## Joanna Jankowicz-Cieslak Ivan L. Ingelbrecht *Editors*

# Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana

Protocols

Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana

Joanna Jankowicz-Cieslak • Ivan L. Ingelbrecht Editors

# Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana

Protocols

Editors

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

Ivan L. 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

ISBN 978-3-662-64914-5 ISBN 978-3-662-64915-2 (eBook) https://doi.org/10.1007/978-3-662-64915-2

© IAEA: International Atomic Energy Agency 2022. This book is an open access publication. The opinions expressed in this publication are those of the authors/editors and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent.

Open Access This book is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

The images or other third party material in this book are included in the book's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the book'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.

The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer-Verlag GmbH, DE part of Springer Nature.

The registered company address is: Heidelberger Platz 3, 14197 Berlin, Germany

## Preface

Globally, bananas are the fourth most important food crop and are consumed both as a fruit and as a staple. Around 85% of the global production is destined for local markets in Southern countries while 15% enters the international trade. As such, bananas play a major role as a source of food, income and employment for smallholder farming systems in the South, in addition to being an important internationally traded commodity.

Currently, a deadly fungal disease, Fusarium wilt, is a major threat to banana production in all parts of the world. Fusarium wilt (FW) is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). The fungus enters the banana plant through the roots and travels into the trunk and leaves where it causes premature wilting and eventually kills the plant. Since the nineteenth century, several strains of Foc have emerged. In the 1990s, a new strain of FW appeared in Asia that devastated banana plantations that were hitherto resistant to FW. This new strain is referred to as 'Tropical Race 4' or TR4. From Asia, TR4 spread to Australia, Africa and, very recently, also to Latin America, the most important banana exporting region globally. Foc TR4 is the most devastating of all FW strains because it not only affects Cavendish bananas but also many other bananas grown by smallscale farmers in Africa, Asia and Latin America. Therefore, Fusarium wilt TR4 has become a matter of international importance not only for the banana export industry but also for income generation and food security in smallholder farming systems in Southern countries.

Host plant resistance is a fundamental component for integrated management of Fusarium wilt in banana. Conventional cross-breeding of banana is hindered by several constraints, including polyploidy and low reproductive fertility in nearly all domesticated bananas. Mutagenesis techniques using physical or chemical mutagens offer an attractive, alternative approach to generate novel genetic diversity for banana genetic improvement, given that these methods do not require seed for the mutagenesis treatment.

To help address the banana Fusarium wilt TR4 pandemic, the FAO/IAEA Plant Breeding and Genetics subprogram launched a Coordinated Research Project (CRP) titled 'Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana'. The CRP brought together leading banana breeders and experts from across the world who used mutagenesis techniques in combination with innovative biotechnology tools to develop and screen mutant populations for resistance to Fusarium wilt TR4.

This book comprises a collection of protocols ensuing from their efforts. The protocol chapters cover conventional and innovative methods for the preparation and mutagenesis of target explants or cells for mutation induction together with lab-, greenhouse- and field-based screening techniques specifically for Fusarium wilt. Low-cost methodologies for in vitro multiplication are also described as well as advanced genomics and bioinformatics tools for mutation discovery. While the CRP and protocols were mainly focused on Cavendish bananas, similar technologies can be adapted to other banana varieties, including cooking banana, an important staple food in tropical countries.

The main purpose of this protocol book is to widely disseminate the methods and techniques developed under this CRP with FAO/IAEA Member States involved in banana breeding generally and especially those countries faced with the new threat of Fusarium wilt TR4.

Ivan L. 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

## Acknowledgements

We would like to thank the entire Plant Breeding and Genetics team, past and present, especially Dr Bradley J. Till and Dr Stephan Nielen, 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 the CRP participants for sharing their unique expertise in banana breeding for Fusarium wilt TR4 resistance and contributing the protocols. We further acknowledge the reviewers of the book chapters for their valuable inputs. Funding for this CRP and supporting research at the PBG Laboratory 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, with additional support from Belgium through the Peaceful Uses Initiative "Enhancing climate change adaptation and disease resilience in banana-coffee cropping systems in East Africa" (code: EBR-BEL01-18-03) and the USA through the support of an associate research officer (plant pathology) (code: EBR-USA07-13-13).

## Contents

#### Part I Mutation Induction



## Contributors

Faiz Ahmad Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

Zaiton Ahmad Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

Mustapha Akil Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

Sakinah Ariffin Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

Sheryl Bothma Department of Plant Pathology, Private Bag X1, Stellenbosch University, Matieland, South Africa

Catherine Breton Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France

Alberto Cenci Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France

Rachel Chase Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France

Babita Jhurree-Dussoruth Food and Agricultural Research and Extension Institute, Reduit, Mauritius

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

Affrida Abu Hassan Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

Chunhua Hu Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences; Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization; Ministry of Agriculture and Rural Affairs, Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research, Guangzhou, China

Rusli Ibrahim Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

Ivan L. 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

Behnam Naserian Khiabani Plant Breeding Department, Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran

Gaudencia A. Lantican Dole Philippines, Inc., Davao City, Philippines

Anis Nadia Mohd Faisol Mahadevan Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

George Mahuku International Institute of Tropical Agriculture (IITA) Regional Hub, Dar-es-Salaam, Tanzania

Diane Mostert Department of Plant Pathology, Private Bag X1, Stellenbosch University, Matieland, South Africa

Privat Ndayihanzamaso Department of Plant Pathology, Private Bag X1, Stellenbosch University, Matieland, South Africa

Norazlina Noordin Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia

Mathieu Rouard Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France

Max Ruas Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France

Nicolas Roux Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France

Julie Sardos Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France

Bradley J. Till Veterinary Genetics Laboratory, University of California, Davis, Davis, CA, USA

Altus Viljoen Department of Plant Pathology, Private Bag X1, Stellenbosch University, Matieland, South Africa

Yuanli Wu Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences; Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization; Ministry of Agriculture and Rural Affairs, Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research, Guangzhou, China

Ganjun Yi Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences; Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization; Ministry of Agriculture and Rural Affairs, Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research, Guangzhou, China

## Chapter Reviewers

Souleymane Bado Plant Science (PHYTOPRISE GmbH), PHYTONIQ GmbH, Oberwart, Austria

Nadiya Akmal Baharum Faculty of Biotechnology and Biomolecular Sciences, Department of Cell and Molecular Biology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Ratri Boonruangrod Department of Horticulture, Faculty of Agriculture at KPS, Kasetsart University, Nakhon Pathom, Thailand

Azam Borzouei Agricultural Research School, Nuclear Science and Technology Research Institute, Karaj, Iran

Christopher Cullis Department of Biology, Case Western Reserve University, Cleveland, Ohio, USA

Jessada Doungkeow Department of Horticulture, Faculty of Agriculture at KPS, Kasetsart University, Nakhon Pathom, Thailand

Ilona Czyczylo-Mysza The Franciszek Górski Institute of Plant Physiology Polish Academy of Sciences, Krakow, Poland

Alexandra zum Felde Independent Consultant, Witzenhausen, Germany

Stanley Freeman Department of Plant Pathology and Weed Research, ARO, The Volcani Center, Rishon LeZion, Israel

Prateek Gupta Department of Genetics, Hebrew University of Jerusalem, Jerusalem, Israel

Sobri Hussein Agrotechnology and Biosciences, Malaysian Nuclear Agency, Kuala Lumpur, Malaysia

Rusli Ibrahim Agrotechnology and Biosciences, Malaysian Nuclear Agency, Kuala Lumpur, Malaysia

Praphat Kawicha Kasetsart University, Sakon Nakhon, Thailand

Sofia Khan Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland

Andrea Kodym Department of Forest Biodiversity and Nature Conservation, Federal Research and Training Centre for Forests, Natural Hazards and Landscape, Vienna, Austria

Ales Lebeda Faculty of Science Department of Botany, Palacky University, Olomouc-Holice, Czech Republic

Freddy Arturo Magdama Tobar Facultad de Ciencias de la Vida, Escuela Superior Politécnica del Litoral, Guayaquil, Ecuador

Iza Marcinska The Franciszek Górski Institute of Plant Physiology Polish Academy of Sciences, Krakow, Poland

Bernard Pecheur Plant Pathology Division, Food and Agricultural Research and Extension Institute (FAREI), Reduit, Mauritius

Raman Thangavelu Plant Pathology Division, ICAR-National Research Centre for Banana, Tiruchirappalli, Tamil Nadu, India

Pumiphat Tongyoo Center for Agricultural Biotechnology, Kasetsart University, Bangkok, Thailand

Vivian Vally Plant Pathology Division, Food and Agricultural Research and Extension Institute (FAREI), Reduit, Mauritius

Sijun Zheng Banana Pest and Disease and Production Systems, Banana Asia Pacific Network (BAPNET), Alliance of Bioversity International and CIAT, Kunming, Yunnan, China

## Part I Mutation Induction

## Chapter 1 Induced Mutagenesis and In Vitro Mutant Population Development in Musa spp.

Joanna Jankowicz-Cieslak, Florian Goessnitzer, Bradley J. Till, and Ivan L. Ingelbrecht

Abstract Mutagenesis of in vitro propagated bananas is an efficient method to introduce novel alleles and broaden genetic diversity. Mutations can be induced by treatment of plant cells with chemical mutagens or ionizing radiation. The FAO/IAEA Plant Breeding and Genetics Laboratory established efficient methods for mutation induction of in vitro shoot tips in banana using physical and chemical mutagens as well as methods for the efficient discovery of EMS-induced single nucleotide mutations in targeted genes or amplicons and identification of large genomic changes, including deletions and insertions. Mutagenesis of in vitro propagated tissues requires large populations serving as starting material, and a long process to dissolve genetic mosaics (chimeras) resulting from the mutagenesis of multicellular tissues. However, treating shoot apical meristems of tissue cultured bananas with a mutagen is a commonly used practice for banana mutation breeding programmes, and still the most effective. In our previous studies, we showed that chimeras, unique mutations accumulated in different cells of the plant propagule, could be rapidly removed via isolation of shoot apical meristems and subsequent longitudinal bisection. Further, induced mutations were maintained in mutant plants for several generations. We established such systems for inducing and maintaining both point mutations caused via EMS mutagenesis as well as insertions and deletions caused by gamma irradiation and describe hereafter methods for dose selection, gamma irradiation and chimera dissolution.

Keywords Mutation induction · Gamma mutagenesis · Banana · Polyploidy · Fusarium TR4

© The Author(s) 2022

J. Jankowicz-Cieslak (\*) · F. Goessnitzer · I. L. 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

B. J. Till Veterinary Genetics Laboratory, University of California, Davis, CA, USA

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_1

#### 1 Introduction

Bananas and plantains are among the most important staple food crops for people living in tropical and subtropical countries. They are herbaceous monocots belonging to the genus Musa; most are seedless, polyploid, sterile and clonally propagated. The majority of banana and plantains are consumed locally.

Vegetatively propagated crops (VPCs) such as banana pose unique problems compared to cereals because they have a reduced genetic diversity as they can't be cross pollinated to enhance variation. Furthermore, because of its triploid parthenocarpic nature, bananas do not produce seeds and conventional breeding is thus a long process. Advances in biotechnology for crop improvement have had a great impact on vegetatively propagated crops (Gosal et al. 2010). Biotechnology based on tissue culture is complementary to conventional breeding technology.

One advantage of vegetatively propagated crops is that methods have been developed for rapid clonal propagation. Micropropagation is currently used in many countries for rapid propagation of disease-free planting material for distribution on a large scale. Such tissue culture techniques that ensure genetic stability (e.g. using shoot tip/nodal cultures for propagation) are particularly useful for in vitro mutation induction and maintenance of mutant plant populations. This technique is also of particular interest to breeders since the multiplication of the new lines for field trials and evaluation could be hastened, thereby shortening the time required for the release of new cultivars. In vitro techniques also offer possibilities to use induced mutation for further manipulation aimed at improvement. New genetic variation conferring a desirable trait can be fixed, and identical material rapidly deployed.

