto de Investigaciones de Viandas Tropicales (INIVIT), Santo Domingo, Villa Clara, Cuba Andrea M € uller Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Seeland/OT, Gatersleben, Germany Malgorzata Nawrot Department of Genetics, Faculty of Biology and Environ- mental Protection, University of Silesia, Katowice, Poland Kathie Ngo Genome Center, University of California, Davis, CA, USA Department of Plant Biology, University of California, Davis, CA, USA Ingrid Otto Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland/OT, Gatersleben, Germany Ebrahim Razzazi-Fazeli VetCore Facility, University of Veterinary Medicine Vienna, Vienna, Austria Ayme ́ Rayas Instituto de Investigaciones de Viandas Tropicales (INIVIT), Santo Domingo, Villa Clara, Cuba Arletys Santos Instituto de Investigaciones de Viandas Tropicales (INIVIT), Santo Domingo, Villa Clara, Cuba Banumaty Saraye Food Agricultural Research and Extension Institute, Reduit, Mauritius Ays ̧e S ̧ en Faculty of Science, Department of Biology, Istanbul University, Vezneciler, Istanbul, Turkey Michal Slota Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland Contributors xix Iwona Szarejko Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland Miriam Szurman-Zubrzycka Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland Thomas H. Tai Crops Pathology and Genetics Research Unit, USDA-ARS, Davis, CA, USA Phuong Tan Tran Department of Agriculture and Rural Development, SocTrang, VietNam Dennis R. Taylor Rokupr Agricultural Research Centre (RARC), Sierra Leone Agricultural Research Institute (SLARI), Freetown, Sierra Leone Bradley J. Till Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Divi- sion of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria Hannes Trautwein Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Seeland/OT, Gatersleben, Germany Helen Tsai Genome Center, University of California, Davis, CA, USA Department of Plant Biology, University of California, Davis, CA, USA Johann Vollmann Plant Breeding Division, Department of Crop Science, Uni- versity of Natural Resources and Life Sciences Vienna (BOKU), Tulln an der Donau, Austria Justyna Zbieszczyk Department of Genetics, Faculty of Biology and Environ- mental Protection, University of Silesia, Katowice, Poland xx Contributors Part I Introduction Chapter 1 Mutagenesis for Crop Breeding and Functional Genomics Joanna Jankowicz-Cieslak, Chikelu Mba, and Bradley J. Till Abstract Genetic variation is a source of phenotypic diversity and is a major driver of evolutionary diversification. Heritable variation was observed and used thousands of years ago in the domestication of plants and animals. The mechanisms that govern the inheritance of traits were later described by Mendel. In the early decades of the twentieth century, scientists showed that the relatively slow rate of natural mutation could be increased by several orders of magnitude by treating Drosophila and cereals with X-rays. What is striking about these achievements is that they came in advance of experimental evidence that DNA is the heritable material. This highlights one major advantage of induced mutations for crop breeding: prior knowledge of genes or gene function is not required to successfully create plants with improved traits and to release new varieties. Indeed, mutation induction has been an important tool for crop breeding since the release of the first mutant variety of tobacco in the 1930s. In addition to plant mutation breeding, induced mutations have been used extensively for functional genomics in model organisms and crops. Novel reverse-genetic strategies, such as Targeting Induced Local Lesions IN Genomes (TILLING), are being used for the production of stable genetic stocks of mutant plant populations such as Arabidopsis , barley, soybean, tomato and wheat. These can be kept for many years and screened repeatedly for different traits. Robust and efficient methods are required for the seamless integra- tion of induced mutations in breeding and functional genomics studies. This chapter provides an overview of the principles and methodologies that underpin the set of protocols and guidelines for the use of induced mutations to improve crops. Keywords Mutation breeding • Reverse-genetics • Forward-genetics • Phenotyping • Genotyping • Technology packages J. Jankowicz-Cieslak • B.J. Till ( * ) Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, PO Box 100, 1400 Vienna, Austria e-mail: b.till@iaea.org C. Mba Seeds and Plant Genetic Resources Team, Plant Production and Protection Division, Food and Agriculture Organization of the United Nations, Rome, Italy © International Atomic Energy Agency 2017 J. Jankowicz-Cieslak et al. (eds.), Biotechnologies for Plant Mutation Breeding , DOI 10.1007/978-3-319-45021-6_1 3 1.1 Inducing Genetic Variation The genetic improvement of crops is a crucial component of the efforts to address pressures on global food security and nutrition (Ronald 2011). It is estimated that food production should be at least doubled by the year 2050 in order to meet the needs of a continually growing population (Ray et al. 2013; Tester and Langridge 2010; FAO 2009). The availability of heritable variation is a prerequisite for genetic improvement of crops. Where sufficient variation does not exist naturally, it can be created through either random or targeted processes (Fig. 1.1). Aside from recom- bination, the treatment of plant materials with chemical or physical mutagens is the most commonly reported approach for generating novel variation. While various mutagens have different effects on plant genomes, and some positional biases have been reported, irradiation and chemical mutagenesis are generally considered random mutagenesis as the location of DNA lesions cannot be effectively predicted in advance (Greene et al. 2003). The effect of different mutagens on the DNA sequence also varies with mutagen type and dosage. Once sufficient genetic vari- ation is induced, the next step is to select materials that have the desired altered traits (see Fig. 1.1 and Sects. 1.2 and 1.3). 1.1.1 Practical Considerations in Induced Crop Mutagenesis Mutation breeding is a three-step process consisting of (a) inducing mutations, (b) screening for putative mutant candidates and (c) mutant testing and official release (Fig. 1.2). The last step tends to be standardised in specific countries and is not an area where research and development can (easily) improve efficiencies. While not trivial, mutation induction has been widely used and highly successful in most species. Screening of mutants and selection of desired variants remain the most intensive step. Incredible advances have been made in the field of phenomics over the past 5 years, however, phenotyping remains more specialised and labour intensive than genotypic selection (Fiorani and Schurr 2013; Cobb et al. 2013). The choice of which type of mutagen to use for mutation breeding is often based on past successes reported for the species and other considerations such as the availability of mutagens, costs and infrastructure (Bado et al. 2015; Mba 2013; MVD 2016). Mutant varieties produced with ionising radiation, specifically gamma rays, predominate in the database of registered mutant varieties (MVD 2016). This may be due primarily to the active promotion of the use of gamma irradiation by the Food and Agriculture Organisation of the United Nations and the International Atomic Energy Agency (FAO/IAEA) Joint Programme, but also may be biologi- cally significant as physical mutagens tend to induce larger genomic aberrations than some chemical mutagens, and more dominant or more easily observable traits could be created at a higher frequency (Jankowicz-Cieslak and Till 2015). Standardised protocols and general considerations for induced mutations in seed 4 J. Jankowicz-Cieslak et al.