Ivan L. W. Ingelbrecht Maria do Céu Lavado da Silva Joanna Jankowicz-Cieslak Editors

# Mutation Breeding in Coffee with Special Reference to Leaf Rust

Protocols

Mutation Breeding in Coffee with Special Reference to Leaf Rust

Ivan L. W. Ingelbrecht · Maria do Céu Lavado da Silva · Joanna Jankowicz-Cieslak Editors

# Mutation Breeding in Coffee with Special Reference to Leaf Rust

Protocols

*Editors* 

Ivan L. W. Ingelbrecht Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture IAEA Laboratories Seibersdorf International Atomic Energy Agency Vienna International Centre Vienna, Austria

Joanna Jankowicz-Cieslak Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture IAEA Laboratories Seibersdorf International Atomic Energy Agency Vienna International Centre Vienna, Austria

Maria do Céu Lavado da Silva Associated Laboratory TERRA LEAF—Linking Landscape, Environment Agriculture, and Food Research Center CIFC—Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia Universidade de Lisboa Lisbon, Portugal

ISBN 978-3-662-67272-3 ISBN 978-3-662-67273-0 (eBook) https://doi.org/10.1007/978-3-662-67273-0

© IAEA: International Atomic Energy Agency 2023. This book is an open access publication.

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# **Preface**

Since its establishment in 1964, the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture has played a significant role in fostering the use of induced mutagenesis for crop improvement in FAO and IAEA Member States to help tackle transboundary plant pests and diseases, enhance food and nutrition security and adapt to climate change. This is being done primarily by coordinating and supporting demand-driven R&D and technology transfer, by providing crop irradiation services, and by collecting and disseminating information on plant mutation breeding.

Mutation induction in plants aims to generate novel genetic diversity for plant breeders targeting increased yield, improved quality and enhanced resistance to biotic and abiotic stresses. Plant mutation breeding has a track record of success with global impact on agricultural productivity. To date, over 3300 mutant varieties have been released in more than 220 crop species as listed in the Mutant Variety Database of the Joint FAO/IAEA Centre (https://mvd.iaea.org/). Since the turn of the twentieth century, mutation breeding has become fully integrated with advanced biotechnology, genomics and informatics tools adding precision, speed and efficiency to the mutation breeding process.

A Coordinated Research Project (CRP) titled "Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana" (D22005; December 2015–November 2020) was launched by the Joint FAO/IAEA Centre to develop innovative R&D tools and protocols and investigate whether induced mutagenesis could generate useful genetic variation in banana and coffee leading to resistance to Fusarium Wilt Tropical Race 4 and Coffee Leaf Rust, respectively. A first protocol book ensuing from this CRP on mutation induction and screening techniques in banana for resistance to the devastating Fusarium Wilt Tropical Race 4 strain was published in 2022 (Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana: Protocols | SpringerLink). This second CRP protocol book is focused on mutation induction and screening techniques of Arabica coffee with special reference to Leaf Rust. Arabica coffee provides a source of income to nearly 125 million people worldwide. Over 90% of the production takes place in developing countries. The first chapter introduces general principles and practices for mutation-assisted breeding along with current breeding limitations of Arabica coffee. A second introductory chapter provides an overview of the Coffee Leaf Rust disease, a major threat to Arabica coffee cultivation, especially in Latin America (Climate Change and Coffee: Combatting Coffee Rust | IAEA).

Since the 1920s, natural spontaneous mutant traits of significant economic value have been found in Arabica coffee plantations or collections such as dwarfism, fruit size and colour, and reduced caffeine content. However, to our knowledge, no Arabica coffee variety has been released following induced mutagenesis and studies on mutation-assisted breeding of Arabica coffee are scarce. Hence, a major objective of the CRP was to establish robust protocols and conditions for mutation induction using physical and chemical mutagens. Within the genus Coffea, Arabica coffee is unique because it is a self-pollinating, amphidiploid species unlike the other species such as *Coffea canephora* (aka Robusta coffee) which is a cross-pollinating diploid. Thus, mutagenesis techniques and methods for population advancement applicable to annual, diploid, seed-propagated crops can be equally followed for Arabica coffee. In vitro cell culture of Arabica coffee started in the 1970s, primarily as an alternative method for multiplication besides seeding. Since then, in vitro methods for mass propagation of coffee from single cells have been published. This opens exciting opportunities to integrate induced mutagenesis with advanced cell culture techniques to produce chimera-free mutant plants, a major bottleneck for mutation breeding of perennial crops with a long juvenile phase such as coffee. Hence, different propagules were used as targets for mutagenesis studies of Arabica coffee under this CRP. In this book, protocols for mutation induction and dose optimization of seed, seedlings, cuttings and in vitro cells are presented by Costa Rica, Nigeria, China and Austria. Screening techniques for Leaf Rust resistance are presented by Portugal, Costa Rica and P. R. China. Towards the end of this CRP, several mutant populations were under development that can be further advanced and screened for Leaf Rust resistance using these protocols. Finally, molecular methods for mutation detection are described, including the use of a coffee Exome Capture kit and High Resolution Melt analysis which can aid the selection process.

It is our hope that this book will serve as a timely resource for breeders and researchers interested in broadening the genetic base or improving Arabica coffee for enhanced Leaf Rust resistance or other targeted traits through induced mutagenesis. Preface vii

We also hope that this book will stimulate the integrated use of single-cell mutagenesis with advanced molecular techniques for accelerated breeding of perennial crops and trees which so far have lagged, behind the annual seed crops.

> Stephan Nielen EMBRAPA Recursos Genéticos e Biotecnologia Brasília, Federal District, Brazil

> Ivan L. W. Ingelbrecht Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture IAEA Laboratories Seibersdorf, Austria

# **Acknowledgements**

We would like to thank all staff of the Plant Breeding and Genetics Laboratory and Section, past and present, who were involved in the development and implementation of the IAEA Coordinated Research Project (CRP): "Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana" (code: D22005). Special thanks to Mr. Till Brad and Mr. Nielen Stephan who led the CRP during its first three years as IAEA Scientific Secretary. We further thank the CRP contract and agreement holders Mr. Yi Kexian (P. R. China), Mr. Wu Weihuai (P. R. China), Mr. Dada Keji Emmanuel (Nigeria), Mr. Melgarejo Gutierrez Tomas Adan (Peru), Ms. do Céu Lavado da Silva Maria (Portugal), Mr. Varzea Vitor (Portugal), Ms. Schwarzacher Trude (UK) and Ms. Laimer Margit (Austria) for their valuable contributions throughout this CRP including the protocols presented in this book. We further acknowledge Mr. Gatica-Arias Andrés (Costa Rica) for stimulating discussions on coffee and his contributions to this protocol book. Thanks also to the consultants who were involved at the inception of this project for their guidance and inputs into the final CRP proposal. Funding for this CRP and supporting research at the FAO/ IAEA PBG Laboratory work was provided by the FAO and the IAEA through the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, with additional support from Belgium through the Peaceful Use Initiative: "Enhancing Climate Change Adaptation and Disease Resilience in Banana-Coffee Cropping Systems in East Africa" (code: EBR-BEL01-18-03).

# **Contents**

### **Introduction**




### Contents xiii


# **Contributors**

**Emanuel Araya-Valverde** Centro Nacional de Innovaciones Biotecnológicas, San José, Costa Rica

**Helena Gil Azinheira** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal; LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

**Souleymane Bado** Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria

**Xuehui Bai** Dehong Institute of Tropical Agriculture, Ruili, Yunnan, P. R. China

**Miguel Barquero-Miranda** Phytoprotection Laboratory, Costa Rican Coffee Institute, Coffee Center Research, San Pedro, Heredia, Costa Rica

**Dora Batista** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal;

LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

**Alejandro Bolívar-González** Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, San Pedro, Costa Rica

**Rashmi Boro** Plant Biotechnology Unit, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

**Eduviges G. Borroto Fernandez** Plant Biotechnology Unit, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

**María José Cordero-Vega** Phytoprotection Laboratory, Costa Rican Coffee Institute, Coffee Center Research, Heredia, Costa Rica

**Reina Céspedes** Coffee Research Centre, Costa Rican Coffee Institute, San Pedro, Barva, Heredia, Costa Rica

**Keji Dada** Plant Breeding Unit, Cocoa Research Institute of Nigeria (CRIN), Ibadan, Oyo State, Nigeria

**Inês Diniz** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal;

LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

**Fabián Echeverría-Beirute** Escuela de Agronomía, Instituto Tecnológico de Costa Rica-San Carlos, San Carlos, Costa Rica

**Noel Arrieta Espinoza** San Francisco Bay Gourmet Coffee, Lincoln, CA, USA

**Andrés Gatica-Arias** Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, San Pedro, Costa Rica

**Thomas Gbokie Jr** College of Plant Protection, Nanjing Agricultural University, Nanjing, P. R. China

**Abdelbagi Mukhtar Ali Ghanim** Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria

**Florian Goessnitzer** Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria

**Leonor Guerra-Guimarães** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal; LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

**Tieying Guo** Dehong Institute of Tropical Agriculture, Ruili, Yunnan, P. R. China

**Veronika Hanzer** Plant Biotechnology Unit, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

**Chunping He** Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Xing Huang** Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Ivan L. W. Ingelbrecht** Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria

**Joanna Jankowicz-Cieslak** Plant Breeding and Genetics Laboratory, Joint FAO/ IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria

**Margit Laimer** Plant Biotechnology Unit, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

**Yanqiong Liang** Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Le Li** Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China;

NHC Key Laboratory of Tropical Disease Control/Key Laboratory of Tropical Translational Medicine of Ministry of Education, School of Tropical Medicine, Hainan Medical University, Haikou, Hainan, P. R. China

**Jinhong Li** Dehong Institute of Tropical Agriculture, Ruili, Yunnan, P. R. China

**Rui Li** Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Xiaoyu Zoe Li** Key Laboratory of Plant Resources Conservation and Sustainable Utilization/Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Tianhe District, Guangzhou, P. R. China

**Qing Liu** Key Laboratory of Plant Resources Conservation and Sustainable Utilization/Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Tianhe District, Guangzhou, P. R. China

**Andreia Loureiro** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal; LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

**Ying Lu** Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Ramón Molina-Bravo** Laboratorio de Cultivo de Tejidos y Células Vegetales, y Laboratorio de Biología Molecular, Universidad Nacional, Heredia, Costa Rica

**Stephan Nielen** EMBRAPA, Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil

**Radisras Nkurunziza** Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria;

Laboratory for Applied in Vitro Plant Biotechnology, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

**Emmanuel Ogwok** Department of Science and Vocational Education, Faculty of Education, Lira University, Lira, Uganda

**J. S. Pat Heslop-Harrison** Department of Genetics and Genome Biology, University of Leicester, Leicester, UK;

Key Laboratory of Plant Resources Conservation and Sustainable Utilization/ Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Tianhe District, Guangzhou, P. R. China

**Ana Paula Pereira** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Quinta do Marquês, Oeiras, Portugal;

LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associated Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal

**Jorge Rodríguez-Matamoros** Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, San Pedro, Costa Rica

**José Andrés Rojas-Chacón** Escuela de Agronomía, Instituto Tecnológico de Costa Rica-San Carlos, San Carlos, Costa Rica

**Trude Schwarzacher** Department of Genetics and Genome Biology, University of Leicester, Leicester, UK;

Key Laboratory of Plant Resources Conservation and Sustainable Utilization/ Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Tianhe District, Guangzhou, P. R. China

**Maria do Céu Lavado da Silva** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal; LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal

**Elodia Sánchez-Barrantes** Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, San Pedro, Costa Rica

**Samira Tajedini** Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria

**Shibei Tan** Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Sílvia Tavares** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal; Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark

**Paulina Tomaszewska** Department of Genetics and Genome Biology, University of Leicester, Leicester, UK;

Department of Genetics and Cell Physiology, Faculty of Biological Sciences, University of Wrocław, Wrocław, Poland

**Kimberly Ureña-Ureña** Phytoprotection Laboratory, Costa Rican Coffee Institute, Coffee Center Research, Heredia, Costa Rica

**César Vargas-Segura** Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, San Pedro, Costa Rica

**Vítor Várzea** CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Quinta do Marquês, Oeiras, Portugal;

LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associated Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal

**Norman Warthmann** Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna, Austria

**Stefaan P. O. Werbrouck** Laboratory for Applied in Vitro Plant Biotechnology, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

**Weihuai Wu** Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Jingen Xi** Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Kexian Yi** Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Hongbo Zhang** Dehong Institute of Tropical Agriculture, Ruili, Yunnan, P. R. China

**Jinlong Zheng** Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, P. R. China

**Hua Zhou** Dehong Institute of Tropical Agriculture, Ruili, Yunnan, P. R. China

# **Introduction**

# **Mutation Breeding in Arabica Coffee**

**Ivan L. W. Ingelbrecht, Noel Arrieta Espinoza, Stephan Nielen, and Joanna Jankowicz-Cieslak** 

**Abstract** Coffee is a perennial (sub)tropical crop and one of the most valuable commodities globally. Coffee is grown by an estimated 25 million farmers, mostly smallholders, and provides livelihoods to about 125 million people. The Coffea genus comprises over 120 species. Two species account for nearly the entire world coffee production: *C. arabica* L. (Arabica coffee) and *C. canephora* Pierre ex A. Froehner (Canephora coffee) with the former supplying about 65% of the world's consumption. Arabica coffee is a self-pollinated, amphidiploid species (2n = 4x = 44) whereas other *Coffea* species are diploid (2n = 2x = 22) and generally cross-pollinated. Induced mutagenesis using physical and chemical mutagens has been a successful strategy in producing over 3,300 mutant varieties in over 220 crop species with global impact. Spontaneous Arabica coffee mutants of significant economic importance have been found since the early 1900s, following the spread of Arabica coffee cultivation across the globe. However, Arabica coffee has so far not been improved through induced mutagenesis and studies on coffee mutagenesis are scarce. In this chapter, principles and practices of mutation-assisted breeding along with current breeding limitations of Arabica coffee are briefly reviewed, as an introduction to subsequent protocol chapters on mutation induction, advanced cell and tissue culture, Leaf Rust resistance screening and the application of novel molecular/ genomics tools supporting mutation-assisted improvement and genetics research of Arabica coffee.

S. Nielen EMBRAPA, Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil

I. L. W. Ingelbrecht (B) · J. Jankowicz-Cieslak

Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria e-mail: i.ingelbrecht@iaea.org

N. A. Espinoza San Francisco Bay Gourmet Coffee, 173 Aviation Blvd, Lincoln, CA 95648, USA

### **1 General Principles of Plant Mutation Breeding**

Mutations can be defined as sudden heritable changes in the DNA of living organisms, not caused by genetic recombination or segregation. Mutational events can be easily produced in the laboratory with two principal types of mutagens: physical or chemical. Among the physical methods gamma and X-rays are the most frequently used (reviewed by Spencer-Lopes et al. 2018). Alkylating agents, especially EMS, and sodium azide are the most frequently used chemical mutagens (reviewed by Ingelbrecht et al. 2018). Mutations induced through physical or chemical mutagens occur randomly throughout the genome. Induced mutagenesis has generated a vast amount of genetic variability with a significant role in plant breeding, genetics, and functional genomics. Applied mutagenesis has been particularly successful for the genetic improvement of annual seed crops such as barley, rice, wheat, and sorghum amongst many others. Records from the Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Austria, maintained in the Mutant Variety Database (MVD), show that over 3,300 crop varieties with one or more traits resulting from induced mutagenesis have been released since the 1960s (IAEA 2023).

The history of plant mutation breeding has been reviewed in several publications (van Harten 1998; Forster and Shu 2012; Bado et al. 2015) and thus will only be briefly described here. In 1901, Hugo de Vries, a Dutch botanist and one of the first plant geneticists, coined the term "mutation" to describe seemingly new forms that suddenly arose in his experiments on the evening primrose. Proof of the mutation theory of de Vries was firmly established by the pioneering work of Lewis John Stadler who induced mutations in barley and maize using X-rays (Stadler 1928). Up to that time, only natural spontaneous mutations were selected to generate novel genetic diversity in plants.

Following the spread of Arabica coffee from Africa to other continents, natural spontaneous Arabica coffee mutants appeared within the widely grown plantations. Several of these mutants attracted the attention of breeders and were described as new varieties in the different regions where they were grown. The most important spontaneous mutants are those affecting plant height, fruit shape and -colour, and leaf colour. Examples include Caturra Vermelho and Caturra Amarelo, dwarf, high-yielding mutants of Bourbon Amarelo observed in the 1930s in Brazil and officially registered as varieties in 1999 (Guimarães Mendes et al. 2007); Pacas, a dwarf mutant of Bourbon found in 1949 in El Salvador; Villa Sarchi, a dwarf mutation of Bourbon found in Costa Rica and released in 1957; and, Maragogype, a large bean size mutant within the Typica variety discovered in Brazil. In addition, a few male sterile plants have been found in Brazil and in Ethiopian accessions in the CATIE collection in Costa Rica (Wintgens 2012; Arabica Coffee Varieties | Variety Catalog; (worldcoffeeresearch.org) (https://varieties.worldcoffeerese arch.org/varieties)). More recently, natural mutations conferring very low caffeine content were discovered at the Instituto Agronômico de Campinas, Brazil. Out of 3,000 coffee trees representing 300 *C. arabica* accessions from Ethiopia, three plants contained only 0.07% caffeine, in contrast to the normal caffeine content of 1.2% in *C. arabica* (Silvarolla et al. 2004). These plants have the potential of being the basis for the development of a new coffee varieties giving rise to "naturally decaffeinated" coffee.

The Joint FAO/IAEA Centre of Nuclear Applications in Food and Agriculture has been promoting and disseminating the efficient use of mutation techniques as a tool for crop improvement since the 1960s. Several authors have documented the global impact of induced mutant varieties (Ahloowalia et al. 2004; Kharkwal and Shu 2009). From the 1980s onwards plant mutagenesis has become increasingly integrated into a range of enabling biotechnology and genomics/bioinformatics tools to fast-track the breeding process, mutant selection or mutant trait discovery (Mokry et al. 2011; Schneeberger and Weigel 2011; Ghosh et al. 2018; Knudsen et al. 2022).

Merits of induced mutagenesis as a complementary tool for crop improvement are:


Limitations of plant mutation breeding include:


Mutation breeding has been especially successful with annual, inbreeding, diploid crops that are seed-propagated, because it is relatively quick to advance populations from the initial mutant population (M1) to advanced mutant lines. However, vegetatively propagated crops and perennial species—including Arabica coffee—have lagged, due to the limitations listed above. Recent advances in in vitro cell culture offer new opportunities and strategies for vegetative crops and trees through singlecell mutagenesis as described further in this protocol book for Arabica coffee. Likewise, new genomic, bioinformatics and genotyping tools enable screening mutant populations in early generations and can provide a means for short-cutting generations and fast-tracking mutation breeding. This is especially relevant for perennial crops and trees with long juvenile periods.

### **2 Breeding Limitations in Arabica Coffee**

Arabica coffee production is facing multiple threats including the interrelated challenges of climate change and transboundary pests and diseases such as Coffee Leaf Rust (CRL) and Coffee Berry Disease (CBD) (Bunn et al. 2015; Läderach et al. 2017; Solymosi and Techel 2019). Pests and diseases affecting Arabica coffee cultivation with special reference to CLR are reviewed in Chapter "Coffee Leaf Rust Resistance: An Overview". Protocols for Leaf Rust screening and molecular diagnostics are presented in Chapters "Screening for Resistance to Coffee Leaf Rust", "Inoculation and Evaluation of *Hemileia vastatrix* Under Laboratory Conditions", "Evaluation of Coffee (*Coffea arabica* L. var. Catuaí) Tolerance to Leaf Rust (*Hemileia vastatrix*) Using Inoculation of Leaf Discs Under Controlled Conditions" and "A PCR-Based Assay for Early Diagnosis of the Coffee Leaf Rust Pathogen *Hemileia vastatrix*".

Nearly all coffee is grown between the Tropics of Cancer and Capricorn where conditions for coffee cultivation are ideal. This band of latitudes is known as the coffee belt. Arabica coffee is usually cultivated in relatively cool mountain climates at 400–2800 m asl. Arabica coffee is sensitive to environmental factors such as exposure to direct sunlight, temperature, and rainfall (Muschler 2001). It is within the coffee belt that the most drastic changes in climate have occurred in recent years. The implications of these changes in coffee production can range from physiological and phenological disorders of plants, to the reduced adaptability of plants to areas with limiting conditions. In addition, pests and diseases also see their physiology and phenology altered, sometimes promoted favorably which implies greater pressure on the production systems. Overall, climate change is impacting coffee production both through changes in weather patterns, viz. rising temperatures, excessive rainfall, or longer droughts, and through changed/expanded habitats of important coffee diseases such as CLR (Avelino et al. 2015; van der Vossen et al. 2015). Coffee Berry Disease, still limited to the African continent, is a latent threat for the Americas in view of the favorable agroecological conditions offered by Latin America for this fungus.

Plant breeding requires genetic variation of useful traits to improve crops. However, the genetic diversity within the primary gene pool of *C. arabica* is very narrow (Scalabrin et al. 2020) so the required genetic variability to address abovementioned constraints is lacking. Most of the genetic diversity is found in Ethiopia and South Sudan, the centres of origin of *C. arabica* (Sylvain 1958; Thomas 1942). Since the 1960s coffee yields have stagnated in all coffee producing countries except Brazil, Colombia and Vietnam (Montagnon et al. 2019). Other challenges of Arabica coffee breeding are inherent to the perennial nature of this crop. The generation time factor—3–5 years from seed to seed—remains a major issue for coffee breeding programs.

Currently, two main approaches are followed for the genetic improvement of Arabica coffee. Since the 1950s the traditional varieties formed the basis for pedigree breeding mainly with the 'Timor Hybrid' (an interspecific hybrid of *C. arabica*  and *C. canephora* resulting from a natural cross) that has resistance to CLR (Bettencourt 1973; Silva et al. 2018). However, pedigree breeding is a long process requiring 30 years or more to release a stable, homogeneous and distinguishable variety. To date, most Arabica coffee plantations around the world are established with the varieties resulting from breeding efforts initiated some 50 years ago. However, these varieties are susceptible to disease outbreaks, especially CLR, and are poorly adapted to the changing climatic conditions observed in many coffee growing regions during the past decade. Obtaining CLR resistant varieties will allow to produce coffee with reduced pesticide use or in organic farming systems (Arrieta 2014). Since the 1990s, F1 hybrids are being developed as an alternative breeding strategy in view of their improved performance over traditional varieties in terms of yield and disease resistance, and because of the reduced timeframe of 10–20 years from breeding to commercial release (Frédérick et al. 2019). However, unlike the traditional varieties, F1 hybrids are not true breeding and thus require a different mechanism for mass production, typically via clonal propagation (World Coffee Research | F1 Hybrid Trials (https://worldcoffeeresearch.org/programs/next-generation-f1-hybridvarieties)). More recently, male sterility has been used for F1 hybrid seed production in Arabica coffee (Frédérick et al. 2019).

### **3 Mutation Breeding in Arabica Coffee**

### *3.1 Background*

Arabica coffee production is threatened by disease outbreaks and climate change while conventional breeding is hampered by the very narrow genetic base within its primary gene pool, as summarized above. Induced mutagenesis may have significant value for Arabica coffee by increasing genetic variability for genetic studies and breeding purposes. The plant Mutant Variety Database (MVD) lists over 3300 released mutant varieties in a wide range of crop plants. Over 80% of these resulted from exposure to physical mutagens. Ionizing radiation such as gamma rays and X-rays have been the most widely used techniques for mutation induction. Example successes in mutation breeding of woody plants reported in the MVD are shown in Table 1. Note that Arabica coffee is not listed in the MVD. To our knowledge no Arabica mutant variety has been released following induced mutagenesis. Thus, Arabica coffee remains a major crop that has not been improved by mutation breeding, though Arabica coffee varieties resulting from natural, spontaneous mutations are being grown commercially.


**Table 1** Examples of perennial crops and trees improved using radiation breeding (IAEA 2023)

According to the MVD, induced mutagenesis has been successful for inducing resistance to fungal diseases in 334 cases (Fig. 1). These include tree crops such as pear (Sanada et al. 1993; Saito 2016) and crops with polyploid genomes such as wheat (Sigurbjörnsson and Micke 1974) and sugarcane (IAEA 2023).

**Fig. 1** Released mutant varieties with induced resistance to fungal diseases according to the FAO/ IAEA Mutant Variety Database (IAEA 2023). Only plant species with at least three released varieties are listed with rice, wheat, barley and maize having the highest counts; 'other' includes plant species with one or two released mutant varieties

So far, there has been limited research on induced mutagenesis of Arabica coffee. The first attempt to induce new mutations in *C. arabica* was reported by Carvalho et al. (1954) using X-ray irradiation of seeds with doses up to 1,500 Gray (Gy, see below Sect.3.2). Main effects established were early termination of seedling growth if treatment was higher than 125 Gy and a general slow growth of mutagenized seedlings as compared to controls. Among the surviving seedlings, variation in the form of abnormal leaves was also observed. Moh and Orbegosos (1960) used thermal neutrons, X-rays and gamma-rays for induced mutagenesis in *C. arabica*  and frequently obtained angustifolia (*ag*) mutants characterized by long and narrow leaves. Appearance of this phenotype already in the M1 generation was explained by possible chromosomal aberrations. Interestingly, the mutant leaf type was similar for the entire plant and not sectorial, which excludes the presence of chimerism. Recently, similar observations were made in M1 mutant coffee plants derived from gamma-ray irradiation at the FAO/IAEA Plant Breeding and Genetics Laboratory, Seibersdorf, Austria (Fig. 2). Moh (1961) speculates that the lack of chimerism in the M1 plants indicates that the coffee plant originates from only one initial cell in the embryo shoot apex. This would, however, be one of very rare cases among the angiosperms. It is also conceivable that the uniform mutant leaf phenotype is the result of diplontic selection between cells of the meristem or that only one initial cell survived after irradiation. A final answer to the question of M1 uniformity is yet to be given. In later induced mutation breeding experiments, analysis of traits of economic importance such as yield were put forward and monitored over several

**Fig. 2** Arabica coffee M1 mutants obtained from gamma-ray irradiation of seed at the FAO/IAEA Plant Breeding and Genetics Laboratory, Seibersdorf, Austria, 29 months after irradiation. **a** Wild type; **b** dwarf and leaf morphology mutant; **c**, **d** leaf morphology mutants. Note that the mutant leaf morphology characteristic is not sectorial but is similar for the entire plant

mutant generations. However, apart from the occurrence of leaf mutations, no correlation between varying yield and radiation dose could be established (Carvalho et al. 1984). These early experiments in coffee mutation breeding however, do suffer from the relatively small number of plants analyzed. A protocol for phenotypic characterization of an M1 Arabica coffee greenhouse-based mutant population is presented in Chapter "Use of Open-Source Tools for Imaging and Recording Phenotypic Traits of a Coffee (*Coffea arabica* L.) Mutant Population".

# *3.2 The Need for Radiosensitivity Testing*

Treatment of a plant or plant part with a mutagen affects its vigour, growth rate, germination, and fertility. Mutation rates vary with mutagen dosage. The higher the dosage of a mutagen, the more frequent the mutations and hence also, the greater the chance of undesired damage and lethality. The optimal dose is the one that, on the one hand, limits adverse effects that prevent the creation of a sufficiently large and vigorous mutant population, and, on the other hand, produces sufficient mutations to have a reasonable chance of recovering the desired mutation or mutant trait in the population, while preserving the (elite) genetic background. Hence, dose optimization is typically the first step in experimental or applied mutagenesis. Here, key principles and considerations for optimizing physical mutagenesis relevant to Arabica coffee will be briefly described. General principles and protocols for dose optimization using physical (Spencer-Lopes et al. 2018) and chemical mutagens (Ingelbrecht et al. 2018; Jankowicz-Cieslak and Till 2016) and subsequent mutant population development (Ghanim et al. 2018) have been published.

Common units in physical mutagenesis include Gray (Gy), used to quantify the dose of radiation absorbed by the plant material and Gy/s or Gy/min, which is the unit for absorbed dose rate, a characteristic of the radiation source and irradiator used for mutagenesis. Radiosensitivity is a property of the target material, e.g., seed versus vegetative tissues, and of the species/variety. In addition, radiosensitivity is subject to external factors, such as, for example, the water content of the target material. Depending on the explant, the water content can be regulated. For example, the water content of seed can be adjusted to ca 12–14% through equilibration in a desiccator containing a 60% glycerol solution prior to irradiation, which is standard procedure at the FAO/IAEA PBG Laboratory, Seibersdorf, Austria.

Radiosensitivity testing refers to the determination of the optimum dose(s) of radiation of a particular plant propagule to be used as a basis for selecting the dose levels for bulk irradiation. In practice, radiosensitivity testing is performed across a series of mutagen doses in the lab or greenhouse over a short period of time. Growth responses or lethality is measured compared to a non-radiated control, to determine the GR30 (30% Growth Reduction) and GR50 (50% Growth Reduction) or LD30 (30% Lethal Dose) and LD50 (50% Lethal Dose) values respectively. These ranges have been observed to preserve the fitness of the M1 plants (first mutant generation ) while inducing sufficient stable, genetic variability for genetic studies or breeding purposes. In case of radiosensitivity testing of seed, the GR value is usually determined from the reduction of seedling height or leaf growth of the M1 plants compared to untreated M0 controls. In case of radiosensitivity testing of lethality of seed, seedling survival is measured over a range of doses compared to untreated controls. Importantly, the biological effects observed at the M1 stage are the result of transient physiological effects and from genetic effects that are passed on to the next M2 generation. Mutations are single cell events and thus mutagenic treatment of seed or other multicellular tissues may carry one or several mutations, each occupying a small part of the resulting M1 plant. Such M1 plants are therefore chimeras. Plant scientists or breeders need to be aware of the complications caused by chimerism and apply techniques to resolve them. Mutagenic treatment of single cells followed by plant regeneration does in principle result in chimera-free plants.

The doses chosen for bulk irradiation and development of the M1 population, depend on different factors, such as the available resources to grow out and screen the mutant populations. The breeding system of the species under study plays a key role (van Harten 1998). For example, for annual diploid, self-fertile crops such as barley or sorghum, background mutations can be relatively easily removed through backcrossing. This is much more challenging or impossible for (obligately) vegetatively propagated crops or trees as in the case of Arabica coffee due to their long juvenile phase. Ideally up to three different doses are applied for bulk irradiation, including doses lower than LD50 or GR50, to ensure that at least one level will yield a sufficient number of the required mutant types. The frequency of induced mutations depends on the type of mutagen, the applied dose and the target materials. The plant species or variety, ploidy level, developmental stage, physiological state, etc. may all result in differences in response to radiation. Therefore, standardization of the target material and keeping records of all relevant information about the radiation source and treatment conditions is critical.

### *3.3 Choice of Material for Mutation Induction*

Since Arabica coffee is self-fertilizing, the cheapest and most appropriate propagation system, especially in a commercial setting, is through seeding. However, for research and experimentation purposes, other plant propagules such as seedlings, cuttings or grafts can also be applied. In vitro cells and tissues are another attractive target in case of Arabica coffee given the availability of methods for de novo regeneration through somatic embryogenesis. Different plant propagules that can be used as targets for mutation induction in Arabica coffee with their advantages and limitations are summarized in Fig. 3.

In choosing the target material for dose optimization and mutagenesis treatments, it is important to consider the life cycle of a coffee tree, the seed and germination process. The coffee plant takes approximately three years from seed germination to produce the first fruit. It takes 6–9 months from flowering to mature cherries ready for harvest. The coffee cherry is the whole fruit, and has a skin, pulp, and parchment that cover the seed of the coffee. Inside the fruit are usually two seeds. Protocols for the establishment of in vitro tissue culture systems for Arabica coffee and methods

**Fig. 3** Target materials for mutagenesis treatment of Arabica coffee with limitations and advantages

for mutation induction using in vitro tissues and cells are described in Chapters "In Vitro Plantlet Establishment of *Coffea arabica* from Cut Seed Explants", "Somatic Embryogenesis and Temporary Immersion for Mass Propagation of Chimera-Free Mutant Arabica Coffee Plantlets", "Protocol on Mutation Induction in Coffee Using In Vitro Tissue Cultures", "Mutation Induction Using Gamma-Ray Irradiation and High Frequency Embryogenic Callus from Coffee (*Coffea arabica* L.)", "Chemical Mutagenesis of Embryogenic Cell Suspensions of *Coffea arabica* L. var. Catuaí Using EMS and NaN3", "Chemical Mutagenesis of *Coffea arabica* L. var. Venecia Cell Suspensions Using EMS" and "Chemical Mutagenesis of Zygotic Embryos of *Coffea arabica* L. var. Catuaí Using EMS and NaN3". Protocols for mutation induction of seed and ex vitro vegetative propagules are described in Chapters "Physical Mutagenesis of Arabica Coffee Seeds and Seedlings", "Mutation Induction in *Coffea arabica* L. Using In Vivo Grafting and Cuttings", "Chemical Mutagenesis of Mature Seed of *Coffea arabica* L. var. Venecia Using EMS" and "Chemical Mutagenesis of Coffee Seeds (*Coffea arabica* L. var. Catuaí) Using NaN3".

### *3.4 Enabling Biotechnology and Genomics Tools*

In vitro plant cell and tissue culture techniques offer the possibility of rapid trueto-type multiplication. Somatic embryogenesis (SE) is an in vitro vegetative propagation technique that can produce clones of plants in large quantities. Research on in vitro tissue culture of Arabica coffee began in the 1970s. Since then, protocols for in vitro regeneration of Arabica coffee through SE have been developed, both direct and indirect methods have been reported (Barry-Etienne et al. 2002; Murvanidze et al. 2021). Somatic embryos can be produced from leaves of trees as starting material. Two innovations aimed at developing commercial scale multiplication systems include the use of bioreactors and direct sowing of somatic embryos in nurseries (Barry-Etienne et al. 1999; de Rezende Maciel et al. 2016; Etienne and Berthouly 2002; Etienne et al. 2013, 2018; Menendez-Yuffa et al. 2010). These micropropagation techniques are intended to enable mass propagation of elite Arabica coffee materials, such as F1 hybrids which cannot be propagated by seeding. The availability of protocols for in vitro regeneration of Arabica coffee offers exciting opportunities to integrate advanced cell culture techniques with induced mutagenesis to produce chimera-free mutant plants, a major bottleneck in induced mutagenesis of perennial crops with a long juvenile phase such as coffee. Some horticultural techniques, such as cuttings, can also enable cloning and multiplication of coffee plants.