The structure of meristematic regions and the development of new meristems from differentiated tissue are particularly important when investigating radiationinduced mutation of vegetatively propagated crops (VPCs). In most cases, the new shoots originate from a single epidermal cell from a tissue, and this could directly lead to homohistant mutant plants whose genetics may be investigated further (Spencer-Lopes et al. 2018).

One of most critical prerequisites for successful mutation breeding is the determination of the optimal mutagen dose. The dose required for a particular experiment depends on the desired effects but may be restricted by undesirable effects of the mutagenic treatment, which could lead to lethality. There is a strong correlation between the genotype and the sensitivity of the plant material to the mutagenic treatments in plants (Jankowicz-Cieslak et al. 2012). The dose increase causes severe mutations, such as chromosomal aberrations, and can cause cell damage and subsequently death. While little is known for VPCs, the data from seed propagated plants suggests that fine-tuning of dose applied may be needed. Therefore, radiation sensitivity tests should be carried out to determine the mutagen dose that results in a 50 percent reduction in e.g., plant height or plant weight. In practice, a breeder applying irradiation treatment on vegetatively propagated crops may decide to settle for a growth reduction of 30–50 percent (GR30–50) for M1V1 plants or a survival rate of 40–60 percent depending on the sensitivity of the plant material. An equal number of control materials for the comparison should be planted at the same time.

Fig. 1.1 Mutation induction pipeline using banana in vitro shoot tip cultures. The first step of the process consists of establishment of banana cultures, either via sucker dissection from the field or alternatively obtaining accessions of interest from germplasm repository. Upon sufficient multiplication of banana shoot tips, the bulk mutagenesis can be performed or, if the knowledge on radiosensitivities of this particular genotype doesn't exist, a radiosensitivity test should be carried out. After mutagenic treatment of a bulk number of shoot tips, chimeras need to be dissolved and at the M1V3 stage plantlets can be rooted and hardened for field or screenhouse selection

Measurement of growth reduction of in vitro treated material should be recorded 30 days after the treatments.

For VPCs, various methods have been developed which involve tissue isolation and dissection during post-mutagenesis aiming at reducing the genotypic complexity of the resulting plants.

The following procedures are routinely used in the Plant Breeding and Genetics Laboratory to micropropagate banana and plantain by shoot tip culture and perform mutagenesis (Fig. 1.1).

#### 2 Materials

#### 2.1 In Vitro Culture Media

	- (a) Thiamine hydrochloride Stock: 100 mg in 100 ml dest. Water


2. Initiation medium / Maintenance medium composition (1 l).


3. Rooting medium composition (1 l).



#### 2.2 Gamma-Ray Mutagenesis


#### 2.3 Calculation of Lethality and Growth Reduction


#### 2.4 Chimera Dissolution


#### 2.5 Hardening


#### 3 Methods

#### 3.1 Preparation of Liquid Maintenance Medium S-27


#### 3.2 Preparation of Solid Culture Medium

	- (a) For initiation/maintenance medium: 4.4 g of Murashige and Skoog basal salt with minimal organics, 10 ml L-Cysteine, 20 ml BAP and 1 ml of thiamine stock solutions.
	- (b) For rooting medium: 4.4 g of Murashige and Skoog basal salt with minimal organics, 1 ml B5 Vitamin, 5 ml 2iP, 0.1 mL IBA and 1 ml of L-Cysteine HCl stock solution.

#### 3.3 Initiation and Maintenance of Banana Cultures


#### 3.4 Establishment of the Radiosensitivity Curve and Calculation of Growth Reduction (GR)

#### 3.4.1 Tissue Irradiation



Table 1.1 Example of doses chosen for mutagenic treatment of in vitro banana


#### 3.4.2 Data Collection


#### 3.4.3 Data Analysis


Fig. 1.2 Effect of irradiation on propagules weight of six different banana accessions expressed as percentage of non-irradiated (control) and percentage reduction in mean weight. Blue, orange, green and red lines indicate measurements taken with weekly intervals (1 week – blue, 2 weeks – orange, 3 weeks – green and 4 weeks – red post mutagenesis). A clear trend in response stabilisation takes place already after 2 (orange) to 3 (green) weeks

Table 1.2 Examples of banana sensitivities expressed as GR30 and GR50 values calculated from radiosensitivity graphs (Fig. 1.2 and see Note 19)


#### 3.5 Bulk Mutagenesis by Physical Agents


#### 3.6 Post-Mutagenesis Handling

#### 3.6.1 Chimera Dissolution


Fig. 1.3 Dissolution of chimeras in bananas. Plants after the mutagenic treatment are allowed to grow for 30 days in a liquid culture media at a constant rotary shaking (60 rpm). Each mutated plant is given a unique line number and assigned a population stage starting with M1V1. Chimeras may exist after 30 days recovery period if surviving meristematic cells harbour different mutations. In order to dissolve potential chimeras, meristems are isolated and divided into two parts through a longitudinal cut which results in most cases in generation of 2 daughter plants. These are allowed to recover and grow for another 30 days. The procedure is repeated 3 times. At the M1V3 stage, plants are being transferred into the solid culture media for long-term storage


#### 3.6.2 Shoot Elongation and Rooting


#### 3.6.3 Hardening of In Vitro Mutant Population


#### 3.7 Phenotypic and Genotypic Selection

1. Screen mutagenized population using reverse or forward genetics methods (see Note 28 and Chaps. 5, 7, 8, 9).

#### 4 Notes


irradiation for the triploid 'Cachaco' (ABB genome) (Jhurree-Dussoruth et al. 2014; Jain 2010).


screening could be undertaken or delayed until more advanced generations. From M1V4 to M1V9 uniform clones may be propagated and planted in experimental trials to test their performance for desired traits such as biotic/abiotic stress. As early as in M1V4 generation, replicated trials of selected mutants may be conducted using parental or local varieties as checks. The M1V5 and M1V6 generations can be used in multi-locational trials and tested for performance in a range of environments and agronomic traits. Final assessment can be made in M1V9 to M1V10 generations depending on plant species, the desired mutant clone or clones will be released as a new improved mutant variety (Spencer-Lopes et al. 2018).


Acknowledgments Authors wish to thank Mr. Danilo Moreno for assistance in performing radiosensitivity experiments. 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. This work is part of IAEA Coordinated Research Project D22005.

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Chapter 2 Gamma Irradiation of Embryogenic Cell Suspension Cultures from Cavendish Banana (Musa spp. AAA Group) and In Vitro Selection for Resistance to Fusarium Wilt

Chunhua Hu, Yuanli Wu, and Ganjun Yi

Abstract In this chapter, the establishment of embryogenic cell suspension (ECS) cultures using immature male flowers of triploid banana (Musa AAA Cavendish subgroup cv. 'Brazil'), followed by somatic embryogenesis and plantlet regeneration is described. Mutation induction is achieved by exposing the ECS to gamma irradiation with the dose of 80 Gy. The mutagenized cell population is transferred to solid long-term suspension culture medium for 96 h to recover from mutagen treatment shock, followed by somatic embryo induction and development medium containing 20% crude culture filtrates from Fusarium oxysporum f. sp. cubense (Foc). After 90 days, the somatic embryos that survive are transferred to the germination medium containing 25% crude culture filtrates. The surviving mature somatic embryos are transferred to rooting medium after the fourth subculture on the germination medium containing 50% crude culture filtrates. Before transplanting in a Foc infected field, the in vitro plantlets are acclimatized and screened for resistance to Foc using a pot-based greenhouse bioassay.

Keywords Cavendish banana · Embryogenic callus · Gamma irradiation · Germplasm resistant to Fusarium wilt

e-mail: yiganjun@vip.163.com

C. Hu · Y. Wu · G. Yi (\*)

Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences; Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization; Ministry of Agriculture and Rural Affairs, Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research, Guangzhou, China

#### 1 Introduction

Bananas and plantains (Musa spp.) are not only the most widely consumed fruits in the world, but also a staple food for over 500 million people. In recent years, the world faced a sharp decline in banana production, due to extreme weather patterns and the outbreak of pests and diseases, especially Fusarium wilt. Today's global banana production is seriously threatened by a new strain of Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4). There is an urgent need to introduce resistance against Foc TR4 into commercial banana cultivars. The most common cultivars for commercial production belong to Musa AAA Cavendish subgroup, which are sterile and seedless. Attempts to develop new banana genotypes resistant to Fusarium wilt using traditional cross breeding techniques face significant hurdles. Thus, induced mutagenesis or genetic engineering offers practical alternatives to create new varieties or novel germplasm and has become a dominant approach for breeding disease-resistant banana (Dita et al. 2018).

Somatic embryos originate from single cells, and the chimera frequency is very low. To accelerate genetic improvement of banana, it is important to establish embryogenic cell suspension (ECS) cultures, followed by plantlet regeneration through somatic embryogenesis. So far, different source explants have been used to establish ECS in banana, including basal leaf sheaths and corm sections (Novák et al. 1989), highly proliferating meristems (Dhed'a et al. 1991; Strosse et al. 2006), zygotic embryos (Marroquin et al. 1993), immature male (Escalant et al. 1994; Côte et al. 1996; Navarro et al. 1997; Becker et al. 2000; Pérez-Hernández and Rosell-García 2008; Kulkarni and Bapat 2012; Elayabalan et al. 2013; Namanya et al. 2014; Morais-Lino et al. 2016) and female flowers (Grapin et al. 2000). Among the various explants used, immature male flowers appear to be the most widely applicable starting material for the establishment of regenerable ECS.

Plant mutation breeding is an effective method for creating novel germplasm. Briefly, seeds, pollen, the whole plant, vegetative organs, or callus are subjected to irradiation, followed by the process of selection and identification of a new variety (Spencer-Lopes et al. 2018). Physical mutagenesis is also applied to improve horticultural crops, it is reported that novel germplasm with agriculturally valuable traits has been developed in apple, peach, pear, and citrus (Source: FAO/IAEA Mutant Varieties Database). Gamma irradiation has been widely used as a physical mutagen for breeding of many crops including banana (Novák et al. 1990; Mak et al. 1996; Guo et al. 2003).

In this chapter, we present a detailed protocol for the establishment of a cell suspension culture and plantlet regeneration via somatic embryogenesis of Cavendish banana (Musa spp. AAA group). The ECS cultures are then subjected to 80 Gy gamma irradiation, followed by in vitro selection for resistance to Fusarium wilt.

#### 2 Materials

#### 2.1 In Vitro Media for Induction of Embryogenic Callus (MI), Long Term Suspension Culture (ML), Somatic Embryo Induction and Development (MSD), Somatic Embryo Germination (MG), and Rooting of Somatic Embryos (MR)


#### 2.2 Materials for the Induction of Embryogenic Callus, Establishment of ECS Cultures, Induction and Maturation of Somatic Embryos


#### 2.3 Mutation Induction of ECS via Gamma-Irradiation


#### 2.4 In Vitro Selection for Resistance to Fusarium Wilt


#### 3 Methods

#### 3.1 Preparation of MI, ML, MSD, MG, and MR Medium


#### 3.2 Isolation of Immature Male Flowers


#### 3.3 Induction of Embryogenic Callus and Development of ECS


Fig. 2.1 Steps for cell suspension culture and plantlet regeneration via somatic embryogenesis of Cavendish banana (Musa spp. AAA group cv. 'Brazil'). (a) Embryogenic callus induced on MI medium; (b) Well established embryogenic cell suspension; (c) Induction of somatic embryo on MSD medium; (d) Maturation of somatic embryo; (e) Germination of somatic embryo; (f) Regenerated plantlet

#### 3.4 Induction and Maturation of Somatic Embryos


#### 3.5 Plantlet Regeneration


#### 3.6 Gamma Irradiation of ECS and In Vitro Selection for Resistance to Fusarium Wilt

#### 3.6.1 Determination of Irradiation Dose


#### 3.6.2 Preparation of Crude Culture Filtrates from Foc TR4


#### 3.6.3 In Vitro Selection for Resistance to Fusarium Wilt

1. Put a sterile filter paper on the surface of the ML agar medium, then transfer mutagen treated cells to the medium. The cultures are maintained for 96 h, which allows the cells to recover from mutagen treatment shock.


#### 4 Notes


#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Part II Fusarium TR4 Screening Technologies

## Chapter 3 Pre-Screening of Banana Genotypes for Fusarium Wilt Resistance by Using an In Vitro Bioassay

Yuanli Wu and Ganjun Yi

Abstract In the process of breeding and selection of banana for resistance to Fusarium wilt, it is important to conduct an efficient resistance screening test by artificial inoculation with Fusarium oxysporum f. sp. cubense (Foc) Tropical Race 4. So far, there are two types of early bioassays for screening Musa genotypes against Foc: a greenhouse and an in vitro bioassay. The most commonly used greenhouse bioassay is a pot-based system followed by a hydroponic system. Here we describe an in vitro bioassay characterized by in vitro inoculation of rooted banana plantlets grown on medium consisting of half-strength MS macronutrients and MS micronutrients. The disease response and evaluation results obtained through this in vitro bioassay correlates with that from a greenhouse screen and/or field evaluation. Given the importance of in vitro cell and tissue culture techniques for banana (mutation) breeding, promising resistant clones could be screened directly. This in vitro bioassay is a totally contained system compared with greenhouse methods and does not require an acclimatization step, thereby improving banana breeding efficiency. The in vitro pre-screening protocol and bioassay for Fusarium wilt resistance presented here is fast, space-effective, and accurate.

Keywords Bioassay · Fusarium oxysporum f. sp. cubense · Musa · Resistance

#### 1 Introduction

Fusarium wilt or Panama disease caused by the pathogenic fungus Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive diseases of banana and is found in all areas where banana is grown (Ploetz 2015). The soil-borne

e-mail: yiganjun@vip.163.com

Y. Wu · G. Yi (\*)

Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences; Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization; Ministry of Agriculture and Rural Affairs, Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research, Guangzhou, China

<sup>©</sup> The Author(s) 2022

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_3

pathogen infects the roots of banana plants and colonizes the xylem vessels, which leads to typical external symptoms such as wilting and yellowing of the foliage, eventually leading to plant mortality. Currently, there is still a lack of economically viable measures for managing Fusarium wilt in an infected field (Dita et al. 2018). It is generally accepted that breeding and selection for disease resistance is the only effective and sustainable management option.

Promising resistant clones acquired through conventional and non-conventional breeding techniques should be screened for resistance to Fusarium wilt using artificial inoculation with Foc. So far, there are two types of early bioassays for screening Musa genotypes against Foc: a greenhouse and an in vitro bioassay (Wu et al. 2010; Ghag et al. 2012; Hu et al. 2013). The most commonly used greenhouse bioassay is a pot system (Dita et al. 2011; Ribeiro et al. 2011; Li et al. 2015; Reboucas et al. 2018; Zhang et al. 2018; Zuo et al. 2018) followed by a hydroponic system.

Although some progress has been achieved, attempts at developing new banana genotypes resistant to Fusarium wilt using conventional breeding techniques face significant hurdles, mainly because most cultivars of Musa AAA Cavendish subgroup are sterile and seedless (Ortiz 2013). Nowadays, non-conventional breeding approaches for banana improvement such as somaclonal variation and genetic transformation have received more attention. Somaclonal variation caused by long-term in vitro propagation is considered an important source of genetic variability, through which several tolerant clones have been acquired (Hwang and Ko 2004). In addition, a genetic transformation protocol has been well established in different banana genotypes, which can be used to create transgenic plants resistant to Foc Tropical Race 4 (Foc TR4) (Dale et al. 2017). Given the fact that non-conventional breeding techniques are based on banana cell and tissue culture, promising resistant clones could be screened directly using an in vitro bioassay. Based on the modification of previous work (Wu et al. 2010), we present here a pre-screening protocol for Fusarium wilt resistance by using an in vitro bioassay that is fast, space-effective, and accurate.

The in vitro bioassay is characterized by in vitro inoculation of rooted banana plantlets grown in a half-strength MS medium without a carbon source. Twenty-four days after inoculation with Foc TR4 at 10<sup>6</sup> conidia/ml, the disease severity was rated on a scale from 1 to 6 (Wu et al. 2010). Results of the disease score were then subjected to ordinal logit model analysis, which is also known as proportional odds model (McCullagh 1980). According to symptom rating probability distribution, the reaction of banana genotypes against Fusarium wilt was divided into five categories: highly resistant (HR), resistant (R), moderately resistant (MR), susceptible (S), and highly susceptible (HS). Compared with the greenhouse bioassay, this in vitro bioassay is a totally closed system. Since acclimatization of in vitro plantlets is not required, the application of the bioassay improves banana breeding efficiency.

#### 2 Materials

#### 2.1 Medium for Interaction System (MIS)


#### 2.2 Plant Material Preparation


#### 2.3 Inoculum Preparation


#### 2.4 In Vitro Inoculation


#### 3 Methods (See Note 3)

#### 3.1 Preparation of MIS Medium


#### 3.2 Plant Material Preparation


#### 3.3 Inoculum Preparation


#### 3.4 In Vitro Inoculation (See Fig. 3.1 and Note 4)


Fig. 3.1 In vitro inoculation protocol


#### 3.5 Disease Severity Rating and Statistical Analysis


Fig. 3.2 A scale of 1–6 is used to measure disease severity of banana rooted plantlets 24 days after in vitro inoculation with Fusarium oxysporum f. sp. cubense tropical race 4 at 10<sup>6</sup> conidia/ml

$$\begin{aligned} \ln\left(\frac{p\_1}{1-p\_1}\right) &= \beta\_{01} - \sum\_{k=1}^k \beta\_k x\_k \\ \ln\left(\frac{p\_1+p\_2}{1-p\_1-p\_2}\right) &= \beta\_{02} - \sum\_{k=1}^k \beta\_k x\_k \\ \ln\left(\frac{p\_1+p\_2+p\_3}{1-p\_1-p\_2-p\_3}\right) &= \beta\_{03} - \sum\_{k=1}^k \beta\_k x\_k \\ \ln\left(\frac{p\_1+p\_2+p\_3+p\_4}{1-p\_1-p\_2-p\_3-p\_4}\right) &= \beta\_{04} - \sum\_{k=1}^k \beta\_k x\_k \end{aligned}$$

$$\ln\left(\frac{p\_1 + p\_2 + p\_3 + p\_4 + p\_5}{p\_6}\right) = \beta\_{05} - \sum\_{k=1}^{k} \beta\_k x\_k$$

Wherein p1, p2, p3, p4, p<sup>5</sup> and p<sup>6</sup> are event probabilities, which respectively represent disease grades of 1–6, and the basal level for comparison is grade 6; xk (<sup>k</sup> <sup>¼</sup> 1,2..., <sup>K</sup>) represents banana cultivar; <sup>β</sup>0j ( <sup>j</sup> <sup>¼</sup> 1,2..., 5) represents an intercept term of regression; and <sup>β</sup><sup>k</sup> (<sup>k</sup> <sup>¼</sup> 1,2..., <sup>K</sup>) represents a regression coefficient; each logit function has the same coefficient term and different intercept terms, and the regression lines of each cumulative logit are parallel to each other.

The estimation method used for the Logistic regression model is a maximum likelihood method, according to the aforementioned Logistic model function designed for predicting the disease severity of the rooted plantlet of banana, the accumulated Logistic regression model obtained is described as follows:

$$\mathbf{y}' = a + \sum\_{k=1}^{K} \beta\_k \mathbf{x}\_k + \varepsilon\_k$$

Wherein y' represents the disease incidence of the rooted plantlet of banana, α represents an intercept term; <sup>β</sup><sup>k</sup> (<sup>k</sup> <sup>¼</sup> 1,2..., <sup>K</sup>) represents a regression coefficient; xk (<sup>k</sup> <sup>¼</sup> 1,2..., <sup>K</sup>) represents banana cultivar, and <sup>ε</sup> is an error term.


$$P(\mathbf{y} \le j | \mathbf{x}) = P(\mathbf{y}' \le \mu\_j | \mathbf{x}) = \frac{1}{1 + e^{-\left[\mu\_j - \left(a + \sum\_{k=1}^k \beta\_k \mathbf{x}\_k\right)\right]^2}}$$

Therefore, the probability value of the rooted plantlet of a banana cultivar in a certain disease grade can be obtained:

$$P\left(\mathbf{y} = 1\right) = P\left(\mathbf{y} \le 1\right)$$

$$P\left(\mathbf{y} = 2\right) = P\left(\mathbf{y} \le 2\right) - P\left(\mathbf{y} \le 1\right)$$

$$P\left(\mathbf{y} = 3\right) = P\left(\mathbf{y} \le 3\right) - P\left(\mathbf{y} \le 2\right)$$

$$P\left(\mathbf{y} = 4\right) = P\left(\mathbf{y} \le 4\right) - P\left(\mathbf{y} \le 3\right)$$

$$P\left(\mathbf{y} = 5\right) = P\left(\mathbf{y} \le 5\right) - P\left(\mathbf{y} \le 4\right)$$

$$P\left(\mathbf{y} = 6\right) = 1 - P\left(\mathbf{y} \le 5\right)$$

The sum of the probability values of each grade is 1, that is, <sup>P</sup> (<sup>y</sup> <sup>¼</sup> 1) + <sup>P</sup> (<sup>y</sup> <sup>¼</sup> 2) + ... <sup>+</sup> <sup>P</sup> (<sup>y</sup> <sup>¼</sup> 6) <sup>¼</sup> 1.

6. Open the spreadsheet at user interface of statistic software (e.g. IBM SPSS Statistics), then execute ordinal logit model analysis and obtain symptom rating probability distribution of each banana cultivar or clone. As an example, the result of statistical analysis is given in Fig. 3.3.

Fig. 3.3 In vitro inoculation of six banana cultivars with Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4). Twenty-four days after in vitro inoculation of rooted plantlets (<sup>n</sup> <sup>¼</sup> 15), disease symptoms were scored on an ordinal scale as illustrated: 1, no discoloration of the pseudostem; 2, 1/2 the height of the pseudostem discolored; 3, >1/2 the height of the pseudostem discolored and (or) leaf stalk discolored; 4, 50% of the leaves wilted or yellowed; 5, >50% of the leaves wilted or yellowed; and 6, the whole plantlet wilted. Control plantlets were treated in the same manner except that Foc TR4 was replaced with sterile distilled water. Data obtained from three independent experiments were subjected to ordinal logit model analysis, which is also known as proportional odds model


Table 3.1 Criteria for grading resistance against Fusarium oxysporum f. sp. cubense

Table 3.2 Screening of banana cultivars for resistance to Fusarium wilt under field, greenhouse, and in vitro conditions, respectively



#### 4 Notes

1. MIS medium is composed of half-strength MS macronutrients and MS micronutrients without a carbon source. Media commonly used for the growth and sporulation of Foc are carbohydrate-rich, and carbon has been shown to be the first limiting substrate of Foc growth in sterilized soil (Couteaudier and Alabouvette 1990). In this bioassay, the subtle balance between the growth of Foc and the carbon source is achieved by removing sucrose from the MS medium and by employing a filter paper disc as carbon source. Since the rooted banana plantlets with leaves are able to photosynthesize, the use of MIS medium in this bioassay also guarantees the normal growth of the plantlets.