The analysis of segregating molecular markers has confirmed earlier genetic and cytogenetic evidence that *C. arabica* is a functional diploid (Lashermes et al. 2008). Alkimim et al. (2017) and Saavedra et al. (2023) reported the use of marker-assisted selection for pyramiding multiple CLR and CBD resistance alleles. Genomic tools and large-scale sequencing enable a better understanding and characterization of the diversity and function of the *Coffea* genetic resources. This knowledge can then be utilised by breeders to select the best parental materials for incorporation into breeding programmes. Genomic selection (GS) allows breeders to select traits that are influenced by large numbers of small-effect alleles in a wide range of genotypes. Using GS in the context of resistance breeding for perennial crops increases the efficiency of breeding programs by shortening the breeding cycles (Alves et al. 2015). The release of reference genomes of *C. canephora* and *C. arabica* broadened the possibilities and facilitated significant progress for *C. arabica* genomic analysis (Denoeud et al. 2014; Dereeper et al. 2015; https://worldcoffeeresearch.org/resour ces/coffea-arabica-genome; Scalabrin et al. 2020). Sant'Ana et al. (2018) used the *C. canephora* reference genome to find SNP markers in the *C. arabica* genome associated with lipids and di-terpenes composition in a GWAS study of 107 diverse *C. arabica* genotypes. Knowledge of the molecular genetic structure of genes of interest to coffee breeders can then be applied for molecular breeding of Arabica coffee, including for example, for gene-based selection in mutation breeding programs. Protocols on the development and use of a Coffee Exome Capture kit, the application of High-Resolution Melt analysis, and the use of molecular cytogenetics for the detection of induced mutations in coffee are described in Chapters "Targeted Sequencing in Coffee with the Daicel Arbor Biosciences Exome Capture Kit", "High Resolution Melt (HRM) Genotyping for Detection of Induced Mutations in Coffee (*Coffea arabica* L var. Catuaí)" and "Protocols for Chromosome Preparations: Molecular Cytogenetics and Studying Genome Organization in Coffee", respectively.

### **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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# **Coffee Leaf Rust Resistance: An Overview**

### **Leonor Guerra-Guimarães, Inês Diniz, Helena Gil Azinheira, Andreia Loureiro, Ana Paula Pereira, Sílvia Tavares, Dora Batista, Vítor Várzea, and Maria do Céu Lavado da Silva**

**Abstract** Coffee is one of the most important cash crops and beverages. Several diseases caused by fungi, bacteria, and viruses can affect coffee plantations and compromise production. Coffee leaf rust (CLR), caused by the biotrophic fungus *Hemileia vastatrix* is the top fungal disease, representing a permanent threat to sustainable Arabica coffee production for more than a century. This review provides a comprehensive survey of the most common coffee diseases, their importance, and geographic distribution, with an emphasis on coffee leaf rust. Summing up the progress obtained so far from different research fields on the coffee–*H. vastatrix*  interaction, we revisited the pathogen genetic diversity and population dynamics, and the complex mechanisms underlying plant resistance/immunity. We also highlight how new advanced technologies can provide avenues for a deeper understanding of this pathosystem, which is crucial for devising more reliable and long-term strategies for disease control.

H. G. Azinheira e-mail: hmga@edu.ulisboa.pt

L. Guerra-Guimarães · I. Diniz · H. G. Azinheira · A. Loureiro · A. P. Pereira · D. Batista · V. Várzea · M. do C. L. da Silva

L. Guerra-Guimarães · I. Diniz (B) · H. G. Azinheira (B) · A. Loureiro · A. P. Pereira ·

S. Tavares · D. Batista · V. Várzea · M. do C. L. da Silva

CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Quinta do Marquês, 2784-505 Oeiras, Portugal e-mail: inesdiniz@isa.ulisboa.pt

LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal

S. Tavares

Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, 1871 Frederiksberg, Denmark

# **1 Introduction**

Coffee is one of the most widely consumed beverages in the world and one of the most traded commodities globally. The main coffee-producing countries are Brazil, Vietnam, and Colombia, while the European Union and the United States of America are the largest consuming and importing markets globally (FAO 2022).

The two cultivated species, *Coffea arabica* L. (Arabica) and *Coffea canephora*  Pierre ex A. Froehner (Robusta) accounted in 2020, on average, for about 60% and 40% of the world's coffee production, respectively (ICO 2020). *C. arabica* is predominantly cultivated in the highlands and preferred by consumers due to its low bitterness, its aromatic characteristics, and its low caffeine content. *C. canephora* is more suitable for intertropical lowlands and characterized by a stronger bitterness and higher caffeine content (Lécolier et al. 2009).

Coffee, like other crops, is affected by several factors, including diseases, which may cause considerable yield losses. Moreover, there is clear evidence that the geographical distribution of several pathogens is expanding due to climate change and increasing global trade (Nnadi and Carter 2021). There are several ways to control diseases, ranging from chemical and biological control to good cropping practices. However, breeding for disease resistance is considered the most efficient and sustainable disease control strategy (Silva et al. 2022 and references therein).

Following a brief description of the most common coffee diseases, this review focuses on the advances in coffee leaf rust (CLR) research, mainly regarding pathogen infection, pathogen genetic diversity and population dynamics, and plant defense mechanisms. This knowledge is of utmost importance as an informed base to breed efficiently for durable resistance and devise innovative crop protection approaches.

### **2 Coffee's Main Diseases**

A plant disease results from the interaction between a susceptible host plant, a virulent pathogen, and favorable environmental conditions (Agrios 2005). Diseases caused by fungi, bacteria, and viruses (Table 1) are the major limiting factors in coffee production. According to Maghuly et al. (2020), approximately 26% of the global annual coffee production is lost due to diseases, threatening the income of nearly 125 million people worldwide.

Coffee leaf rust (CLR), caused by the biotrophic fungus *Hemileia vastatrix*  Berkeley & Br. (phylum Basidiomycota, class Pucciniomycetes, order Pucciniales), is the major disease affecting Arabica coffee (Talhinhas et al. 2017; Silva et al. 2022 and references therein) inducing losses of over \$1 billion annually (Kahn 2019). CLR was first recorded in 1861 near Lake Victoria (East Africa), but its first major outbreak was in 1869 in Ceylon (now Sri Lanka), leading to the eradication of coffee cultivation in this country, with devastating social and economic consequences. Nowadays, CLR is present in all the coffee-growing regions (Wellman 1952; Rodrigues Jr. et al.


**Table 1** List of the main diseases of *Coffea* spp.

1975; McCook and Peterson 2020; Keith et al. 2021). In the last decade, the epidemic resurgence of CLR, known as "the big rust", had strong economic and social impacts on several countries across Latin America and the Caribbean (Baker 2014; Avelino et al. 2015). Moreover, CLR has been reported to have expanded its area of distribution to regions of higher altitude where previously it was not detected, namely above 1000–1100 m in Central America and above 1600 m in Colombia (Rozo et al. 2012; Avelino et al. 2015). It has led to food security issues as a result of the high dependence on coffee production by most coffee farmers and laborers (Avelino et al. 2015). There is little evidence that the big rust was caused by the evolution of new virulence in *H. vastatrix*. Rather, a combination of more conducive weather patterns, changing climatic conditions, and recurring economic shocks appear to be responsible (Rhiney et al. 2021 and references therein).

*H. vastatrix* infects the lower surface of the leaves, where it produces chlorotic spots preceding the differentiation of suprastomatal, bouquet-shaped, orangecoloured uredinia (Fig. 1a). Infected leaves fall off, leaving long expanses of twigs without any foliage (Fig. 1b). Another coffee rust [powdery rust (or grey rust) of coffee] is caused by the fungus *Hemileia coffeicola* Maublanc and Roger. Both *H. vastatrix* and *H. coffeicola* have *C. arabica* and other *Coffea* species as hosts, but *H. coffeicola* is only of local importance in some West African countries (Rodrigues Jr. 1990; Ritschel 2005). The symptoms of the disease are characterised by a dusty or powdery coating of yellow uredosori covering the underside of the coffee leaves (Rodrigues Jr. 1990).

Coffee berry disease (CBD) caused by the hemibiotrophic fungus *Colletotrichum kahawae* J.M. Waller & P.D. Bridge (phylum Ascomycota, class Sordariomycetes,

**Fig. 1** Coffee leaf rust (CLR) symptoms and urediniospore infection. **a** Chlorotic spots and uredosporic sori on the lower leaves surface; **b** severe defoliation associated with CLR in one coffee plant contrasting with resistant ones in the background; **c** *Hemileia vastatrix* infection process. Photos taken by the authors. Created with Biorender.com

order Glomerellales) is the most devastating disease affecting Arabica coffee production in Africa at high altitudes (Silva et al. 2006; van der Vossen and Walyaro 2009; Loureiro et al. 2012; Maghuly et al. 2020). It was first reported in 1922 in Kenya (McDonald 1926) and is still restricted to Africa, but represents a threat to highaltitude coffee areas of Latin America and Asia (Silva et al. 2006 and references therein; van der Vossen and Walyaro 2009). *C. kahawae* infects all stages of the crop, but maximum crop losses occur following infection of green berries with the formation of dark sunken lesions with sporulation causing their mummification and premature dropping. Under adequate climatic conditions and if no control measures are applied, this disease can destroy 50–80% of the developing green berries (Firman and Waller 1977; Silva et al. 2006; Hindorf and Omondi 2011).

Cercosporiosis, or brown eye spot (BES), is currently one of the main diseases of coffee in Brazil. It is caused by *Cercospora coffeicola* Berk. and Cooke (phylum Ascomycota, class Dothideomycetes, order Mycosphaerellales). The pathogen causes lesions on leaves and fruits, resulting in defoliation, decreased productivity, diminished coffee quality, and yield loss. In the nursery, this defoliation reduces the seedling's growth rate, which becomes inappropriate for planting and marketing. Also, in field conditions, the disease could be harmful to young trees (Botelho et al. 2017 and references therein). BES can appear as two distinct symptoms on leaves and in field conditions, the 'brown eye spot' and the 'black spot' (Andrade et al. 2021). The first one is the typical symptom caused by *C. coffeicola* on coffee leaves, which can be described as small necrotic spots consisting of a light-colored center and sometimes surrounded by a purple, brown ring with yellow edges, giving rise to the name brown eye spot. The atypical symptom is characterized by a black spot with the lesions being black, without the yellow halo.

Coffee wilt disease (CWD), or tracheomycosis, is caused by the vascular wilt pathogen *Fusarium xylarioides* Steyaert (teleomorph = *Gibberella xylarioides* R. Heim & Saccas) (phylum Ascomycota, class Sordariomycetes, order Hypocreales). CWD spreads across Africa, destroying coffee trees, reducing yields, and significantly impacting producer livelihoods. (Pinard et al. 2016; Maghuly et al. 2020; Flood 2021). It is frequently found in older and densely planted coffee trees (Assefaa et al. 2022). Through systematic sanitation and the establishment of breeding programs in affected countries, CWD appeared to have declined. However, in the 1990s, the disease re-emerged and increased to epidemic proportions affecting Robusta coffee in the Democratic Republic of the Congo, Uganda, and Tanzania and Arabica coffee in Ethiopia (Flood 2021). The first symptoms of CWD are yellowing of the leaves, which then wilt and develop brown necrotic lesions. The leaves then curl, dry up and fall off. This process may start on one part of the tree but eventually spreads to the rest of the plant. Once a tree is infected, there is no remedy other than to uproot the tree and burn it in situ to reduce the chances of spreading the infection. No new trees should be planted in the same place for at least six months to prevent remnants of the root system in the soil, which retain viable spores of the disease, from reinfecting new plants (Phiri and Baker 2009).

American leaf spot disease, also known as "Ojo de Gallo" is caused by the fungus *Mycena citricolor* (Berk. & M.A. Curtis) Sacc. (Phylum Basidiomycota, class Agaricomycetes, order Agaricales) and has been reported in Latin America. This fungus can grow on all plant organs, including leaves, stems, and fruits. Subcircular spots, initially brown, becoming pale-brown to straw-colored, are produced mainly on leaves. The spots have a distinct margin and are 6–13 mm in diameter but with no halo. The centers of older leaf spots may disintegrate, giving a shot-hole appearance. The main effect of the disease is leaf fall, with a consequent reduction in the growth and yield of the coffee plants (Wang and Avelino 1999; Krishnan 2017).

The bacterial halo blight (BHB) of coffee caused by the Gram-negative bacterium *Pseudomonas syringae* pv. *garcae* (Psgc) Young, Dye & Wilkie of the family *Pseudomonadaceae*, was first described in 1955 by Amaral et al. (1956) in the municipality of Garça in the Brazilian state of São Paulo, and later in Paraná and Minas Gerais states (Badel and Zambolim 2019 and references therein). Bacterial halo blight has been reported in the African Continent, in Kenya, Ethiopia, and Uganda (Ramos and Shavdia 1976; Korobko and Wondinagegne 1997) and in China (Bai et al. 2013). It has been estimated that BHB can cause losses up to 70% in nurseries and in the field, predominantly in regions above 1000 m in the presence of severe wind (Zoccoli et al. 2011). Necrotic spots surrounded or not by chlorotic haloes in leaf borders are the most common symptoms of BHB disease. However, flowers, fruits, and branches can also be affected (Badel and Zambolim 2019). The bacterium survives mainly as an epiphyte associated with plant debris. It penetrates the host tissue through natural openings (stomata) or wounds and is disseminated by water and wind-driven aerosol particles (Zoccoli et al. 2011). A recent study suggests that seeds may also be a source of inoculum (Belan et al. 2016).

Coffee leaf scorch (CLS), also referred to as atrophy of branches, is caused by *Xylella fastidiosa* subsp. *pauca (Xfp)*, a Gram-negative bacterium belonging to the family *Xanthmonadaceae*. CLS has been reported in Brazil, Costa Rica, and Porto Rico (Beretta et al. 1996; Rodriguez et al. 2001; Bolaños et al. 2015). Strains of the bacterium isolated from coffee and citrus are closely related, and both share the sharpshooter insect of the *Cicadellidae* family as a dissemination vector. The bacterium colonizes the xylem vessels of host plants, as well as the foregut of its insect vectors (Badel and Zambolim 2019). CLS symptoms include apical and marginal leaf scorch, defoliation, reduction of the internode length, the leaf size, the plant height, fruit size and quantity, terminal clusters of small chlorotic and deformed leaves, lateral shoot dieback, and overall stunting (Li et al. 2001). CLS disease is widespread and often occurs if coffee is adjacent to citrus orchards affected by *X. fastidiosa* (citrus variegated chlorosis disease). Although there appears to be some degree of host specialization within the subspecies of *X. fastidiosa*, cross-infection has been reported in commercial grapevine cultivars and olive trees (Badel and Zambolim 2019).

Coffee ringspot virus (CoRSV), currently classified as Coffee ringspot dichorhavirus by the International Committee on Taxonomy of Viruses (ICTV), belong to the genus *Dichorhavirus*, of *Rhabdoviridae* family (Genus: Dichorhavirus, ICTV). CoRSV has been reported in the main coffee-growing states of Minas Gerais (Brazil) (Ramalho et al. 2014, 2016) and some regions of Costa Rica (Rodrigues et al. 2002). The virus is transmitted by *Brevipalpus phoenicis* (Geijskes) (Acari: *Tenuipalpidae*) and can infect coffee leaves and fruits. Symptoms on leaves are typical concentric chlorotic rings, sometimes forming bands on the veins. On coffee berries, CoRSV develops chlorotic or necrotic lesions that are frequently invaded by secondary fungal or bacterial opportunists. In severely affected trees, leaves fall and fruit drops, which can affect coffee production and quality (Ramalho et al. 2014). The severity of the disease has been attributed to ecological disturbances associated with expanding coffee areas and to chemical pest control favoring the vector.

### **3 CLR Causal Agent:** *Hemileia vastatrix*

### *3.1 Life Cycle and Infection Process*

*Hemileia vastatrix* is a hemicyclic fungus producing urediniospores, teliospores, and basidiospores. Urediniospores and teliospores are produced in the same sorus but at different times. Urediniospores are dikaryotic and represent the asexual cycle, reinfecting the leaves whenever environmental conditions are favorable. Teliospores rarely occur and germinate in situ, producing a promycelium from which four basidiospores are formed. Basidiospores cannot infect coffee, but no other host plant has been identified (Talhinhas et al. 2017 and references therein).

As an obligate biotroph, *H. vastatrix* can only feed, grow and reproduce on living coffee leaves by the differentiation of a specific hypha called haustorium. This organ forms after penetration of the wall of a live host cell, expanding on the inner side of the cell wall while invaginating the surrounding host plasma membrane (Garnica et al. 2014). In addition to their role in nutrient uptake, haustoria are actively involved in establishing and maintaining the biotrophic relationship through the secretion of effector proteins (Voegele and Mendgen 2011; Kemen et al. 2005; Bozkurt and Kamoun 2020).

The *H. vastatrix* infection process on coffee leaves (Fig. 1c), like other rust fungi (Heath 1997; Voegele and Mendgen 2011), involves specific events, including urediniospore germination, appressorium formation over stomata, penetration, and interand intracellular colonization (Rodrigues Jr. et al. 1975; Silva et al. 1999, 2006 and references therein). Urediniospore germination requires water and is optimal at about 24 °C. After appressorium formation, the fungus penetrates, forming a penetration hypha that grows into the substomatal chamber. This hypha produces at the advancing tip two thick lateral branches; each hypha and its branches resemble an anchor. From each lateral branch of the anchor is borne a hypha, the haustorial mother cell (HMC), that gives rise to a haustorium, which primarily infects the stomatal subsidiary cells. The fungus pursues its growth with the formation of more intercellular hyphae, including HMCs, and many haustoria in the spongy and palisade parenchyma cells and even of the upper epidermis. A dense mycelium is observed below the penetration area, and a uredosporic sorus protrudes like a "bouquet" through the stomata about 21 days after inoculation.

### *3.2 Genetic and Physiological Diversity*

The first experimental evidence for the physiological specialization of *H. vastatrix*  was identified in India by Mayne (1932), who differentiated the local rust samples into four physiologic races. The world surveys of coffee rust races initiated in the 1950s at the Coffee Rusts Research Center (Centro de Investigação das Ferrugens do Cafeeiro—CIFC) in Portugal enabled the characterization of more than 55 rust races from about 4000 rust samples received from different coffee-growing countries. These races have been identified according to their spectra of virulence on a set of 27 coffee plant differentials (Silva et al. 2022 and references therein).

Coffee rust race identification relies on applying Flor's gene-to-gene theory to the *Coffea* sp.–*H. vastatrix* interaction (Noronha-Wagner and Bettencourt 1967). The resistance genes characterized on coffee plants were designated by "SH genes". Major genes SH1, SH2, SH4, and SH5 were found in *C. arabica*, the gene SH3 is considered to derive from *C. liberica*, and SH6, SH7, SH8, and SH9 were only found in "Timor hybrid"—HDT (natural *Coffea arabica* × *canephora* hybrid) derivatives, therefore coming from the Robusta side of the hybrid (Rodrigues Jr. et al. 1975; Bettencourt and Rodrigues Jr. 1988). Concomitantly, it was possible to infer nine virulence genes (v1–v9) on *H. vastatrix*.

The race genotypes comprise v1–v9 virulence genes from isolates derived from *C. arabica* and tetraploid interspecific hybrids, whereas the virulence genes from some isolates that attack diploid coffee species are not known due to the unavailability of genetic studies in these plants (Rodrigues Jr. et al. 1975; Bettencourt and Rodrigues Jr. 1988; Talhinhas et al. 2017). However, virulence profiling, particularly of isolates infecting tetraploid interspecific hybrids, like HDT derivatives, can only go as far as the available collection of coffee differential genotypes allows, leaving many virulence profiles incomplete or entirely unidentified (Talhinhas et al. 2017; Silva et al. 2022).

Work carried out at CIFC allowed to find rust races with the ability to infect all known coffee genotypes of *C. arabica*, as well as some genotypes of HDT, like CIFC HDT 832/1 and CIFC HDT 832/2 used as sources of resistance in the coffee populations of Catimor and Sarchimor that were spread to the coffee world (Talhinhas et al. 2017; Silva et al. 2022; CIFC records).

The evolution of virulence in *H. vastatrix* is parallel to the existent resistance genotypes. For example, in a coffee country that grows only pure Arabica like Geisha (SH1), Kent's (SH2), Agaro (SH4), and Bourbon and Typica (SH5), the probability of finding different associations of v1, v2, v4, v5 is 100%. Contrarily, the probability of finding the virulence genes v6, v7, v8, and v9 associated with the interspecific tetraploid hybrids, like HDT, is very low or inexistent (CIFC records).

# *3.3 Implications of Rust Molecular Diversity and Population Dynamics in Coffee Breeding*

The global dissemination of *H. vastatrix* across continents seems to be intimately linked to the historical evolution of the global coffee industry. It is no surprise that coffee hosts act as a major selective pressure shaping the geographical distribution and local prevalence of rust races, leading to the recorded large spectrum of virulence profiles and the recurrent emergence of new ones. However, it is puzzling how *H. vastatrix* can so rapidly evolve to overcome resistance in coffee cultivars, considering that no sexual phase in the pathogen's life cycle has yet been identified. Numerous studies have been engaged to characterize population genetic variability in coffee rust, failing to detect any clear genetic structuring pattern or a direct link between phenotypic diversity and molecular diversity (Talhinhas et al. 2017 and references therein; Kosaraju et al. 2017; Santana et al. 2018; Quispe-Apaza et al. 2021; Bekele et al. 2022). Those studies using several kinds of DNA markers (AFLP, RFLP, rDNA-ITS, SRAP, SSRs) and mainly addressing local populations reported different levels of genetic variability, from low to high, but consistently no evidence of population structure could be found, either relating to race, host or geographical origin. The first insights on the population evolutionary history of this pathogen arouse only recently from efforts on genomic research with the analysis of genome-wide SNP data. For the first time, *H. vastatrix* populations were found to be clearly structured

into three divergent genetic lineages with marked host specialization, differentiating rusts infecting diploid coffee species from those infecting tetraploid coffee species (Silva et al. 2018). Moreover, evidences of recombination and footprints of introgression were also found, alerting to the possibility of virulence factors exchange between rust lineages. Episodes of introgression by hybridization have probably been rare in time, but the risk of having different rust lineages within cruising range should be taken into account to guide disease control and breeding strategies. While recombination has been detected both from DNA marker and genomic data (Maia et al. 2013; Cabral et al. 2016; Silva et al. 2018), albeit through unknown mechanisms, probably of a parasexual nature. Both these events may, however, be contributing to virulence increase and could explain the high pace of pathotype emergence. More recently, additional genomic data allowed to further discover a population genetic subdivision in a worldwide sampling of rusts from *C. arabica* and interspecific hybrids, revealing three divergent genetic subgroups: a low differentiated and globally distributed rust lineage, as well as two other highly differentiated rust lineages, one occurring specifically in Africa and the other in East Timor (Rodrigues et al. 2022). Most interestingly, the divergence of these lineages could be explained by only a small set of SNP loci putatively under the effect of positive selection (Rodrigues et al. 2022). These results suggest that genetic variation underlying host adaptation relies on a small portion of the genome and that the genes associated with these *loci* may be critically important for the species to survive in novel environments/coffee hosts. Important genome reference assemblies for *H. vastatrix* have been created in the last years (Cristancho et al. 2014; Porto et al. 2019). The recent availability of a chromosome-level genome resource for *H. vastatrix* (Tobias et al. 2022) offers renewed prospects for characterizing virulence loci, envisioning the future development of candidate diagnostic markers associated with rust pathotypes and alternative strategies for selective breeding.

### **4 Coffee Rust Resistance**

### *4.1 Concepts of Plant Disease Resistance*

Plants' perception and response to pathogen invasion evolved alongside the pathogen infection strategy as a sophisticated multilayer surveillance system leading to complete host immunity, partial resistance, or susceptibility (Bettgenhaeuser et al. 2014; Li et al. 2020). Plant defenses are structured in constitutive/passive and induced responses. The first comprises preformed barriers, such as waxy epidermal cuticles, rigid cell walls, and antimicrobial secondary metabolites that offer protection against pathogens. The second one results from a suite of plasma membrane receptors that detect the pathogens or their molecules and is based on the recognition of conserved microbe-associated or pathogen-associated molecular patterns (MAMPs/PAMPs) and host danger-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) that activate PAMP-triggered immunity (PTI) (Delplace et al. 2022). Alongside this broad-range defense system, plants developed intracellular resistance proteins to detect specific effector proteins secreted by pathogens activating the effector-trigger immunity (ETI) (Andersen et al. 2018). This two-branch immunity system (PTI and ETI) leads to the induction of a continuous and overlapping downstream response, such as mitogen-activated protein kinase (MAPK) cascades, G-proteins, calcium flux, reactive oxygen species (ROS), transcriptional reprogramming, phytohormones, pathogenesis-related (PR) genes/ proteins and epigenetic modifications (Meng and Zhang 2013; Zhang et al. 2012; Lecourieux et al. 2006; Robert-Seilaniantz et al. 2011; Amorim et al. 2017; Zhu et al. 2016). These responses seem stronger and more prolonged during ETI when compared to PTI. ETI is typically associated with the hypersensitive reaction (HR), a form of programmed plant cell death localized at the infection sites (Torres et al. 2006; Jones and Dangl 2006). HR is one of the most important factors in the restriction of pathogen growth, particularly of obligate biotrophs (Heath 2000; Andersen et al. 2018) such as rust fungi (Periyannan et al. 2017).

ROS can trigger HR (Torres et al. 2006; Martins et al. 2020) and may act as a damaging or signaling molecule depending on the balance between ROS production and scavenging mechanisms. When the ROS-scavenging mechanisms fail, this leads to an excess of ROS accumulation responsible for oxidative damage, promoting lipid peroxidation, and damaging macromolecules (e.g., proteins, lipids, and sugars) and DNA (Das and Roychoudhury 2014). Membrane damage by peroxidation of polyunsaturated fatty acids (e.g., linoleic acid) can be initiated not only by ROS but also by lipid radicals or by lipoxygenases (LOXs) during the HR. Also associated with the rapid loss of cell membrane integrity during HR is an increase in oxidizing enzymes, such as superoxide dismutase (SOD) and peroxidases (Heath 2000; Daudi et al. 2012).

Plant peroxidases (POD) function in plant defense against pathogens through the production of antimicrobial quantities of H2O2, as well as in cell wall lignification or cross-linking with cell wall proteins (Penel 2000; Torres et al. 2006).

Moreover, several studies suggest that plant phenolic compounds are strongly involved in plant–pathogen interaction and may restrict pathogen spread (Vermerris and Nicholson 2006). As well phenylalanine ammonia-lyase (PAL), a key enzyme of the phenylpropanoid pathway, catalyzes the deamination of phenylalanine to transcinnamic acid, a precursor for several phenolic compounds such as salicylic acid (SA), phenylpropanoids, flavonoid, and lignin seems to be involved in immunity responses (Bagal et al. 2012).

Other proteins also reported as being involved in plant resistance are hydrolases, particularly sugar hydrolases (GH) and peptidases/proteases. The proteases (together with phosphatases) can lead to a complex regulation of cell wall proteins through posttranslational modification and GHs confer high plasticity to cell wall polysaccharides and/or are directly involved in antifungal activity (Guerra-Guimarães et al. 2015). Indeed, ß-1,3-glucanase and chitinase (considered as PR-proteins) hydrolyze ß-1,3 glucan and chitin, respectively, the cell wall components of most fungal pathogens (as revised by Silva et al. 1999). Additionally, Germins and germin-like proteins (GLPs) which are homohexamer glycoproteins, have also been implicated in biotic and abiotic stress responses (Liao et al. 2021). Studies related to GLP and plant immunity showed their association with jasmonic acid (JA)-mediated defense response (Liu et al. 2016; Pei et al. 2019) or the connection between GLP overexpression and the improved resistance to fungal pathogens and the accumulation of ROS (Beracochea et al. 2015; Sultana et al. 2016).

# *4.2 Mechanisms of Coffee Resistance to* **H. vastatrix**

Over the years, there has been considerable progress in understanding the mechanisms of coffee's complete resistance to *H. vastatrix* at the cellular and biochemical levels, and more recently via analytical chemistry, gene expression analysis, and the use of omics approaches.

Coffee resistance to *H. vastatrix* is characterized by the arrest of fungal growth, which may occur at pre-haustorial stages (pre-haustorial resistance) or after the formation of at least one haustorium (post-haustorial resistance). In both types of resistance, the hypersensitive reaction (HR) is one of the first cytological responses induced by *H. vastatrix*. This response occurs initially in stomatal cells associated with pre-haustorial fungal stages and later in plant cells invaded by haustoria, and spreads to adjacent noninvaded cells (Silva et al. 2006, and references therein; Silva et al. 2008; Diniz et al. 2012; Guerra-Guimarães et al. 2015).

The early perception of the pathogen invasion by a repertoire of recognition kinases at the plasma membrane, such as RLK and *LRR-RLK2*, or intracellularly by nucleotide-binding site–leucine-rich repeat (*NBS-LRR*) are successful steps in triggering host defense (Fernandez et al. 2004; Guzzo et al. 2009; Diniz et al. 2012; Diola et al. 2013; McCook and Peterson 2020). During coffee resistance to *H. vastatrix*, the up-regulation of two MAPKs (*MAPK2—Mitogen-activated protein kinase 2* and *MEK2—Dual specificity mitogen-activated protein kinase kinase 2*) suggests that they are important signaling elements of the defense response during the infection process (Diola et al. 2013). In addition, two calcium-related genes, *calciumdependent protein kinase 5* (*CDPK5*) and *calmodulin-binding protein* (*CaMBP*) have been associated as part of the Ca2+ signaling in coffee resistance response (Diola et al. 2013). In coffee plants, a small list of transcriptional factors has been associated with resistance to *H. vastatrix*: *Ap2* (*AP2 type transcription factor*) (Fernandez et al. 2004), *bHLH* (*basic helix-loop-helix DNA-binding protein*) (Florez et al. 2017), and *bZIP56* (*bZIP transcription factor*) (Diola et al. 2013). However, the WRKY family is the most well-studied transcriptional regulators in coffee plants, with 17 out of 22 genes that seem to be linked to *H. vastatrix* resistance. Of all known coffee WRKYs, CaWRKY1 (*CaWRKY1a* and *CaWRKY1b*) is activated as early as *H. vastatrix* penetration into host tissues and deployment of HR (Ganesh et al. 2006; Petitot et al. 2008, 2013a; Diniz et al. 2012). The role of phytohormones, like JA and ethylene (ET), in coffee defense against *H. vastatrix*, remains unclear (Guzzo et al. 2009; Ramiro et al. 2010; Diniz et al. 2012; Diola et al. 2013; Florez et al. 2017). However, several

studies point out that SA is a key hormone in coffee defense against *H. vastatrix*, by the increased expression of SA pathway-related genes, such as SA-biosynthesis gene *PAL*, by SA-induce PR genes, *CaPR1*, *CaPR2*, *CaPR5*, *CaPR10* (Couttolenc-Brenis et al. 2020; Diniz et al. 2012, 2021; Diola et al. 2013; Guzzo et al. 2009; Ramiro et al. 2009) and by SA-mediated protein–protein interactions gene *NPR1* (*Non-expressor of pathogenesis-related*) (Diniz et al. 2012; Petitot et al. 2013b; Couttolenc-Brenis et al. 2020). NPR1 is a transcription co-factor and a *bone fide* SA receptor and, consequently, a positive regulator of several SA downstream responses such as HR (Saleem et al. 2021).

Light and transmission electron microscopic observations further suggest the involvement of ROS, such as H2O2 and O2 − in the HR of coffee-resistant genotypes. Additionally, deposition of phenolic-like compounds in cell walls and cytoplasmic contents, plant cell wall lignification, haustoria encasement with callose and β-1,4-glucans; accumulation of intercellular pectin-like material containing polysaccharides and phenols, and plant cell hypertrophy were also observed (Rodrigues Jr. et al. 1975; Silva et al. 2001, 2002, 2006, 2008; Ramiro et al. 2009; Diniz et al. 2012).

Biochemical studies with coffee-resistant genotypes, revealed the early increase in the activity of several oxidative enzymes associated with ROS homeostasis, namely, POD, LOX, and SOD, as well as the enzyme PAL (Rojas et al. 1993; Silva et al. 2002, 2008; Guerra-Guimarães et al. 2009a). The early increase in the SA levels, quantified by HPLC/ESI-MS/MS, reinforces the involvement of an SA-dependent pathway in coffee resistance to *H. vastatrix* (Sá et al. 2014). Additionally, the evaluation of chlorogenic acid, an abundant polyphenol in coffee, performed by HPLC-DAD and LC-MS also revealed an early and significant increase in its content associated with the resistance (Leitão et al. 2011). Chitin and β-1,3-glucan, are the main components of *H. vastatrix* cell walls, including those from intercellular hyphae (infection hyphae and HMCs) and haustoria (Silva et al. 1999). An early increase of β-1,3-glucanase (PR2) and chitinase (PR3) activity was observed in crude extracts and in the apoplast of resistant coffee leaves (Maxemiuc-Naccache et al. 1992; Guerra-Guimarães et al. 2009b). Furthermore, basic isoforms specific to class I chitinases were detected earlier and only in the resistance, suggesting its involvement in the defense response of the coffee plants (Guerra-Guimarães et al. 2009b).

Going deep into the study of the coffee leaf apoplast, a proteomic analysis was performed revealing the increase in abundance of several cell wall glycohydrolases (GH3, GH31, and GH38 family), PR proteins [PR1, PR2, PR3, thaumatin/osmotin (PR5), GPLs (PR15 and PR16)], proteases (serin, cysteine and aspartic peptidases) and other enzymes (e.g.; metallophosphatases) playing a role in the coffee defense response (Azinheira et al. 2013; Guerra-Guimarães et al. 2015; Possa et al. 2020; Silva et al. 2022).

The resistant and susceptible coffee genotypes share most of the described host responses when infected by *H. vastatrix*. However, they are observed earlier and with greater magnitude during the resistance response, particularly in pre-haustorial resistance.

# **5 Conclusions**

This chapter reviews decades of scientific knowledge accumulated on coffee–*H. vastatrix* interactions. From the first studies back in the sixties until today, a wealth of data has been gathered about this binomial relationship. What we know today has evolved significantly from Flor's gene-for-gene model to rust races' candidate markers within the current "omics" era (genomics, transcriptomics, metabolomics, and proteomics). The income of new data continuously challenges what we know and raises further questions. What is the true nature of the coffee resistance genes, and how are they regulated? How are the recently discovered small non-coding RNA (sRNA, miRNAs) involved in gene regulation? How can miRNA be related to coffee resistance to *H. vastatrix*? The answer to these and more questions relies on our ability to continue to explore the coffee–*H. vastatrix* interaction and adventure ourselves to go deep into barely explored coffee resistance research fields such as functional characterization and epigenetics. Despite all the significant progress made to date, a thorough exploration of *Coffea*–*Hemileia vastatrix* interactions using advanced technologies remains critical for developing new and efficient disease control strategies.

**Acknowledgements** Funding for this work was provided by the Food and Agriculture Organization of the United Nations and the International Atomic Energy Agency through their Joint FAO/ IAEA Research Contract nº 20902/R0 of the IAEA Coordinated Research Project D22005 and by Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES ref. PTDC/ASP-PLA/29779/2017 and by FCT UNIT (UID/AGR/04129/2020) of LEAF—Linking Landscape, Environment, Agriculture and Food, Research Unit.

### **References**

Agrios GN (2005) Plant pathology. Academic Press, New York


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Induced Mutagenesis of in Vitro Tissues and Cells of** *Coffea arabica* **L.**

# **In Vitro Plantlet Establishment of** *Coffea arabica* **from Cut Seed Explants**

**Florian Goessnitzer, Joanna Jankowicz-Cieslak, and Ivan L. W. Ingelbrecht** 

**Abstract** Arabica coffee is one of the most important products in the world market. As a perennial crop, conventional breeding of Arabica coffee is challenged by its long reproductive cycle and narrow genetic base. In vitro tissue culture in combination with mutation induction techniques provides an attractive alternative approach for the genetic improvement of coffee. In this chapter we describe a simple and robust method to rapidly establish in vitro Arabica coffee plantlets from cut seed explants. The method streamlines the germination process under in vitro environmentally controlled conditions and overcomes microbial contamination, often associated with coffee seed lots harvested from the field or greenhouse. Using this protocol, diseasefree in vitro coffee plantlets can be generated within 5–6 weeks, useful for downstream tissue culture manipulations such as the production of friable embryogenic callus and cell suspension cultures or induced chemical or physical mutagenesis.

# **1 Introduction**

World coffee production relies mostly on two species: *Coffea arabica* and *Coffea canephora*, of which Arabica is the most widely cultivated, primarily because of its superior quality. Somatic embryogenesis in coffee has been used for mass propagation, genetic engineering and, more recently, also for induced mutagenesis studies (Los Santos-Briones and Hernández-Sotomayor 2006; Menéndez-Yufá et al. 2010; Bolívar-González et al. 2018). Since the 1990s, somatic embryogenesis (SE) techniques have enabled clonal propagation of both *Coffea arabica* and *Coffea canephora*  (Etienne et al. 2018). In coffee, indirect SE through an intermediate callus phase using semi-solid and liquid media is well established while direct somatic embryogenesis

e-mail: f.goessnitzer@iaea.org

F. Goessnitzer (B) · J. Jankowicz-Cieslak · I. L. W. Ingelbrecht (B)

Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria

I. L. W. Ingelbrecht e-mail: i.ingelbrecht@iaea.org

methods have also been described but to a lesser extent (Quiroz-Figueroa et al. 2006; Murvanidze et al. 2021). In combination with techniques for induced mutagenesis, in vitro cell and tissue culture methods can provide an alternative strategy for enhancing genetic diversity and improvement of Arabica coffee.

Coffee seeds have limited viability. When stored at ambient temperature viability decreases rapidly; after two months for *Coffea canephora* and after six months in case of *Coffea arabica* (Wintgens 2012). In addition, the germination of coffee seeds is a slow process, taking 30–60 days under the most favourable conditions. In practice, favourable conditions may not always be available, hence the need for standardized conditions for coffee seed germination. This is particularly relevant for induced mutagenesis studies and related radiosensitivity testing where lethal doses or growth reduction values need to be determined. Coffee cell and tissue culture applications require that starting explants, frequently leaves, are free from microbial contamination. Depending on the environment and conditions of storage, however, coffee seed can become contaminated with fungi or other microbes. Due to the shape and morphology of the coffee seed, such microbial contamination can be challenging to remove through classical sterilization procedures.

Here we present a simple and robust in vitro protocol that streamlines the germination of coffee seed using cut seed explants. This method is effective for overcoming potential fungal/microbial contamination often associated with coffee seed batches. Germination proceeds under stable environmental conditions. Under in vitro conditions the coffee cut seed explants germinated, i.e., the radicle breaking through the endosperm, within 14 days. Overall, the procedure generated in vitro plantlets within 5–6 weeks, useful for downstream in vitro experiments such as somatic embryogenesis or induced mutagenesis studies.

### **2 Materials**

### *2.1 Plant Material*

1. Seed from three Arabica coffee varieties; Venecia, Caturra, and Catuai.

### *2.2 Chemicals*


# *2.3 Tools and Labware*


# *2.4 Lab Equipment*


# **3 Methods**

# *3.1 Media Preparation*



# *3.2 Seed Preparation*


**Fig. 1** *Coffea arabica* seed preparation for the isolation of cut seed explants

**Fig. 2** Cut seed explant isolation from coffee seed; arrows indicate cuts; crosses indicate seed parts to be discarded

# *3.3 Explant Isolation*


# *3.4 Surface Sterilization of the Cut Seed*


**Fig. 3** Cut seed explants in the sieve

**Fig. 4** Transfer of the cut seed explant to culture tubes

# *3.5 Transfer of Explants and Culture Conditions*


# *3.6 Germination and Plantlet Development*


**Fig. 5** Germination and plantlet development

### **4 Notes**


**Acknowledgements** The authors would like to thank Plant Breeding and Genetics Laboratory colleagues and participants of the Coordinated Research Project (CRP) D22005 'Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana' for stimulating discussions. We would like to thank Dr Noel Arrieta Espinoza, Instituto del Café de Costa Rica (ICafe), Costa Rica for providing seed of Arabica coffee used in these experiments. This work was supported by the FAO and IAEA through the CRP D22005.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Somatic Embryogenesis and Temporary Immersion for Mass Propagation of Chimera-Free Mutant Arabica Coffee Plantlets**

**Samira Tajedini, Florian Goessnitzer, and Ivan L. W. Ingelbrecht** 

**Abstract** Coffee is one of the most valuable cash crops providing employment for millions of people worldwide. Arabica coffee is widely grown in Latin America where it is under threat of leaf rust. Conventional breeding of Arabica coffee is challenged by its narrow genetic base and long reproductive cycle, and it can take up to 30 years for variety development and release. In vitro somatic embryogenesis is a propagation technique whereby a single plant somatic cell can give rise to a somatic embryo under appropriate culture conditions. For tree crops such as Arabica coffee, single-cell mutagenesis using embryogenic cell cultures provides a powerful approach to produce chimera-free mutant lines directly from cells. Here we describe protocols to induce friable embryogenic callus, establish embryogenic cell suspensions, and convert somatic embryos into plantlets using a RITA® bioreactor for *Coffea arabica* var. Venecia. In addition, methods for gamma-ray mutagenesis of regenerable cell suspensions are described.

# **1 Introduction**

Coffee is a global commodity providing employment for millions of people worldwide (FAOSTAT 2021). The *Coffea* genus belongs to the family *Rubiaceae* and the two main cultivated species are *Coffea arabica* L. (2n = 4x = 44) and *Coffea canephora* Pierre ex A. Froehner (2n = 2x = 22). Arabica coffee is a self-pollinating species, widely cultivated in South America, Africa and Asia. During the past decade, coffee leaf rust, a fungal disease caused by *Hemileia vastatrix*, has devastated *C. arabica* plantations across Latin America (Avelino et al. 2015).

S. Tajedini (B) · F. Goessnitzer · I. L. W. Ingelbrecht (B)

Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria e-mail: stajedini@gmail.com

I. L. W. Ingelbrecht e-mail: i.ingelbrecht@iaea.org

Conventional breeding of Arabica coffee is challenging due to its long reproductive cycle and narrow genetic base (Wintgens 2012; Scalabrin et al. 2020). Mutationassisted breeding offers an attractive alternative to induce genetic diversity useful for coffee breeding and genetic studies. Since the 1990s in vitro tissue culture technologies have been developed for coffee, including methods to regenerate plants through somatic embryogenesis (Campos et al. 2017; Etienne et al. 2018). Both direct and indirect methods for somatic embryogenesis in coffee have been described (Quiroz-Figueroa et al. 2006; Murvanidze et al. 2021). However, to our knowledge so far, Arabica coffee single-cell micropropagation techniques have not been integrated with mutation induction techniques using gamma-ray irradiation.

Single cells or cell clusters derived from embryogenic callus or somatic cell suspensions are attractive targets for induced mutagenesis given that they are expected to (mostly) produce chimera-free, homo-histont plants, as opposed to mutagenesis of multicellular tissues such as seed which results in chimeras. Dissolving chimeras through successive cycles of selfing is possible in *C. arabica* as it is a self-compatible species, however this is a lengthy process given its long reproductive cycle. Here we present protocols to produce friable embryogenic callus, establish embryogenic cell suspensions and convert somatic embryos into plantlets using a RITA® bioreactor in Arabica coffee var. Venecia. Induced mutagenesis of embryogenic cell suspensions using gamma-ray irradiation is also described.

### **2 Materials**

### *2.1 Plant Material*

1. Mature coffee plants of *C. arabica* var. Venecia (see Note 1).

### *2.2 Supplies, Reagents and Basic Equipment*


### *2.3 Equipment*


### *2.4 Stock Solutions and Tissue Culture Media*



**Table 1** Media composition (mg/l) for somatic embryogenesis and plantlet regeneration of *Coffea arabica* var. Venecia (van Boxtel and Berthouly, 1996)

# **3 Methods**

The procedures described below utilize leaf discs as starting material to produce friable embryogenic callus on semi-solid medium. The friable embryogenic callus is then transferred to a liquid medium in Erlenmeyer flasks to establish a homogenous embryogenic cell (cluster) suspension culture. The cell suspension culture serves to multiply the embryogenic cells and cell clusters and is used for gamma-ray irradiation. Next, globular-stage somatic embryo cell cultures are transferred to a 1-L RITA® bioreactor for further development of the somatic embryos and conversion to rooted plantlets. The different steps of the procedure are illustrated in Table 2 and Figs. 1, 2, 3.


**Table 2** Steps in the in vitro regeneration and mutagenesis procedure with indicative timeline

**Fig. 1** Somatic embryogenesis process in *Coffea arabica* from ex vitro leaf disks. **a** Leaf disk in callus induction medium. **b** Leaf disk with primary calli. **c**–**d** Close-up of friable embryogenic calli

**Fig. 2** Preparation of embryogenic cells/cell clusters for irradiation treatment. **a** 40-Mesh filter. **b** Embryogenic cell suspension after sieving. **c** Cell suspension in microcentrifuge tubes ready for gamma-ray irradiation

# *3.1 Media Preparation*


# *3.2 Tissue Collection and Disinfection*


**Fig. 3** *Coffea arabica* somatic embryogenesis process. **a** Friable embryogenic callus indicated with the arrow. **b** SE in proliferation medium. **c** Embryogenic cells for radiation treatment. **d** Germination of somatic embryos in a RITA® bioreactor. **e**–**g** Rooted plantlets

# *3.3 In Vitro Leaf Disc Preparation and Primary Callus Induction*


# *3.4 Embryogenic Callus Induction*


# *3.5 Cell Multiplication and Establishing the Embryogenic Cell Suspension*


# *3.6 Gamma-ray irradiation of the Embryogenic Cell Suspension*


# *3.7 Somatic Embryo Differentiation and Germination*


# *3.8 Development of Somatic Embryos and Conversion to Plantlets*


# **4 Notes**


**Acknowledgements** The authors thank PBG Laboratory colleagues and participants of the Coordinated Research Project (CRP) D22005 'Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana' for stimulating discussions. We would like to thank Dr Noel Arrieta Espinoza, ICafe, Costa Rica for providing seed of *Coffea arabica* and sharing insights on coffee breeding and challenges. We further thank the IAEA and the Belgian Government for financial support through the CRP D22005 and the Peaceful Use Initiative 'Enhancing Climate Change Adaptation and Disease Resilience in Banana-coffee Cropping Systems in East Africa', respectively.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Protocol on Mutation Induction in Coffee Using In Vitro Tissue Cultures**

**Margit Laimer, Rashmi Boro, Veronika Hanzer, Emmanuel Ogwok, and Eduviges G. Borroto Fernandez** 

**Abstract** Pathogens are the major limiting factors in coffee production. Approximately 26% of the global annual coffee production is lost to diseases, threatening the income of approx. 125 million people worldwide. Therefore, reducing coffee yield losses by improving coffee resistance to diseases and insect attacks through breeding can make a major contribution to agricultural sustainability. Mutation breeding in vegetatively propagated and perennial crops is hampered in large part due to bottlenecks in the induction of variation (lack of recombination) and challenges in screening. Tissue culture approaches using alternative types of material were developed. This offers a clear advantage of providing the required sample size for mutation induction and subsequent screening within a reasonable time frame. The protocols developed compare different tissue culture systems for mutation induction involving unicellular and multicellular explants requiring different numbers of subsequent subcultures to reduce the impact of chimerism: (a) axillary shoot culture for the provision of donor material for mutation induction and regeneration; (b) leaf disc cultures for the induction of calli; (c) direct and indirect somatic embryogenesis for the production of somatic embryos; (d) the irradiation of somatic embryos at the globular and cotyledonary stage. Mutagenesis was induced by irradiation with a Cobalt-60 Gamma-source at the FAO/IAEA Laboratories in Seibersdorf, Austria. A comparison of the time required for the regeneration of high numbers (hundreds) of plantlets from irradiated in vitro shoots versus irradiated embryogenic calli is clearly in favor of embryogenic calli, since the plantlets regenerate from individual cells and can be used for genotypic and phenotypic analyses directly. This chapter describes the general methods for mutation induction using gamma irradiation and the procedures that can be used to generate large numbers of induced mutants in different tissues of coffee under in vitro conditions.

E. Ogwok

M. Laimer (B) · R. Boro · V. Hanzer · E. G. Borroto Fernandez

Plant Biotechnology Unit, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190 Vienna, Austria e-mail: margit.laimer@boku.ac.at

Department of Science and Vocational Education, Faculty of Education, Lira University, 1035, Lira, Uganda

# **1 Introduction**

After petroleum, coffee beans are the second most economically important product traded worldwide. Approximately 11 million ha of coffee trees are cultivated in tropical regions (Déchamp et al. 2015), providing income to 125 million people. Despite the importance of coffee, several factors, which could be amplified by changing climatic conditions, hamper its production and influence the extent of yield losses, including agronomic management, growing environment, cultivar selection affected by diseases and pathogens. Pathogens are the major limiting factors in coffee production. Approximately 26% of the global annual coffee production is lost to diseases.

Cultivated coffee originated from wild populations in Africa, Madagascar, the Comoro and the Mascarene islands, the Indian subcontinent, Southern tropical Asia, South-East Asia and Australasia (Razafinarivo et al. 2013; Davis et al. 2006, 2007).

Among different coffee species, the two economically most important are the diploid *C. canephora* Pierre ex Froehn (2n = 2x = 22), native to Central and Western sub-Saharan Africa, and the allotetraploid *C. arabica* L. (2n = 4x = 44) from the Southwestern Ethiopian highlands, Mount Marsabit of Kenya and the Boma Plateau of Sudan (Anthony et al. 2002). *C. arabica* resulted from ancestral hybridization—dated approximately 50,000 years ago—between two diploid ecotypes, namely *C. eugenioides* and *C. canephora* (Anthony et al. 2010; Lashermes et al. 1997; 1999; Ribas et al. 2011). *C. arabica* is self-pollinated, while *C. canephora* is cross-pollinated. A recent study by Sant'Ana et al. (2018) showed a high allelic variation in wild accessions from Ethiopia, however, the mode of pollination and the history of coffee cultivation resulted in a reduction of genetic diversity in *C. arabica*. According to different authors, coffee was introduced from Ethiopia to Yemen between 1,500 and 300 years ago. From this point, the first reduction of diversity happened within Arabica cultivars. Genetic data analyses showed that two genetic bases spread from Mocha (Yemen) in the early eighteenth century (Chevalier and Dagron 1928; Haarer 1956). *C. arabica* var. *arabica* (also called var. *typica* Cramer) originated from a single plant that was introduced to Java (Indonesia) and later cultivated in the Botanical Garden of Amsterdam. *C. arabica* var. Bourbon (B. Rodr.) Choussy (Carvalho et al. 1969; Krug et al. 1939) was introduced to the Bourbon Island (Réunion). These were the starting points of coffee cultivars spreading rapidly to the American continent and Indonesia by the use of seeds produced by the auto-fertilization of coffee trees, which caused a further reduction in genetic diversity.

There are several major constraints in coffee breeding. As already mentioned, the vast majority of the world coffee production is based on two species, *Coffea arabica*  (2n = 4x = 44 chromosomes) and *C. canephora* (2n = 2x = 22 chromosomes). This results in low genetic diversity among coffee cultivars and represents a massive limitation in case of control and management of pest and disease under climatic changes. The absence of pest resistance in the most preferred *Coffea arabica* cultivars can be overcome by cross-breeding, but due to the long juvenile period of tree crops this is a time-consuming process (Silva et al. 1999; 2006; Várzea et al. 2000).

Plant biotechnological interventions in coffee improvement are used to develop uniform planting material through cell and tissue culture (Krishnan 2011) since the pioneering attempts in mutation induction by Carvalho in the 1950s (Carvalho 1988).

In recent years mutation breeding programs have been initiated within the FAO/ IAEA funded Coordinated Research Project (CRP) D22005 on mutation induction for coffee improvement (Dada et al. 2014, 2018; Bolívar-González et al. 2018; Bado et al. 2018a, b). In contrast to conventional breeding, taking at least 20 years to release a new cultivar, biotechnological methods offer valuable tools for coffee improvement and for speeding up the selection process of superior plants (Bado et al. 2017; Campos et al. 2017). Micropropagation by organogenesis is used for plant multiplication mainly from shoot tips and axillary buds allowing the production of large-scale populations for mutation induction and subsequent mutant line propagation and is mainly suitable for vegetatively propagated crops with a long juvenile period. This allows to reduce the time required and to accelerate mutation breeding when using single cell explants. A considerable acceleration of mutation breeding can be achieved by using single cell explants like double haploids or somatic embryos, which mark the shortest route to produce homozygous lines from heterozygous plant material.

Somatic embryos can be produced on a large-scale in suspension cultures and in bioreactors. As a matter of fact, somatic embryogenesis is an excellent system for mutation induction, since somatic embryos originate from single cells (da Câmara Machado et al. 1995).

Among the physical mutagens, gamma rays are the most commonly used for mutation breeding (Mba et al. 2012), resulting in small to large deletions, point mutations, single and double strand brakes and even chromosome deletions. When applying physical mutagens to different types of plant material, care should be taken with soft material such as in vitro shoot cultures as well as callus and embryogenic callus cultures, which require lower doses in comparison to seeds. In fact, the water content, storage time, applied mutagen dose and temperature represent important factors influencing mutagens in all types of plant material (Mba et al. 2010).

Depending on the explant type subjected to mutation induction different approaches are required for chimera dissolution. Plants originating either from unicellular or multicellular explants require different time frames for chimera dissolution ranging from 0 for plantlets stemming from somatic embryos to several generations up to M1V4 for plantlets originating from multicellular explants (Novak and Brunner 1992). Entire mutant populations are screened by either phenotypic evaluation to select the phenotype of interest or by genotypic evaluation to detect novel alleles in genes of interest (Fig. 1).

Pathogens are the major limiting factor in coffee production. New approaches are available to breed varieties that are resistant to a broad-spectrum of pathogens, genetically stable and high-yielding. Recently developed tools in genomic technologies allow to better understand coffee-pathogen interaction and help to identify the genes and mechanisms involved in pathogen resistance or susceptibility. Understanding the influence of individual factor and their interaction will help to select realistically interesting accessions and to accelerate breeding strategies.

**Fig. 1** Mutation breeding scheme for mutagenesis in *Coffea* sp. **1** Mature donor plants provide vegetative buds, flower buds, leaves, while seeds may be established directly in vitro. **2** In vitro cell and tissue cultures may involve somatic and gametic cells. **3** Coffee explants for mutation induction may be of uni- or multicellular origin. Note that multicellular systems require an additional step for chimera dissolution, while single cell systems do not require this step. **4** Screening of mutants can be conducted either before acclimatization through early screening of irradiated cells and plantlets or after acclimatization of plantlets to greenhouse or under open field conditions. **5** Selected improved cultivars can be released as direct mutants or be further used in breeding programs by hybridization

# **2 Materials**

# *2.1 Establishment of a Collection of Donor Material In Vivo*

A collection of starting material of the two species of coffee—both as seeds and potted plants—should be initiated in the greenhouse to allow the maintenance of mother plant under controlled conditions (Fig. 2). In order to rule out major genotypic differences, in our experiments this included fourteen different cultivars of the leaf rust susceptible *C. arabica* (self-pollinating) as well as two genotypes of the leaf rust resistant *C. canephora* (self-incompatible) with different climate requirements and tolerance/susceptibility to different pathogen races. The collection of *C. arabica*  cultivars (https://sca.coffee/research/coffee-plants-of-the-world) comprised:

• **Bourbon**: A common cultivar *C. arabica* that developed naturally on Île Bourbon (an island in the Indian Ocean, east of Madagascar, now known as Réunion) from coffee brought to the island from Yemen by the French. Depending on the specific sub-group, this coffee can be red (Vermelho) or yellow (Amarelo). These plants generally have broader leaves and rounder fruit and seeds than Typica varieties. Stems are stronger and stand more upright than Typica. They are susceptible to all major diseases and pests.


**Fig. 2** Maintenance of donor plants of *Coffea* sp. under greenhouse conditions

For the maintenance of donor plants of *Coffea* sp. under greenhouse conditions the following items should be available:


# *2.2 Establishment of In Vitro Shoot Cultures as Donor Material*

Axenic cultures were established and prepared as source material for the specific regeneration and mutation programs. Different in vivo donor material was used with the intention to establish micropropagation for media optimization and induction of callus, somatic embryogenesis and suspension cultures: seeds, seedlings, leaves, stems, roots, flowers. Serving both as material for mutagenesis treatment, as well as for the recovery of individual mutagenized cells, these culture systems should be maintained throughout the entire period of the experiment.

The first 4 nodes of orthotropic shoots growing in the greenhouse under controlled conditions were excised as explants. Surface sterilization was achieved with a 15% sodium hypochlorite solution containing 1% Tween 80 for 20 min followed by 4 washes with distilled water. To carry out these steps, the following facilities and items are required:


**Fig. 3** Axenic in vitro cultures of *Coffea* sp. serving as donor material for mutation induction

Axillary shoot cultures of different cultivars were established from 2 genotypes of *C. canephora*, Niaoulli (14 clones) and Quillou (6 clones), as well as from 15 genotypes of *C. arabica*: HDT (4 clones), Caturra, Catimor, Kent, Sarchimor, Typica (3 clones), Villa Sarchi, Java (6 clones) and Bourbon (Fig. 3).

# *2.3 Establishment of Tissue Culture Material for Mutation Induction*

Somatic embryogenesis is an excellent system for plant propagation and mutation induction, since somatic embryos originate from single cells and therefore reduce chimerism. Somatic embryos can be produced on a large-scale in suspensions in Erlenmeyer flasks and in bioreactors. Somatic embryos of *Coffea* can be obtained either by direct or by indirect somatic embryogenesis, the difference being the intermediate callus induction.

For the induction of coffee callus cultures and somatic embryogenesis additional multicellular explants, like leaves, stems and roots of in vitro grown seedlings (Fig. 4) and from in vitro shoots from selected cultivars are used.

The conversion of embryos to plantlets from a number of selected cultivars after transfer of emerging embryos from embryogenic calli to regeneration medium yielded a high number of mutant plantlets (Fig. 6).

To establish an efficient regeneration from embryogenic calli into plantlets the following items are needed:


**Fig. 4** Induction of direct and indirect embryogenesis from different in vitro explants of *Coffea* sp.

**Fig. 5** Induction of direct or indirect embryogenesis from different in vitro explants


**Fig. 6** Regeneration of plantlets/somatic embryos from irradiated embryogenic calli

# *2.4 Mutagenesis by Physical Agents*

Gamma ray mutagenesis may be performed using different facilities, such as gamma cell irradiator, gamma phytotron, gamma house, gamma field. The gamma cell irradiator with Cobalt-60 (or Cesium-137) as radioactive source is the most commonly available equipment worldwide (IAEA 1975, 1977).

However, the radioactive source remains the major consideration and constraint in plant mutagenesis (Bado et al. 2015).


# **3 Methods**

### *3.1 Mutagenesis by Physical Agents*

Mutations were induced by gamma-irradiation of different explants of selected genotypes of *C. arabica* and *C. canephora* at different intervals with several repetitions. A Cobalt-60 Gamma irradiator was used and the irradiation was performed at the FAO/

**Fig. 7** Working steps for irradiation using a Cobalt-60 gamma cell

IAEA Laboratories in Seibersdorf, Austria. The workflow shown in Fig. 7 can be carried out with different plant cell and tissue cultures either in Magenta boxes or petri dishes.

# *3.2 Selection and Treatment of Explants*

The first type of explants to be subjected to irradiation are axenic shoot cultures in order to determine the radiosensitivity of different *C. canephora* and *C. arabica*  cultivars (Fig. 3, see Notes 6 and 7).


After having determined the dose range for entire shoots, callus cultures, embryogenic callus cultures, somatic embryos at the globular, torpedo or cotyledonary stage can be irradiated in a similar way.


**Fig. 8** Callus cultures of *Coffea* sp. prepared for mutation induction

# *3.3 Regeneration of Mutant Plant Lines*

Plant cell and tissue cultures from these irradiation experiments were cultivated further and resulted in shoot formation and plantlet regeneration. These tissues have to undergo rigorous scrutiny for visual detection of altered phenotypes and are evaluated for a range of parameters (Table 1). Additional parameters to be evaluated for regenerated plantlets are active shoot growth, axillary bud formation, secondary root formation.



**Table 1** Overview of recommended Gamma doses used for different explants of *Coffea* sp. and evaluation parameters applied within this study


**Table 2** GR30 and GR50 determined according to effects observed from different Gamma doses used for *Coffea* sp. shoot cultures

Values are calculated in relation to control plants *Note GR* growth reduction

**Fig. 9** Phenotypic analyses of in vitro development of irradiated shoots with focus on root development at 0, 10, 15, 20, 40 and 60 Gy

Since roots are known to respond more sensitively to different stresses, their development was carefully evaluated. The optimal dose range for shoot cultures was identified between 20 and 42 Gy (Table 2).

From the original 75 irradiated shoots finally after a period of approx. 18 months more than 600 plants could be recovered (Table 3). Interestingly, no shoot survived the treatment with 60 Gy.

**Fig. 10** Irradiated embryogenic callus of coffee



Data are given from 3 replicates

Following the dose range determined for shoot cultures, single cell explants should be handled (Fig. 10).


After irradiation, initial growth was observed only in untreated calli for the first month. However, with a delay, calli from all treatments recovered and survived. All treated calli showed a change in colour as response to gamma irradiation compared to control which maintained the yellow colour. In the third month of incubation cotyledonary embryos were observed with the doses up to 20 Gy, whereas from 40 to 80 Gy no embryo development was observed. The irradiation of embryogenic callus of *Coffea canephora* irradiated on 24.01.2018 led to the recovery of hundreds of shoots, this time of single cell origin (Table 4). Again, it was noted that only very few shoots survived the treatment with 60 Gy.

Irradiation of different developmental stages of somatic embryos revealed, that globular stage and cotyledonary stage embryos besides not growing anymore after being irradiated with 40 and 60 Gy, did not develop directly into actively growing plantlets. However, the circuit through a repetitive embryogenesis allows to recover plantlets also through this process. In fact, irradiation of globular and cotyledonary embryos of *Coffea arabica* cv. Java after 9 months led to recallusing and from there again to embryogenic calli producing new embryos and finally after 12 months approximately 200 shoots.

Globular embryos were relatively more resistant to gamma irradiation than cotyledonary and torpedo shaped embryos (Fig. 11).


**Table 4** Number of shoots of N20 (*Coffea canephora* cv. Quillou) recovered after irradiation of embryogenic calli with 0, 15, 20 and 40 Gy

**Fig. 11** Irradiated somatic embryos of coffee **a** globular stage, **b** cotyledonary stage


**Table 5** Response of different in vitro explants to gamma irradiation

As anticipated, the experiments allowed to confirm the higher radio-sensitivity of multi-cellular when compared to uni-cellular explants under in vitro conditions (Table 5). It was possible to:


According to the mutagenesis objectives starting from the second generation and higher after chimera dissolution, in vitro plants can be screened for the selection of candidate based on phenotypes or genotypes. Mutations can be detected with various direct and indirect methods. Direct methods such as sequencing, exome capture sequencing, restriction site associated DNA (RAD) sequencing and genotyping by sequencing (GBS) provide the necessary information for mutation detection and confirmation (Denoeud et al. 2014, Dereeper et al. 2015). Additionally, the generation of various EST sequences in *C. arabica* (Anthony et al. 2001; Mishra and Slater 2012; de Moro et al. 2009; Krishnan 2014; Vieira et al. 2006; Leroy et al. 2005; Lin et al. 2005; Noir et al. 2004) will allow to identify genes and their regulatory sequences responsible for mutated traits and estimate their value for further breeding programs.

### **4 Notes**


**Acknowledgements** This work was supported by the Food and Agriculture Organization of the United Nations and the International Atomic Energy Agency through their Joint FAO/IAEA Program of Nuclear Techniques in Food and Agriculture as Coordination Research Project D22005. We also thank Dr. Bado S. for technical assistance and the Plant Breeding and Genetics Laboratory (PBGL) Seibersdorf, Austria for the irradiation services provided.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Mutation Induction Using Gamma-Ray Irradiation and High Frequency Embryogenic Callus from Coffee (***Coffea arabica* **L.)**

**Miguel Barquero-Miranda and Reina Céspedes** 

**Abstract** Mutation induction through chemical or physical mutagenesis has been widely used for crop improvement for more than 70 years. Coffee is one of the most important crops in Latin-America, and, as any other crop, it can be affected by pests and diseases. Coffee leaf rust (CLR), caused by the biotrophic fungus *Hemileia vastatrix*, is the most important disease affecting Arabica coffee leading to significant losses for growers. As a perennial crop, conventional breeding of Arabica coffee is time-consuming. Plant tissue culture in combination with mutation induction techniques can provide an alternative approach to increase genetic variability of Arabica coffee for breeding applications. The present chapter describes protocols to establish embryogenic callus suspensions from Arabica coffee cv Venecia and for gamma ray irradiation of callus suspension cultures to achieve genetic improvement in the crop.

# **1 Introduction**

*Coffea arabica* L. (coffee) belongs to the Rubiaceae family which comprises about 500 genera and more than 6000 species, mostly tropical trees and shrubs (Jiménez and Carril 2014). The *Coffea* genus includes more than 100 species from which only *C. arabica* and *C. canephora* are grown commercially (Mishra and Slater 2012). Central America is the world's fifth largest Arabica coffee producer, where Costa Rica stands out in terms of production and quality (ICAFE 2016).

More than 80% of Arabica coffee produced in Latin America comes from varieties derived from a narrow genetic base, being highly susceptible to diseases and pests, caused by microorganisms such as fungi, bacteria, viruses and nematodes (Bertrand et al. 2011). In the region, the majority of the diseases are caused by phytoparasitic fungi and around 300 diseases affecting the crop have been detected worldwide (Canet Brenes et al. 2016). Coffee leaf rust (CLR), caused by the fungus *Hemileia*

M. Barquero-Miranda (B) · R. Céspedes

Coffee Research Center, 37-1000, Heredia San Pedro, Barva, Costa Rica e-mail: mbarquero@icafe.cr

I. L. W. Ingelbrecht et al. (eds.), *Mutation Breeding in Coffee with Special Reference to Leaf Rust*, https://doi.org/10.1007/978-3-662-67273-0\_6

*vastatrix*, is one of the main limiting factors of Arabica coffee production in all coffee growing countries (Mishra and Slater 2012).

Despite ongoing efforts for resistance breeding, chemical control is still the most widely used method to contain pests and diseases, including CLR (Neto and da Cunha 2016). Therefore, the development of alternative, environmentally friendly solutions for control of CLR is important. A long-term solution is through the development of resistant varieties, which is the focus of many breeding programmes in Arabica coffee (Mishra and Slater 2012). However, due to the perennial nature of coffee it can be difficult and time consuming to breed for disease resistance through conventional breeding methods (Barrueto Cid et al. 2004).

Plant breeders can use different tools to induce genetic variation in crops (Bermúdez-Caraballoso et al. 2016). Given the perennial nature of Arabica coffee, an effective way to induce variability, can be plant tissue culture in combination with mutation induction (Muthusamy et al. 2007). Combined, these techniques could increase genetic variability and reduce the time needed to develop new plant varieties (Bolívar-González et al. 2018).