#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Chapter 4 In Vitro Based Mass-Screening Technique for Early Selection of Banana Mutants Resistant to Fusarium Wilt

Behnam Naserian Khiabani

Abstract Banana and plantains are among the most valuable agricultural commodities in the world. Banana Fusarium wilt, caused by the soil-borne Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana globally. In the 1990s a new strain of Fusarium oxysporum called tropical race 4 (TR4) emerged in Southeast Asia that affected commercial Cavendish plantations. The development of resistant cultivars is an effective strategy for management of the disease. Field-based screening to identify Foc-resistant plants is time-consuming, expensive and is often challenged by variable environmental conditions. Here we present an early selection protocol enabling evaluation of the disease under in vitro conditions. This method provides a preliminary screening and allows evaluation of a large number of in vitro plantlets. Using this method, within a short time and in a small laboratory, breeders can evaluate thousands of banana plantlets, produced via irradiation. Subsequently, putative, disease-resistant mutant lines can be identified and evaluated in the field.

Keywords Banana · Fusarium oxysporum · In vitro bioassay · Mass selection

#### 1 Introduction

Banana and plantains belong to the genus Musa and are important agricultural products in developing countries. More than 1000 varieties of bananas are produced and consumed locally. Cavendish banana (AAA) are the main commercial variety for export and international trade and account for around 47% of global production (FAO 2019a). Approximately 50 million tons of Cavendish bananas are being produced globally every year. In 2017 the global banana production reached

B. N. Khiabani (\*)

Plant Breeding Department, Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Tehran, Iran e-mail: bnaserian@aeoi.org.ir

<sup>©</sup> The Author(s) 2022

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_4

114 million tons (FAO 2019b). Bananas are locally consumed as vital staple food or as a significant addition to the diets in Africa, southern Asia, and tropical America (FAO 2019b).

Fusarium oxysporum is a soil-borne fungus that is ranked fifth on the list of top fungal plant pathogens (Ploetz 2005; Dean et al. 2012). Over 120 formae speciales (ff. spp.) of Fusarium oxysporum have been described based on host specificity (Baayen et al. 2000). Differences in pathogenicity on specific host cultivars is being defined as physiological races among isolates (Kistler 1997; Baayen et al. 2000; Takken and Rep 2010; Meldrum et al. 2012). Fusarium oxysporum f. sp. cubense (Foc) refers to strains that infect bananas and plantains and cause Fusarium wilt or Panama disease (Ploetz 2005). It has been recognized that Foc has a polyphyletic origin (Lievens et al. 2009), hence comprises a suite of genetically distinct lineages (Ordonez et al. 2015). Maryani et al. (2019) have recently revised the taxonomy of Foc and designated different species names to strains affecting banana and merged them into the Fusarium of Banana Species Complex.

The disease cycle of this Fusarium spp. starts with infection of the root system and subsequent colonization within the vascular tissue, leads to water stress, severe chlorosis, and wilting (Ploetz 2015). Infected plants frequently die before they produce bunches, hence Fusarium wilt significantly reduces yields in infested fields (Dita et al. 2010).

A variant of Foc, called tropical race 4 (TR4) was first identified in Taiwan in 1989 but was probably the cause of banana wilt in the country since 1960. In the 1990s, Foc TR4 was identified in Malaysia and Indonesia, and the strains are thought to have originated from Taiwan (Buddenhagen 2009; Maryani et al. 2019). Foc TR4 has spread to many countries of Asia, as well as Australia and Africa and recently has reached Colombia and Peru in Latin America. Since its appearance, TR4 has severely affected Cavendish plantations in Malaysia, Indonesia, South China, Philippines, and the Northern Territory of Australia (Ploetz 2006; Molina et al. 2010; Buddenhagen 2009; Chittarath et al. 2018). TR4 is considered one of the most destructive Foc strains because of its broad host range. This pathogen is attacking the important cultivars of Cavendish but also all other cultivars that are sensitive to Foc (Cheng et al. 2019). The disease predominantly affects the Cavendish varieties, which not only primarily meets the international market demand but also is important for local consumption in developing countries. Cavendish varieties are the cornerstone for international trade, therefore TR4 threatens the entire global production (FAO 2019b).

Strategies controlling TR4 spread are based on visual monitoring of early symptom appearance, eradication of infected plants and isolation of infested areas to reduce pathogen dissemination. Pérez Vicente et al. (2014) reported that once plants are infected with TR4, there is no way to eradicate the disease. In this case the affected plants and all plants in the surrounding 7.5 m radius should be destroyed. Host resistance is a basis for sustainable disease management in most crops and this is usually achieved by intensive breeding programs (Ploetz 2006). Therefore, breeding for resistant/tolerant banana plants is the best way to overcome the disease.

To develop new, resistant cultivars, breeders need reliable and rapid phenotyping methods enabling selection of improved lines (García-Bastidas et al. 2019). Different approaches can be pursued for resistance screening e.g. in the field or under greenhouse conditions (Dhingra and Sinclair 1986; Trigiano et al. 2004; Singh and Singh 2005). Field screening encounters problems such as time, costs, variable environmental conditions, and unspecified biodiversity of soil-borne pathogens (Mert and Karakaya 2003; Subramaniam et al. 2006; Sutanto et al. 2013). In contrast, greenhouse-based phenotyping facilitates high-throughput selection under controlled conditions with specific fungal genotypes, leading to more reproducible results (Smith et al. 2008). Greenhouse assessments have been reported as a reliable method by several researchers (Smith et al. 2008; Pérez Vicente et al. 2014). In vitro screening is one of the most high-throughput and efficient method (Švábová and Lebeda 2005; Pillay 2002; Naserian Khiabani et al. 2018; Wu et al. 2010). Compared to selection in an experimental field, in vitro selection can considerably reduce the space needed for screening. However, some factors influencing in vitro selection may differ from those in field selection (Matsumoto et al. 2010). The most used selection agents in the tissue culture medium are metabolites of pathogens or similar chemicals. There have been several reports of the use of fusaric acid to select Fusarium resistance in in vitro culture system (Matsumoto et al. 2010; Wu et al. 2010; Švábová and Lebeda 2005). Daub (1986) used a crude filtrate of a Fusarium suspension as a selection agent under the in vitro condition. Wu et al. (2010) and Naserian Khiabani et al. (2018) used suspensions containing pathogenic components including micro and macro-conidia and mycelium of Fusarium oxysporum, as a selection agent for in vitro screening. Using methionine sulfoximine as a selective agent, Carlson (1973) demonstrated for the first time the possibility of selecting disease-resistance plants using an in vitro tobacco protoplast system. Since 1980, the theoretical and practical approaches of in vitro selections and their usefulness for plant breeding have been addressed (Shepard 1981; Wenzel 1985; Daub 1986; Buiatti and Ingram 1991; Švábová and Lebeda 2005). According to Lebeda and Svábová (2010) the ideal system for in vitro selection for disease resistance should comprise: (1) an in vitro explant culture that can generate genetic variations (or an in vitro mutation induction system) with efficient recovery of genetically stable and fertile resistant/tolerant plants; (2) a selection agent that can be readily produced and which induces similar biochemical reactions in vitro as the pathogen in vivo; and, (3) molecular tools to characterize the selected resistant lines at the DNA level.

Several successful experiments have been carried out in vitro with live inoculums. For example: Clavibacter michiganensis (Bulk et al. 1991); Xanthomonas campestris (Hammerschlag 1990); Mycosphaerella musicola (Trujillo and Garcia 1996); Alternaria alternata (Takahashi et al. 1992); Fusarium solani (Huang and Hartman 1998); and Phytophthora cinnamoni (McComb et al. 1987; Cahill et al. 1992). Matsumoto et al. (2010) used fusaric acid as a selection agent in an in vitro culture system to select banana plants resistant to Fusarium wilt race 1.

Basic knowledge about the biology of the causal agent and its relationship with the host plant is necessary to develop suitable methods for resistance screening and selection (Russell 1978). Usually, wilting is either caused by blockage of plant vessels due to the accumulation of spores and mycelium of pathogenic fungi or due to a toxic element produced by the pathogen. In case of Fusarium, the use of fusaric acid or a crude filtered fungal extract does not lead to blockage of the xylem vessels. In this case, it is the toxicity of the extract that leads to the appearance of symptoms. This is distinct from disease resistance screening under field conditions, where plants are affected by the live pathogen, spores, mycelia in addition to toxins. In vitro plantlets inoculated with mycelium and spores of the pathogen are expected to show symptoms very similar to those observed in the field.

To accelerate Fusarium wilt resistance screening in banana breeding programs, bioassays that can efficiently and accurately differentiate resistant from susceptible cultivars are required. Two prerequisites should be met for effective in vitro disease resistance screening using a pathogen-derived selection agent: (1) One or more compounds found in the selection agent should be present in infected plants; and (2) the agent should at least partially induce disease symptoms when inoculated into healthy plants. When an in vitro plantlet is directly inoculated by the pathogen (conidia and mycelium), the above-mentioned requirements are met.

Traditional banana breeding is faced with several impediments, primarily the sterile nature of the triploid cultivars; seedless fruits are required to meet consumer demands but hampers breeding (Pillay and Tenkouano 2011; Pillay 2002). Banana breeders have incorporated non-classical breeding approaches, such as mutation breeding, to induce diversity in their elite germplasm. Mutagenic agents, such as radiation or certain chemicals, can be used to generate genetic variation from which desired mutants may be selected. The combination of mutation breeding and in vitro culture (also called in vitro mutagenesis) is effective for the induction and selection of somatic mutations (Roux 2004). Novák et al. (1989) described the dose-response of tissue-cultured shoot tips to gamma irradiation. Since edible bananas are vegetatively propagated and heterozygous, mutation breeding is an ideal approach for their genetic improvement (Jain 2010). In addition, mutagenic treatments need to be optimized and efficient screening techniques developed to select desirable mutants (Jain 2000, 2006, 2007; Van Harten 1998). In vitro techniques can improve the effectiveness of mutation induction, especially when handling large mutant populations (Predieri 2001; Jain 2000; Jain and Maluszynski 2004). We present here a protocol for inoculating in vitro banana plants with the live agent Fusarium wilt (spores and mycelium) and for screening mutant banana seedlings under in vitro conditions.

#### 2 Materials

#### 2.1 Plant Tissue Culture Medium


#### 2.2 Culture Media for the Isolation and Culture of Fusarium oxysporum


#### 2.3 Biological Materials


#### 2.4 Gamma Irradiation


#### 3 Method

#### 3.1 Preparation of Micropropagation and Rooting Medium


#### 3.2 Preparation of Streptomycin Stock


#### 3.3 Preparation of PDA (Potato Dextrose Agar) Medium


#### 3.4 Preparation of Solid Inoculation Medium (SIM)


#### 3.5 Preparation of Inoculum


Fig. 4.1 Preparation of the inoculum (a) Fusarium oxysporum colony (b) collection of conidia. (c) Counting cells in a haemocytometer (Source: http://insilico.ehu.eus/counting\_chamber/thoma.php)


#### 3.6 Preparation of Filter Paper Disks


#### 3.7 Establishment of In Vitro Cultures


#### 3.8 In Vitro Mutagenesis


Fig. 4.2 Prepare the mutant population for in vitro bioassay by pedigree method, each genotype is evaluated for disease resistance, while the same genotype is being kept for subsequent studies, as well as for the reproduction of putative resistant mutants

#### 3.9 In Vitro Inoculation


#### 3.10 Disease Evaluation


Fig. 4.3 Appropriate seedlings for in vitro evaluation of disease. Two-month-old seedlings 4–5 cm height of the pseudostem, have more than two fully expanded leaves and at least three white healthy roots

Fig. 4.4 Steps of inoculation: (a) selected plantlet incubated for 2 weeks on SIM media before inoculation, (b) autoclaved filter paper disks, (c) soaked filter disk in the inoculum suspension and placed on the surface of the SIM, (d) to (f) development of disease symptoms

Table 4.1 Disease severity-rating scale used to record symptoms caused by Fusarium oxysporum f.sp. cubense in banana plants (Wu et al. 2010)


#### 4 Notes


Fig. 4.5 An example of in vitro bioassay of Fusarium oxysporum. M1V4 banana plantlets (local dwarf Cavendish CV) were used. The numbers (1 to 6) indicate disease severity scored according to Wu et al. (2010) after 21–30 days of inoculation

enter into the final extract, and for the same reason, potatoes should not mash during boiling and filtering. A clear extract is needed for preparation of PDA.


necrotic leaves is more pronounced. Therefore, for disease scoring in the in vitro bioassay, one should especially score the appearance of necrotic and discolored leaves and pseudostem.