Mutations can be induced by physical mutagens such as X-rays, gamma rays, neutrons and by chemical mutagens such as ethyl methanesulfonate (EMS). Physical mutagens appear more frequently used than chemical mutagens (Beyaz and Yildiz 2017). Since the 1960s gamma-ray mutagenesis has been the most commonly used method in plant mutation breeding (Li et al. 2019). Gamma rays are ionizing radiation (Beyaz and Yildiz 2017); they interact with atoms or molecules producing free radicals in cells that induce physiological, biochemical, cytological, genetic and morphogenetic changes in cells and tissues of plants (Chusreeaeom and Khamsuk 2019).

Somatic embryogenesis (SE) is a plant tissue culture technique where embryos are obtained from cells that are not the product of gametic fusion. Through SE thousands of seedlings identical to the mother plant can be produced (Bartos et al. 2018). Induced mutations are single cell events and thus the mutagenic treatment of seeds will result in chimeric M1 plants, i.e., they may carry different mutations, each occupying a (small) part of the plant. Since somatic embryos regenerate from single cells, somatic embryogenesis is considered to be an effective method for eliminating chimeras (Roux et al. 2004).

The optimal irradiation dose(s) leading to genetic improvement of a specific crop or trait may vary depending on the genetic constitution of the plant species and cultivar. Until now only the work of Sari et al. (2019) has referred to the use of gamma rays for mutation induction of Robusta coffee embryogenic callus suspensions. It is necessary and essential to conduct radiosensitivity testing to determine the optimal dose(s) of gamma-ray irradiation of Arabica coffee embryogenic cell cultures before conducting bulk irradiation experiments (Bermúdez-Caraballoso et al. 2016; Spencer-Lopes et al. 2018). The present chapter describes a protocol on how to obtain the embryogenic callus suspensions of *Coffea arabica* and to determine the optimal irradiation dose.

# **2 Materials**

# *2.1 Plant Material*

1. In these experiments *Coffea arabica* cv Venecia.

# *2.2 Explants Collection and Disinfection*


## *2.3 Induction of Embryogenic Callus*


### *2.4 Embryogenic Callus Multiplication*



**Table 1** Composition of media culture (mg/l) for indirect somatic embryogenesis in Coffee (*Coffea arabica*) (see Note 2)

*Source* Van Boxtel and Berthouly (1996)

# *2.5 Embryogenic Callus Irradiation*


# *2.6 Regeneration of the Irradiated Embryogenic Callus*


# *2.7 Embryo Germination*


# *2.8 Development of Somatic Embryos into Plantlets*


# *2.9 Media Culture: Preparation*


# **3 Methods**

# *3.1 Explants Collection and Disinfection*


# *3.2 Induction of Embryogenic Callus*


# *3.3 Embryogenic Callus Multiplication*


# *3.4 Irradiation of the Embryogenic Callus*


# *3.5 Regeneration of the Irradiated Embryogenic Callus*


### *3.6 Embryo Germination*


**Fig. 1** *Coffea arabica* embryogenic callus irradiation; **a** embryogenic suspension; **b** filtration system; **c** filtrated embryogenic callus; **d** embryogenic callus divided into microcentrifuge tubes to irradiate; **e** callus irradiation on Ob-ServoIgnis irradiator; **f** regeneration of the irradiated material

# *3.7 Regeneration of Plantlets*


# **4 Notes**

1. The irradiation of the materials was conducted at the Gamma radiation laboratory at the facilities of the Instituto Tecnológico de Costa Rica (TEC), using a Ob-ServoIgnis irradiator (Cobalt 60 radioactive source and an activity of 4.4 × 1014 Bq) (Becquerel).

**Fig. 2** Somatic embryogenesis process in *Coffea arabica*


**Acknowledgements** Funding for this work was provided by the Costa Rican Coffee Institute-Coffee Center Research and the IAEA. This work is part of the IAEA Coordinated Research Project D22005 titled "Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana", Contract Number 20475.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chemical Mutagenesis of Embryogenic Cell Suspensions of** *Coffea arabica* **L. var. Catuaí Using EMS and NaN3**

**Andrés Gatica-Arias and Alejandro Bolívar-González** 

**Abstract** Chemical mutagens, such as ethyl methanesulfonate (EMS) and sodium azide (NaN3), interact with DNA and can primarily induce single base modifications along the genome. Populations derived from chemical mutagenesis experiments are presumed to harbor high density of point mutations in the genome. Therefore, this technique, along with in vitro culture methods such as somatic embryogenesis (SE), can introduce genetic variation in otherwise genetically homogeneous populations. In vitro mutagenesis of embryogenic cell suspension cultures represents an efficient method to quickly develop mutant plantlets of unicellular origin. The development of mutant populations in this important crop represents a fundamental steppingstone in the development of novel varieties and the characterization of candidate genes involved in traits such as disease resistance, grain metabolite content and flowering induction. This chapter describes the protocol for establishment of embryogenic cell suspension cultures as well as methods of mutation induction using EMS and NaN3 on embryogenic cell suspensions of *C. arabica*, variety Catuaí. Furthermore, this chapter includes a protocol for mutant plant regeneration in in vitro conditions.

# **1 Introduction**

The combination of chemical mutagenesis with in vitro culture techniques offers advantages to improve the efficiency of mutagenic treatments. The easier management of large populations of plants and the independence of agronomic and environmental factors can be listed among these advantages (Xu et al. 2011). In vitro selection procedures may also be applied to accelerate some screening steps and mutant lines can be quickly micropropagated. Success of these protocols depends on the establishment of robust in vitro regeneration procedures. It is advisable to apply mutagenic treatments on culture methods that involve regeneration via individual cells. This way, chimeric events could be avoided in most cases, or at least, can

A. Gatica-Arias (B) · A. Bolívar-González

Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, 2060, San Pedro, Costa Rica

e-mail: andres.gatica@ucr.ac.cr

<sup>©</sup> The Author(s) 2023 I. L. W. Ingelbrecht et al. (eds.), *Mutation Breeding in Coffee with Special Reference to Leaf Rust*, https://doi.org/10.1007/978-3-662-67273-0\_7

be dissolved more rapidly. Somatic embryogenesis (SE) is one of the ideal systems which could be incorporated in a mutation breeding program. It can be described as a morphogenic process characterized by the formation of embryos from somatic cells without fecundation (Campos et al. 2017). Somatic embryos or embryogenic calli usually are formed from a limited number of cells on the plant tissue, thus rendering a mostly unicellular origin for the regenerated plantlets. Chimeric events can be reduced when embryogenic cultures are mutagenized and time limitations in breeding programs can be overcome. The predominant unicellular origin of embryogenic structures facilities the early and direct screening of M1V1 plantlets regenerated from M1V1 treated calli or tissues without the need to develop an M2 generation (Serrat et al. 2014).

When using embryogenic cultures, variables such as survival of cells and regeneration capacity after mutagenic treatment must be assessed to optimize the mutagen dose(s). Dual tests that allow qualitative and quantitative viability analysis are advised. One of the most widely used assays for checking the viability of in vitro cultures is the 2,3,5-triphenyltetrazolium chloride (TTC) test used to differentiate between metabolically active and inactive tissues. In living tissues TTC is converted to a red colored precipitate 1,3,5-triphenylformazan (TPF) that can be easily detected and quantified. Somatic embryos regenerated from mutagenized tissues can show germination and growth delay, germination inhibition can also appear.

Coffee is a key driver in social development and cultural identity of many tropical and subtropical regions. Worldwide production of coffee relies on two species, *Coffea arabica* L. (60%) and *C. canephora* (40%). The better cup quality and higher market value are associated to *C. arabica* L., the only allotetraploid (2n = 4x = 44) species among *Coffea*. *C. arabica* is an autogamous plant mostly incompatible with the remainder of *Coffea* species (Anthony et al. 2002). These characteristics, along with severe bottlenecks that happened during coffee domestication led to reduced genetic variability in *C. arabica* populations; this reduction enhances the general susceptibility of many *C. arabica* L. genotypes to diseases (Hendre and Aggarwal 2007).

SE has been developed in coffee, both directly through proembryogenic cells and indirectly through embryogenic calli from leaf explants. Embryogenic cultures are induced on an auxin containing medium, thereafter, subculture on auxin free medium induces embryo regeneration (Gatica-Arias et al. 2008; Quiroz-Figueroa et al. 2002; van Boxtel and Berthouly 1996). Coffee SE has been widely studied and represents an useful tool in breeding (reviewed by Campos et al. 2017). In this chapter, we describe methods for the mutagenic treatment of in vitro coffee embryogenic cultures to induce genetic variability. The chapter covers the application of the chemical mutagens EMS and sodium azide on the *C. arabica* var. Catuaí embryogenic cell suspensions as well as the regeneration of mutant plantlets.

# **2 Materials**

# *2.1 Plant Material*


# *2.2 Reagents*


# *2.3 Glassware and Minor Equipment*


# *2.4 Equipment*


# *2.5 Tissue Culture Media*


# *2.6 Software*

1. Standard spreadsheet software (e.g. Microsoft Excel or Open Office Excel).

# **3 Methods**

# *3.1 Preparation of Stock Solutions*


# *3.2 Preparation of Tissue Culture Media*

	- (a) For 1 L of callus induction medium: half-strength MS salts, 10 ml thiamine HCl, 1 ml pyridoxine HCl, 1 ml nicotinic acid, 1 ml glycine, 100 mg myoinositol, 100 mg casein hydrolysate, 400 mg malt extract, 0.5 ml 2,4-D, 1 ml IBA, 2 ml 2-iP, 30 g sucrose, 2 g Phytagel™, pH 5.6.
	- (b) For 1 L of embryo induction medium: half-strength MS salts, 20 ml thiamine HCl, 20 ml glycine, 40 ml l-cysteine, 200 mg myo-inositol, 6 ml adenine hemisulfate salt, 200 mg casein hydrolysate, 800 mg malt extract, 1 ml 2,4-D, 4 ml BAP, 30 g sucrose, 2 g Phytagel™, pH 5.6.
	- (c) For 1 L of liquid proliferation medium (CP): half-strength MS salts, 5 ml thiamine HCl, 0.5 ml pyridoxine HCl, 0.5 ml nicotinic acid, 10 m lcysteine, 50 mg myo-inositol, 100 mg casein hydrolysate, 200 mg malt extract, 2 ml 2,4-D, 1 ml KIN, 30 g sucrose, pH 5.6.
	- (d) For 1 L of regeneration medium (R): half-strength MS salts, 10 ml thiamine HCl, 1 ml pyridoxine HCl, 1 ml nicotinic acid, 2 ml glycine, 200 ml myoinositol, 4 ml adenine hemisulfate salt, 400 mg casein hydrolysate, 400 mg malt extract, 4 ml BAP, 40 g sucrose, 2.5 g Phytagel™, pH 5.6.
	- (e) For 1 L of germination medium (EG): half-strength MS salts, 8 ml thiamine HCl, 3.2 ml pyridoxine HCl, 100 mg myo-inositol, 0.45 ml IAA, 0.25 ml BAP and 2.5 g Phytagel™, pH 5.6.
	- (f) For 1 L of development medium (DEV): full-strength MS salts 1 ml thiamine HCl, 1 ml pyridoxine HCl, 1 ml nicotinic acid, 1 ml calcium pantothenate, 0.01 ml biotin, 100 mg myo-inositol, 0.3 ml BAP, 30 g sucrose, 2.5 g Phytagel™, pH 5.6.
	- (g) For 1 L of TEX medium (Teixeira et al. 2004): half-strength MS salts, 10 ml thiamine HCl, 1 ml pyridoxine HCl, 1 ml glycine, 250 mg citric acid, 10 ml l-cysteine, 100 mg/L myo-inositol, 100 mg/L casein hydrolysate, 200 mg/ L malt extract, 1 ml 2,4-D, 1 ml IBA, 2 ml 2-iP, 20 g sucrose, pH 5.6.

# *3.3 Germination of Coffee Zygotic Embryos Under In Vitro Conditions*


# *3.4 Protocol for Plant Regeneration via Somatic Embryogenesis*

This protocol involves a series of sequential stages: callus formation with embryogenic structures; establishment and multiplication of embryogenic suspension cultures*;* formation, maturation, and germination of somatic embryos; and conversion to plants; field evaluation (van Boxtel and Berthouly 1996) (see Fig. 1).

### **3.4.1 Embryogenic Callus Culture Initiation**


**Fig. 1** Schematic representation of the steps for indirect somatic embryogenesis in coffee (*Coffea arabica* L. var. Catuaí)


### **3.4.2 Establishment of Embryogenic Suspension Cultures**


# **3.4.3 Regeneration of Somatic Embryos and Development into Plantlets**


# **3.4.4 Hardening of In Vitro Plantlets in the Greenhouse**


# *3.5 Determination of the Viability of the Embryogenic Calli*


# *3.6 Sodium Azide Dose Determination*



**Table 1** NaN3 concentrations chosen for the toxicity test in coffee embryogenic calli

a Prepare a fresh solution immediately before the experiment

**Fig. 2** Effect of NaN3 concentration on survival and viability of coffee (*C. arabica* L*.* var. Catuaí) embryogenic calli. **a** Survival percentage (solid line) and absorbance (490 nm) (dotted line) versus NaN3 concentrations. Each value represents the mean ± SD of two repetitions, **b** cell viability versus NaN3 concentrations

# *3.7 Ethyl Methanesulphonate Dose Determination*



**Table 2** EMS concentrations and incubation time chosen for the toxicity test in coffee embryogenic calli

a Prepare fresh immediately before experiment

**Fig. 3** Effect of EMS concentration on survival and viability of coffee (*C. arabica* L*.* var. Catuaí) embryogenic calli. **a** Survival percentage (solid line) and absorbance (490 nm) (dotted line) after 60 min and 120 min of exposure time to different EMS concentrations. Each value represents the mean ± SD of two repetitions. **b** Cell viability versus EMS concentrations. *Bar*, 0.5 cm

# *3.8 Bulk Mutagenesis*


**Fig. 4** Bulk mutagenesis process under in vitro conditions of embryogenic calli of coffee (*C. arabica* L*.* var. Catuaí)

# **4 Notes**


**Acknowledgements** Funding for this work was provided by the University of Costa Rica, the Ministerio de Ciencia, Innovación, Tecnología y Telecomunicaciones (MICITT), the Consejo Nacional para Investigaciones Científicas y Tecnologicas (CONICIT) (project No. 111-B5-140; FI-030B-14) and the Cátedra Humboldt 2023. A. Gatica-Arias acknowledged the Cátedra Humboldt 2023 of the University of Costa Rica for supporting the dissemination of biotechnology for the conservation and sustainable use of biodiversity.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chemical Mutagenesis of** *Coffea arabica*  **L. var. Venecia Cell Suspensions Using EMS**

**Joanna Jankowicz-Cieslak, Florian Goessnitzer, and Ivan L. W. Ingelbrecht** 

**Abstract** Arabica coffee is widely grown in Latin America where it is under threat of leaf rust, a fungal disease caused by *Hemileia vastatrix*. As a perennial crop, conventional breeding of Arabica coffee is challenged by its long juvenile period and narrow genetic base. Plant mutants are important resources for crop breeding and functional genomics studies. The ethylating agent ethyl methanesulfonate (EMS) is widely used for inducing random point mutations. In a wide range of species, treatment with EMS causes GC-to-AT transitions with great efficiency. These properties, combined with ease of use, make EMS a mutagen of choice for induced mutagenesis. In vitro cell and tissue culture integrated with mutation induction provide an attractive approach for broadening the genetic base and breeding purposes, especially for perennial crops such as Arabica coffee. Embryogenic cell cultures are suitable targets for mutation induction and can accelerate the development of chimera-free mutant plantlets. Here we describe a robust protocol for EMS mutagenesis of embryogenic cell suspensions of *Coffea arabica* var. Venecia. Dose-response curves were established within 3–4 weeks and showed LD30 and LD50 values in the range of 0.5% and 0.6% EMS respectively. Methods and media used for development of the treated cell suspensions and conversion to in vitro plantlets are also described.

Joanna Jankowicz-Cieslak, Florian Goessnitzer—Contributed equally.

J. Jankowicz-Cieslak (B) · F. Goessnitzer (B) · I. L. W. Ingelbrecht

Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria e-mail: j.jankowicz@iaea.org

F. Goessnitzer e-mail: f.goessnitzer@iaea.org

# **1 Introduction**

Coffee is one of the most valuable cash crops and provides employment for millions of people worldwide, especially in Latin America and parts of Africa and Asia (FAOSTAT 2021). Coffee belongs to the family *Rubiaceae* and the two main species of cultivated coffee are *Coffea arabica* L. and *Coffea canephora.* Coffee leaf rust (CLR) caused by the airborne fungus *Hemileia vastatrix* and coffee berry disease are among the most important diseases affecting coffee production. *C arabica*  is the most severely affected by leaf rust. Leaf rust epidemic has hit countries in Mesoamerica, including Colombia, Peru, Ecuador, and Guatemala amongst others, in the past decade (Avelino et al. 2015).

Resistant varieties are perhaps the most appropriate means to manage CLR. Improvement of Arabica coffee using conventional cross breeding is challenged by its long juvenile phase and narrow genetic base (Wintgens 2012; Scalabrin et al. 2020). Induced mutagenesis is widely used an efficient method to induce genetic variability useful for genetic studies and breeding. Since the 1970s in vitro tissue culture technologies have been developed for coffee, including methods to regenerate plants from single cells through somatic embryogenesis (see Etienne et al. 2018 for a review). Both direct and indirect methods for somatic embryogenesis in Arabica coffee have been described (Quiroz-Figueroa et al. 2006; Murvanidze et al. 2021 and references therein). Single cells or cell clusters are attractive targets for mutagenesis given the high likelihood for directly yielding chimera-free, homohistont plants. In addition, in vitro systems could offer significant efficiency gains in terms of space and labour compared to greenhouse- or field-based experiments to establish large mutant populations for perennial crops and trees such as Arabica coffee. Here, a fast and reproducible protocol for EMS mutagenesis of embryogenic cell suspensions of Arabica coffee var. Venecia is presented. Protocols for converting the EMS treated somatic embryos into in vitro plantlets are also provided.

### **2 Materials**

### *2.1 Culture Medium*



**Table 1** Media composition (mg/l) for somatic embryogenesis and plantlet regeneration of *Coffea arabica* var. Venecia


# *2.2 Chemical Toxicity Test*


# *2.3 Calculation of Lethal Dose (LD)*


# **3 Methods**

# *3.1 Preparation of Liquid Culture Medium*


# *3.2 Mutagenesis of Coffee Cell Suspensions: Chemical Toxicity Test*


**Fig. 1** Procedure for EMS chemical mutagenesis of in vitro coffee cell suspensions. **a** A 10% EMS stock mixture is prepared under the fume hood. **b** Final dilutions are prepared by adding appropriate volumes of the 10% EMS stock solution into the cell culture maintenance media. **c** The EMS dilutions are aliquoted into 50 ml falcon tubes containing 20 ml cell suspensions, replicates of 3 per treatment are prepared. **d** Cells are incubated for a specific time (here 1 h) under orbital rotation. **e** Shortly before the end of the incubation, falcon tubes containing mutagenized cells are removed from the shaker and put aside to allow cells to settle. **f** The supernatant is carefully decanted, not to lose the pellet. **g**, **h** Washing of mutagenized material with 40 ml maintenance liquid media, repeated at least 3 times. **i** After the washing step, a set volume of culture media is added to the mutagenized material. Tubes are transferred to the in vitro laboratory and 100 µl aliquots of the mutagenized cells are transferred to the regeneration media

# *3.3 Calculation of Lethal Dose (LD)*


**Fig. 2** Example data showing the response of Arabica coffee embryogenic cell suspension treated with different EMS concentrations, observed 3 weeks post EMS treatment. Growth inhibition of 100% was observed for cultures treated with 1.5% and 2% EMS

**Fig. 3** Example of the survival count of cultured coffee cells taken 3 weeks after treatment with EMS. For the control material, 90/90 cultured tubes maintained growth. In the case of treated cultures, 0.2% EMS had similar growth rate to the control, a slight drop is being observed for 0.5% EMS treated cultures (66/90 tubes survived). A clear drop occurred for the material subjected to 0.8% EMS for which only 4 out of 90 cultured cell suspension tubes maintained the growth

**Fig. 4** Survival calculated as percentage of the control, whereby the control is 100%. The kill curve indicates the LD30 (the dose causing the death of 30% of the population) and LD50 (the dose causing the death of 50% of the population) values in the range of 0.5% and 0.6% EMS respectively

# *3.4 Development of Somatic Embryos and Conversion into Plantlets*


**Fig. 5** Conversion of *Coffee arabica* somatic embryos to plantlets, control cultures are shown. **a** The embryogenic cells are cultured on a solid medium for the induction of somatic embryos, **b** torpedo shape embryos are selected and transferred to individual culture tubes for **c**, **d** plantlet development

**Table 2** The averages of torpedo shape embryos and regenerated plantlets per 100 µl cultured volume


Percentage of the control has been also calculated

# **4 Notes**

1. This protocol describes EMS chemical mutagenesis of *Coffea arabica* var. Venecia, a late maturing variety with excellent cup quality. The Venecia coffee variety has its origin in San Carlos, Alajuela where it was discovered on a coffee plantation of 100% Caturra. It was selected due to its increased productivity, larger fruit size and increased resistance to fruit drop in the rain. The procedures described here utilize coffee cell suspensions generated in the Plant Breeding and Genetics Laboratory, Seibersdorf, Austria. For details on establishing the cell suspension culture, see Chap. "Somatic Embryogenesis and Temporary Immersion for Mass Propagation of Chimera-Free Mutant Arabica Coffee Plantlets". Briefly, leaf discs served as the starting material to produce embryogenic callus. The embryogenic callus was then transferred to a liquid medium to establish a homogenous embryogenic cell suspension culture. The cell suspension culture served to maintain and multiply embryogenic cell/cell clusters and was used for EMS treatments. The EMS-treated cultures were regenerated on semi-solid media described here.


It is important that the bottle does not leak. Test the bottle with water first and mimic the shaking procedure in the fume hood.


**Acknowledgements** The authors wish to thank Dr. Noel Arrieta Espinoza, the Coffee Institute of Costa Rica (ICAFE), Costa Rica for providing the seed of *Coffea arabica* var. Venecia. Funding for this work was provided by the Food and Agriculture Organization of the United Nations and the International Atomic Energy Agency through their Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chemical Mutagenesis of Zygotic Embryos of** *Coffea arabica* **L. var. Catuaí Using EMS and NaN3**

**Andrés Gatica-Arias and Jorge Rodríguez-Matamoros** 

**Abstract** The genetic improvement of *C. arabica* L. is challenged by its low genetic diversity and autogamous reproductive biology. Induced mutagenesis offers an alternative approach to conventional cross-breeding to increase genetic variability in wild and cultivated Arabica coffee germplasm for further use in breeding programs and genetic studies. Here protocols are described for the preparation of zygotic embryos from *C. arabica* seed and for toxicity testing of zygotic embryos using two chemical mutagens, sodium azide (NaN3) and ethyl methanesulfonate (EMS). Zygotic embryos were immersed for 10 min in a solution of NaN3 (0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0 and 20. 0 mM) and for 2 h in a solution of EMS (0, 0.5, 1, 1.5, 2, 4 and 6 % v/v). The percentage survival was evaluated and the LD values for NaN3 and EMS were determined at 12.5 mM (51.6%) and 1 % v/v (48.3%), respectively. Our protocols indicate that coffee zygotic embryos are suitable propagules for NaN3 and EMS mutagenesis and expand the types of propagules suitable for induced mutagenesis, breeding and genetic studies in Arabica coffee.

# **1 Introduction**

Coffee is one of the most important products around the world. Global coffee imports in 2021–2022 amounted to 133.59 million 60-kg bags with a global market value of US \$107.93 billion in 2021, taking second place in international trade after crude oil. Coffee is cultivated in more than 80 countries in tropical and subtropical regions of the globe, especially in Africa, Asia, and Latin America. Coffee production generates directly or indirectly income to more than 100 million people around the world (Mishra and Slater 2012).

e-mail: andres.gatica@ucr.ac.cr

A. Gatica-Arias (B) · J. Rodríguez-Matamoros

Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, 2060, San Pedro, Costa Rica

Approximately 60% of the world coffee production is derived from *C. arabica*  L. because of its superior quality, aromatic characteristics, and low caffeine content compared to Robusta coffee (Mishra and Slater 2012; Alpízar 2014; Ahmed et al. 2013).

The cultivated varieties of *C. arabica* L. are derived from the "Typica" or "Bourbon" coffee lineages, resulting in low genetic diversity (Mishra and Slater 2012). The reproductive biology of Arabica coffee, being approximately 90% autogamous, along with historical and geographic data indicating the occurrence of genetic bottlenecks due to domestication and global spread from its center of origin in Ethiopia and a polyploidization event are responsible for the low genetic variation in Arabica coffee (Romero et al. 2010; Mendonça 2014; Scalabrin et al. 2020; Montagnon et al. 2021). Consequently, *C. arabica* varieties are often highly susceptible to different diseases and pests (Romero et al. 2010).

Induced mutagenesis offers a promising alternative to improve current coffee cultivars for enhanced tolerance to pathogens, as previously shown in other crops (Gressel and Levy 2006). Induced mutagenesis using chemical or physical mutagens typically introduces random changes throughout the genome and can thus generate a variety of mutations within a single plant. As opposed to cross-breeding, induced mutagenesis can be applied to a wide variety of plant propagules.

Ethylmethanesulfonate (EMS) is today the most widely used chemical mutagen. It selectively adds alkyl groups to guanine bases causing random point mutations; most of the changes (70–99%) are base-pair transitions from G/C to A/T (Jankowicz-Cieslak et al. 2016; Sikora et al. 2011). Another chemical mutagen is sodium azide (NaN3), whose mutagenic effect is mediated through the production of an organic metabolite of the azide compound: l-azidoalanine. It creates point mutations, mostly transitions, of the type: G/C to A/T or vice versa (Prina et al. 2010; Srivastava et al. 2011).

This chapter describes protocols for the preparation and mutagenic treatment of zygotic embryos of *C. arabica* L. var. Catuaí using the chemical mutagens sodium azide and ethyl methanesulfonate, including methods for bulk treatment, germination, and development of mutant plantlets.

### **2 Materials**

### *2.1 Plant Material*

1. *Coffea arabica* L. var. Catuaí seeds (see Note 1).

### *2.2 Reagents*


# *2.3 Glassware and Minor Equipment*


# *2.4 Equipment*


# *2.5 Software*

1. Standard spreadsheet software (e.g., Microsoft Excel or Open Office Excel).

# *2.6 Bulk Mutagenesis of Zygotic Embryos*

All materials as listed in Sects. 2.1, 2.2, 2.3, 2.4, and 2.5.

# **3 Methods**

# *3.1 Preparation of Stock Solutions*


# *3.2 Preparation of Tissue Culture Medium*

	- For 1 L of germination medium (EG): full-strength MS salts, 1.0 ml BAP, 1 ml GA3, and 300 mg/l activated charcoal, and 2.5 g Phytagel™.
	- For 1 L of development medium (DEV): full-strength MS salts 1 ml thiamine HCl, 1 ml pyridoxine HCl, 1 ml nicotinic acid, 1 ml calcium pantothenate, 0.01 ml biotin, 100 mg myo-inositol, 0.3 ml BAP, 30 g sucrose, 2.5 g Phytagel™.

# *3.3 Seed Disinfection and Excision of Zygotic Embryos*


**Fig. 1** Schematic representation of the mutagenesis of coffee (*Coffea arabica* L. var. Catuaí) zygotic embryos. **a** For chemical mutagenesis and toxicity testing, normal shaped, disease-free seeds are selected and NaN3 and EMS dosage and incubation time are optimized using seed lots with a germination rate equal to or above 90%. These steps take approximately 8 weeks. **b** Bulk irradiation of zygotic embryos. For both **a** and **b**, the zygotic embryos are prepared by manually removing the pulp, the mucilage, and the parchment of the seed, disinfecting the seeds, excising the zygotic embryo, and incubating the zygotic embryos using the appropriate NaN3 or EMS concentration followed by incubating, rinsing and planting the mutagenized zygotic embryos


### *3.4 Determination of the Viability of the Zygotic Embryos*


**Fig. 2** Germination and viability test of zygotic embryos of coffee (*Coffea arabica* L. var. Catuaí). **a** Excised zygotic embryos germinated under in vitro culture conditions. **b** TTC viability test

# *3.5 Sodium Azide Dose Determination*



**Table 1** Concentrations chosen for the toxicity test in coffee seeds

a Prepare a fresh solution immediately before the experiment


# *3.6 Ethyl Methanesulphonate Dose Determination*


**Fig. 3** Effect of NaN3 concentration on survival and viability of coffee (*C. arabica* L*.* var. Catuaí) zygotic embryos. **a** Survival percentage (solid line) versus NaN3 concentrations. Each value represents the mean ± SD of three repetitions. **b** Zygotic embryo viability versus NaN3 concentrations. Zygotic embryos that stain red are considered viable. *Bar*, 1.0 cm



**Table 2** Concentrations chosen for the toxicity test in coffee zygotic embryos

**Fig. 4** Effect of EMS concentration on survival/viability of coffee (*C. arabica* L*.* var. Catuaí) zygotic embryos. **a** Survival percentage (solid line) versus EMS concentrations. Each value represents the mean ± SD of three repetitions. **b** Zygotic embryo viability versus EMS concentrations. Red stained zygotic embryos were considered as viable. *Bar*, 1.0 cm

# *3.7 Bulk Mutagenesis*


# *3.8 Acclimatization of M1 Plantlets*


# **4 Notes**


**Acknowledgements** Funding for this work was provided by the University of Costa Rica, the Ministerio de Ciencia, Innovación, Tecnología y Telecomunicaciones (MICITT), the Consejo Nacional para Investigaciones Científicas y Tecnologicas (CONICIT) (project No. 111-B5-140; FI-030B-14) and the Cátedra Humboldt 2023. A. Gatica-Arias acknowledged the Cátedra Humboldt 2023 of the University of Costa Rica for supporting the dissemination of biotechnology for the conservation and sustainable use of biodiversity.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Induced Mutagenesis of Seed and Vegetative Propagules of** *Coffea arabica* **L.**

# **Physical Mutagenesis of Arabica Coffee Seeds and Seedlings**

**Abdelbagi Mukhtar Ali Ghanim, Souleymane Bado, and Keji Dada** 

**Abstract** Coffee, a perennial tropical crop, can be grown from seed or from cloned plants in the form of cuttings, grafts or tissue cultured plants. Arabica coffee is most commonly grown from seeds while Canephora is mostly grown vegetatively from cuttings and other propagules. Improving Arabica coffee through conventional breeding is seriously limited by the lack of genetic variation within the cultivated and wild species. Mutation breeding provides great potential to induce the novel genetic variation needed for Arabica coffee improvement. Here we present protocols for mutation induction of coffee seed and seedlings using the FAO/IAEA in-house gamma (60Cobalt) and X-ray (RS2400 irradiator) sources. Methods for mutation induction using gamma- and X-ray mutagenesis techniques are described. Methods for the preparation, seed quality control and post-radiation treatment of materials are also provided along with example data for radio-sensitivity testing of *Coffea arabica*  seed under laboratory conditions.

# **1 Introduction**

Coffee is a perennial crop belonging to the genus *Coffea* in the family *Rubiacea*. There are about 125 species within this genus, with *Coffea arabica* and *Coffea canephora*, representing approximately 70% and 30% of coffee production, respectively (Lashermes et al. 2008). Arabica coffee is a tetraploid and self-pollinating (autogamous), while Robusta coffee is a diploid and allogamous (Wintgens 2004). The efficiency of traditional Arabica coffee breeding approach is greatly reduced by the lack of sufficient genetic diversity and the long time needed for coffee to flower and bear

A. M. A. Ghanim (B) · S. Bado

Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria e-mail: abdmali@yahoo.com

K. Dada Plant Breeding Unit, Cocoa Research Institute of Nigeria (CRIN), Ibadan, Oyo State, Nigeria

fruits (Scalabrin et al. 2020). Mutation breeding provides great potential to induce the novel genetic variation needed for Arabica coffee improvement.

Coffee can be grown from seed or from cloned plants in the form of cuttings, grafts or tissue cultured plants. Arabica coffee is most commonly grown from seeds while Canephora is mostly grown vegetatively from cuttings and other propagules. Despite widely reported spontaneous mutants of Arabica coffee, there are very few studies on induced mutagenesis in coffee. The first attempt to induced mutation breeding of *C. arabica* was reported by Carvalho et al. (1954) using X-ray irradiation. The process of optimizing dose involves dose-response experiments where the pattern of reduction in germination (Lethal Dose, LD) or growth rate (Growth Reduction, GR) is determined in relation to an increase of absorbed dose. From these experiments, the LD/GR30 and LD/GR50 is calculated. In case of coffee after adjustment trials of seed and vegetative part using our in-house gamma and X-ray irradiators, we came to a range of (0, 50, 100, 150, 200, 400 Gy) for *C. arabica* seeds and (0, 5, 10, 15, 20, 30 and 40 Gy) for seedlings and cuttings of *C. arabica* and *C. canephora*. The protocol for seed treatment follows the general procedure which starts with sorting clean and viable seeds, moisture equilibration in a desiccator with 60% glycerol, irradiation treatments, planting the treated material in suitable set-up such as moist filter papers in petri-dish, soil in trays or pots and incubate at appropriate condition under warm condition 28–30 °C. Germination or growth rate after 30 days is recorded and plotted relative to the untreated seeds over the series of the doses. From the plotted graph the doses for LD50, GR50 and LD30, GR30 are estimated. The same follows for vegetative propagules (cuttings, seedling, embryo etc.) except that the applied doses here are relatively low in the order of 0–40 Gy. The estimated dose can be used for bulk treatment. Induced mutations are random events, implying that even adherence to published irradiation conditions might not result in the same mutation events. A way of overcoming this is to work with large populations. In case of seed, it is generally recommended to target the production of an M2 population of a minimum of 5,000–10,000 individuals.

In this chapter, step-by-step procedures for seed quality control, irradiation treatment, and radiosensitivity testing of seed and seedlings is described. Example LD/ GR values resulting from radiosensitivity testing of seed germination and growth rate under laboratory conditions are presented.

### **2 Materials**


# **3 Methods**

# *3.1 Seed Treatment*

### **3.1.1 Preparation and Quality Control of Seed**


**Fig. 1** Viability test of the seed lot by germination test on moist filter paper in petri dish (**a**), viable seed lot is divided into number of samples equal to the planned irradiation treatment, seed sample for each treatment is placed in small paper bag and labelled (**b**), bags with similar treatment are grouped in a larger bag and placed in a desiccator with 60% glycerol for moisture equilibration (**c**), after moisture equilibration seeds are ready for irradiation (**d**)


### **3.1.2 Gamma and X-ray Irradiation Treatment**

The in-house Cobalt gamma-ray Cell and RAD-Source (RS 2400) was used for gamma-ray and X-ray irradiation treatment respectively, using previously described procedures (Spencer-Lopes et al. 2018). Briefly, the RS 2400 X-ray machine source

**Fig. 2** Post treatment handling and radiosensitivity testing of irradiated seed. Steps of water soaking of treated seeds at 28 °C and 12 h photoperiod in incubator for 3–7 days, transfer to either petri dishes or soil for monitoring and recording germination (viability) and growth responses (growth reduction) of the irradiated seeds

is an upgraded irradiator for research and industry used by the FAO/IAEA Insect Pest Control Laboratory for the Sterile Insect Technique (Mastrangela et al. 2010; Mehta and Parker 2011) and adapted for mutation induction of plant propagules by the FAO/IAEA Plant Breeding and Genetics Laboratory. It is important in X-ray irradiation that samples are tightly packed to minimize air space and to maintain a near uniform field of X-rays through the entire sample. The dose rate of the RS2400 X-ray is about 12 Gy/min. After irradiation, the seeds and resulting plants are known as M1 stage.

### **3.1.3 Post-treatment Handling and Radio-Sensitivity Testing**

Viability and Growth Rate Testing of the M1 Seeds in Petri Dish in an Incubator


Viability and Growth Rate Testing of the M1 Seeds in Soil in the Glasshouse

Repeat steps 1 and 2 as described above under Sect. 3.1.3. Then, follow below steps:


# *3.2 Coffee Seedlings Treatment*



# *3.3 Example Radio-Sensitivity Testing of Arabica Coffee Seed Under Laboratory Conditions*

Seed from three *C. arabica* varieties (Kent, Geisha and Mundo Novo) from the Cocoa Research Institute of Nigeria were freshly harvested and sent within less than one month to the FAO/IAEA PBG Laboratory, Austria. Upon arrival, the seeds were immediately inspected, cleaned, and used for gamma and X-ray radiosensitivity testing as described above. Germination was scored using the laboratory-based procedure about 2 weeks after treatment and data analysed to estimate LD30 and LD50 values as well as measurement of seedling growth rate relative to the untreated seeds (control) to estimate GR30 and GR50 values (Note 4). The viability of the seeds of

**Fig. 4** Radio-sensitivity testing based on germination percent of the M1 seeds using gamma-ray (**a**) and X-ray (**b**) in-house irradiation sources in three *C. arabica* varieties. As shown, for gamma treatment, the LD30 ranged between 38 and 75 Gy, compared to 16–52 Gy for the X-ray treatment, while the LD50 ranges between 118 and 150 Gy for gamma, and between 105 and 136 Gy using X-ray

**Table 1** Comparing LD30 and LD50 using the FAO/IAEA in-house gamma and X-ray sources and their relative biological effect (RBE) in three *C. arabica* coffee varieties


the three coffee varieties exceeded 95% of germination on moist filter paper after soaking in warm water for 3 days.

As show in Fig. 4, the LD30 ranged between 38 and 75 Gy among the three varieties for gamma treatment, compared to 16–52 for the X-ray, while the LD50 ranged between 118 and 150 Gy for Gamma and between 105 and 136 using X-ray (Table 1).

The relative biological effect (RBE) of gamma to X-ray ranged from 0.21 to 0.74 for LD30 and from 0.7 to 0.95 for LD50 indicating that Gamma and X-ray were relatively closer in their effect in LD50 (Table 1).

### **4 Notes**

1. When performing a radio-sensitivity test 15–20 seeds are placed in paper bag per replication (3 replications). In case of X-ray irradiation, bags of the same dose are rolled together with the seed well distributed at the bottom and then placed in the center of the container to ensure uniform radiation. The remaining space is filled with instant rice for vacuum establishment. For bulk irradiation large amounts of seeds are used and these may be placed in an appropriate container for uniform irradiation without the use of a filler.


**Acknowledgements** The protocols and findings described here were developed at the FAO/IAEA PBG Laboratory in the context of an Internship for Mr. Keji Dada, Cocoa Research Institute of Nigeria (CRIN), Ibadan, Nigeria. Seeds of the different *C. arabica* varieties used in this study were provided by CRIN.

### **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Mutation Induction in** *Coffea arabica* **L. Using in Vivo Grafting and Cuttings**

**Weihuai Wu, Xuehui Bai, Kexian Yi, Xing Huang, Chunping He, Jinhong Li, Hongbo Zhang, Hua Zhou, Thomas Gbokie Jr, Tieying Guo, and Jingen Xi** 

**Abstract** Coffee leaf rust (CLR) caused by the obligate parasite, the biotrophic *Hemileia vastatrix* Berk. & Broome (Basidiomycetes: Pucciniales), is the most devastating disease of *Coffea arabica* L. Breeding resistant varieties is one of the most economic and environment friendly means to control the disease. However, this is challenged by the loss of resistance after a short period in commercial production. Catimor CIFC7963, an elite, leaf rust resistant *Coffea arabica* L. variety, has been cultivated in China for decades, which has resulted in the breakdown of its disease resistance. Due to the lengthy breeding process of coffee, the development of new resistant varieties is arduous. Physical and chemical mutagenesis offers an alternative means to more rapidly create novel and beneficial genetic variations. Bud grafting is a propagation technique frequently used for woody plants whereby a bud of one plant is attached to the rootstock of another plant. Likewise, cutting is a frequently used propagation technique. In coffee, physical irradiation of the bud followed by grafting or cutting can accelerate the mutation breeding process, as cutting or grafting increases the growth rate without affecting the major traits of the background varieties. Here, we present protocols to induce mutations on buds of the *C. arabica* variety Catimor CIFC7963 by gamma-ray irradiation and their subsequent propagation through cutting or bud grafting.

W. Wu · K. Yi (B) · X. Huang · C. He · J. Xi

Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China e-mail: yikexian@126.com

X. Bai · J. Li · H. Zhang · H. Zhou · T. Guo Dehong Institute of Tropical Agriculture, Ruili 678600, Yunnan, China

T. Gbokie Jr College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

# **1 Introduction**

Coffee is one of the most important beverages in the world. The genus *Coffea* has over 70 different species. Of these, only two species, namely *Coffea arabica* (Arabica) and *C. canephora* (Robusta) dominate global coffee production. *C. arabica* is a dominant species with a demand for genetic improvement, especially for disease resistance such as coffee leaf rust (CLR) (Tran et al. 2018). CLR caused by the fungus *Hemileia vastatrix* is one of the most important diseases of *C. arabica*.A few rust-resistant varieties have been developed, *e.g.* Oeiras MG 6851 (Pereira et al. 2000), however, the resistance was soon broken down by the newly emerged race XXXIII of *H. vastatrix* (Capucho et al. 2012). Thus, widening the genetic base would be key for developing sustainable disease resistance in coffee, given that Coffee-*H. vastatrix* rust interactions follow the gene-for-gene relationship (Flor 1942). Transcriptome and proteome methods have been used to identify leaf rust resistance genes (Guerra-Guimarães et al. 2015; Florez et al. 2017). While various *H. vastatrix*  effector candidate genes (*HvECs*) have been reported, the knowledge of leaf rust resistance genes in coffee (*SH 1-SH 9*) is still limited (Maia et al. 2017). There is still insufficient knowledge to develop functional markers for marker-assisted selection for CLR resistance. Mutation breeding seems to be a promising approach for coffee improvement to help address the leaf rust resistance breakdown due to rapidly evolving *H. vastatrix* races.

Compared to conventional breeding techniques such as traditional crossing, induced mutagenesis can more efficiently generate novel variations and introduce new traits (Harten 1998). Gamma irradiation has proven to be effective in improving important agronomic traits such as yield, quality and disease resistance. The FAO/ IAEA Joint Centre has significantly promoted the application of mutation breeding in agriculture, which generated more than 3400 varieties in 210 plant species and commercially planted in more than 70 countries (https://mvd.iaea.org/).

It takes over three years for *C. arabica* from planting until fruit production. This makes its propagation and breeding an extremely lengthy process. For tree crops, grafting offers an efficient way to propagate and maintain elite germplasm (Parlak 2017). Grafting technique has already been successfully applied in coffee for preventing damage by root-lesion nematodes (Villain et al. 2000). In addition, cutting is also widely used to propagate tree plants for maintaining outstanding traits. Cutting is also applied for clonal propagation of coffee plants, which is more stable than seed propagation for maintaining yield traits (Priyono et al. 2010). The survival rate of cutting is influenced by environmental factors such as exogenous phytohormones, humidity, temperature, etc. It results in a lower survival rate compared to grafting. On the other hand, cutting is a simpler method than grafting as it does not require a rootstock. Here, we present a protocol for gamma irradiation of coffee in combination with grafting and cutting techniques.

# **2 Materials**

# *2.1 Grafting*


# *2.2 Cutting*


# **3 Methods**

# *3.1 Mutagenesis and Propagation of Coffee Through Bud Grafting*

# **3.1.1 Preparation of Straight Branches**


**Fig. 1** Grafting procedure. **a** Straight shoot; **b** the floral foam for arrangement of the coffee shoots; **c** irradiation; **d** preparation of the rootstock; **e–g** grafting; **h** successful graft

### **3.1.2 Mutagenic Treatment**


### **3.1.3 Preparation of the Rootstock**


### **3.1.4 Preparation of the Scion**

1. Take the irradiated shoots, retain the green unpinched cork with 1–2 buds on the top by cutting off the lower part.

2. Cut the scion with a grafting knife by two chops. The length of the two cutting faces should be about 3 cm, each side should be uneven with the other side in length as well as thickness. The width of the cutting side should be slightly narrower or equal to the diameter of the rootstock (Fig. 1f).

### **3.1.5 Grafting Method**


# **3.1.6 Post-grafting Management**


# **3.1.7 Survival Rate Statistics**

1. One to two months after grafting, the successful graft is identified by the presence of emerged leaves on the scion and healing of the contact point between the scion and rootstock (*see* Note 6 and Fig. 1h).

# **3.1.8 Unbundling**

1. Remove the wrapping tape when the scion tip is over 10 cm long and the interface is completely healed.

# *3.2 Mutagenesis and Propagation of Coffee Through Cuttings*

### **3.2.1 Preparation of Straight Branches**


**Fig. 2** Cutting procedures. **a** Straight shoot; **b** arrange coffee shoots in the floral foam; **c** irradiation; **d** 0.1% difenoconazole sterilizing solution; **e** NAA solution treatment; **f** stick cutting into the most rooting substrate; **g–h** set up wire support and cover with plastic bag to maintain moisture and humidity; **i** shading; **j** established cutting

### **3.2.2 Mutagenesis**


### **3.2.3 Preparation of the Cutting Substrate**


# **3.2.4 Sterilization**


# **3.2.5 Cultivation**


# **3.2.6 Survival Rate Statistics**

1. After 6 months, successful cuttings are identified by the newly emerging leaves and the presence of fibrous roots (Fig. 2j).

# **4 Notes**


**Acknowledgements** Funding for this work was provided by the FAO/IAEA Coordinated Research Project (Contract No. 20380), and the International Exchange and Cooperation Project funded by the Agricultural Ministry Construction of Tropical Agriculture Foreign Cooperation Test Station and Training of Foreign Managers in Agricultural Going-Out Enterprises (SYZ2019-08) and the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630042017021).

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chemical Mutagenesis of Mature Seed of** *Coffea arabica* **L. var. Venecia Using EMS**

**Joanna Jankowicz-Cieslak, Florian Goessnitzer, Stephan Nielen, and Ivan L. W. Ingelbrecht** 

**Abstract** Chemical mutagens are a major tool to generate novel genetic variation in crops, functional genomics and breeding. They are advantageous because they do not require any specialized equipment and can induce a high mutation frequency. Compared to physical methods, chemical mutagens cause point mutations rather than deletions or translocations. Point mutations can have varying effects on gene expression ranging from knockouts to changes in amino acids that may have subtle effects on protein function. Many important gene functions have been uncovered by evaluating the in vivo effect of mutated genes in a broad range of model and crop plants. Chemical mutagens have been successfully applied to induce tolerance to fungal diseases in cereals such as barley and wheat. Among the chemical mutagens used for plant mutagenesis, ethyl methanesulfonate (EMS) is the most widely applied. This protocol chapter describes the utilization of EMS for establishing kill curves and generating a mutagenized population of *Coffea arabica* var. Venecia via treatment of mature seed. The different steps of the mutagenesis process are described, including quality control and preparation of the seed batches, procedures for determining Lethal Dose (LD) and Growth Reduction (GR) values, and for post-treatment handling of the chemically mutagenized seed, specific for Arabica coffee.

# **1 Introduction**

Mutation breeding has proven to be an efficient tool to develop improved crop varieties whereby various novel traits such as e.g., yield, growth, and disease resistance can be induced (Spencer-Lopes et al. 2018). Among various mutation induction techniques, mutagenesis using chemical agents has become an efficient and

J. Jankowicz-Cieslak (B) · F. Goessnitzer · I. L. W. Ingelbrecht

Plant Breeding and Genetics Laboratory, Vienna International Centre, Joint FAO, IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna, Austria e-mail: j.jankowicz@iaea.org

S. Nielen EMBRAPA Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil

robust tool to induce point mutations (Ingelbrecht et al. 2018; Jankowicz-Cieslak and Till 2016). Mutations in coding regions of genes can be silent, missense, or nonsense, while mutations can equally occur in non-coding, regulatory sequences affecting gene expression, e.g., at intron splice sites. Among the different chemical mutagens, ethyl methanesulfonate (EMS) has been frequently used to induce random mutations because it is highly effective and relatively easy to handle. To date, mutation induction using EMS has been described for a wide range of plant species covering both seed and vegetatively propagated plants (Jankowicz-Cieslak et al. 2011; Jiang et al. 2022). Chemical mutagens have been successfully applied to induce tolerance to fungal diseases in cereals such as powdery mildew-resistance in barley (Molina-Cano et al. 2003) and resistance to leaf rust *Puccinia* sp. in wheat (Mago et al. 2017).

The workflow for chemical mutagenesis involves choosing the genotype and the appropriate tissue type to be mutated, the mutagen, and optimizing the treatment conditions and dosage. Once the optimal dose(s) have been determined, bulk mutagenesis can proceed. EMS mutagenesis of seed is widely used in diploid, self-pollinating cereals and legumes, amongst other plant species. Briefly, seed are pre-treated by soaking in water, EMS is added, and mutations are induced during a specific treatment period. Following this, seed are thoroughly washed and sown. This results in the M1 generation which can harbor a high density of induced point mutations.

Growth and survival measurements remain the simplest route to dosage optimization and have the added appeal that they can be applied in almost any facility as little infrastructure or expertise are required. A reduction of growth rate in the early seedling stage, typically two weeks for small-seeded cereals, serves as an easy indicator (Jankowicz-Cieslak and Till 2016; Mba et al. 2010). A range between 30 and 50% growth reduction is typically chosen as optimal dose for bulk irradiation in cereals. As with any mutagenesis treatment, a compromise needs to be made between achieving a sufficiently high mutations frequency to have a reasonable chance to recover the desired mutations and suitable level of survival and fecundity (Jankowicz-Cieslak and Till 2015). It is advisable to use up to three doses of the mutagen, corresponding to ± 20% of the optimal dose found through the toxicity test.

For chemical mutagens such as EMS, both the concentration and duration of the treatment are evaluated during dose optimization. Different treatments are tested, and germination and survivability as well as growth reduction typically measured. Where possible, embryo lethality in the M2 seed can be used as an indicator for the efficiency of mutagenesis (Till et al. 2003). There are additional issues with chemical mutagenesis that one needs to consider. Cytotoxicity may limit the efficacy of specific mutagens for certain plant species or genotypes, necessitating trials with different mutagens (Till et al. 2007). Following the mutagenesis procedure, M2 mutant populations can be evaluated for phenotypic or genotypic variation distinct from the non-mutagenized parental line.

The advantages and limitations of different propagules for mutagenesis treatment of Arabica coffee are briefly described in Chap. Mutation Breeding in Arabica Coffee. Since Arabica coffee is self-fertilizing and mostly propagated through seeding, seed can serve as starting material for mutagenesis treatments. As with other seed crops, M1 chimeric plants are expected which can be resolved through successive cycles of selfing (Mukhtar Ali Ghanim et al. 2018). In this chapter, the susceptibility of Arabica coffee seed var. Venecia to the chemical mutagen EMS was evaluated and optimal doses for EMS bulk irradiation were determined. Methods of evaluation of mutagenic effect at the seedling stage are presented. Further, example data on morphological and chlorophyl variegations observed at the M1 stage are shown.

# **2 Materials**

# *2.1 Chemical Toxicity Test*


# *2.2 Calculation of Lethal Dose (LD) and Growth Reduction (GR)*


# *2.3 Bulk Mutagenesis*


# *2.4 Phenotyping*


# **3 Methods**

# *3.1 Seed Quality Control and Pre-treatment*


**Fig. 1** Overview of EMS mutagenesis of mature coffee seed. The procedure involves three steps: (i) quality control and pre-treatment of the seeds, (ii) seed mutagenesis; (iii) post-treatment handling. **a** Ahead of chemical mutagenesis high quality coffee seeds are selected that are uniform, homozygous and isogenic, while discoloured, small or damaged seeds are discarded. **b** After removal of the parchment and the silverskin, the seeds are pre-soaked in sterile, distilled water for 48 h at room temperature. **c** After incubation, the water is decanted from the seed batches. **d** Seeds are placed into labelled beakers for treatment. A dilute mixture of EMS plus DMSO is prepared and stored under the fume hood. **e** The seeds are incubated for a specific time under orbital rotation. **f** The mutagen is removed, and seeds are thoroughly washed, minimum three times. **g** Coffee seeds are then transferred to the glasshouse and immediately planted in a light soil


# *3.2 Coffee Seed Mutagenesis*


**Table 1** Example calculations for solutions containing different EMS concentrations and 2% DMSO at a final volume of 1 L used for mutagenic treatment of var. Venecia



# *3.3 Post-treatment Handling of Seeds*


**Fig. 2** Coffee plants grown in the FAO/IAEA Plant Breeding and Genetics Laboratory, Seibersdorf, Austria. **a** Mutagenized coffee seeds are sown in multiwell trays filled with light soil immediately after EMS treatment. **b** Seven months post-mutagenesis, plants are transplanted to bigger pots

**Fig. 3 a** Coffee seed are germinated in the glasshouse and the germination date as well as germination rate is scored. **b** At the seedling stage (*e.g.,* 104 days post-mutagenesis) survival count is taken along with hypocotyl/seedling height and number/dimension of leaves. **c** At the plant stage (*e.g.,*  208 days post-mutagenesis) the survival rate, plant height, number of leaves and leaf morphology and other variegations are recorded

**Fig. 4** Coffee seed germination scored for estimation of 50% lethality (LD50): **a** Mutagenized materials are first grown, and the germination date is registered which serves as the basis for the calculation of the Days to Germination values (DTG). **b** The germination of EMS treated seed occurred between 34 and 63 days after sowing. Here the average of DTG values for each treatment is displayed which ranges between 35 days for the control and 0.5% EMS and 48 days for 4% and 6% EMS. **c** The number of germinated seed was recorded at the stage of the seedling emerging from the soil. **d** Nearly 80% of planted control seeds emerged. Germination was visibly enhanced for 4 EMS treatments (0.2% up to 2%) and then dropped to 69% at 4% EMS and to 17% at 6% EMS

# *3.4 Calculation of Lethal Dose (LD): Data Collection and Analyses*


# *3.5 Bulk Mutagenesis*


**Fig. 5** Example of chemical toxicity test performed on the coffee seed. Coffee seed are exposed to 6 concentrations of EMS (0.2; 0.5; 1, 2, 4 and 6% EMS) plus the control and incubated for 2 h. Here, the percentage germination in relation to the control (100%) was plotted for each EMS concentration (blue line). The germination was recorded between 34 and 63 days after treatment. Note that germination is enhanced for 0.2, 0.5, 1 and 2% compared to the control, whereby a clear drop is visible for 4% and 6% EMS. Same for the survival scored 104- (orange line) and 208-days (grey line) post-mutagenesis, it drops as EMS concentration increases

**Fig. 6** Examples of coffee leaf phenotypes scored 8 months post-mutagenesis for a nonmutagenized control (labelled as 'control') and mutant plants (no labels)

# *3.6 Phenotyping*


# **4 Notes**


**Acknowledgements** The authors wish to thank Dr Andrés Gatica-Arias, Universidad de Costa Rica, San Pedro, Costa Rica for guidance on coffee germination and growth, Dr Noel Arrieta Espinoza, The Coffee Institute of Costa Rica (ICAFE), Costa Rica for providing the coffee seed and Mr Islam Tazirul for technical support during these studies. Funding for this work was provided by the Food and Agriculture Organization of the United Nations and the International Atomic Energy Agency through their Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Chemical Mutagenesis of Coffee Seeds (***Coffea arabica* **L***.* **var. Catuaí) Using NaN3**

**Andrés Gatica-Arias and César Vargas-Segura** 

**Abstract** Coffee (Coffea arabica L.) is one of the most important crops in the world and one of the main export products in several developing countries. Coffee is a perennial crop threatened by multiple, serious diseases and pests. Induced mutagenesis of seeds is widely used for increasing the genetic diversity and improvement of annual seed crops and could equally be applied to Arabica coffee breeding and genetic studies. Here we describe protocols to induce genetic variability in Arabica coffee seeds through mutagenesis using sodium azide (NaN3). Methods for NaN3 chemical toxicity testing and bulk irradiation are described. Briefly, the coffee seeds were immersed for 4, 8 and 12 hours in a NaN3 solution at different concentrations (0, 25, 50, 75, and 100mM). Two controls were used: one with distilled water and the other with the phosphate buffer (KH2PO4). Effects of the chemical mutagen on seed germination, seedling height, and root length were evaluated. As the concentration of applied NaN3 increased, the germination, seedling height, and root length decreased. Eight hours exposure was determined as an adequate immersion time. The LD50 values for NaN3 were between 50–75 mM. Our results indicate that NaN3 is an effective mutagen for Arabica coffee seeds and can be applied to coffee breeding and to study gene function in coffee.

# **1 Introduction**

Mutations can be induced in plants by exposure of seeds or meristematic cells, tissues, and organs, to both physical and chemical agents with mutagenic properties (Mba et al. 2010). Chemical mutagenesis is the exposure of plant material to chemical agents such as alkylating agents and azides under optimized doses. The mutagenic effect of sodium azide (NaN3) is mediated through the creation of an organic intermediate (*L-azidoalanine*) which incorporates the azide group and interacts with DNA to mainly produce simple base substitutions (Gruszka et al. 2012). Sodium azide's

e-mail: andres.gatica@ucr.ac.cr

A. Gatica-Arias (B) · C. Vargas-Segura

Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, 2060 San Pedro, Costa Rica

<sup>©</sup> The Author(s) 2023

I. L. W. Ingelbrecht et al. (eds.), *Mutation Breeding in Coffee with Special Reference to Leaf Rust*, https://doi.org/10.1007/978-3-662-67273-0\_13

mutagenic effect greatly depends upon the acidity of the working solution, which must be prepared at low pH values (Gruszka et al. 2012).

Reducing the time required to develop improved plants through mutation breeding is a desirable characteristic, especially in long-life cycle plant species such as coffee. Coffee cultivation has great socio-economic impact; it is positioned as the world's second most exported commodity, only surpassed by oil (FAOSTAT 2018). The better cup quality and higher market value are associated to *Coffea arabica* L., the only allotetraploid (2n = 4x = 44) species among Coffea. Arabica coffee is an autogamous plant mostly incompatible with the remainder of Coffea species (Anthony et al. 2002). These characteristics, along with severe bottlenecks that happened during coffee domestication led to reduced genetic variability in *C. arabica* populations; this reduction enhances the general susceptibility of many *C. arabica* genotypes to diseases (Hendre and Aggarwal 2007). Conventional breeding faces limitations due to the long-life cycle of the coffee plant, requiring nearly 30 years to develop new cultivars.

This chapter describes the application of the chemical mutagen sodium azide for mutagenesis of *C. arabica* seeds as well as the germination, and development of mutant plantlets.

### **2 Materials**

### *2.1 Plant Material*

1. All experiments were conducted using seeds from *Coffea arabica* L. var. Catuaí (*see* Note 1).

### *2.2 Reagents*


# *2.3 Glassware and Minor Equipment*


# *2.4 Equipment*


# *2.5 Software*

1. Standard spreadsheet software (*e.g.* Microsoft Excel or Open Office Excel).

# **3 Methods**

# *3.1 Preparation of Stock Solutions*


# *3.2 Seed Germination Test*


**Fig. 1** Schematic representation of the process of mutagenesis of coffee seeds. **a** In advance of chemical mutagenesis, the NaN3 concentration and incubation time are optimized using seeds having germination rate close to or above 90% (preferably normal shaped seeds that are diseasefree). These steps take approximately 8 weeks. **b** bulk mutagenesis of coffee seed is a 6 steps procedure that consists of the seed sorting and selection, processing (manual removal of the pulp, the mucilage, and the parchment), disinfection of seeds, incubation of seeds with the appropriate NaN3 concentration, post-treatment washing and planting of mutagenized seeds

# *3.3 Sodium Azide Toxicity Test and Dose Optimization*



**Table 1** NaN3 concentrations and incubation time evaluated for the toxicity test of Arabica coffee seeds

\* Prepare a fresh solution prior to each experiment

**Fig. 2 a** Germination percentage, **b** hypocotyl emergence percentage, **c** aerial length (cm), and **d** root length (cm) of the arabica coffee plantlets 8 weeks after incubation of seeds treated with different concentrations of NaN3 and immersed for 4, 8, and 12 h. Each value represents the mean ± SD of four repetitions


### *3.4 Bulk Mutagenesis*


**Fig. 3** Effect of NaN3 treatment on germination of coffee seeds 8 weeks after incubation. Different concentrations of NaN3 (25, 50, 75, and 100 mM) and incubation times (4, 8, and 12 h) were used. *Bar*, 1 cm


# *3.5 Planting Mutagenized M1 Seedling*


# **4 Notes**


7. Lab safety precautions: read the Materials Safety Data Sheet (MSDS) of materials being used and follow the recommendation of the manufacturer. Pay careful attention to the information on sodium azide and what to do in case of exposure. It is very important to wear personal protective clothing: gloves must be compatible with chemical mutagens, for instance PVC or neoprene gloves; safety glasses with side shields or chemical goggles; lab coat, closed-toe shoes, shoe protections, and full-length pants. A double glove system is advised.

**Acknowledgements** Funding for this work was provided by the University of Costa Rica, the Ministerio de Ciencia, Innovación, Tecnología y Telecomunicaciones (MICITT), the Consejo Nacional para Investigaciones Científicas y Tecnologicas (CONICIT) (project No. 111-B5-140; FI-030B-14). A. Gatica-Arias acknowledged the Cátedra Humboldt 2023 of the University of Costa Rica for supporting the dissemination of biotechnology for the conservation and sustainable use of biodiversity.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Mutant Phenotyping and CLR Resistance Screening Methods**

# **Use of Open-Source Tools for Imaging and Recording Phenotypic Traits of a Coffee (***Coffea arabica* **L.) Mutant Population**

### **Radisras Nkurunziza, Joanna Jankowicz-Cieslak, Stefaan P. O. Werbrouck, and Ivan L. W. Ingelbrecht**

**Abstract** Mutation breeding in *Coffea arabica* offers a powerful tool to induce novel genetic variability for breeding and genetic studies. The success of a mutation breeding program depends largely on the ability to screen large populations for target traits. There is also a need to accurately record induced mutant traits at the individual plant level. Comprehensive phenotyping requires measuring and tracking traits of interest during the crop growth cycle and subsequent generations. Therefore, efficient and accurate data collection and recording of traits is essential, both at the individual plant level and populations. In recent years, various high-throughput plant phenotyping platforms have been developed. However, these are typically proprietary, and/ or require costly infrastructures. In this chapter we illustrate the use of Field Book and ImageJ, two public domain software tools, for phenotyping and documenting growth and yield traits of a greenhouse-grown Arabica coffee mutant population. Example data of M1 and M2 mutant phenotypes induced through EMS and gamma-ray mutagenesis are presented. We further demonstrate the use of these tools for quantifying the canopy of mutants and non-mutagenized controls. These tools can be more widely applied to other visual phenotypes including plant or tissue responses to biotic or abiotic stresses. The use of free, open-access tools for integrating electronic data recording with image processing can greatly improve the efficiency, precision and speed of data collection for screening large mutant populations and is especially useful in resource-limiting settings.

R. Nkurunziza · S. P. O. Werbrouck

R. Nkurunziza (B) · J. Jankowicz-Cieslak (B) · I. L. W. Ingelbrecht

Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, IAEA Laboratories Seibersdorf, International Atomic Energy Agency, Vienna International Centre, Vienna, Austria e-mail: radisras.nkurunziza@ugent.be

J. Jankowicz-Cieslak e-mail: j.jankowicz@iaea.org

Laboratory for Applied in Vitro Plant Biotechnology, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

# **1 Introduction**

Coffee (*Coffea* spp.) is an indispensable source of income in Asia, Africa, South and Central America. It is ranked among the world's most valuable export commodities, on which more than 125 million people in the coffee growing areas derive their income directly or indirectly (FAO 2015). *C. arabica* contributes about 75% of the world coffee production due to its superior cup quality and aromatic characteristics. *C. arabica* has a unique biology compared to other species in the genus Coffea because it is a self-fertile, allotetraploid (2n = 4x = 44) species while almost all other coffee species are diploids (2n = 2x = 22) and generally selfincompatible (Carvalho et al. 1991). Due to its reproductive biology, *C. arabica*  varieties tend to remain genetically stable. However, *C. arabica* varieties are typically low yielding and highly susceptible to a myriad pests and diseases and abiotic stresses, including Coffee Leaf Rust (CLR). Genetic improvement of *C. arabica* to withstand the afore mentioned constraints and with higher yield through classical breeding is laborious and can require up to 25–30 years to release an improved variety (Lashermes et al. 1996, 2009).

Mutation breeding offers a powerful tool to enhance genetic variation in the *C. arabica* gene pool which is very narrow. Random mutagenesis has been used in different crops to induce novel agronomic traits that were absent in their primary gene pool (Ulukapi and Nasircilar 2018). Induced mutagenesis can enhance stable, genetic variability not only in seed but also in vegetatively propagated plants (Jain 2005; Pathirana et al. 2009). Major traits improved through mutation-assisted breeding include plant architecture, early flowering and maturity, yield and quality traits, and tolerance to pests and diseases (Pathirana 2011; FAO/IAEA 2018). For a successful coffee mutation breeding program, phenotyping large mutant populations is paramount. Likewise, it is important to identify, and document induced mutant phenotypes at the individual plant level. Comprehensive phenotyping requires that traits of interest can be accurately and rapidly measured and documented during the crop growth cycle and subsequent generations (Sabina 2022). Recent technological advances have enabled accurate, high-throughput plant phenotyping. Commercial and open-source digital phenotyping technologies and methods have been developed to increase the precision and speed of data collection and analysis useful for plant breeders. However, high-throughput phenotyping methods typically require highly automated and sophisticated systems for image acquisition and analysis. Also, highthroughput technologies require significant infrastructural investment in the field or greenhouse facilities. The use of simple image capture tools such as manually operated cameras and downstream open-source image analysis tools such as ImageJ, Fiji and MATLAB provide affordable alternatives (Hartmann et al. 2011; Schindelin et al. 2012; Singh et al. 2017). In recent years several open-access applications for data recording have been developed. Examples include Field Book, an open-source application for taking phenotypic notes (Rife and Poland 2014), OneKK, an app designed to analyze seed lots, Coordinate, an open-source Android app used to collect and organize data into a predefined grid (Prasad et al. 2018) and Open Data Kit (ODK) for seed tracking (Ouma et al. 2019). Such public domain software tools facilitate a digital migration from manual methods of data capture and recording that are associated with unstandardised data that is difficult to process for analysis (Mechael 2009).

In this chapter, we illustrate the use of two public domain software tools, Field Book and ImageJ, for phenotyping and documenting growth and yield component traits of *C. arabica* M1 and M2 populations and plants developed and maintained in the greenhouse of the FAO/IAEA Plant Breeding and Genetics Laboratory in Seibersdorf, Austria. These tools are simple, user-friendly and especially useful for plant scientists, breeders or data collectors in resource-limiting environments where advanced, high-throughput phenotyping facilities and/or expertise is missing.