Acknowledgments I gratefully acknowledge the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture Vienna, Austria, and Nuclear Science and Technology Research Institute, Karaj, Iran for their financial support. The author would like to acknowledge the Plant Breeding research group of Nuclear Agriculture Research School for their support and contribution to this study. I also thank Dr. Hamideh Afshar Manesh for assistance with the isolate F. oxysporum and Mr. Cyrus Vedadi for valuable discussions.

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Chapter 5 An Optimised Greenhouse Protocol for Screening Banana Plants for Fusarium Wilt Resistance

#### Privat Ndayihanzamaso, Sheryl Bothma, Diane Mostert, George Mahuku, and Altus Viljoen

Abstract Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is considered one of the most devastating diseases of banana in the world. Effective management of Fusarium wilt is only achieved by planting banana varieties resistant to Foc. Resistant bananas, however, require many years of breeding and field-testing under multiple geographical conditions. Field evaluation is reliable but time consuming and expensive. Small plant screening methods are, therefore, needed to speed up the evaluation of banana varieties for Foc resistance. To this end, a small plant screening method for resistance to banana Fusarium wilt is presented. The method proposes the planting of 2- to 3-month-old banana plants in soil amended with 10 g Foc-colonised millet seeds. Rhizome discoloration is then evaluated to rank the disease resistance response. The optimized millet seed technique could be useful in mass screening of newly developed genotypes for resistance to Foc.

Keywords Musa · Fusarium oxysporum f. sp. cubense · Evaluation · Resistant varieties

#### 1 Introduction

Fusarium wilt is considered one of the most devastating diseases of banana in the world (Stover 1962; Ploetz 2006). The disease is caused by a soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc), which infects the plants through the roots, colonises the rhizome and xylem, and causes a lethal wilting of plants (Stover 1962;

P. Ndayihanzamaso · S. Bothma · D. Mostert · A. Viljoen (\*)

Department of Plant Pathology, Private Bag X1, Stellenbosch University, Matieland, South Africa e-mail: altus@sun.ac.za

G. Mahuku International Institute of Tropical Agriculture (IITA) Regional Hub, Dar-es-Salaam, Tanzania

Guo et al. 2015; Warman and Aitken 2018). Foc originated in Southeast Asia, and was disseminated globally with Foc-propagating and planting material as banana production expanded during the twentieth century (Stover 1962; Ploetz 2015a; Dita et al. 2018). By the 1950s, Fusarium wilt was so widespread in Latin America that it became impossible to sustain the international banana export industry that was almost exclusively based on the Gros Michel (AAA) bananas. Gros Michel was thus replaced with Cavendish bananas as export variety in the 1960s, which currently account for nearly 50% of production globally (Lescot 2015; Ploetz 2015a). Cavendish bananas, however, are now also severely affected by a different strain of Foc in Asia, the Middle East, Mozambique in Africa and Colombia in Latin America, called Foc Tropical Race 4 (TR4) (Ploetz and Pegg 2000; Mostert et al. 2017; Dita et al. 2018; García-Bastidas et al. 2019a, b; Thangavelu et al. 2019; Viljoen et al. 2020).

Foc comprises of three races based on their pathogenicity to a group of differential cultivars, with Foc races 1, 2 and 4 causing disease to Gros Michel, Bluggoe and Cavendish bananas, respectively (Pegg and Langdon 1987; Ploetz and Pegg 2000). Foc race 4 is further subdivided into Foc subtropical race 4 (Foc STR4) and tropical race 4 (Foc TR4) strains. The former causes disease on Cavendish bananas in the subtropics when plants are stressed by adverse climatic conditions, while Foc TR4 affects Cavendish bananas both under tropical and subtropical conditions (Ploetz and Pegg 2000). Foc TR4 has a wider host range than Foc races 1 and 2, and causes disease to Cavendish banana cultivars as well as most Foc race 1- and race 2-susceptible cultivars (Ploetz 2015b). Foc strains have been further classified using vegetative compatibility group (VCG) analysis, which is based on the ability of fungal hyphae to anastomose and form a stable heterokaryon (Leslie and Summerell 2006). Twenty-four VCGs have been described for Foc (Pegg et al. 1993; Bentley et al. 1995; Ploetz and Correll 1998; Fourie et al. 2009).

The most effective method to control banana Fusarium wilt is the use of Foc-resistant varieties (Ploetz 2015b). If existing varieties with resistance are not available to replace susceptible ones, the susceptible bananas can be improved by conventional breeding, mutation breeding, genetic engineering and genome editing (Rowe 1987; Hwang and Tang 2000; Hwang and Ko 2004; Bakry et al. 2009; Ortiz 2013; Dale et al. 2017; Naim et al. 2018). Bananas resistant to Foc race 1 that have been developed by conventional breeding include the hybrids FHIA-01, FHIA-18 and FHIA-25 developed in Honduras; RG1, Bodles Altafort, 2390-2 and 72-1242 bred in Jamaica; Pacovan Ken, Preciosa and Tropical in Brazil; and BRS-01 and BRS-02 in India (Bakry et al. 2009; Lorenzen et al. 2012; Ortiz 2013). Several partially resistant and/or resistant clones have also been developed by mutation breeding through chemical and radiation mutagenesis techniques and through somaclonal variation. For instance, GCTCV-215, GCTCV-105, GCTCV-218 and GCTCV-219 are Giant-Cavendish derived somaclones, which were selected through somaclonal variation, and Dwarf Parfitt mutant DPM-25 is a banana mutant generated with gamma irradiation (Tang and Hwang 1994; Hwang and Ko 2004; Smith et al. 2006). Dale et al. (2017) developed two transgenic Cavendish lines, RGA2-3 and Ced9-21, which did not develop any Fusarium wilt after 3 years of evaluation in banana fields infested with Foc TR4. Apart from GCTCV-218, most improved bananas with Fusarium wilt resistance have, however, not satisfied export market requirements (Bakry et al. 2009; Ortiz 2013).

Before local varieties or improved bananas can replace susceptible bananas in Foc-infested fields, they need to be evaluated for resistance against local Foc strains (Morpurgo et al. 1994a; Ploetz 1994; Carlier and De Waele 2002; Mak et al. 2004; Dita et al. 2011). Field evaluation is more accurate when identifying Foc-resistant plants under natural environmental conditions, but the process is time-consuming and expensive. Inoculum levels in the soil might also be unequally distributed. In addition, banana varieties can only be tested against Foc strains present in the country or region where the tests are performed. Greenhouse screening can be performed on small plants, achieved in a short time, and can be screened against quarantine pathogens in restricted environments (Smith et al. 2008; Dita et al. 2011). Yet, greenhouse screening methods seldom correlate well with field screening due to a number of factors, including inoculum preparation, inoculum concentration, inoculation method, the effect of temperature and photoperiod, type of planting material tested, and the potting soil used (Brake et al. 1995; Smith et al. 2008; Dita et al. 2011). Greenhouse screening methods, thus, need optimisation.

Several greenhouse evaluation methods have been developed for banana Fusarium wilt (Morpurgo et al. 1994b; Brake et al. 1995; De Ascensao and Dubery 2000; Mohamed et al. 2001; Smith et al. 2008; Wu et al. 2010; Dita et al. 2011; Viljoen et al. 2018; Chen et al. 2019; García-Bastidas et al. 2019a, b). The most common inoculation methods include the dipping of plant roots in a conidial suspension, the drenching of soil with a conidial suspension, and the replanting of plants in Foc-infested soil or sand. In the dipping method, plants are carefully removed from soil and their roots dipped in a conidial suspension for a few minutes before replanting (Mohamed et al. 2001; Dita et al. 2011; Ribeiro et al. 2011). The soildrenching method consists of pouring a spore suspension on the surface of potting soil (Smith et al. 2008). In the infested soil technique, bananas are planted in soil mixed with millet seeds or maize kernels that were pre-colonized with Foc (Smith et al. 2008; Dita et al. 2011). A combination of the dipping method and replanting in infested soil was reported to result in quick and consistent disease development (Dita et al. 2011). The screening of banana plants grown in vitro has also been reported, but Hamill (2018) considered these not suitable for resistance screening, as the fungus may kill both susceptible and resistant varieties due to high inoculum pressure.

The type of planting material used can influence Fusarium wilt development in the greenhouse. Tissue culture-derived plants are more susceptible and have a shorter incubation period compared to suckers and bits (Hwang and Ko 1987; Smith et al. 2008). Tissue culture plants are also free of other pests and diseases, apart from plant viruses, which may influence disease development. Tests with tissue culture plants smaller than 5 cm did not reflect field results, but plants of 10–15 cm (around 2 months old) did (Brake et al. 1995; Mohamed et al. 2001; Smith et al. 2008). Further investigations of the effect of plant age on Fusarium wilt development are thus needed to improve the reliability of small plantlet screening methods (Brake et al. 1995; Smith et al. 2008; Ribeiro et al. 2011).

In this chapter, we present an optimised greenhouse screening method for resistance to banana Fusarium wilt, considering the effect of inoculum concentration, inoculation methods and plant age on disease development.

#### 2 Materials

#### 2.1 Preparation of Plant Material


#### 2.2 Culture Medium


#### 2.3 Preparation of Inoculum and Greenhouse Infection


#### 2.4 Disease Rating


#### 3 Methods

#### 3.1 Planting Material

1. Banana accessions to be tested are prepared together with resistant and susceptible control plants. Resistant and susceptible control varieties are selected according to the Foc strain to be used for the inoculation (Table 5.1) (Viljoen et al. 2017).

Table 5.1 Banana varieties used as resistant and susceptible checks for evaluation against races of Fusarium oxysporum f. sp. cubense


a EAHB East African Highland Bananas

Fig. 5.1 Construction of a humidity chamber used for the weaning of tissue culture plants. The humidity chamber is constructed out of a metal framework covered in clear polyethylene sheeting. The humidity chamber is covered with shade netting and fitted with an automatic misting system. Seedling trays are placed on a lifted metal mesh to ensure the drainage of excess water


Fig. 5.2 Plants during the weaning and hardening phases. (a) Tissue culture plant ready for weaning. (b) A plant at the end of the weaning phase that is ready to be hardened-off. (c) Hardened-off plants ready to be screened for Fusarium wilt resistance

#### 3.2 Preparation of Culture Media


#### 3.3 Millet Seed Inoculum Preparation


Fig. 5.3 Preparation of Fusarium oxysporum f. sp. cubense (Foc) inoculum on millet seed. (a) Autoclaved millet seeds ready for inoculation, (b) white fungal growth on the surface of the millet seeds. At this time, the flasks/Schott bottles need to be shaken to ensure proper distribution and thorough colonisation of millet seeds, and (c) the plating out of Foc-colonized millet seeds onto PDA plates to ensure proper colonization with the Foc inoculum, and no contamination with other micro-organisms


#### 3.4 Greenhouse Inoculation


Fig. 5.4 Inoculation of banana plants for resistance screening against Fusarium oxysporum f. sp. cubense (Foc). (a) Weighing-off of millet seed inoculum, (b) mixing Foc-millet seeds with potting soil, (c) uprooted plantlet ready for replanting in bags/pots containing Foc-infested soil, (d) inoculated plants set up in a screen house and (e) plants ready for disease development rating


#### 3.5 Scoring Disease Severity


Fig. 5.5 Evaluation of Fusarium wilt development. (a) Rhizomes of inoculated plants are cut open for rating, (b) the position where the cut is made, and (c) rating is based on the discoloration of the inner rhizome as follows: 1 ¼ no internal symptoms, 2 ¼ few internal spots, 3 ¼ <1/3 of the inner rhizome affected, 4 ¼ 1/3–2/3 of the inner rhizome discolored 5 ¼ >2/3 of the inner rhizome discolored and 6 ¼ entire inner rhizome discolored

Acknowledgments The authors would like to acknowledge the University of Stellenbosch, International Institute of Tropical Agriculture (IITA) and the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture for financial assistance.

#### References


resistance to Fusarium wilt tropical race 4. Nat Commun 8:1496. https://doi.org/10.1038/ s41467-017-01670-6


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Chapter 6 Lab-Based Screening Using Hydroponic System for the Rapid Detection of Fusarium Wilt TR4 Tolerance/Resistance of Banana

Norazlina Noordin, Affrida Abu Hassan, Anis Nadia Mohd Faisol Mahadevan, Zaiton Ahmad, and Sakinah Ariffin

Abstract Field-based screening and evaluation of banana plant tolerance or resistance to Fusarium oxysporum f. sp. cubense (Foc) Tropical Race 4 (TR4) or also known as Fusarium wilt TR4 is ideal though not always feasible. Alternatively, screening of banana plantlets at lab-stage seems to be an effective method for early detection of Foc TR4 tolerance. We present a simple hydroponic system, that allows plant to grow in a water-based condition. The system has two layers, the upper layer is a tray that has holes for plantlets to be placed where the root system is supported using an inert medium such as rock-wool. The lower layer is a perforated container filled with a water-based nutrient solution. For this lab-based screening, ex vitro gamma irradiated banana cv. Berangan (AAA) rooted plantlets with a pseudostem height of 10–15 cm were inoculated by soaking in a Foc TR4 conidial suspension (10<sup>6</sup> spores/ml) for 2 h under room temperature. The Foc TR4 inoculated rooted plantlets were screened using the hydroponic system and disease symptoms were scored. In this chapter, protocols on acclimatization of ex vitro irradiated rooted plantlets, inoculation with a Foc TR4 conidial suspension, lab- screening using hydroponic system, observation for early detection of disease symptoms and scoring of disease severity are presented.

Keywords Banana · Gamma irradiation · Fusarium oxysporum f. sp. cubense · Labbased screening · Hydroponic system · Disease severity scale · Resistance scoring

N. Noordin (\*) · A. A. Hassan · A. N. M. F. Mahadevan · Z. Ahmad · S. Ariffin Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation Malaysia (MOSTI), Kajang, Selangor, Malaysia e-mail: azlina@nuclearmalaysia.gov.my

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_6

#### 1 Introduction

Banana is the second most commonly grown fruit crop in Malaysia. About 50% of the banana growing area is cultivated with Pisang Berangan and the Cavendish type. However, in the recent years, the overall banana production and cultivation areas have decreased due to the increasing threat of Fusarium wilt and Moko diseases. This alarming issue has led to increasing prices and limited availabilities of banana fruits for local consumption and export (MOA report 2017).

Fusarium wilt of bananas, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana in most parts of the world including Malaysia. This severe disease almost crippled the world banana plantation, production and export trade of 'Gros Michel' circa 1940s and 1950s due to high susceptibility of this cultivar to Fusarium Race 1 (Pegg and Langdon 1987). Introduction of the resistant Cavendish group of cultivars provided an effective and economical solution but the emergence of Foc TR4 and its dissemination in the tropics and sub-tropics poses an immediate threat (Asif and Mak 2001). In Malaysia, we have experienced a total wipe out of a commercial scale Cavendish plantation in the southern part of Malaysia during the 1990s due to the destructive Foc TR4 disease.

Various control measures such as injection of chemicals, soil treatments including fumigation and incorporating soil amendments may reduce the severity of the disease, but none of them is commercially applicable (Hwang and Ko 1987). It was also reported that fungicides, fumigants, flood fallowing, crop rotation, and organic amendments have rarely provided long-term control in any production area (Pegg et al. 1996). Among three races of Foc namely Race 1, Race 2 and Tropical Race 4 which attack bananas, Tropical Race 4 is the most pathogenic and affects many banana cultivars including Cavendish. The pathogen, Foc, can be disseminated through suckers, soil, water, and by farming practices when farmers use contaminated tools. Chemical control, such as soil fumigation, is a promising measure but is hazardous to the environment. The pathogen persists in the contaminated soil by producing chlamydospores even in the absence of the host bananas or sometimes by infection of roots of some weeds (Pegg et al. 1996). As a result, once the field is invaded by Foc, the field cannot be used for banana production for up to 30 years (Asif and Mak 2001). It is therefore of high importance to develop and select new banana varieties that are resistant to Fusarium wilt to overcome this problem (Rusli 2011). One of the techniques to produce new varieties is through in vitro mutagenesis with the application of gamma irradiation (Mak et al. 1995). Gamma irradiation is a reliable and popular physical mutagen to increase genetic diversity and induce mutations and has led to the establishment of new plant varieties (Norazlina et al. 2014).

Field evaluation is the most reliable method for screening disease-resistant lines, but both manpower and space requirements are the limitations that add to the cost of screening (Pegg et al. 1996). It is also essential to maintain strict quarantine measures to avoid pathogen spread and cross-contamination. In addition, plants tend not to show disease symptoms until after 4–5 months (Morpurgo et al. 1994). The uneven distribution of the pathogen in the field can lead to 'disease escape' while many variables that can affect infection and symptom expression cannot be altered nor controlled (Asif and Mak 2001). Alternatively, methods that are simpler, cost effective, low maintenance with space requirements that can show early detection against Fusarium wilt are more attractive and have been developed by many laboratories.

Earlier studies reported the need for improved methods like pre-screening or early detection of tolerance against Fusarium wilt using ex vitro or in vitro rooted plantlets. This early detection is not only for screening for tolerance/resistance but also for comparative virulence and pathogenicity studies (Buddenhagen 1987; Pegg and Langdon 1987). This early detection can be carried out at lab-based or nurseybased stages prior to field-screening.

An earlier study reported roots inoculated by dipping or soaking with a fungal spore suspension before being transferred to an infested field (Mak et al. 2001; Vakili 1965). Susceptible banana plantlets showed external symptoms of leaf yellowing within 2 weeks and wilted within 4 weeks of inoculation (Vakili 1965). Earlier, a double-cup sand-culture containment method had been developed for testing pathogen virulence (Liew 1996). Modification was done and it was replaced by a 'double compartment' apparatus, which contains two plastic trays, one fitting inside the other. This double-tray technique has the capacity for pathogen containment to eliminate cross-contamination (Mak et al. 2001). This technique can be further modified to investigate the effects of various inoculum concentrations and environment variables on infection and disease expression. A bigger size tray could screen a greater number of plants. However, this system must be done in a nursery that still requires a large space with special containment, manpower for the preparation of the system and the cost for the set-up and modification is also less effective.

In this study, a lab-based screening using hydroponic system has been developed. This system is simpler, soilless, and easy to set up, cost effective, portable, requires less space and maintenance. The system can be modified to suit the requirements of the work in the laboratory. In this chapter, protocols on acclimatization of ex vitro irradiated rooted plantlets, inoculation with Foc TR4 conidial suspension, lab- based screening using hydroponic system and observation for early detection of disease symptoms and scoring of disease severity are presented. This work also describes the methodology, reliability of this lab-based screening by considering several factors, including the concentration and duration of Foc TR4 conidial suspension inoculation, type of host plants, and the ability to show differential disease symptoms similar to nursery-based and field evaluations.

#### 2 Materials

#### 2.1 Acclimatization of Banana Plantlets


#### 2.2 Fusarium oxysporum f. sp. cubense (Foc) Tropical Race 4 (TR4) Cultures


#### 2.3 Foc Conidial Suspension


#### 2.4 Soaking in Foc TR4 Conidial Suspension


#### 2.5 Screening in Hydroponic System


#### 3 Methods

#### 3.1 Preparation of Acclimatized Ex Vitro Plantlets


Fig. 6.1 Berangan in vitro rooted plantlets selected for acclimatization. (a) Rooted Berangan cultures in rooting media. (b) In vitro rooted plantlets with the pseudostem height of 5–7 cm. (c) Washing and removal of adhering agar/media from in vitro rooted plantlets. (d) Transfer of plant material into ventilated culture containers containing perlite

Fig. 6.2 Acclimatization of Berangan ex vitro rooted plantlets. (a) Ventilated culture containers placed in hardening room. (b) 3-weeks old acclimatized Berangan rooted plantlets. (c) Berangan rooted plantlets with pseudostem height of 10–15 cm ready to be advanced to lab-based screening protocol using hydroponic system


#### 3.2 Subculturing Foc TR4


Fig. 6.3 Foc TR4 cultures. (a) Subculture of Foc TR4. (b) One agar slab (0.5 0.5 cm) cut. (c) Growth of Foc mycelium onto PDA medium

#### 3.3 Preparation of Foc Conidial Suspension


Fig. 6.4 Foc TR4 suspension (10<sup>6</sup> spores/ml) ready to be used for lab-based screening

#### 3.4 Soaking of Rooted Plantlets in Foc TR4 Conidia Suspension (10<sup>6</sup> Spore/ml)

	- (a) T0: Control (non-inoculated with Foc TR4);
	- (b) T1: 10<sup>6</sup> spore/ml Fusarium solution for 1 h;
	- (c) T2: 10<sup>6</sup> spore/ml Fusarium solution for 2 h;
	- (d) T3: 10<sup>6</sup> spore/ml Fusarium solution for 3 h.

#### 3.5 Screening in Hydroponic System


Fig. 6.5 Acclimatized rooted plantlets ready to be soaked in Foc TR4 conidial suspension. (a) Ex vitro rooted plantlets removed from perlite. (b) Washing roots to remove adhering perlite. (c) Rooted plantlets with pseudostem height of 10–15 cm ready for pathogenicity test


Fig. 6.6 Inoculation of roots with Foc TR4. (a) Soaking the irradiated rooted plantlets with Foc TR4 conidia suspension (106 spore/ml). (b) Soaking with different intervals (hours) under biosafety cabinet level 2

Fig. 6.7 Screening in hydroponic system. (a) Fill 15 l water in the lower compartment of the hydroponic system. (b) Transfer inoculated plantlets into thumb-pots of the hydroponic system

#### 3.6 Observation of Disease Symptoms


Fig. 6.8 Screen for early detection of resistance against Foc TR4


Table 6.1 Disease severity scale (Wu et al. 2010)

#### 3.7 Observation and Results from Lab-Based Screening Using Hydroponic System


Fig. 6.9 Disease scoring (Wu et al. 2010). (a) Rooted plantlets after 28 days of inoculation. (b) Conduct scoring of disease symptoms for all treatments. (c) Plantlets cut-open for scoring. (d) Corm completely clean, no vascular discoloration (control). (e) 1/2 the height of the pseudostem was discolored (T3). (f) >1/2 the height of the pseudostem was discolored and (or) there was discoloration of the leaf stalk (T1). (g) Total discoloration of vascular tissue (T2)

	- (a) Score 1: No streaking or yellowing of leaves. Plant appears healthy.
	- (b) Score 2: Slight streaking and/or yellowing of lower leaves.