# **2 Materials**

# *2.1 Establishment of the* **C. Arabica** *Mutant Populations*


# *2.2 Phenotyping of the Mutant Populations*


# *2.3 Data Analysis Tools*


# **3 Methods**

# *3.1 Establishing the M1 Mutant Population*


**Fig. 1** Developmental stages of coffee mutants. **a** Coffee seedlings with 4–5 true leaves, ready for transplanting. Just before transplanting, data can be recorded on traits like number of leaves, size of canopy and leaf dimensions (leaf length and leaf width) (*see* Chap. "Chemical Mutagenesis of Mature Seed of Coffea arabica L. var. Venecia Using EMS"). **b** Mutant plants established in pots at about six months after transplanting. Growth traits including plant height can be measured to monitor plant growth

# *3.2 Phenotypic Characterisation of the M1 Mutant Population*

### **3.2.1 Electronic Phenotyping Tools**


**Fig. 2** Schematic representation of the steps taken while using Field Book application for data collection on a coffee mutant population. **a** The overview of the layout of the field (import file) displaying information on coffee mutant labels only. No trait information is required. **b** Field Book logo. **c** Option for importing the experimental layout as a field into the application. **d** Option to input and define trait. **e** Appearance of the coffee traits as defined in the application. **f** Option to begin data collection. **g** An interface during data collection. It displays the trait (e.g., plant height), plant ID (e.g. Ca-2020–001 Gy 20) information as previously determined. Forward and backward arrows guide data collection. **h** Option to export data after collection for storage. **i** Selected procedure to export the data after collection




**Fig. 3** A screenshot of the exported data file from the Field Book application for the coffee mutant population. The file is retrieved as an Excel file (csv). Compared to the import file, the extra columns show traits with the corresponding data


### **3.2.2 Measurement of Growth Traits**


**Fig. 4** M2 seedlings five months after sowing. Abnormal phenotypes were identified at early stages of seedling development. The red-circled seedlings indicate aberrant leaf shapes. The seedlings within white circles show yellowing, an indicator of chlorophyll deficiencies

**Fig. 5** Image capture of the seedlings at transplanting. **a** Camera stand with three support stands. **b** Camera fixed in one position to capture all images. **c** Acclimatized potted seedling ready for transplanting

**Fig. 6** Example images of M1 seedlings captured using the fixed camera setting for ImageJ analysis (*see* Chap. "Chemical Mutagenesis of Mature Seed of Coffea arabica L. var. Venecia Using EMS")


# **3.2.3 Measurement of Yield Component Traits**


21. Screen the mutants for resistance to major pests and diseases or tolerance to abiotic stresses (for leaf rust resistance screening, *see* Chaps. "Screening for Resistance to Coffee Leaf Rust", "Inoculation and Evaluation of *Hemileia vastatrix* Under Laboratory Conditions" and "Evaluation of Coffee (*Coffea arabica*  L. var. Catuaí) Tolerance to Leaf Rust (*Hemileia vastatrix*) Using Inoculation of Leaf Discs Under Controlled Conditions").

# *3.3 Image Analysis to Estimate the Canopy Size*


**Fig. 7** Schematic representation of the steps taken to analyse images using ImageJ. **a** ImageJ software menu bar. **b** ImageJ software logo. **c** A picture of the seedling to be imported to the software for analysis. **d** Calibration line drawn from the internal diameter (100 mm) of the pot. **e** Red pixels adjusted using sliders to cover the seedling. **f** Output of the first image analysis with noise. **g** Output after noise removal

# *3.4 Statistical Analysis*


**Fig. 8** Example data demonstrating the induced phenotypic variability in the coffee M1 population treated with different doses of EMS (%EMS) and gamma-rays (gray—Gy). **a** Plant height measured three years after planting. **b** Leaf length measured from the petiole to the apex. **c** Leaf width measured at the widest part of the leaf. **d** Estimated Leaf Area (ELA) based on leaf length and leaf width measurements using allometric model, ELA = 0.99927\*(L\*(– 0.14757 + 0.60986\*W)) according to Unigarro-Muñoz et al. (2015)

# *3.5 Establishment of M2 Population*


# **4 Notes**


9. In most cases, the goal of a mutation breeding program is to improve one particular trait. However, one can simultaneously screen the mutant population for other traits of interest including quality, resistance or tolerance to major pests and diseases, drought, etc.

**Acknowledgements** The authors wish to thank the International Atomic Energy Agency and the Government of Belgium for their financial support through the CRP D22005 'Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana' and the Peaceful Use Initiative project 'Enhancing Climate Change Adaptation and Disease Resilience in Banana-Coffee Cropping Systems in East Africa', respectively.

### **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Screening for Resistance to Coffee Leaf Rust**

### **Vítor Várzea, Ana Paula Pereira, and Maria do Céu Lavado da Silva**

**Abstract** Coffee leaf rust (CLR), caused by *Hemileia vastatrix* (*Hv*), is one of the main limiting factors of Arabica coffee production worldwide. Breeding for rust resistance is the most appropriate and sustainable strategy to control CLR. The characterization of coffee resistance to *Hv*, initiated in the 1930s in India, expanded with the creation of Coffee Rusts Research Center (CIFC) in 1955, in Portugal. Since then, the screening of coffee resistance to *Hv* races, from different geographical origins, has been carried out assisting breeding programmes of coffee growing countries and originating over 90% of the resistant varieties cultivated worldwide. However, the high adaptability of *Hv* has resulted in the gradual loss of resistance of some varieties. Thus, the characterization of new sources of resistance is crucial, also to face the recent epidemic resurgence of CLR across Latin America and the Caribbean.

Here, we provide a protocol for the screening of coffee resistance to *Hv* using different methods of inoculation on attached and detached leaves and on leaf disks. Information on environmental and pathogenicity factors that may affect the assessment of coffee resistance is also presented. This protocol allows the characterization of rust resistance on coffee mutants at laboratories, greenhouses, and field conditions.

# **1 Introduction**

Coffee, the most important agricultural commodity, is crucial for the economy of more than 70 countries and is a livelihood source for between 12 and 25 million farmers worldwide (ICO 2019). The value of coffee exports amounted to USD 20 billion in 2017/18 being the revenue of the coffee industry estimated to surpass USD 200 billion (Samper et al. 2017; ICO 2019).

V. Várzea (B) · A. P. Pereira · M. do C. L. da Silva

CIFC, Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Quinta do Marquês, 2784-505 Oeiras, Portugal e-mail: vitorvarzea@isa.ulisboa.pt

LEAF, Linking Landscape, Environment, Agriculture and Food Research Center, Associate Laboratory TERRA, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisbon, Portugal

The two main cultivated coffee species, *Coffea arabica* (Arabica) and *C. canephora* (Robusta) account, on average, for 60% and 40%, respectively, of the world's coffee production (ICO 2020).

Coffee leaf rust (CLR), caused by the biotrophic fungus *Hemileia vastatrix*  Berkeley and Broome, is considered the main disease of Arabica coffee. Since the historical first burst of CLR in the nineteenth century that caused the eradication of coffee cultivation in Sri Lanka, the disease gained a worldwide distribution, becoming practically endemic in all regions where coffee is grown (Wellman 1957; McCook 2006; Silva et al. 2006; Talhinhas et al. 2017; Keith et al. 2021). The disease produces economic losses over USD 1 billion annually (Kahn 2019).

*H. vastatrix* is a hemicyclic fungus producing urediniospores, teliospores and basidiospores, but only the dikaryotic urediospores, which form the asexual part of the cycle, reinfect successively the leaves whenever environmental conditions are favourable (Talhinhas et al. 2017 and references therein). After urediospore germination and appressorium differentiation over stomata, the fungus penetrates and colonizes the mesophyll tissues inter-and intracellularly giving rise to sporulation about 21 days after inoculation (Silva et al. 1999 and references therein; Silva et al. 2006; Talhinhas et al. 2017).

Breeding for resistance has been the most appropriate and sustainable strategy to control crop diseases.

Plant resistance to pathogens has been grouped into two different categories (Vanderplank 1968): complete resistance conditioned by single genes with major effects and incomplete resistance conditioned by multiple genes with minor and additive effects. A variety of terms have been used to refer to this perceived dichotomy, vertical versus horizontal, major-genes versus minor-genes, oligogenic versus polygenic, qualitative versus quantitative, race-specific versus race nonspecific*,* hypersensitive versus non-hypersensitive, narrow-spectrum versus broadspectrum (Parlevliet and Zadocks 1977; Roelfs et al. 1992). This diversity of terms reflects the assumptions made by the respective authors, but it also adds an element of confusion to the literature because some terms are used in different ways by different authors (Poland et al. 2009). Here the term incomplete resistance is considered as any form of resistance which allows for at least some reproduction of a given pathogen isolate on a given host plant (Eskes 1983).

Complete resistance results in phenotypes that fit into discrete classes of resistant and susceptible individuals according to Mendelian ratios (qualitative resistance). On the other hand, incomplete resistance cannot be easily categorized into distinct groups but in a continuous distribution of susceptible and resistant individuals (quantitative resistance) (Corwin and Kliebenstein 2017).

The traditional recording system for complete resistance on coffee to rust was developed at Coffee Rusts Research Center (CIFC) by d'Oliveira (1954–57) and consists of the identification of eight lesion types grouped into 4 classes of resistance and susceptibility. The incomplete resistance can be measured by its components, like infection frequency, lesion size, latent period and sporulation intensity (Browning et al. 1977; Parlevliet 1979; Eskes 1983).

The first effective characterization of coffee resistance to CLR, in experimental bases, was initiated in the 1930s in India (Mayne 1932, 1942). This work was greatly developed and broadened with the creation of CIFC in 1955, in Portugal. Inheritance studies have demonstrated that coffee-rust interactions follow the gene-for-gene relationship of Flor (1971) within a race-specific resistance system (complete resistance), being the resistance of coffee plants conditioned at least by nine major dominant genes (SH1- SH9) singly or associated. Reversely, it was possible to infer 9 genes of virulence (v1-v9) on *H. vastatrix* (Noronha-Wagner and Bettencourt 1967; Bettencourt and Rodrigues 1988). More than 55 *H. vastatrix* physiological races, from different geographic origins, were also identified over 60 years of world surveys carried out at CIFC (Rodrigues et al. 1975; Várzea and Marques 2005; Silva et al. 2006, 2022; Talhinhas et al. 2017; CIFC records), which allowed the characterization of coffee germplasm to support breeding programmes at coffee research institutions.

For many years, selection for *H. vastatrix* resistance has been based on highly specific complete resistance derived from major introgressed genes from *C. arabica*  (SH1, SH2, SH4 and SH5) as well as from diploid species such as *C. canephora*  (SH6-SH9) or *C. liberica* (SH3). To date, some of the most widely used sources of resistance to CLR are the Timor hybrids – HDTs (natural *C. arabica* x *C. canephora* hybrids) characterized and supplied by CIFC to research institutions of coffee growing countries (Rodrigues et al. 1975; Bettencourt and Rodrigues 1988).

The recent loss of resistance in some HDT-derived varieties, due to the appearance of more virulent rust races (Várzea and Marques 2005; Silva et al. 2006, 2022; Prakash et al. 2010; Talhinhas et al. 2017, CIFC records), as well as the current epidemics in Latin America and Caribbean, highlights the importance of the discovery and characterization of new sources of resistance.

Based on the CIFC's routine activities, we present a detailed protocol focused on the screening of complete resistance to CLR. A description of the qualitative scale used for the assessment of the reaction types and the environmental and pathogenicity factors that may affect this evaluation is also reported. The methods described here can be used in a greenhouse, laboratory or in field conditions and are useful for screening coffee mutants for leaf rust resistance.

### **2 Materials**

### *2.1 To Collect and Store Inoculum*


# *2.2 Spores Viability*


# *2.3 Inoculation*


### *2.4 Phenotyping of Coffee-Rust Interactions*


# **3 Methods**

### *3.1 Procedures to Collect and Store Inoculum*

For disease resistance screening, urediniospores collected with gelatin capsules are used as inoculum. The urediniospores must be collected from well sporulated young lesions. Note that spores from lesions in fallen leaves lose their viability very soon.

If enough spores cannot be harvested in the field for reliable screening tests, we can increase this amount with inoculations on vars. Caturra, Catuaí, Mundo Novo, Typica, Bourbon, etc. (carrying the resistance gene SH5, i.e., with susceptibility to all the rust races infecting Arabicas) in greenhouse conditions.

Storage of rust samples must be done using recently collected spores thus to ensure high viability.

Rust samples can be stored for short and long term: (*i*) urediospores in gelatin capsules placed above sulfuric acid solution in a desiccator (50% relative humidity) and kept in refrigerator (4 °C) should retain good viability for about 180 days; (*ii*) in a freezer, at −80 °C, the spores keep the viability for more than 15 years; (*iii*) in liquid nitrogen, at −196 °C, spores can be stored for more than 20 years with high viability (CIFC records).

After storage at a negative temperature, a heat shock treatment (40 °C for 10 min) is required to break dormancy of the urediniospores and to recover their germination ability (CIFC records).

### *3.2 Spores Germination Tests*

Before each experiment, laboratory germination tests are recommended to check the spores' viability. The urediniospore germination may be evaluated in vitro (glass slides) or in vivo (leaf pieces), being the last more accurate.

The germination in vitro is evaluated by placing aliquots of 100 µl of the urediniospore suspension (prepared as described in 3.3.1.2.) in glass slides which are kept in a moist chamber during 16 h at 23 °C. After this time, the germination is stopped with an aqueous solution of 3% formaldehyde. The glass slides are then covered with cover slips and observed under the microscope and the percentage of germinated spores counted on a minimum of ten fields of 100 urediniospores each (Silva et al. 1985).

The germination in vivo is evaluated on leaf pieces (5cm2) cut from the previously inoculated leaves (*see* Sect. 3.3.), 16–24 h after inoculation (Silva et al. 1999). After let dried, the fragments are painted with transparent nail polish on the lower surface. About 24 h later, the dried nail polish (leaf replica) is removed with the help of tweezers and dipped into cotton blue lactophenol to stain the fungal structures (urediniospores, germ tubes and appressoria). The leaf replicas are placed in glass slides containing the same staining, covered with cover slips and observed under the microscope. With this technique it is possible to evaluate the rates of both the germinated urediniospores and the appressoria differentiated over stomata. Countings are made on a minimum of six microscope fields of 100 urediniospores each.

### *3.3 Inoculation Techniques*

The screening of disease resistance is usually carried out by artificial inoculations by spreading fresh urediniospores on the lower surface of the coffee leaves with the help of a sterilized soft hairbrush or by using a urediniospore suspension on attached leaves in greenhouses or at field conditions, as well as on detached leaves and leaf disks at laboratory conditions.

Young, fully expanded leaves of the terminal node are used. In the day before inoculation, the plants are abundantly watered, and the turgid leaves are inoculated on the plant (leaves attached to the plant) or removed from the plant (detached leaves or leaf disks).

### **3.3.1 Attached Leaves**

### Brushing

This method can be used in attached leaves in the greenhouse or in the field. Following the routine procedure used at CIFC, fresh urediniospores of *H. vastatrix* (about 1 mg per pair of leaves) are placed with a scalpel on the lower surface of the leaf (*see* Fig. 1a) and then brushed gently with a camel's hairbrush (*see* Fig. 1b). The inoculated leaves are sprayed with distilled water (*see* Fig. 1c) and the plants are placed for 24 h under darkness at room temperature (18 °C to 24 °C) in moist chambers, (*see* Fig. 1d) after which they are placed in the greenhouses*.*

When the moist chambers are too small to allow the incubation of plants, the inoculated leaves after sprayed with distilled water are enveloped with a humid plastic bag during 24 h (*see* Fig. 1e). To avoid direct incidence of the sun rays, the plastic bags are covered with ordinary paper or newspaper sheets (*see* Fig. 1f) (D'Oliveira 1954–57). The same procedure can be used in field conditions.

**Fig. 1** Inoculation of attached leaves using the brushing technique. *H. vastatrix* urediniospores on the scalpel (**a**) and then brushed on the lower surface of the leaf with a camel's hairbrush (**b**). Inoculated leaves are sprayed with distilled water **(c)** and placed in a moist chamber (**d**). For large plants, the inoculated leaves are enveloped in a humid plastic bag **(e)** and covered with newspaper sheets (**f**)

Urediniospore Suspension

The lower surface of the leaves is inoculated with an urediniospore suspension, with a concentration of 0.8 to 1.2 mg ml−1, using a manual or electric sprayer (*see* Fig. 2), in a greenhouse or field conditions. These suspensions are prepared by suspending urediniospores in distilled water (to which 1–2 drops of Tween 0.02% is previously added). In the absence of Tween the following procedure is suggested: a spore mass is placed in a small test tube; one drop of distilled water is then added and kneaded into the spore mass with the help of a vortex mixer. This process is repeated by

**Fig. 2** Inoculation of the lower surface of the coffee leaves with an urediniospore suspension using an electric sprayer

adding one drop of water at a time until the moistened spore mass has the pasty consistency of heavy cream. At this point, the bottom of the test tube is placed in a vortex mixer for several minutes and the remaining volume of water required for the final suspension is added during the stirring process. This step degasses the spore surfaces, which improves spore viability and yields almost complete dispersal of the spores in water. Good but incomplete suspensions leave a film of unwetted spores on the water surface if the stirring is omitted. Spores in suspensions prepared by this method germinate normally (CIFC records).

The upper inoculated leaves and part of the branch to which the leaves are attached are sprayed with distilled water and enveloped with a humid plastic bag. To avoid direct incidence of the sunrays, the plastic is covered with paper/newspaper sheets. The plastic bags are removed about 24 h after. Inoculations at field conditions are carried out in the late afternoon and the bags are removed early in the morning (Eskes 1989).

### **3.3.2 Detached Leaves**

The leaves are placed with the abaxial surface upwards in trays lined on the bottom with a nylon sponge saturated with distilled water. Each leaf is inoculated with droplets (10–20 µl) from the urediniospores suspension (with a concentration of 250–500 spores/droplet). The droplets are deposited between the veins, using a micropipette (*see* Fig. 3). The trays are covered with glass plates and placed in the dark for 24 h at 22 ± 2 °C. After this time, the drops are dried out with small pieces

**Fig. 3** Detached leaves inoculated with droplets of an urediniospore suspension

of filter paper, and the trays, covered with glass plates, are placed under moderate light conditions (fluorescent or indirect daylight of 500–1,000 lx) with a photoperiod of 12 h under similar temperatures (Eskes 1983).

### **3.3.3 Leaf Disks**

Leaf disks are cut with cork borers from 1 to 2 cm in diameter and placed in Petri dishes or in trays with the upper leaf side down, on a sponge saturated with tap water. The disks are inoculated with droplets, from 10 to 20 µl of urediniospores suspension (with a concentration of 250–500 spores/droplet). After inoculation the boxes are closed with glass lids and incubated in the dark in the same conditions and environment described above for the detached leaves (Eskes 1982a).

### *3.4 Phenotypic Scoring Method for Disease Resistance*

At greenhouse conditions, with a range of temperatures from 18 to 24 °C, the reading of the reaction types takes place usually 30–35 days after the inoculations, by a qualitative scale developed at CIFC by D'Oliveira (1954–57). However, the time to score the reactions can be extended to 45 days or more in the following situations: at higher or lower temperatures during the colonization process and with low aggressiveness of the fungal isolates.

This recording system has been followed at CIFC for more than 60 years to identify complete resistance on *Coffea* spp to CLR and to characterize rust races.

Qualitative scale used at CIFC to score the reaction types on attached leaves (D'Oliveira 1954–57; Bettencourt and Rodrigues Jr. 1988) (*see* Fig. 4).

**i** = immune (no visible symptoms).

**fl** = Flecks: small chlorotic flecks at the penetration sites, well visible with a pocket lens or when holding the leaf against the light.

**;** = Necrotic spots, visible macroscopically at the penetration site or dispersed over the infected area.

**t** = Punctiform tumefactions, often associated with flecks.

**0** = Chlorotic spots, more or less intense, in the infected area, sometimes associated with small necrotic areas, but without spore production.

**Fig. 4** Reaction types, according to the qualitative scale used at CIFC. Flecks visible when holding the leaf against the light (**a**); tumefactions (**b**); reaction 0 (**c**); reaction 1 (**d**); reaction 2 (**e**); reaction 3 (**f**); reaction 4 (**g**)

**1** = Rare sporulating sori, always very small, sometimes only visible with a pocket-lens, in areas which are mainly chlorotic, sometimes associated with necrosis.

**2** = Small or medium-sized pustules, diffused but visible macroscopically, in areas with intense chlorosis.

**3** = Medium-sized or large pustules, surrounded by chlorosis.

**4** = Large sporulating pustules, without true hypersensitivity, but sometimes surrounded by a slight chlorotic halo (highly susceptible or compatible).

**X** = Heterogeneous reaction with urediosporic pustules very variable in size associated with resistant reaction types.

The reaction types **i**, **fl**, **t** and **0** are jointly referred to as resistant (**R**), **1** as moderately resistant (**MR**), **2** as moderately susceptible (**MS**), and **3** and **4** as susceptible (**S**).

Detached leaves and leaf disks are useful to identify very susceptible genotypes to rust. However, intermediate levels of resistance expressed by a low reaction type (reactions 1 and 2) on attached leaves, at greenhouse or field conditions, may not be observable in leaf disks or detached leaves. In this way, whenever lesions without sporulation are found on detached leaves and leaf disks, we suggest to repeat the inoculations on attached leaves.

### **4 Notes**

### 1. **To collect inoculum**

When collecting rust samples in plants, either in the field or in the greenhouse it is important to avoid the presence of mycoparasites like the fungus *Lecanicillium lecanii* (Zimm.) Zare and W. Gams, with the ability of reducing spore viability and disease severity (Vandermeer et al. 2010; James et al. 2016; CIFC records). The first evidence of *L. lecanii* in rust lesions is in the form of small white spots at the center of the rust sori. The spots gradually enlarge; the cotton-like, white colored mycelium of the mycoparasite covered the rust sori. The development of this mycoparasite is restricted to the rust infected leaf parts, but never grow to over the entire width of the rust lesions (CIFC records).

### 2. **Inoculum**

When the virulence of rust local populations is not known, the source of inoculum to be used to detect resistance in coffee mutants should be gathered from the same plants or similar genotypes where they come from. If resistance is found in the first inoculations, the screening on coffee mutants should continue with inoculum collected from different coffee genotypes in different regions to try to get rust samples with higher spectra of virulence. In general, the origin and distribution of rust races follow the resistance genes present in coffee populations.

### 3. **Factors influencing the infection process and the resistance symptoms**

### 3.1. *Moisture*

The urediniospores do not germinate, even at high relative humidity, if the free water is absent. If the water dries off before penetration, then the process is inhibited (Nutman and Roberts 1963; Rayner 1972; CIFC records).

	- (i) The temperature, while the leaf surface is wet, is one of the most important factors that determine the amount of spore germination and penetration. The optimum temperatures for germination are 20 to 25 °C (CIFC records).
	- (ii) Extreme temperatures after inoculation causes some depressive effect on fungal colonization and sporulation, with the slight lower reaction types on susceptible plants and in extreme may kill the fungus inside the leaves (Montoya and Chaves 1974; Ribeiro et al. 1978; Silva et al. 1992). The small chlorotic lesions, developed in these conditions, are likely to be confused with resistant reactions (CIFC records).
	- (iii) The enlargement of lesions on leaves and the sporulation are limited by temperatures over 35 °C and lower than 10 °C.
	- (i) Leaves exposed to higher light intensities before inoculation show more lesions than those exposed to lower intensities (Eskes 1982b, 1983, 1989).
	- (ii) In screening tests, the light intensity should preferably be kept at medium levels before inoculation and medium to low levels after inoculation (Eskes 1982b, 1983, 1989, CIFC records).
	- (iii) Some derivatives of interspecific tetraploid hybrids (*C. arabica* x *C. canephora*) like Icatu and Timor Hybrid (HDT) show lower and even resistant reaction type lesions at a lower light intensity under greenhouse conditions (Marques and Bettencourt 1979; CIFC records).
	- (iv) Studies on the effect of leaf age and light intensity on CLR found higher resistance on young leaves growing in the shade, and lower resistance for old leaves exposed to sunlight (Eskes 1983).

### 4. **Phenotypic scoring for disease resistance**


node (Quintana et al. 2019). These changes may influence the expression of resistance to leaf rust. Irradiated coffee plants should be inoculated, if possible, on leaves of different ages.


### 5. **Incomplete resistance**

The contribution of Albertus B*.* Eskes (1989 and references therein) for the characterization of incomplete resistance on coffee to CLR, using leaf disks and detached leaves, was of paramount importance and his works are a reference to those who intend to develop studies on this kind of resistance.


Infection frequency: Number of lesions per leaf or leaf area unit, or the percentage of disks with lesions.

Latent period: The time from inoculation to spore production. Normally it is calculated as the time taken for 50% of the lesions to sporulate or the time between the inoculation and the formation of the first spores.

Incubation period: The number of days between the inoculation and the appearance of the first chlorotic lesions per leaf or disk

Proportion of sporulating lesions: Percentage of sporulated lesions in relation to the total number of lesions by leaf or disk.

Sporulation intensity: The number of spores produced per sporulating lesion or per infected leaf area, over a certain time interval.

Lesion size: Normally evaluated at the end of the experiment

5.4 Relations amongst reaction types (RT) and components of incomplete resistance

The majority of the components of incomplete resistance are a quantitative extension of the scale used for RT. These components, as well as the RT's are related to the same basic criteria, like lesion size, sporulation intensity, and the occurrence of chlorosis or necrosis. The latent period is related to lesion size when fungal growth is slow, the sporulation will generally be delayed, and the lesions will be smaller. The reaction types "0" (chlorosis is without sporulation) or necrotic spots will reduce the sporulation intensity and/or the duration of sporulation (Eskes 1981).

**Acknowledgements** Funding for this work was provided by the Food and Agriculture Organization of the United Nations and the International Atomic Energy Agency through their Joint FAO/IAEA Research Contract nº 20902/R0 of IAEA Coordinated Research Project D22005 and Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project UIDB/04129/ 2020 of LEAF-Linking Landscape, Environment, Agriculture and Food, Research Unit.

### **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Inoculation and Evaluation of** *Hemileia vastatrix* **Under Laboratory Conditions**

**Miguel Barquero-Miranda, María José Cordero-Vega, and Kimberly Ureña-Ureña** 

**Abstract** The coffee leaf rust, a disease caused by the biotrophic fungus *Hemileia vastatrix*, is one of the main limitations in coffee production today as it causes significant economic losses to the coffee production sector. Genetic improvement is an option to solve these problems. The Arabica varieties have a very narrow genetic base therefore the induction of mutations, through e.g. physical methods such as gamma rays, could be an efficient tool to increase the genetic diversity of the crop. This would allow to obtain desirable agronomic characteristics such as resistance to pests and diseases. To determine the effect of irradiation on the plants, protocols enabling evaluation of improved traits must be applied. In the case of the assessment of plant resistance to pests and diseases, screening protocols that take into account their biology should be considered. This chapter provides a detailed protocol for the inoculation and evaluation of *Hemileia vastatrix* under laboratory conditions.

# **1 Introduction**

Coffee is the second most commercialized product worldwide. It is produced in over 50 countries and secures livelihoods for millions of farmers (Vega et al. 2003; ICAFE 2017). *Hemileia vastatrix* Berk. & Broome, the causal agent of coffee leaf rust, is one of the biotic factors that affects coffee, causing significant economic losses due to the defoliation, subsequent harvest losses, and renewal needs due to severe damage caused in plants (Barquero 2013; ICAFE 2013).

This disease caused a widespread impact in 2012 in Central America, mainly due to the susceptibility of planted varieties of *Coffea arabica* such as Caturra and Catuaí (Avelino and Rivas 2013). Due to the origin, domestication process, reproduction and evolution of the genome, Arabica varieties are characterized by a low genetic diversity (Hendre et al. 2008; Prakash et al. 2002). As a result of a natural hybridization process between *C. eugenioides* and *C. canephora*, *C. arabica* is the only tetraploid

Phytoprotection Laboratory, Costa Rican Coffee Institute, Coffee Research Center, 37-1000, San Pedro, Heredia, Costa Rica

M. Barquero-Miranda (B) · M. J. Cordero-Vega · K. Ureña-Ureña

e-mail: mbarquero@icafe.cr

<sup>©</sup> The Author(s) 2023

I. L. W. Ingelbrecht et al. (eds.), *Mutation Breeding in Coffee with Special Reference to Leaf Rust*, https://doi.org/10.1007/978-3-662-67273-0\_16

**Fig. 1** Coffee leaf rust symptoms and signs, chlorotic spots and urediniosporic sori on the lower leaf surface

species of the genus *Coffea* (2n = 4x = 44). This generates a limitation for genetic improvement of resistance genes due to the homogeneity of the varieties (Jefuka et al. 2010; Naranjo Zúñiga 2018).

*Hemileia vastatrix* is a biotrophic fungus that penetrates the plant through the stomata located on the abaxial side of the leaf. Its taxonomic classification is as follows: Phylum: *Basidiomycota*, Class:Pucciniomycetes, Order: Pucciniales, Family: Zaghouaniaceae, Genus: *Hemileia* (gbif 2022).

*H. vastatrix* is a hemicyclic fungus producing urediniospores, teliospores and basidiospores, but only the dikaryotic urediniospores, which form the asexual part of the cycle, reinfect coffee leaves successively and are responsible for the disease (as revised by Talhinhas et al. 2017). As a first symptom, small yellow chlorotic spots are observed in the foliage that subsequently, as the infection progresses, produce masses of orange urediniosporic sori (*see* Fig. 1) (Arauz Cavallini 2011). An epidemic of coffee leaf rust can be divided into two stages: stage of the production of the initial inoculum, whose main source is the residual inoculum, and a second stage that comprises the production of the secondary inoculum, which is the result of the successive repetition of the infection process on the same leaf (Avelino and Rivas 2013, Naranjo Zúñiga 2018).

The disease cycle of coffee leaf rust consists of five stages which can be affected by factors such as fruit load, plant resistance, microclimate and plant nutrition (Avelino 2004; Rhiney et al. 2021).

These stages include:


Genetic improvement is an attractive approach that enables solving production constraints caused by pests and diseases. Induced mutagenesis is one of the tools that can be used to increase genetic diversity (ICAFE 2011; Novak and Brunner 1992; Shu et al. 2011). Gamma rays have proven to be an efficient tool to improve traits of agronomic importance such as resistance to pests and diseases (Borzouei et al. 2010; Yadav and Singh 2013; Shu et al. 2011). An efficient screening protocol is therefore required for evaluation of mutant populations developed via induced mutagenesis.

This protocol describes the procedures for the inoculation and evaluation of the defense response of the coffee genetic material infected with *Hemileia vastatrix*  under laboratory conditions, or the biological efficacy of molecules for the control of the disease*.* This protocol is based on Eskes and Toma-Braghini (1982), with modifications made by the Costa Rican Coffee Institute-Coffee Research Center, Phytoprotection Laboratory.

# **2 Materials**

### *2.1 Preparation of Rust Inoculum*


### *2.2 Rust Inoculation*


# *2.3 Rust Evaluation*


# **3 Methods**

# *3.1 Preparation of Rust Inocula*


*Urediniospore concentration/ml* = *urediniospores counted* × 5 × 1*e*10<sup>4</sup>

**Fig. 2** Urediniospores count points in Neubauer chamber

**Fig. 3** A general description of the preparation stage of the inoculum and of the plant material, of the process for the germination and incubation of the pathogen and of the quantification of the presence or absence of the disease

# *3.2 Rust Inoculation*


# *3.3 Rust Evaluation*



**Table 1** Scale used to measure the severity of rust in coffee segments (Eskes and Toma-Braghini 1982) (*see* Note 5)

4. The incidence rate of the disease is determined by the formula:

# % incidence = Number of segments with presence of uredospores × 100 total segments inoculated

5. Determine the presence and abundance of signs and symptoms (Table 1**)**.

This inoculation technique can also be used to evaluate the response of different natural or chemically synthesized molecules for the defense of plants susceptible to the disease. To do this, the use of Table 1 allows us to understand the mechanism of action of the products according to the incubation and latency periods of the pathogen.

# **4 Notes**


the sporulation is more abundant and successful when dark conditions remain for three days. Therefore, it is recommended that this step is being tested and adjusted to the conditions of the laboratory in which the evaluation is going to be performed.

5. In this protocol, in addition to the rust incidence the scale described in Table1 is being used. The inoculation technique presented in this chapter can also be used to evaluate the response of different natural or chemically synthesized molecules for the defense of plants susceptible to the disease. To do this, the use of Table 1 allows us to understand the mechanism of action of the products according to the incubation and latency periods of the pathogen.

**Acknowledgements** Funding for this work was provided by the Costa Rican Coffee Institute-Coffee Research Center and the FAO/IAEA Joint Center. This work is part of the Coordinated Research Project D22005 titled "Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana", Contract Number 20475.

### **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Evaluation of Coffee (***Coffea arabica* **L. var. Catuaí) Tolerance to Leaf Rust (***Hemileia vastatrix***) Using Inoculation of Leaf Discs Under Controlled Conditions**

**José Andrés Rojas-Chacón, Fabián Echeverría-Beirute, and Andrés Gatica-Arias** 

**Abstract** Coffee leaf rust (CLR), caused by the obligate biotrophic fungus *Hemileia vastatrix*, is considered one of the most devastating diseases of Arabica coffee. The use of leaf rust resistant or tolerant coffee varieties is a critical component for effective management of this disease at the farm level. Conventional breeding of Arabica coffee for leaf rust resistance requires many years of breeding and field-testing. Induced mutagenesis is an effective tool to increase genetic variability and generate new alleles with potential benefit for addressing abiotic and biotic stresses such as leaf rust in Arabica coffee. Efficient screening methods are required to evaluate coffee germplasm or mutant populations for resistance to *H. vastatrix*. Here, we present a screening method that uses inoculation of leaf discs in a controlled environment. The method was evaluated using M1V1 and M2 plants derived from chemically mutagenized Arabica coffee cell suspensions. In this method, the first rust symptoms appear on the leaf discs approximately 29 days after inoculation while the disease severity and incidence can be scored about 47 days after inoculation. Our results show that the methodology is simple, efficient and suitable to rapidly screen large mutant populations in a small area.

J. A. Rojas-Chacón · F. Echeverría-Beirute

Escuela de Agronomía, Instituto Tecnológico de Costa Rica-San Carlos, 21002 San Carlos, Costa Rica

A. Gatica-Arias (B) Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, 2060 San Pedro, Costa Rica e-mail: andres.gatica@ucr.ac.cr

# **1 Introduction**

Coffee (*Coffea arabica* L.) is one of the most important beverages in the world and the second most important commercial product exported by developing countries (Alemayehu 2017). Coffee leaf rust (CLR), caused by the biotrophic fungus *Hemileia vastatrix* Berk. and Broome, is one of the main limiting factors of Arabica coffee production worldwide (Waller et al. 2007). The disease can reduce global coffee production by 20 to 25%, with losses of over \$ 1 billion annually (McCook 2006; Talhinhas et al. 2017).