Fig. 6.10 Average severity index and average plant height for different inoculation time based on the disease scoring (Wu et al. 2010). Severity at the scale of 1 to 2 for T1 and T2. Average severity index was at 0.50 for treatment 1 and 0.88 for treatment 2. Scoring of 0 to 1 was observed in T3 treatment with average severity index of 0.13. Plant development (height) was decreased for both T1 and T2 treatment, with an average of 9.72 cm as compared to the non-inoculated plantlets; 13.63 cm

#### 4 Notes

1. Mutagenesis of banana (cv. Berangan Intan) plantlets was performed by irradiating meristem tissues/shoot tips with acute gamma irradiation using BioBeam GM8000 (Germany) with a Caesium-137 source and chronic gamma irradiation using Gamma Greenhouse with a Caesium-137 source. The meristem pieces (about 1 cm 2 mm) were aseptically excised from in vitro plantlets of Pisang Berangan. Each meristem was cut longitudinally into two pieces. A total of 50 meristem pieces for each dose were transferred into sterile moist Petri dishes and sealed with parafilm. The meristems in the Petri dishes were irradiated with acute gamma ray using gamma cell BioBeam GM8000 and chronic gamma irradiation using Gamma Greenhouse with a Caesium-137 source at 0, 10, 20, 30, 50, 70, 90, 120 Gy. Each growing irradiated shoot from all the optimal doses (chronic irradiation: 30, 50, 70 Gy and acute irradiation: 10, 20, 30 Gy) were separately sub-cultured to M1V4 generation (three subcultures at monthly interval) to minimize chimerism.

Fig. 6.11 Disease scoring observation on leaves symptomatic responses after inoculation in Foc suspension. (a) Non-inoculated plantlets showed no leaves yellowing. (b) T1 – 1 h soaking period. (c) T2 – 2 h soaking period. (b) T3 – 3 h soaking period


nursery-based condition and later proceed to field-screening for selection of possible mutant lines.


Acknowledgments Authors wish to thank Mr. Shuhaimi Shamsudin, Mr Ayub Mohamad and Mr. Norhafiz Talib for their assistance and services for the acute and chronic gamma irradiation. Our heartfelt thanks and appreciation for Miss Nashimatul Adadiah Yahya, Miss Nurhayati Irwan and Mr. Mohamed Najli Mohamed Yasin for their dedication, endless support and assistance in the plant tissue culture laboratory. We would also like to thank the Plant Pathology Unit of Malaysian Agricultural Research and Development Institute (MARDI) for providing us the initial Foc TR4 mother-culture. Special thanks to the management of Agrotechnology and Biosciences Department and Malaysian Nuclear Agency for their continuous support of our R&D. 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. This work is part of IAEA Coordinated Research Project D22005.

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Chapter 7 Field Screening of Gamma-Irradiated Cavendish Bananas

Gaudencia A. Lantican

Abstract In our search for Cavendish bananas to withstand Fusarium oxysporum f. sp. cubense (Foc TR4) and other diseases, field screening of tissue-cultured Grand Nain banana seedlings derived from gamma-irradiated shoot tips was explored. Six months after irradiation and multiplication in the laboratory, the plantlets (M1V6) were individually grown in seedling bags under screen house conditions for 8 weeks, side-by-side with non-irradiated plantlets of the same clone. Once acclimatized, the banana plants were grown in an area confirmed positive of Foc TR4 (based on previous farm records stating that more than 50% of the plant population succumbed to the disease). Seedlings from each treatment (dose of radiation) were divided into four replicates, regardless of the number of plants. Each plant was given a unique identification code for traceability during disease monitoring, bunch and fruit quality evaluation.

Incidences of Foc TR4, Moko disease (Ralstonia solanacearum) and virus diseases were monitored weekly. Plants found positive of any disease were eradicated immediately. The plant population for the succeeding generation was managed by removing the unwanted suckers, 12 weeks from planting using a spade gouge and keeping only one sucker per plant for the next generation. Agronomic characters of each plant were taken at the flowering stage. These included age to flower, height, pseudostem circumference, number of leaves and height of the sucker. The bunch was harvested 12 weeks from flowering. The number of hands in a bunch, the number of fingers and weight of a hand were recorded. The same agronomic characters of the plant were taken for the succeeding generations.

Plants left standing in the field without any disease symptoms 3 years after planting were considered as putative mutants and were selected as candidate lines for multiplication and second-generation field screening. Only healthy suckers (free from viruses) were further multiplied via tissue culture technique to reach M1V6. Clean suckers from each line free of soil debris or dirt were sent to the laboratory for multiplication. At least 1000 plantlets were produced from each line for the

G. A. Lantican (\*)

Dole Philippines, Inc., Davao City, Philippines e-mail: Gaudencia.Lantican@doleintl.com

<sup>©</sup> The Author(s) 2022

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_7

second-generation field screening. These were grown in two locations – with and without records of Foc TR4. Field monitoring activities including plant population management, disease incidence assessment and fruit quality evaluation were carried out following the same protocols used in the establishment of the first-generation plants. Lines with population showing 10% Foc TR4 after the first harvest, with good vigor, fruit quality and productivity were considered as candidates for further multiplication, farmers distribution and field planting under semi-commercial scale.

Keywords Gamma irradiation · Shoot tips · Field screening · Foc TR4 · Cavendish

#### 1 Introduction

The choice of planting Cavendish bananas in the Philippines particularly Grand Nain resides in its high-yielding capacity, fruit quality and export market demands. After decades of cultivation, the fungal disease, Fusarium oxysporum f. sp. cubense (Foc TR4) debilitated the plant population, and its productivity per unit area. In some locations, the disease affects more than 50% of the population in a short period of time.

To date, there is no single effective treatment against the disease. The variety GCTCV 218 introduced from Taiwan Banana Research Institute (TBRI) through the Bureau of Plant Industry has helped to improve some devastated farms. Yet it has been found that this variety shows susceptibility to Foc TR4 at varying degrees, depending on the location. The availability of a resistant clone therefore, with qualities at par or better than Grand Nain is a welcome development to help manage, if not totally address the complexities of banana pest and disease problems. This will help growers and farmers especially in regions that are vulnerable and are positive of the disease.

Bhagwat and Duncan (1998) reported banana mutation breeding for tolerance to Foc TR4 using gamma irradiation. In 2006, Damasco et al. published a paper "Banana Bunchy Top Virus (BBTV) Resistance in Cultivar Lakatan Developed via Gamma Irradiation of Shoot Tips". This prompted Dole Research to seriously engage in experiments using Grand Nain clone of Cavendish. We have coordinated with the Philippine Nuclear Research Institute (PNRI) equipped with facilities to cover the gamma irradiation treatments. The protocol of Novak et al. (1990) served as an important guide in this project. Radiation treatment is at M1V1 stage of the shoot tips while inside the culture bottles and further multiplied to reach M1V6. Some aspects of Novak et al.'s (1990) protocol have been modified to better understand the characteristics of the plants in the field in addition to its susceptibility to diseases.

The field screening project started in year 2013 in Malandag, Malungon, Sarangani Province, and after 4 years, it was extended to two other regions namely, Maragusan and, Compostela Valley. Both locations are situated in the islands of Mindanao and Philippines.

This protocol describes the field screening tests of plants exposed to gamma irradiation treatments, their multiplication and further field screening tests of those that escaped from Foc TR4 and other diseases (putative mutants) after completing at least three harvest cycles. Disease incidence, plant vigor, growth and development patterns, productivity, and fruit quality were recorded. These parameters describe the tolerance of a putative mutant to a certain disease and ensure that fruit quality and productivity are within farmers' and consumers' level of acceptability.

#### 2 Materials

#### 2.1 Planting Material

1. Hardened banana seedlings (Fig. 7.1 and see Note 1).

#### 2.2 Land Preparation and Planting

1. Farm record showing the historical Foc TR4 cases of the area, 50% of the population.

Fig. 7.1 Establishment of healthy seedlings in the screen house for 8 weeks (a) and a healthy seedling during field planting (b)


#### 2.3 Disease Survey and Eradication


#### 2.4 Bunch Care


#### 2.5 Harvest and Transport


#### 2.6 Fruit Quality Assessment


#### 2.7 Selection and Transport of Putative Mutant Suckers from Field to Laboratory


#### 3 Methods

#### 3.1 Selection of Land for Planting

1. Select an area which is confirmed to have Foc TR4 (see Note 2).

#### 3.2 Land Preparation and Planting


#### 3.3 Disease Monitoring


#### 3.4 Disease Eradication


#### 3.5 Plant Care and Population Management


#### 3.6 Bunch Care

	- (a) Number of leaves
	- (b) Plant height
	- (c) Height of the selected sucker (next generation)
	- (d) Pseudostem circumference (1-m from the base)

#### 3.7 Harvest

	- (a) Number of hands
	- (b) Number of fingers in a hand
	- (c) Weight of each hand

#### 3.8 Fruit Quality Reading


#### 3.9 Overall Plant Population and Farm Status


#### 3.10 Handling of Putative Mutant Plants from an Infected Field for Laboratory Multiplication and Second-Generation Field Screening



Table 7.1 An illustration of survived plants in the field after 3 years of screening seedlings from shoot tips subjected to gamma irradiation


Table 7.2 Seedlings multiplied from the putative mutant plants for second generation field screening

a Selected putative mutant plants showing desirable agronomic characteristics and fruit quality


Fig. 7.2 Sample fruit under second generation field screening of putative mutant lines originally from one Cavendish clone (Grand Nain) at different doses of gamma radiation – (a) 8 Gy and (b) 10 Gy


Table 7.3 Sample field screening data from two different locations showing incidence of diseases, after completing the first-generation harvest


Fig. 7.3 A graphical presentation of the weekly cumulative flowering of putative mutant lines (a– e) in an area negative of Foc TR4, compared with the untreated control

Table 7.4 Program of activities and estimated time frame in the field screening of gamma irradiated Cavendish bananas for disease tolerance, plant vigor and fruit quality


will be produced (see Table 7.4). In order to meet this target, the screening process for disease tolerance need to be planned and implemented correctly.

#### 4 Notes


with 10% Foc TR4 infection in the first cropping (from planting to completion of first harvest cycle) is a better alternative than the susceptible Grand Nain.

14. Samples of putative mutant lines have been submitted to Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre on Nuclear Techniques in Food and Agriculture, Department of Nuclear Applications, International Atomic Energy Agency for molecular analysis together with the untreated control.

Acknowledgments FAO/IAEA Co-ordinated Research Project on Efficient Screening Techniques to Identify Mutants with Disease Resistance for Coffee and Banana.

Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, IAEA – Dr. Ivan Ingelbrecht, Dr. Joanna Jankowicz-Cieslak, Dr. Stephan Nielen, Dr. Bradley Till.

The Philippine Nuclear Research Institute.

Dole Philippines, Inc. – Research, Production and Management Teams.