The application of fungicides has been the most widely used method to control CLR, even when the development of varieties with genetic resistance is the best alternative (Zambolim 2016). The quest for natural resistance to CLR by traditional breeding has been the focus of research for decades (Melese Ashebre 2016; Mishra and Slater 2012). However, conventional genetic control of CLR has been hampered by the prodigious pathological diversity and rapid genetic evolution of the fungus overcoming the plant resistance genes deployed so far (Cabral et al. 2016; Lima et al. 2020). The induction of genetic variability in Arabica coffee through mutagenesis provides an important complementary tool for crop improvement programs, since a range of variants can be generated (Dhumal and Bolbhat 2012; Vargas-Segura et al. 2019).

Chemical mutagens such as sodium azide (NaN3) and ethyl methanesulfonate (EMS), have been used in crop breeding for developing mutants (Bolívar-González et al. 2018; Laskar et al. 2018). These chemical mutagens induce a broad variation of morphological and yield-related traits. Other authors reported cases of crops treated with chemical mutagens and improved for fungal resistance or tolerance, for example, powdery mildew-resistant barley (Khan et al. 2010) and wheat resistant to leaf rust *Puccinia* sp. (Mago et al. 2017).

Genetic studies related to *H. vastatrix* and coffee genotypes pursuing resistance, require periodic inoculation of different uredospores of the fungus into the host. A safe and efficient way to evaluate the resistance to different *H. vastatrix* races is carried out by infection *in situ*, using detached leaves or leaf discs under controlled conditions of humidity, light, and temperature that stimulate the development of the pathogen (Cabral et al. 2016; Eskes 1982).

This chapter presents a Coffee leaf rust resistance screening method based on inoculation of leaf discs under controlled conditions. The method was evaluated using M1V1 mutant plants obtained from M0 embryogenic callus treated with NaN3 and EMS and the resulting M2 population. The method proved suitable to rapidly screen large coffee populations for CLR resistance.

# **2 Materials**

# *2.1 Plant Material*


# *2.2 Other Biological Materials*


# *2.3 Consumables and Minor Equipment*


# *2.4 Reagents and Agrochemicals*


# *2.5 Equipment*


# **3 Methods**

# *3.1 Germination of M2 Seeds*


**Fig. 1** M2 plant development stages. **a** Seedbed preparation and seed germination. **b** Transplanting and planting of seedlings. **c** Establishment of plants under controlled conditions in a greenhouse or growth chamber

# *3.2 Planting Seedlings M2*


# *3.3 Preparation of Coffee Leaf Rust Inoculum*


**Fig. 2** Sample collection for *Hemileia vastatrix* inoculum, **a** leaves with CLR spores, **b** sporulated lesions, **c** uredospores

**Fig. 3** Preparation of the CLR inoculum and counting cells


# *3.4 Inoculation of the Coffee Leaf Discs with CLR*


**Fig. 4** Preparation of the humidity chambers, **a** cylindrical punch to cut circular (10mm) leaf discs, **b** moist chambers, and **c** inoculum in each leaf disc


# *3.5 Evaluation of Plant Resistance Against CLR*


# **4 Notes**


**Acknowledgments** This work was funded by the Consejo Nacional de Rectores (CONARE), project "Evaluation of alternative sources of genetic resistance to coffee rust (*Hemileia vastatrix*)", (project No. 5401-1701-6140). A-Gatica-Arias acknowledged the Cátedra Humboldt 2023 of the University of Costa Rica for supporting the dissemination of biotechnology for the conservation and sustainable use of biodiversity.

### **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **A PCR-Based Assay for Early Diagnosis of the Coffee Leaf Rust Pathogen**  *Hemileia vastatrix*

### **Weihuai Wu, Le Li, Kexian Yi, Chunping He, Yanqiong Liang, Xing Huang, Ying Lu, Shibei Tan, Jinlong Zheng, and Rui Li**

**Abstract** Early detection and identification of plant pathogens is one of the most important strategies for sustainable plant disease management. Fast, sensitive, and accurate methods that are cost-effective are crucial for plant disease control decisionmaking processes. Coffee leaf rust (CLR) caused by *Hemileia vastatrix* is a devastating worldwide fungal disease which causes serious yield losses of coffee, especially relevant for *Coffea arabica*. A rapid PCR assay for detecting and characterizing *H. vastatrix* with high specificity, high sensitivity and simple operation has been developed based on specific amplification of the Internal Transcribed Spacer (ITS) region of ribosomal genes. The specificity of the primers was determined using isolates DNA of *H. vastatrix*, *Coleosporium plumeriae*, and other fungal species that infect coffee plants and are common in coffee leaves, such as *Lecanicillium* sp (the *H. vastatrix* hyperparasite fungi)*, Cercospora coffeicola, Colletotrichum gloeosporioides,* amongst others. Results showed specific amplification of a 396-bp band from *H. vastatrix* DNA with a detection limit of 10 pg/µl of pure genomic DNA of the pathogen. The PCR assay described in the current chapter allows to detect *H. vastatrix* rapidly and reliably in naturally infected coffee tissues, vital for the early detection and diagnostics of *H. vastatrix* and CLR epidemiology.

# **1 Introduction**

Accurate identification and diagnosis of plant diseases are vital for prevention of the spread of invasive pathogens (Balodi et al. 2017). So far, advances in the development of molecular methods have provided diagnostic laboratories with powerful tools for the detection and identification of phytopathogens, among which polymerase chain reaction (PCR) and other DNA-based techniques proved to be rapid and highly suitable approaches to improve the accuracy and efficiency of plant pathogen detection and characterization (Lévesque et al. 1998; Haudenshield et al. 2017). Detection

W. Wu · L. Li · K. Yi (B) · C. He · Y. Liang · X. Huang · Y. Lu · S. Tan · J. Zheng · R. Li Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China e-mail: yikexian@126.com

protocols used for the diagnosis or quarantine measures should be reproducible and cost effective, time saving and simple in procedure (Elnifro et al. 2000; Hayden et al. 2008; Tomkowiak et al. 2019). In addition, sensitivity to pathogen concentration, and specificity to genetic variability within a target pathogen population are also high priorities for molecular detection (Balodi et al. 2017).

The Internal Transcribed Spacer (ITS) of the ribosomal DNA show high interspecies variability and intra-species stability and conservation, and hence is considered a reliable DNA marker to identify and classify the pathogenic fungi (Glynn et al. 2010). PCR assays based on the ITS region have been widely used for the detection of fungal pathogens in different crops such as sunflower, tobacco, soybean, cedar trees, miscanthus and others (Guglielmo et al. 2007; Chen et al. 2008; Torres-Calzada et al. 2011; Capote et al. 2012), relating to the pathogens of *Phytophthora* (Grünwald et al. 2012; Patel et al. 2016), *Puccinia* (Guo et al. 2016), *Verticillium* spp*.* (Nazar et al. 1991), *Pleurotus* spp. (Ma and Luo 2002), *Pyricularia and anthracnose* (Sugawara et al. 2009), *Saccharomyces saccharum* (Anggraini et al. 2019), *Podosphaera xanthii*  (Tsay et al. 2011) and *Golovinomyces cichoracearum* (Troisi et al. 2010). This technique was applied to differentiate two pathotypes of *Verticillium alboatrum* infecting hop, to distinguish 11 taxons of wood decay fungi infecting hardwood trees, and to differentiate multiple *Phytophthora* species from plant material and environmental samples (Shamim et al. 2017; Belete and Boyraz 2019).

Coffee leaf rust (CLR), a major disease of Arabica coffee (*Coffea arabica* L.), is caused by the obligate biotrophic fungus *Hemileia vastatrix Berkeley and Broome*  (Talhinhas et al. 2017). The infection of coffee leaves by *H. vastatrix* starts with urediniospore germination, appressorium formation over stomata, penetration, and inter- and intracellular colonization without any visible symptoms in the early stages of the infection in the field conditions < 10 days (Talhinhas et al. 2017; Silva et al. 2018). In field conditions, the visible rust spores can be observed about 20 days after the first infection of *H. vastatrix* (Schieber 1972). So far, the traditional method for detecting and characterizing CLR was time-consuming and laborious, and relied on conventional morphological examination requiring professional taxonomic knowledge and extensive experience (McCartney et al. 2003; Silva et al. 2012). Hence, rapid and high-throughput identification and detection methods for *H. vastatrix* are required to recognize the infection as early as possible before the appearance and spread of CLR spores in the leaf surface. Early detection methods can facilitate implementing proper management approaches to prevent the development and spread of the coffee leaf rust pathogen (Sankaran et al. 2010).

The present study was undertaken with the objective of early detection of *H. vastatrix* based on the PCR amplification of a specific ITS region in the rDNA of *H. vastatrix*. A simple, accurate and rapid PCR-based assay for CLR is presented as a reliable technique to monitor *H. vastatrix* in the early stages of the infection, as well as to provide scientific basis for the prevention and control of CLR.

# **2 Materials**



**Fig. 1** Primers Hv-ITS-F/R designed for *H*. *vastatrix* PCR assay based on rDNA-ITS sequences

**Fig. 2** Example of specificity test of Hv-ITS-F/R primer sets. The DNA of 4 strains of *H. vastatrix*  (lanes 2–5), 8 other fungi (lanes 6–13) (*see* Note 5) and sterilized ddH2O as the negative control (lane 1) were amplified by PCR using Hv-ITS-F/R primers. Primers for Hv-ITS-F/R amplify a 396-bp specific band from the DNA of *H. vastatrix*, while no bands were observed from the DNA of other fungi. **M:** DL 2000 DNA marker; **1:** ddH2O control; **2–5:** *H. vastatrix*; **6:** *Colletotrichum gloeosporioides*; **7:** *Lecanicillium* sp.; **8:** *Cercospora coffeicola*; **9:** *Coleosporium plumeriae*; **10:**  *Colletotrichum falcatum;* **11:** *Ustilago scitaminea*; **12:** *Leptosphaeria sacchari*; **13:** *Aspergillus niger*

# **3 Methods**

# *3.1 Designing the Specific Primers for* **Hemileia vastatrix**


# *3.2 Total DNA Extraction from Suspected Diseased Leaves or Typical Diseased Samples*

The CTAB method (Siegel et al. 2017) was used to extract DNA from diseased leaves.


# *3.3 Preparation of the PCR Reaction Mixture and PCR Amplification*

1. Prepare a 20 µl PCR reaction mix as follows (*see* Note 4):



# *3.4 Gel Electrophoresis*


**Fig. 3** Example sensitivity test of primer sets Hv-ITS-F/R. Prepare a series of DNA concentrations to determine the sensitivity of the detection system. The initial genomic DNA concentration of *H. vastatrix* was adjusted to 10 ng/µL, with serial tenfold dilutions to reach 10−5 ng/µl. The results showed that samples with DNA concentration of 10 pg/µL or higher yielded a clearly visible 396 bp band while samples with a lower concentration were negative. **M:** DL 2 000 DNA marker; **1:**  ddH2O control; **2:** 10 ng/µl; **3:** 1 ng/µl; **4:** 10−1 ng/µl; **5:** 10−2 ng/µl; **6:** 10−3 ng/µl; **7:** 10−4 ng/ µl; **8:** 10−5 ng/µl

### **4 Notes**


**Acknowledgements** Funding for this work was provided by the National Key R&D Program of China (2018YFD0201100), the IAEA Collaborative Research Project D22005 (No. 20380), the International Exchange and Cooperation Project funded by the Agricultural Ministry 'Construction of Tropical Agriculture Foreign Cooperation Test Station and Training of Foreign Managers in Agricultural Going-Out Enterprises' (SYZ2019-08) and the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630042017021).

### **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Molecular Characterisation of Induced Mutations in Coffee**

# **Targeted Sequencing in Coffee with the Daicel Arbor Biosciences Exome Capture Kit**

### **Norman Warthmann**

**Abstract** Exome Capture is a molecular biology technique that, in combination with Next Generation DNA sequencing technologies (NGS), allows for selectively sequencing the predicted genes of an organism. Such capture sequencing provides a compromise between genome coverage and sequencing cost. The capture reaction is an additional step in an otherwise standard sequencing protocol and exome capture effectively enriches the sequencing library for DNA molecules that overlap with predicted genes (the exome). This enables genome-wide assessments while focusing on the gene space. Capture sequencing is particularly attractive in species with large genomes, where whole genome sequencing in larger numbers of samples would be cost-prohibitive at present prices. Plant Breeding and Genetics Laboratory (PBGL) developed an Exome Capture Kit for *Coffea arabica* in collaboration with Daicel Arbor Biosciences (Ann Arbor, MI, USA). Use of the kit achieves eightfold enrichment, and hence approx. eightfold reduction in sequencing cost for a whole genome assessment of *Coffee arabica* plants. The kit is available as a regular product from Daicel Arbor Biosciences and this protocol describes the kit and gives detailed instructions on how to perform the capture reaction.

# **1 Introduction**

With today's cheap Next Generation DNA sequencing (NGS) virtually all DNA variation in genomes can be readily identified, including new mutations. Such knowledge makes the breeding process more efficient. Being able to comprehensively catalogue genome-wide DNA variation at the population-scale opens the door for genomic prediction as well as for tracking genetic variation through the breeding process.

Despite the low prices, sequencing cost is currently still of concern when applying whole genome approaches on a large number of samples, particularly when high sequencing depth is required. An example is mutation detection in mutant M1 populations, where induced mutations are in hemizygous, and often in chimeric state.

N. Warthmann (B)

Plant Breeding and Genetics Laboratory (PBGL), Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, International Atomic Energy Agency, Seibersdorf, Austria e-mail: n.warthmann@iaea.org; norman@warthmann.com

I. L. W. Ingelbrecht et al. (eds.), *Mutation Breeding in Coffee with Special Reference to Leaf Rust*, https://doi.org/10.1007/978-3-662-67273-0\_19

For many genomics-supported breeding applications it is sufficient to sequence only a representative subset of the genome. This can save cost. There exist several approaches to achieve such complexity reduction. One of them is 'target sequence capture', a molecular biology procedure that enriches for predefined regions of the genome (targets) prior to sequencing. Probes complementary to target DNA sequences are designed at large scale and used to effectively capture, i.e., pull-out, the desired molecules from sequencing libraries, thereby enriching for target molecules. The so enriched libraries are then subjected to Next Generation Sequencing (NGS) and the resulting sequencing data mostly consist of sequences representing the target regions. In the case of exome capture, those target regions are the predicted genes, the exome.

Applying target capture requires an up-front investment: It needs prior knowledge of the DNA sequence of target regions and the production of probes. In case of exome capture, which intends to enrich for (all) genes, the selection of target regions is based on a suitable reference genome and a genome annotation, which has to be available or generated. The number of exons in a eukaryotic genome is large, and the necessary number of probes can be in the hundreds of thousands. In human medical applications, including diagnostics, exome capture sequencing is standard procedure for more than a decade (Choi et al. 2009). Exome capture sequencing has gained traction in plant breeding for important food crops with very large genomes such as wheat (Dong et al. 2020; Gardiner et al. 2019) and barley (Mascher et al. 2013; Russell et al. 2016), with several commercial suppliers offering competing exome capture panels and kits.

To enable cost-effective whole genome approaches in coffee breeding, we developed and provide an Exome Capture Kit for *Coffea arabica*. This is in collaboration with Daicel Arbor Biosciences (Ann Arbor, MI, USA), hereafter "Arbor". *Coffea arabica* is an allotetraploid and the genome is the result of a merger of *C. eugenoides* and *C. canephora* (Scalabrin et al. 2020). The design is based on a public *C. arabica* genome assembly and annotation (Cara\_1.0, NCBI accession number GCF\_003713225.1, derived from cultivar 'Caturra red', isolate CCC135- 36), which we augmented with a public *C. arabica* chloroplast sequence (NCBI accession number: NC\_008535.1).

This chapter details the design of Daicel Arbor Biosciences' Exome Capture Kit, provides a step-by-step protocol for its use, and describes a validation experiment of exome capture sequencing of 41 indexed samples in a single capture experiment.

# **2 Materials**

Main inputs to the exome capture procedure are a whole-genome DNA sequencing library outfitted with respective adaptors and Arbor's Exome Capture Kit. Additional requirements for equipment, consumables, and reagents are listed below. Most of these should already be at hand as they will have been used when preparing the NGS library. For post-capture library amplification, Arbor recommends KAPA HiFi DNA polymerase.

### *2.1 The Exome Capture Kit*

Main component of the Exome Capture Kit are thousands of probes that are complementary to the thousands of target regions. They function as baits to fish their complementary targets from an NGS library in solution. In case of this Arbor kit the baits are biotinylated RNA molecules, and the target is the exome extracted from a publicly available coffee reference genome and annotation (NCBI).

### **2.1.1 Exome Capture Kit Design Details**

Initial target intervals for probe design included a *C. arabica* Chloroplast (NC\_ 008535.1) in its entirety and all annotations containing the string "exon" found in the *C. arabica* genome assembly GCF\_003713225.1 (https://www.ncbi.nlm.nih.gov/ assembly/GCF\_003713225.1).

The exonic intervals of the genome assembly were merged into non-overlapping regions representing 94.5 Mbp total exome space. The regions were padded with 50 nt on either side (i.e., 5'- and 3'-ends) and new overlaps re-merged, which resulted in 121.0 Mbp sequence space for initial probe design. Regions were divided into non-overlapping 100nt intervals and the best 80nt candidate probe hybridization site was chosen using Arbor's proprietary algorithm. Candidate probe sequences with strong predicted affinity to regions outside of the target regions were removed. The final predicted retrievable space of the filtered probe set was estimated by aligning the remaining probes back to the genome (megablastn, BLAST + version 2.6.0 +, default parameters) and padding each probe hit with 200nt on either side. Merging these regions results in 151.8 Mbp total genome space (represented in file DAB\_CoffeeExomeV1\_capspace.bed.gz), of which 87.2 Mbp overlap with the original exon region intervals (overlap represented in file: DAB\_CoffeeExomeV1\_ exonspace.bed.gz). These files can be downloaded from the kit's dedicated section on the Arbor website https://arborbiosci.com/genomics/targeted-sequencing/mybaits/ mybaits-custom-predesigned-community-panels/plants-and-fungi/.

The probes were synthesized in four distinct sets: Subgenome "C" (=*canephora*), Subgenome "E" (=*eugenoides*), Subgenome "O" ("other" = unassigned contigs), and "Chlor" (=chloroplast). The probe sets can be used separately or combined as the user sees fit depending on the application. To generate a pool of all nuclear genome probes, the "C" sub-genome module should comprise 47.4% of the pool by volume, the "E" sub-genome module 49.1%, and the "other" sub-genome module 3.5%. If the user aims to enrich the chloroplast as well, that module can comprise a final 0.1% of the final pool, though optimization for the tissue type might be required.

# **2.1.2 Availability of the Exome Capture Kit**

The Coffee Exome V1 kit is available from Daicel Arbor Biosciences as part of their Community Panels series (https://arborbiosci.com/genomics/targeted-sequencing/ mybaits/mybaits-custom-predesigned-community-panels/plants-and-fungi/). The design ID is D10496CFEXM. Order inquiries should be directed to sales@arbor. daicel.com.

# *2.2 NGS Library Requirements*

In principle, libraries prepared for Illumina short-read as well 3rd-generation longread sequencing technologies can be used. This protocol describes the exome capture reaction for Illumina sequencing libraries with dual-index-barcoded Nextera-type adaptors. For different adaptors, such as 'TruSeq', the protocol is the same, but different blockers and universal amplification primers will be required. Please consult the respective manual from Arbor.


# *2.3 Equipment*


# *2.4 Consumables and Reagents (Non-standard)*


# *2.5 PCR Primers*

Universal amplification primers post-capture amplification of the NGS library must match the respective NGS library type. This protocol uses Nextera-type/Illumina libraries.


athe star (\*) denotes a PTO-binding

# **3 Methods**

Figure 1 provides an overview of the subsequent steps, their approximate duration, and required consumables and equipment.

**Fig. 1** Workflow of the exome capture procedure with time estimates and required consumables

Sequencing libraries are combined with various blockers (=Hybridisation Mix Setup) and then incubated with the baits/probes at 65 °C for the actual capture (=Hybridisation). The hybridisation is usually performed overnight. The next day, buffer and beads for the binding and washes are prepared and the bait/target hybrid molecules are captured with beads (=Bead Binding). A total of 4 washes at 65 °C remove unbound and unspecific DNA molecules (=Washes). The target molecule library is then recovered from the beads and amplified to desired amount (Library Resuspension, Library Amplification) and bead cleaned for sequencing (=Library Cleanup, Library QC).

All reagents required for the actual capture and wash reactions are included in the Daicel Arbor Biosciences Kit. Reagents for resuspension, amplification, final bead clean-up, and QC will have to be provided by the user.

# *3.1 Hybridisation Mix Setup*

The following describes the preparation of the baits, the setup of the hybridization mix. All consumables for the hybridisation are contained in the Exome Capture Kit.

### **3.1.1 Combining Baits**

Pool the different sub-genome probe sets in representative ratios (*see* Note 1). Below table gives the necessary amounts for one capture reaction, scale if required.


### **3.1.2 Set Up the Hybridisation Mix**


### **3.1.3 Set Up the Blockers Mix**

The Blockers Mix has changed between Arbor myBaits kit manuals versions v4 and v5. Version v5 should be used. Version v4 is given for backwards compatibility only.

1. Set up the Blockers Mix.

(Amounts are given for one capture reaction, scale as appropriate)


### *3.2 Hybridisation*

During hybridization the binding of the probes/baits to the complimentary molecules in the NGS library occurs. Hybridisation is performed at 65 °C after denaturation at 95 °C. Use PCR tubes/or strips and perform the Incubation program in a thermal cycler. Use a heated lid to minimize condensation. The hybridisation is a 2-step process, where blockers and library are denatured at 95, and the Hybridisation mix is added after, the library has been cooled down to 65 °C. (Amounts given are per capture reaction).

1. Create the incubation program in a thermal cycler.


2. For hybridisation, combine components and incubate as per table below.


# *3.3 Bead Binding and Washes*

During binding, the bait-target hybrids are collected with streptavidin coated magnetic beads and subsequently washed with warm buffer (65 °C) to remove non-target DNA. 'Wash buffer X' and beads and need to be prepared before use.

### **3.3.1 Prepare 'Wash Buffer X'**

Amounts given are per capture reaction. Scale up if you have more than one.


### **3.3.2 Prepare Beads**

	- Add 200 µl Binding Buffer and thoroughly resuspend the beads,
	- Pellet the beads on the magnet for 2 min,
	- Remove and discard the supernatant.

# **3.3.3 Bead Binding Reaction**

At this point the hybridization reaction should have been in the thermal cycler for the past 16 + hours and still be in the cycler at 65 °C. In the below we will add the prepared magnetic beads to our hybridisation reaction. Those beads will then bind the baits.

1. For bead-binding the baits, combine components and incubate as per table below.



### **3.3.4 Bead Washing**

Repeat the below steps 4 times for a total of 4 washes. After the last wash, remove all wash buffer and proceed without delay to 3.4 Library Resuspension.


Proceed without delay with the next step: 3.4 Library Resuspension.

# *3.4 Library Resuspension*

Add 30 µl of 10 mM Tris-Cl, 0.05% Tween-20 (pH 8.0–8.5) to the washed beads and resuspend the 'enriched library' by pipetting.

# *3.5 Library Amplification*

Set up the PCR reaction mix as per below with universal primers suitable for your library type. The resuspended, 'enriched library' is of sufficient volume to conduct two PCRs as per Arbor protocol. Overamplification of the library should be avoided. Pooling of independent PCRs can reduce error.

### **3.5.1 PCR Primers**


### **3.5.2 PCR Reaction Mix**


### **3.5.3 PCR Program**


aRecommended elongation times (by average insert size): 500 bp: 30 s, 500–700 bp: 45 s, > 700 bp: 1 min

### *3.6 Library Clean-Up*


# *3.7 Library QC and Quantification*

Sequencing service providers will have minimum requirements with respect to DNA amount and quality and often require a minimum 'molarity', which can be calculated from average fragment size and weight. The size distribution should be determined with a Fragment Analyzer and the amount of dsDNA in ng by fluorescence-based DNA quantification. Molarity can then be calculated using the formula below:

$$\frac{concentration \left(\frac{ng}{\mu l}\right) \* 10^6}{660 \* Average \, fragment \, length} = Molarity \left(\frac{n mol}{l}\right)^3$$

The formula was copied from https://bitesizebio.com/23105/quantifying-yourngs-libraries/. Illumina has published a technical note on the quantification of Nextera Libraries of similar content: https://www.illumina.com/documents/products/techno tes/technote\_nextera\_library\_validation.pdf.

# **4 Performance of the Exome Capture Kit—Example Project**

To test the performance of the PBGL/Daicel Arbor Biosciences Exome Capture Kit, we performed exome capture and sequencing on an Illumina/Nextera NGS library pool of 41 DNA samples, aligned the resulting sequencing reads to the reference genome and assessed the fraction of reads that matched the exome and the coverage. We used the same reference genome and annotation that had been used to design the kit.

# *4.1 Example Project: Sequencing a Mutant Population (M1V1)*

The work was performed at the PBG Laboratory, Seibersdorf, Austria and entailed individual DNA isolations from 41 leaf samples derived from *Coffea arabica* plants that had been grown in tissue culture, sequencing library construction for each sample (Nextera), pooling of all samples, performing the exome capture reaction on the pool of 41 samples, and submitting the library pool to a service provider for Illumina short-read sequencing (PE150). During library preparation, each sample received an individual molecular barcode (index), so the sequencing reads could be associated to the respective samples after DNA sequencing. We aligned the raw reads (fastq files) to the *Coffea arabica* reference genome Cara\_1.0 (NCBI assembly GCF\_003713225.1) with software bwa mem (Li and Durbin 2009). From these alignments (bam files) we evaluated the quality of the capture and enrichment with the R-package TEQC (Hummel et al. 2011, 2020).

### **4.1.1 Input NGS Library**

An Illumina DNA sequencing library pool with 41 individually-indexed coffee samples was prepared following a transposase-mediated protocol (Nextera-type) as detailed in the IAEA-PBGL protocol: Library Preparation for Medium- to Highthroughput DNA Sequencing on the Illumina Sequencing Platform, A Laboratory Protocol (IAEA 2022a). The library pool was size selected with Ampure XP beads (one-sided, 0.7×) to an average insert size of ~ 540 bp and a lower size limit of above 300 bp (Fig. 2). Seven microliter (7 µl) containing 300 ng of this Illumina/Nextera sequencing library pool was the input for the exome capture reactions.

**Fig. 2** Size distribution of the input DNA sequencing library pool of 41 individually indexed coffee samples (Illumina/Nextera), assessed with ©Agilent Technologies, Inc. TapeStation, high sensitivity D1000 ScreenTape®

### **4.1.2 Exome Capture**

One capture reaction was performed on this pool of 41 samples following Arbor protocol version 4: Baits (5.5 µl) were combined with the hybridisation components to 20 µl Hybridisation Mix. Blockers (2 µl IDT Blocker, 2.5 µl Block O) were added to 7.5 µl of the Illumina library resulting in 12 µl total. 18.5 µl of Hybridisation mix were combined with the 12 µl library/blocker mix and hybridization was allowed to occur in a PCR machine for 16 h at 65 °C. The bait/library hybrids were captured (with streptavidin-coated beads) and washed with 1× Buffer X (618 µl H2O, 156 µl wash buffer, 6.25 µl Hyb S). Beads were resuspended in 30 µl 10 mM TrisCl, 0.05% TWEEN-20, pH 8.0, and two independent enrichment PCRs (50 µl, KAPA HiFi) were performed, each with 15 µl of the bead suspension as template, 13 PCR cycles with 45 s extension time. Both PCRs were pooled (100 µl total) and subjected to PCR purification (Qiagen MinElute) and two subsequent bead-cleanups for size selection (1 and 0.7× with Ampure XP beads). Final DNA amount was assessed by fluorescence measurement (Qubit). A one in four dilution was assessed for size distribution on the Agilent TapeStation.

### **4.1.3 Output Exome Enriched NGS Library**

DNA amount of the exome enriched library was assessed by fluorescence measurement (Qubit). A one in four dilution was assessed for size distribution on the Agilent TapeStation (Fig. 3). Average fragment size of the library was ~ 570 bp, which corresponds to an average insert size of ~ 460 bp, adaptors subtracted.

**Fig. 3** Size distribution of the Exome Captured library as shipped to the sequencing service provider, 1/4 dilution assessed with ©Agilent Technologies, Inc. TapeStation, high sensitivity D1000 ScreenTape®

### **4.1.4 DNA Sequencing**

The exome-enriched library along with the list of sample indices was submitted to a sequencing service provider for Illumina DNA sequencing PE150 (paired-end reads with 150 bp read length). We shipped 200 ng (50 µl, 4 ng/µl) and requested 400 Gbp raw data output. We received a total of 3.2 billion reads. They were fairly well distributed across the 41 samples (Fig. 4), with between 58 and 113 Mio reads per sample (Median: 75 Mio).

### **4.1.5 Analysis and Results**

We aligned all 3.2 billion sequencing reads to the coffee reference genome; the same annotated reference assembly that had been used to derive the targets (Cara\_1.0, NCBI accession number: GCA\_003713225.1). The reads were aligned with software bwa mem (Li and Durbin 2009) as part of our automated analysis workflow: A Software Workflow for Automated Analysis of Genome (Re-) Sequencing Projects, A Laboratory Protocol (IAEA 2022b). Software and documentation are available on PBGL's github page (https://github.com/pbgl).

The on-target enrichment for each individual sample was assessed from the alignments to the reference, represented in per sample.bam files, with the R-Bioconductor package TEQC (Hummel et al. 2011, 2020). Target definitions were the actual exons of the annotation (*see* Figs. 5 and 6 for results). As an example, a representative genomic region is shown in Fig. 7.

**Fig. 5** For each individual sample we assessed what fraction of the sequencing reads that align to the genome match annotated genes. Counting strictly the region annotated as exons we reach 80% with a very little variation between samples. When extending the target space by 100 or 200 bp to either side this fraction increases. This is expected, because the probes are fishing molecules from a library with an average insert size of 460 bp (Fig. 3). We can conclude that close to 90% of the sequencing reads are matching the target space

**Fig. 6** We assessed what fractions of genes are covered at least 1, 5, 10, 20 or 40-fold. More than 90% of annotated gene is covered at least one-fold and ¾ of the genes are covered more than tenfold


**Fig. 7** Visualization of successful target enrichment by the Exome Capture Kit. Depicted is a representative genomic region (screenshot of the Integrative Genomics Viewer, IGV, *see* Note 4), showing the alignments of sequencing reads (bam file) of 41 coffee samples on the Coffee arabica reference genome. Target regions (red bars) correspond to the exons (thick blue bars) of genes (blue bars). The libraries are effectively enriched for the target regions, reads (grey bars) pile on target regions (red bars) with very little background, i.e., non-target reads

# **5 Manuals**

1. The manufacturer's manuals for performing exome capture reactions with this kit

myBaits, Hybridization Capture for Targeted NGS Manual Version 4.01 April 2018, https://arborbiosci.com/wp-content/uploads/2019/08/myBaits-Manual-v4.pdf.

myBaits, Hybridization Capture for Targeted NGS User Manual Version 5.00 September 2020,

https://arborbiosci.com/wp-content/uploads/2020/08/myBaits\_v5.0\_Manual. pdf.

2. Sequencing library preparation

The custom-indexed Nextera NGS libraries for Illumina Sequencing were prepared following the PBGL protocol: *Library Preparation for Medium- to High-throughput DNA Sequencing on the Illumina Sequencing Platform, A Laboratory Protocol*  (IAEA 2022a).

3. Sequence read mapping

Read mapping with software bwa mem (*see* Note 5) (Li and Durbin 2009) was performed as part of PBGL's automated software workflow: *A Software Workflow for Automated Analysis of Genome (Re-) Sequencing Projects, A Laboratory Protocol*  (IAEA 2022b).

4. Quality assessment of the capture reactions

TEQC: Quality control for target capture experiments, Hummel et al. (2020). DOI:10.18129/B9.bioc.TEQC, TEQC, R package version 4.18.0. https://bioconduc tor.org/packages/release/bioc/html/TEQC.html (Hummel et al. 2011).

# **6 Notes**


**Acknowledgements** This work was funded by FAO/IAEA. The Exome Capture Kit design was a contribution by Daicel Arbor Biosciences (Ann Arbor, Michigan, USA). Mr Florian Goessnitzer (IAEA) provided tissue from in vitro coffee plantlets.

# **References**


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **High Resolution Melt (HRM) Genotyping for Detection of Induced Mutations in Coffee (***Coffea arabica* **L. var. Catuaí)**

**Andrés Gatica-Arias, Alejandro Bolívar-González, Elodia Sánchez-Barrantes, Emanuel Araya-Valverde, and Ramón Molina-Bravo** 

**Abstract** Arabica coffee (*C. arabica* L.) is a highly valued agricultural commodity on the world market. Tons of products are traded internationally, and it has become an extremely valuable resource. However, the species is threatened by the alarmingly low genetic diversity present among its wild populations and agronomic varieties. It is highly relevant to exploit different mechanisms to increase genetic variability in coffee. One of such methods is the induction of variability through chemical or physical mutagenesis. In this work, a population of 320 coffee plants (*Coffea arabica*  L. var. Catuaí) originated from chemically mutagenized embryogenic callus was analysed. Here we describe a protocol for detection of induced mutations using High Resolution Melting (HRM) on a Real Time PCR machine with HRM capabilities. The protocol allows to detect mutations in pooled DNA samples of up to four M2 mutant plants. The procedures and example data are presented for mutation detection in the CaWRKY1 gene. This procedure can be applied for mutation detection in other genes of interest to coffee breeders and scientists.

# **1 Introduction**

The genetic improvement of crops depends on the selection of genotypes with the desired, novel agronomic characteristics. Genetic variation provides the main resource to develop varieties adapted to different scenarios. Arabica coffee (*Coffea arabica* L.) is an allopolyploid species (2n =4x=44) that resulted from hybridization

e-mail: andres.gatica@ucr.ac.cr

A. Gatica-Arias (B) · A. Bolívar-González · E. Sánchez-Barrantes

Laboratorio Biotecnología de Plantas, Escuela de Biología, Universidad de Costa Rica, 2060 San Pedro, Costa Rica

R. Molina-Bravo Laboratorio de Cultivo de Tejidos y Células Vegetales, y Laboratorio de Biología Molecular, Universidad Nacional, Heredia, Costa Rica

E. Araya-Valverde Centro Nacional de Innovaciones Biotecnológicas, San José, Costa Rica

between two species extremely close to *C. eugenioides* and *C. canephora* (Scalabrin et al. 2020). The genetic variation present in Arabica coffee doesn't represent the entire possible conglomerate of spontaneous mutations. Rather, they result from the recombination of genotypes within populations and their continuous interaction with both biotic and abiotic environmental elements (Oladosu et al. 2016). Therefore, the availability of genotypes to introduce into a breeding program is limited.

Through induced mutagenesis it is possible to generate heritable changes in the genome of an organism, without the need for genetic segregation or recombination (Oladosu et al. 2016). These changes can be generated in genes that regulate characteristics of interest and finally allow their improvement or functional analysis. Mutation induction has been carried out in different tissue types through irradiation and exposure to chemical agents (Serrat et al. 2014). One of the most widely used chemical agents, is ethyl methanesulfonate (EMS), which mainly induces C-T substitutions that result in C/G to A/T transitions (Kim et al. 2006).

A fundamental aspect of the genetic improvement through induced mutagenesis is the process of identifying those plants with the mutations of interest in their genome. This can be done in two phases: 1) screening or detection of the mutants, and 2) confirmation of the mutation (Forster and Shu 2012). To achieve this, it is important to have an efficient and scalable detection strategy to increase the probability of detecting new genetic variants within mutant populations. The High-Resolution Melting (HRM) analysis can facilitate the detection of variants in genes. This technique does not involve any enzymes, but rather requires the presence of saturating fluorochromes that interact with the double-stranded DNA. In this way, a heteroduplex structure, with less stability, is denatured at lower temperatures than the DNA copies, a process that is monitored by the decrease in fluorescence emission (Szurman-Zubrzycka et al. 2016). When trying to detect mutations in large populations, it is convenient to pool the DNA of individuals, thus reducing the number of samples to be analyzed and consequently the cost. However, this clustering decreases the sensitivity and makes it difficult to detect low frequency mutations (Simko 2016). COLD-PCR (lower denaturation temperature co-amplification) can be applied to increase the sensitivity of HRM analysis by preferentially amplifying mismatched DNA. This is a modification of PCR where the reaction is carried out at a denaturation temperature at which the heteroduplex DNA is denatured in a greater proportion than the other DNA types (Chen and Wilde 2011). This chapter describes the PCR-HRM based detection of variants in genomic sequences of Arabica coffee plants var. Catuaí developed via chemical mutagenesis.

**Fig. 1** Coffee (*Coffea arabica* L. var. Catuaí) M2 mutant population. **a** M2 mutant plants in the experimental field, **b** fresh material brought from the field to the lab for DNA extraction, **c** young and disease-free leaves used for DNA extraction

# **2 Materials**

# *2.1 Plant Material*

1. M2 mutant coffee population (*e.g., Coffea arabica* L. var. Catuaí) (*see* Fig. 1a *see* Note 1).

# *2.2 Reagents*


# *2.3 Equipment*


# *2.4 Software*


# **3 Methods**

# *3.1 Preparation of Stock Solutions*


# *3.2 DNA Extraction*

The following protocol describes the procedure for the extraction of genomic DNA from young and disease-free coffee leaves. It has been optimized to eliminate or reduce oxidation during the extraction. Although it can be used on dry material, the recommendation is to use fresh tissue, which yields better quality DNA. To transfer the fresh material from the field to the lab, it is recommended to cut the branch and place it in a bag with water until it reaches the laboratory (*see* Fig. 1b).


# *3.3 Determination of DNA Integrity*


# *3.4 PCR Amplification of the 18S Gene*


**Fig. 2** Quantification and determination of DNA integrity, **a** DNA isolated from young and diseasefree leaves. **L:** 1 Kb DNA Ladder, **1–6**: samples, **b** quantification and DNA purity determined using a NanoDrop 2000 (Thermo Scientific) spectrophotometer, **c PCR** amplification of a 480 bp fragment of the 18S gene **L:** 100 bp DNA Ladder, **1–6:** samples

# *3.5 Literature Mining and Selection of Candidate Genes for Mutation Screening*


### *3.6 Primer Design*



**Fig. 3** Literature mining and selection of candidate genes for mutation screening. For details *see* Sect. 3.5


**Fig. 4** Designing primers for the amplification of a specific gene fragment using Primer3. For details *see* Sect. 3.6

	- A length of 18–25 nucleotides.
	- The melting temperature (Tm) between 55 and 65 °C, and not more than 3 °C difference of each other.
	- The GC content between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
	- Balanced distribution of GC-rich and AT-rich domains.

# *3.7 In Silico Analysis of Primer Specificity*


# *3.8 In Silico PCR*





**Fig. 5** Primer specificity in silico analysis performed at NCBI. For details *see* Sect. 3.7


**Fig. 6** In silico PCR analysis performed at NCBI. For details *see* Sect. 3.8

# *3.9 Nested PCR*


primer, 1.5 mM of MgCl2, 1.5 U Taq polymerase, and 1 µl DNA (90 ng/µl). Include a negative control, as well as a no-DNA template.


# *3.10 Mutation Identification Using the HRM Technique*

The mutation identification using PCR-HRM was performed using the nested PCR methodology.

	- Initial denaturation: 95 °C for 10 min.
	- Amplification (30 cycles): 95 °C for 30 s, 57 °C for 15 s, and 72 °C for 20 s
	- High-resolution melting:
		- Formation of homo- and heteroduplexes: 95 °C for 30 s, 40 °C for 60 s with continuous fluorescence acquisition (60–95 °C).

### High Resolution Melt (HRM) Genotyping for Detection of Induced … 287

**Fig. 7** Mutation identification using the HRM technique, **a** example of an HRM-PCR plate, **b** example of an HRM-PCR program of temperatures with melt curve protocol adjusted for posterior analysis

# *3.11 DNA Sanger Sequencing for HRM Validation*


**Fig. 8** Example of a melt file generated with the precision melt analysis, showing the normalized melt curve, the difference curve, the plate, and the classification of species by cluster

# **4 Notes**


range of 2.0–2.2 indicate the absence of contaminants (such as carbohydrates and phenol).


**Acknowledgements** Funding for this work was provided by the University of Costa Rica, the Ministerio de Ciencia, Tecnología y Telecomunicaciones (MICITT), the Consejo Nacional para Investigaciones Científicas y Tecnologicas (CONICIT) (project No. 111-B5-140; FI-030B-14) and a fellowship granted to Alejandro Bolivar-González by Centro Nacional de Alta Tecnología (CeNAT). A. Gatica-Arias acknowledged the Cátedra Humboldt 2023 of the University of Costa Rica for supporting the dissemination of biotechnology for the conservation and sustainable use of biodiversity.

# **References**

Chen Y, Wilde HD (2011) Mutation scanning of peach floral genes. BMC Plant Biol 11:1–8


**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **Protocols for Chromosome Preparations: Molecular Cytogenetics and Studying Genome Organization in Coffee**

### **Le Li, Trude Schwarzacher, Paulina Tomaszewska, Qing Liu, Xiaoyu Zoe Li, Kexian Yi, Weihuai Wu, and J. S. Pat Heslop-Harrison**

**Abstract** Cytological preparations from cell nuclei are required to count the number of chromosomes (including determining ploidy or aneuploidy), to investigate their morphology and organization. The results are valuable for genetic and evolutionary studies, and in breeding programs to understand species relationships, polyploidy, and potential introgression of chromosomes in hybrids between different species. Preparation of good chromosome spreads with well-separated metaphase chromosomes is the foundation of cytogenetic research including chromosomal mapping based on FISH (fluorescence in situ hybridization). FISH combined with specific locus probes correlated with molecular markers to specific chromosomes for integrating physical and linkage maps as well as studying the genetic evolution of allopolyploidization, has rarely been applied in *Coffea* spp. despite being a global high-value crop. Cytogenetic studies of *Coffea* are limited by the small size and similar morphology of the chromosomes, but FISH can help to map sequences to

L. Li

J. S. Pat Heslop-Harrison e-mail: phh4@le.ac.uk; phh@molcyt.com

T. Schwarzacher · Q. Liu · X. Z. Li · J. S. Pat Heslop-Harrison Key Laboratory of Plant Resources Conservation and Sustainable Utilization/Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Tianhe District, Xingke Road 723, Guangzhou 510650, P. R. China

P. Tomaszewska Department of Genetics and Cell Physiology, Faculty of Biological Sciences, University of Wrocław, 50-328 Wrocław, Poland

K. Yi · W. Wu Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Science (CATAS), Longhua District, Xueyuan Road 4, Haikou, Hainan 571101, P. R. China

NHC Key Laboratory of Tropical Disease Control/Key Laboratory of Tropical Translational Medicine of Ministry of Education, School of Tropical Medicine, Hainan Medical University, NO.3 of Xueyuan Road, Haikou, Hainan 571199, P. R. China

T. Schwarzacher (B) · P. Tomaszewska · J. S. Pat Heslop-Harrison (B) Department of Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK e-mail: ts32@le.ac.uk

chromosome arms and identify individual chromosomes. This chapter presents protocols for germinating seeds and growing coffee plants involving pre-treatment and fixation of root-tips where the meristems of actively growing roots have many divisions. Mitotic metaphase chromosome preparation on microscope slides is described, as well as preparing probes of 5S and 18S rDNA to be used for FISH. The FISH experiments involve a two-step protocol with pre-treatments and setting up the hybridization on day 1 and the detection of probe sites on day 2 after overnight hybridization. A final section gives advice about visualization using a fluorescent microscope and capturing images.

# **1 Introduction**

The vast majority of commercial coffee comes from two closely related species, *Coffea canephora* and *Coffea arabica* (Melese and Kolech 2021). *C. canephora*  (known as robusta; 35% of world production) is diploid with two sets of chromosomes, while *C. arabica* (65% of production) is tetraploid, with four chromosome sets from two ancestral species (see other chapters in this volume). Thus, the genome of *C. arabica* has four sets of chromosomes (2*n* = 4x = 44), and four copies of most genes (compared to the two in *C. canephora* (2*n* = 2x = 22) (see Hamon et al. 2009). At meiosis, the tetraploid *C. arabica* behaves as a diploid and chromosomes pair and recombine. The genus *Coffea* includes in total over 90 species, with wild species of coffee including both diploids and polyploids (see Lashermes et al. 1997; Melese and Kolech 2021). The use of this germplasm through hybridization can increase the genetic base of the crop, and may be used to transfer useful genes from wild relatives into a crop variety.

The study of chromosome numbers in an accession of a species is important to give the ploidy-level (Tomaszewska et al. 2021). Identification of individual chromosomes by morphological analysis or by in situ hybridization with DNA probes, can be used to link the genetic map to physical chromosomes (Paesold et al. 2012), and track chromosomes in breeding programmes involving hybridization and recombination (eg, in cereals, Patokar et al. 2016 or Ali et al. 2016). In crosses involving wide species or polyploid species, cytogenetic study of the chromosome numbers and morphology is particularly valuable to define which crosses may be most easy to make, and to determine new combinations of chromosomes in hybrids and backcross derivatives. In some cases, recombination between chromosomes of different species is required to introgress useful agronomic characteristics without undesirable characters. Aneuploidy, involving the loss or gain of one or more chromosomes (e.g., Niemelä et al. 2012 in *Brassica*; Tomaszewska et al. 2023 in *Urochloa*), is found occasionally, along with other types of chromosome rearrangements such as inversions, deletions or translocations (eg, Forsström et al. 2002; Liu et al. 2019; Tomaszewska and Kosina 2021), particularly in irradiated material and after some tissue culture protocols.

The key method for molecular cytogenetics is fluorescent in situ hybridization (FISH) that allows visualizing the location of DNA sequences to be determined along chromosomes, providing cytogenetic maps of chromosomes (Schwarzacher 2003; Heslop-Harrison and Schwarzacher 2011; Baˇcovský et al. 2018). Many repetitive sequences can be used to provide chromosomal landmarks to identify chromosomes, show aspects of genome organization and evolution, and track chromosome presence or rearrangement through evolution and crossing programmes. High-resolution FISH mapping on mitotic chromosomes is a powerful technique to help integrate physical and genetic maps and also to evaluate genome assembly quality (Szinay et al. 2008). Although now less used than in the past, chromosome banding can also offer chromosome differentiation and identification (e.g. Schwarzacher 2003, Kumar et al. 2021 and in coffee Pierozzi et al. 1999). Meiotic analysis can show the pairing of chromosomes and reveals any translocations between chromosomes, either during evolution or following breakage and rejoining (e.g. Lashermes et al. 2000).

Preparation of high quality metaphase, with well-spread chromosomes free of cytoplasm and other cellular material, containing high number of divisions, is a prerequisite for cytogenetic studies such as chromosome counting, morphological analysis and mapping, chromosome banding procedures, and in situ hybridization. In this chapter, we describe the basic methods of chromosome preparations and fluorescent in situ hybridization (FISH) in coffee species, including protocols to obtain root tips with abundant metaphases from seedlings, root tip sampling and fixation, mitotic chromosome spread preparation and the basic steps of FISH for repetitive DNA using rDNA probes. Schwarzacher and Heslop-Harrison (2000) and Schwarzacher (2016) give more details about chromosome preparation and in situ hybridization for many species.

Cytogenetic maps provide an efficient tool for gene localization, validation of contig order from sequence analysis, characterization of gene regions or physical genetic distances, and the presence of chromosomal rearrangements such as inversions or translocations (Heslop-Harrison and Schwarzacher 2011). In the protocol below, we show the use of the repetitive rDNA as probes to identify coffee chromosomes carrying the 35S/45S and 5S rDNA loci, which provide robust and useful landmarks on chromosomes (e.g., Široký et al. 2001; Ali et al. 2016; and for coffee Hamon et al. 2009). Many other repetitive probes can be used to provide landmarks. Generally, short lengths of probes from single-copy genes or groups of genes (singlecopy FISH) do not work reliably in plants. In most cases, repetitive sequences simple sequence repeats, tandem repeats from satellite DNA, or rDNA genes are used as probes to provide chromosomal landmarks (e.g. Liu et al 2019; Agrawal et al. 2020; Rathore et al. 2022). Large insert clones, in particular BACs, or large pools of synthetic oligonucleotide probes (typically 20,000 bp or more) may also be used (see Niemelä et al. 2012; Zaki et al. 2021), particularly when any repetitive sequences within the probes have been removed. Mitotic metaphase chromosome FISH can locate probes with a longitudinal resolution of about 2–20 Mb. Meiotic chromosome preparations can also be used, and in some circumstances may be readily available. In particular, pachytene chromosomes from meiotic prophase may give resolution of in situ signal of 50–100 kb (e.g. Szinay et al 2008, Mandáková et al. 2019). Other systems with statistical analysis of hybridization sites within interphase nuclei, stretched chromosomes (digested with proteinase K), and fibre FISH to DNA fibres from the nucleus extended to nearly their full molecular length can be used to give higher resolution of a few kb (see Schwarzacher and Heslop-Harrison 2000).

The application of FISH to coffee mitotic chromosomes has provided opportunities for identifying chromosomes and mapping genes and sequences of interest in several *Coffea* species. On several cultivated and wild species of coffee, mitotic chromosomes, as well as meiotic pachytene have been used to map using repetitive sequences of 45S rDNA and 5S rDNA or BACs linked to resistance genes as probes for in situ hybridization (Lombello and Pinto-Maglio 2004a, b, c; Pinto-Maglio 2006; Herrera et al. 2007; Hamon et al. 2009; Iacia and Pinto-Maglio 2013). Coffee mitotic chromosomes in metaphase are small (1–3 μm) and have similar morphologies, making their individual identification difficult (Krug 1934, 1937; Mendes 1957). On these small chromosomes, the exact location of a small repetitive sequence by in situ hybridization is also difficult to determine (Lombello and Pinto-Maglio 2003; Herrera et al. 2007), although the repetitive probes and BACs can identify clearly chromosome arms and domains such as terminal, intercalary or centromeric.

Most chromosome analyses rely on mitotic metaphases, so dividing tissue is essential and is best obtained from healthy, disease free, and rapidly growing plants (Schwarzacher and Heslop-Harrison 2000; Schwarzacher 2016). Among plant tissues containing actively dividing cells, root-tip meristems are one of the most commonly used, but other plant tissues such as meristematic cells from young shoots, leaves or emerging buds as well as hairy root cell culture lines or liquid tissue culture cells (Anamthawat-Jónsson and Thórsson 2003; Baˇcovský et al. 2018). Calli or protoplasts (Nishibayashi et al. 1989) from tissue culture can be also used for chromosome preparation but it is difficult to obtain many metaphases, and to spread chromosomes sufficiently that they can be counted. The best source to obtain fresh coffee root tips for chromosome preparation is from seedlings or young plants. When growing tropical species in temperate climates, it is important to understand temperature and light requirements and we give suggestions to achieve best results.

Although several methods for chromosome preparation are available, we recommend to use the squashing method for coffee. Mitotic chromosomes are released with cells from fixed rapidly dividing root tips, and spread onto a microscopic slide into a single layer by gentle pressure on the digested tissue through squashing. Classically, this preparation method was used in combination with acetocarmine staining to analyse the number and shape of metaphase chromosomes. Not discussed below, but preparations for chromosome counting can be made by acid digestion of root-tips, staining with Feulgen's reagent, before spreading of cells and chromosomes on a microscope slide. The squashing method does not require costly equipment and usually gives high quality metaphase spreads. The protocols for labelling probes for rDNA is based on PCR amplification and the two step FISH experiments follow our previous published protocols (Schwarzacher and Heslop-Harrison 2000) and although modifications and optimizations for coffee chromosomes are included below.

# **2 Materials**

It is assumed that a well-equipped laboratory with molecular biology and microscopy facilities and consumables are available; these include microcentrifuge (Eppendorf) tubes, automatic pipettes, tips, micro centrifuge, balance, stirrer and mixer. Only more specialized or essential equipment is described in the following list of materials.

# *2.1 Seed Germination and Plant Cultivation*


# *2.2 Fresh Root Sampling and Fixation*


# *2.3 Chromosome Preparation*

1. Enzyme buffer (10 mM, pH 4.6): for a 100 mM stock solution, mix 100 mM citric acid and 100 mM tri-sodium citrate in a ratio of 2:3 and autoclave (once opened can be stored at 4 °C for a few days to 2 weeks). Before use dilute buffer with distilled water to 10 mM.


# *2.4 Probe Labelling*

	- a. 5S rDNA: DNA of clone containing the 5S rDNA repeat, pTa794 from wheat, *Triticum aestivum* (Gerlach and Dyer 1980), insert length 410 bp.
	- b. 18S-5.8S-26S rDNA (35S or 45S rDNA): total genomic DNA of wheat or rice for amplification (coffee DNA would probably work too).
	- a. For clones M13 sequencing primers; eg. M13 forward (GTA AAA CGA CGG CCA GT) and M13 reverse (GGA AAC AGC TAT GAC CAT G); this will amplify the insert plus about 30–50 bp on each side depending on the cloning site.
	- b. 35S/45S rDNA: Primers based on 18S rDNA sequence of rice (Chang et al. 2010) rice\_18S\_P1 forward (CGA ACT GTG AAA CTG CGA ATG GC) and rice\_18S\_P2 reverse (TAG GAG CGA CGG GCG GTG TG); the product will be about 2.7 kb depending on species of the template DNA. (*see* Note 7).

# *2.5 FISH Day 1: Pre-treatments and Hybridization*

	- a. Formamide (molecular grade).
	- b. Dextran sulfate: 50% (w/v) solution in water, heat to dissolve and sterilize by forcing through a 0.22 μm filter.
	- c. SDS solution: 10% (w/v) sodium dodecyl sulfate (also called sodium lauryl sulfate) in water, filter sterilize; store at room temperature.
	- d. Salmon sperm DNA: 4 μg/μl sonicated or autoclaved DNA (also suitable are herring sperm or *E. coli* DNAs).
	- e. 20X SSC (sterile): see above Sect. 2.1.
	- f. Optional EDTA (Ethylene-diamine-tetra-acetic acid): 100 mM, pH 8.
	- g. Probe DNA from protocol 3.4.
	- h. Molecular grade distilled water.

# *2.6 FISH Day 2: Detection of Hybridization Sites and Mounting of Slides*

	- a. streptavidin (e.g. Alexa Fluor 594 conjugated; 1mg dilute with distilled water to 200 μg/ml; Molecular Probes, Invitrogen).
	- b. anti-digoxigen antibody (e.g. FITC conjugated FAB fragment, 200 μg/ml; Roche).

### *2.7 Microscopy and Image Analysis*


# **3 Methods**

### *3.1 Seed Germination and Plant Cultivation*

Coffee seeds are able to germinate with water when isolated from the yellowishgreen fruits at around 225d after anthesis (Eira et al. 2006; Bytof et al. 2007). Coffee embryos are very sensitive to low temperature and are damaged when seeds are kept at temperatures below 25 °C. However, seed storage for medium periods of a few weeks to months at 25 °C is possible if environmental relative humidity is maintained around 50%, while for conservation at freezing temperatures, a lower moisture content of coffee seeds and hermetic conditions are required (Eira et al. 1999, 2006; Patui et al. 2014).

Once seeds have germinated and seedlings are transferred to soil, favorable temperatures and high air humidity near saturation are the most important conditions required for good growth. *C. arabica* is well adapted to cooler temperatures with the optimum growth at mean annual temperature in natural conditions ranging from 18 to 22 °C, while *C. canephora* is better adapted to higher temperatures, with the optimum growth at annual mean temperature ranging from 22 to 30 °C (Pohlan and Janssens 2010).

In a greenhouse (warm tropical or temperate conditions, 25–28°C, 14h day) or growth chamber, the following method yields coffee plants for molecular biology and cytology experiments and analysis. In order to maintain high humidity of 80–90% needed for good growth, individual plants are covered with plastic bags.


Use either of the following methods in the green house or growth chamber.


# *3.2 Fresh Root Sampling and Fixation*

In order to maximise the number of metaphases, the best period to sample fresh root tips is 4–4.5 h after sunrise or lights coming on. Only collect from healthy and well growing plants and only take roots with white ends indicative of new growth (Fig. 1). Steps are carried out at room temperature unless otherwise stated; use about 3X as much solution as plant material and make sure that roots are well covered by each solution.


**Fig. 1** Coffee seedlings and fresh roots, **a** seedlings in good health have many freshly growing roots to sample. **b** The arrows and the circles mark the fresh roots which are the best choices for chromosome preparation. The sampled roots should be less than 1 cm suitable for the small tubes

# *3.3 Chromosome Preparation*

Having good preparations with plenty well spread metaphase chromosomes that are free of cytoplasm is the most crucial prerequisite of successful chromosome banding or in situ hybridization. Here we describe the method using proteolytic enzymes including pectinase and cellulase to remove cell walls and squashing dissected meristematic tissue in acetic acid between glass slide and cover slip. The method is modified from Schwarzacher et al. (1980) and Schwarzacher and Heslop-Harrison (2000). Steps are carried out at room temperature in a Petri dish if not otherwise stated.


**Fig. 2** Enzyme treatment of the root tips

**Fig. 3** Example of roots with pointed root caps and meristematic cells just behind. Cutting away non-dividing tissue is essential for preparations with high metaphase index

	- a. Put a drop of 60% acetic acid on a clean slide and then place one or two treated root tips in it. Leave for 1–3 min.
	- b. Under a dissection microscope, separate the root cap (<0.1 cm) from the root tip using a clean needle or forceps and discard.
	- c. Dissect the root meristem that contains the dividing cells in the acetic acid and separate individual cells by tapping or squeezing with a fine forceps; remove non-meristematic tissues and mix cells evenly.

**Fig. 4** Examples of the chromosomes under phase contrast microscope. Arrows mark several typical features of the chromosome preparation. **a** Nuclei are at good density; arrow1 shows the chromosomes from a single cell, but not well spreads. **b** Cells are too dense and nuclei overlap; arrow2 shows pro-metaphase chromosomes, that are not well spread. Arrow3 shows metaphase chromosomes, but they are squashed to hard, so some chromosomes are distorted or destroyed. **c** Well separated cells and metaphase chromosomes. Arrows 4 and 5 are good examples of metaphase chromosome preparation, well spreads and very little cytoplasm. The round opaque object is the nucleolus


### *3.4 Probe Labelling*

An important factor for the success of FISH experiments is the choice of probes. The amount of target sequences are critical and low copy sequences present at less than 10–15 kb at one site within the chromosomes are not suitable whereas repetitive sequences in large arrays such as the rDNAs or tandem repeats are ideal targets. Template DNAs to be used for generating probes can be inserts of clones, PCR products or total genomic DNA. Probes after labelling should be 100–300 bp long to allow for sufficient penetration to the DNA within the chromosomes, but shorter probes of 30–100 bp are also suitable, while probes longer than 500 bp are not recommended. Many different labelling kits are available commercially and use DNA polymerases in random priming or nick-translation that automatically generate

**Fig. 5** Fluorescent in situ hybridization of a root tip metaphase of *C. arabica* (2*n* = 4x − 44). **a** The chromosomes are stained with DAPI **(a)** showing centromeres as small gaps or slightly brighter bands **(b)**. Overlay of DAPI image with 18S rDNA signal in green **(c)** and 5S rDNA signal in red **(d)**. One pair of chromosomes has a major 5S rDNA site near the centromere and a terminal 18S rDNA site of the small chromosome arm. Additionally, there is one pair of minor 18S rDNA sites (arrows) and one pair of minor 5S rDNA sites (arrowhead). Bar = 5 μm

probes of suitable lengths from larger templates. However, template DNAs longer than 2 kb do not label efficiently and will need cutting with enzymes, sonication or heat (Schwarzacher and Heslop-Harrison 2000; Salvo-Garrido et al. 2001).

Either labelled dUTP or dCTP are used and dependent on the labelled attached different ratios to unlabelled dTTP or dCTP nucleotides are recommended to allow efficient incorporation by the DNA polymerase. Manufacturers give detailed instructions of the procedure and recommendation for amounts of reagents to be used, but we have found that often the amount of expensive labelled nucleotides can be reduced when they are fresh and have not undergone several freeze-thaw cycles. We recommend to use biotin and digoxigenin as labels and here we give the rDNA probes that we used on coffee chromosomes (Fig. 5), but they can also be used on any plant species. Similarly, any cloned DNA or amplified PCR product that represents the repeats to be visualized in the species of interest are also suitable.

	- a. For 5S rDNA: use miniprep DNA of clone pTa794 with M13 primers to amplify the insert by PCR using an annealing temperature of 56 °C. Expected product insert size plus about 80 bp (Fig. 6).

**Fig. 6** PCR amplification of pTa794 insert using M13 primers. The image shows the following lanes from left to right: 100 bp ladder (with 100, 200, 300, 400, 500, 600, 700, 800, 900 bp, 1, 1.25, 1.5 and 2 kb band), 3 PCR replicas of the same miniprep DNA, empty lane, 3 PCR replicas of a different miniprep DNA, 100 bp ladder. The correct product is a very strong band at about 500 bp as the primers used are outside the cloning site and add 81 bp to the insert length of 410 bp. Some background smear and bands are also visible. It is therefore best to cut the band and purify before labelling

	- a. If there is a single sharp band of the expected size, then the entire PCR product can be used for labelling after purification.
	- b. If there are several bands, or a smear, then cut out the band of the expected size, extract DNA from the band and purify.

### *3.5 FISH Day 1: Pre-treatment and Hybridization*

For in situ hybridization, the protocol of Schwarzacher et al. 1989 and Schwarzacher and Heslop-Harrison (2000) is used with some adaptations to coffee chromosomes reported by Pinto-Maglio (2006) and several optimizations proposed here. Steps are carried out at room temperature unless otherwise indicated. Washing steps and incubation in buffers are carried out in Coplin jars (holding 8 slides and 80–100 ml solution); specific reagents are applied in small volumes of 200–300 μl per slide and covered with a plastic cover slip and incubated in a humid chamber.

	- a. Incubate slides in fixative (ethanol/acetic acid 3:1) for 10–30 min.
	- b. Wash with 100% ethanol 2 times for 5 min each.
	- c. Air-dry.
	- a. Apply 200 μl RNase solution to each slide and cover with a plastic cover slip.
	- b. Incubate for 1 h at 37 °C in a humid chamber.
	- c. Wash 2 times in 2X SSC for 5 min each.
	- a. Incubate slides in 0.01 M HCl for 2 min.
	- b. Shake of excess solution and apply 200 μl Pepsin solution to each slide and cover with a plastic cover slip.
	- c. Incubate at 37 °C for 10 min in a humid chamber.
	- d. Rinse in distilled water for 1 min.
	- e. Wash in 2X SSC for 5 min.
	- a. In the fume hood, incubate slides in 4% paraformaldehyde solution for 10 min.
	- b. Wash in 2X SSC for 5 min.
	- a. Decide on probes (from **Protocol 3.4**) and amounts to be used, normally the final concentration of the probes should be 1–3 ng/μl in a hybridization mixture (*see* Table 1). Each slide can be probed with two different probes (e.g. 5S and 35/45S rDNA), but each probe needs to be labelled with a different hapten (e.g. biotin and digoxigenin) so it can be detected with a different antibody linked to a different fluorochrome (*see* **Protocol 3.6** and Fig. 5).
	- b. Calculate and make master mix for all slides plus one following Table 1. Mix well and keep on ice.
	- c. Prepare the hybridization mixture for each slide in a separate tube by adding master mix, probe and water following Table 1. Mix gently but thoroughly.
	- d. Denature hybridization mixtures at 75 °C for 10 min and stabilize on ice for 10 min.
	- a. Apply hybridization mixture on each slide and cover with a plastic coverslip.
	- b. Denature chromosomes and hybridization mixture in hybridization oven. This step is critical, and time and temperature will need adjusting even if the same species or variety is used. It is influenced by n the way chromosome preparations are made, how plants were grown, how old fixations were when used for preparation and how long slides were stored before FISH. As a guide, use 72–75 °C for 5–8 min.
	- c. Hybridize slides in the hybridization oven or a humid chamber at 37 °C overnight (about 16 h).

# *3.6 FISH Day 2: Detection of Hybridization Sites and Mounting of Slides*

Original methods for FISH used 20 or 50% formamide for washing steps (Schwarzacher and Heslop-Harrison 2000), but to avoid using this toxic chemical, we now routinely use low salt conditions for stringency washes as this also reduces the background created by formamide. Take care that the slides do not dry out during all steps of the protocol. Washes are carried out in a shaking waterbath if available, otherwise gentle shaking by hand is recommended once every 30–60 s. We describe here the use of two probes labelled with digoxigenin and biotin and they must be detected with two different colours, we recommend to use FITC for digoxigenin detection and Alexa594 for biotin detection (*see* Sect. 2.6 step 4), but other fluorochromes can be used too (Schwarzacher and Heslop-Harrison 2000). For visualization of chromosomes, slides are stained with DAPI (4', 6-diamino-2-phenylindole) and mounted in antifade solution.

	- a. Prepare post-hybridization wash solutions and heat in 45 °C waterbath.
	- b. Collect slides from hybridization oven, carefully examine for bubbles, extra water or dried out patches and note down if there are any irregularities.
	- c. Put slides from hybridization in 2X SSC at 35–40 °C to float off coverslips.
	- d. Wash slides in 2X SSC at 42 °C for 2 min.
	- e. Wash twice in 0.1X SSC at 40–45 °C for 5 min; record temperature.
	- f. Wash in 2X SSC for 5 min. Allow to cool to room temperature.
	- a. Transfer slides to detection buffer
	- b. Shake of excess solution and apply 200 μl of blocking solution to each slide and cover with a plastic cover slip. Incubate at RT or 37 °C for 10 min.
	- c. Remove coverslip, drain slides and apply 40–50 μl of appropriate detection solution to each slide (*see* Sect. 2.6 and Table 2).
	- d. Replace the coverslip and incubate at 37 °C for 1 h.
	- e. Wash slides in detection buffer at 40–42 °C 3 times for 5 min each.

# *3.7 Microscopy and Image Analysis*

For visualization of probe hybridization and chromosome staining, an epifluorescence microscope equipped with suitable filters for the fluorochromes used in the detection step. A selection of filters are given in Table 2. Apart from a 20 or 40X lens for scanning the slides, you will need a top of the range 63 or 100X lens for image capture that all need to be specified for UV fluorescence. Also make sure you use immersion oil that is specified for fluorescence analysis (*see* Notes 21 and 22). The microscope should be located on a stable surface in a completely dark room with a comfortable adjustable chair and ideally a small lamp with a dimmer switch, so operation for several hours is possible.


# **4 Notes**



**Table 1** FISH hybridization mixture

a Components of master mix. Make this for the number of slides plus one extra

**Table 2** Common fluorochromes and microscope filter sets used for FISH analysis. When using DAPI and two FISH probes, images can be conveniently displayed in RGB mode with each captured image in a separate channel. All microscope and filter manufacturers have useful descriptions, graphics and often active visualizations of filter/fluorochrome combinations (*see* Note 22)


**Acknowledgements** The work was carried out in the framework of the IAEA Coordinated Research Programme CRP22005 "Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana". P.T. has received support from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreements No 844564 and No 101006417 for analysis of polyploid chromosomal evolution. We thank John Bailey and Adel Sepsi for sharing useful information about some of the reagents and details described in the protocols.

# **References**


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