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Part III Mutation Detection Using Genomics Tools

## Chapter 8 Mutation Detection in Gamma-Irradiated Banana Using Low Coverage Copy Number Variation

#### Joanna Jankowicz-Cieslak, Ivan L. Ingelbrecht, and Bradley J. Till

Abstract Mutagenesis of in vitro propagated bananas is an efficient method to introduce novel alleles and broaden genetic diversity. The FAO/IAEA Plant Breeding and Genetics Laboratory previously established efficient methods for mutation induction of in vitro shoot tips in banana using physical and chemical mutagens as well as methods for the efficient discovery of ethyl methanesulphonate (EMS) induced single nucleotide mutations in targeted genes. Officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic changes such as insertions and deletions (InDels). Such dosage mutations may be particularly important for generating observable phenotypes in polyploids such as banana. Here, we describe a Next Generation Sequencing (NGS) approach in Cavendish (AAA) bananas to identify large genomic InDels. The method is based on low coverage whole genome sequencing (LC-WGS) using an Illumina short-read sequencing platform. We provide details for sonication-mediated library preparation and the installation and use of freely available computer software to identify copy number variation in Cavendish banana. Alternative DNA library construction procedures and bioinformatics tools are briefly described. Example data is provided for the mutant variety Novaria and cv Grande Naine (AAA), but the methodology can be equally applied for triploid bananas with mixed genomes (A and B) and is useful for the characterization of putative Fusarium Wilt TR4 resistant mutant lines described elsewhere in this protocol book.

Keywords Physical mutagenesis · Banana · Polyploidy · TR4 · CNV · NGS

© The Author(s) 2022

J. Jankowicz-Cieslak (\*) · I. L. 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

B. J. Till (\*) Veterinary Genetics Laboratory, University of California, Davis, Davis, CA, USA

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_8

#### 1 Introduction

New developments in the field of molecular biology enable fast and accurate identification of spontaneously occurring or induced changes of DNA sequence. This can allow a more precise use of induced mutations in crop improvement programmes. Mutagens produce various spectrum of changes. Chemical mutagens such as EMS predominantly induce point mutations, whereby physical mutagens such as gamma irradiation produce a broader spectrum of changes ranging from SNPs and small InDels to deletions greater than one million base pairs (Jankowicz-Cieslak and Till 2015; Till et al. 2018; Datta et al. 2018). While phenotypic consequences of large structural variants may be greater, datasets on the spectrum and density of such mutations are currently much smaller than that of EMS.

Mutation breeding may be especially useful in species with a narrow genetic base or those that are recalcitrant to traditional breeding methods such as obligate and facultative vegetatively propagated species. Additionally, mutagens that cause dominant or dosage-based phenotypes can increase the efficiency of generating novel traits in polyploids as the expression of phenotypes arising from recessive mutations requires the combination mutations from homologous sequences (Krasileva et al. 2017). Gamma irradiation has been used widely as a mutagenizing agent for breeding programmes for many crops. In poplar, treatment of pollen with gamma irradiation resulted in InDels varying between small fragments to whole chromosomes (Henry et al. 2015). This work further showed that large genomic InDels could be effectively recovered using low coverage whole genome sequencing (LC-WGS), making mutation discovery more cost-effective and data analyses more streamlined. Larger deletions range in size and may include loss of part of a chromosome (segmental aneuploidy) or loss of an entire chromosome (aneuploidy). Aneuploidy is better tolerated in polyploid plants and may be lethal for diploid plants and animals (Siegel and Amon 2012). These lead to changes in copy number of single genes to whole chromosomes, which have profound effects on phenotypes of the organism. Copy number variations especially affect haploinsufficient genes for which a single functional copy of a gene is not sufficient for normal function. Single copy mutations can potentially knock out the function of genes where only one functional copy is being maintained.

Inducing mutations in triploid banana provides an approach for generating novel variation that is heritable. The logistics of banana mutation breeding including tissue culture propagation, chimerism, polyploidy, heterozygosity, and field space required to find rare favourable mutations makes banana less tractable than seed propagated crops. However, these limitations can be overcome by tissue culture mutagenesis and genomic screening at earlier stages. Previously, we established a system for inducing and maintaining SNP mutations in clonally propagated banana plants. Treating shoot apical meristems of tissue cultured bananas with the chemical mutagen ethyl methanesulphonate (EMS) introduced a high density of GC-AT transitions mutations (Jankowicz-Cieslak et al. 2012). We further showed that mosaicism (chimerism) caused by accumulation of chemically induced mutations in different cells of the plant propagule could be rapidly removed via isolation of shoot apical meristems and subsequent longitudinal bisection. Further, induced mutations were maintained in mutant plants for more than six generations.

We sought to establish a similar system for inducing and maintaining insertions and deletions using physical irradiation. We aimed to develop an efficient pipeline for the generation and recovery of large copy number variations (CNVs) in gamma irradiated Cavendish banana cultivars, employing tissue culture, low coverage whole genome sequencing (LC-WGS) and chromosome dosage analysis (Fig. 8.1). We chose a chromosomal dosage analysis that was previously successful in detecting aneuploidy, insertions and deletions in Arabidopsis, rice and poplar (Tan et al. 2015, 2016). To establish a pipeline for banana, we first adapted sequencing and dosage analysis for the previously released mutant banana variety Novaria. Large genomic deletions of up to 3.8 Mbps were recovered. We next developed a newly mutagenized banana population and tested two different irradiation dosages to establish that new genetic variation can be induced and maintained in vitro (Datta et al. 2018). This work suggests that a large-scale mutagenesis pipeline can be

Fig. 8.1 A pipeline for the generation and recovery of large copy number variations (CNVs) in gamma irradiated Cavendish banana cultivars, employing tissue culture, low coverage whole genome sequencing (LC-WGS) and chromosome dosage analysis. An in vitro mutant population was generated, and a subset was evaluated using the method described in this chapter. This ensures that mutagenesis was successful and mitotically heritable DNA lesions were produced during gamma irradiation and subsequent propagation. Genome sequencing can also be applied to plants showing improved resistance to disease such as Foc TR4 in order to identify mutations causative for the observed phenotype(s). (This figure is modified from Jankowicz-Cieslak et al. 2021)

created for routine production of mutant populations suitable for glasshouse and field evaluations. The efficacy of this approach is being further tested for Foc TR4 resistance (Fig. 8.1). We provide here the methodology for low-coverage DNA sequencing and data analysis to identify large indels in mutant populations of triploid (AAA) banana.

#### 2 Materials

#### 2.1 Library Preparation and Sequencing

#### 2.1.1 DNA Isolation and Quantification


#### 2.1.2 Library Preparation and Sequencing


#### 2.2 DATA Analyses


#### 3 Methods

#### 3.1 Library Preparation and Sequencing

#### 3.1.1 DNA Isolation


#### 3.1.2 Assay DNA Quality and Quantity


#### 3.1.3 Library Preparation for Sequencing

1. Choose library preparation method, sequencing chemistry and read-length (see Note 14).


#### 3.1.4 Data Analysis



./clumpify.sh in ¼ Sample.R1.fq.gz in2 ¼ Sample.R2.fq.gz out ¼ Sample.R1. dedup.fastq.gz out2 ¼ Sample.R2.dedup.fastq.gz dedupe;

Where Sample.R1 and Sample.R2 are the two paired-end reads from one of the samples. For example, G2.R1.fq.gz, and G2.R2.fq.gz in the example data for the cv Grande Naine. Repeat this for all samples.


This step will take many hours on a personal computer. The -t option sets the number of threads. If using Ubuntu, it may be helpful to launch the System Monitor software and select the Resources tab. This will graphically show the CPU usage and allow to monitor your computer to ensure it has not crashed.

12. Sort the sam file using samtools. In the terminal window, enter the following command:


Table 8.1 Partial output from bin-by-sam2.py using example data provided with this protocol

samtools sort -O sam -T sample.sort -o G2\_aln.sam G2.dedup.sam. Where G2 is replaced with the sample name. Note that the output file name

should end in \_aln.sam for the bin-by-sam tool to work.

13. Convert SAM files to BAM format and index it for visual analysis in step 2 of Sect. 3.1.5. In the terminal window, enter the following command: samtools view -b G2\_aln.sam > G2.bam

Where G2 is replaced with the sample name. When complete, enter the following command: samtools index G2.bam. This will create an index file titled G2.bam.bai. Replace G2 with sample name.


#### 3.1.5 Data Visualisation

1. Graph the data. The sample/control columns of the bin-by-sam output can be plotted as an Overlay Plot using a standard spreadsheet software such as Microsoft Excel or LibreOffice Calc, or alternatives such as JMP or R. If using LibreOffice Calc (which comes preinstalled in Ubuntu), open the .txt file created in Sect. 3.1.4.15 step 15, select data from column G (the ratio of mutant to reference in the example) for one chromosome (chromosome 5 in the example data is labelled HE813979.1). Select Insert Chart from the drop-down menu. Select "Line Points Only" to produce a coverage graph (Fig. 8.3).

2. View data with IGV (optional). This tool provides a graphical view of mapped reads and can be a useful visual check of your mapping data. IGV can be used as a web app, which is preferred if the analysis computer has less than 16 Gb RAM. The genome file (.fna) from Sect. 3.1.4 step 8 needs to be renamed and indexed for IGV. Copy the .fna file to a new folder and change the extension from .fna to . fa. Next, open a terminal window, enter (cd) to the new folder and index by typing the following command: samtools faidx genome.fa. Where genome is the name of your genome file. Open a web browser and go to https://igv.org/app/. In the Genome pull down menu, go to the bottom (you may need to expand your browser to full screen in Ubuntu) and select Local File. Select both the .fa file and also the .fa.fai file that was created with samtools. Next, select Tracks, Local File to upload your bam files. This produces a graphical view of mapped reads (Fig. 8.4).

Fig. 8.3 Dosage plot analysis of chromosome 5 of mutant variety Novaria. Each dot represents a bin that is the mean coverage for 100 kb. Relative coverage values less than 3.0 indicate a putative deletion of one or more copies of a chromosome fragment while higher values (>3.0) indicate potential insertional events. The previously identified ~3.8 Mbp single copy deletion is underlined (Datta et al. 2018)

Fig. 8.4 Graphical view of mapped reads of cv Grande Naine (G2) and mutant variety Novaria (N3) example data using IGV

Fig. 8.5 (a) Stably inherited large deletion of 3.8 Mbp identified via LC-WGS in a 'Novaria' mutant. One hundred and eighty-nine genes are affected in the validated region by losing one copy. (b) qPCR verification of identified mutation (CL control left border, CR control right border, CNV Region(s) showing deletion). (Figure modified from Datta et al. 2018)

#### 3.1.6 Validation of Predicted Variants


#### 4 Notes


lowest cost option (in terms of raw Gb per dollar) will be suitable and fragment size and library preparation parameters can be adjusted for this. For the purposes of this protocol, parameters for low input libraries with 2300PE sequencing are described. Different methods are available for DNA fragmentation. It is best to optimize fragment size utilizing a high sensitivity DNA system (e.g. Fragment Analyzer), however fragment sizes can be estimated using gel electrophoresis. Illumina 2300PE sequencing-by-synthesis were used to prepare this protocol. However, higher throughput, shorter read sequencing provides a lower-cost alternative if sequencing is being outsourced.


potential insertional events. Different thresholds can be applied to filter potential false positive signals. In previous work, a threshold of three consecutive bins showing the same trend (below or above 3) was applied. This filter can be applied to the table of data independent of the visualization methods. Such variants were experimentally validated.

Acknowledgments Authors wish to thank Dr. Bernhard Hofinger and Dr. Prateek Gupta for support in setting up the bioinformatic pipelines. 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. This work is part of IAEA Coordinated Research Project D22005.

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Chapter 9 A Protocol for Detection of Large Chromosome Variations in Banana Using Next Generation Sequencing

#### Catherine Breton, Alberto Cenci, Julie Sardos, Rachel Chase, Max Ruas, Mathieu Rouard, and Nicolas Roux

Abstract Core activities of genebank operations include the preservation of germplasm identity and maintenance of genetic integrity. Some organisms such as banana are maintained by tissue culture that can foster accumulation of somatic mutations and loss of genetic integrity. Such changes can be reflected in their genome structure and thus be revealed by sequencing methods. Here, we propose a protocol for the detection of large chromosomal gains and/or losses that was applied to in vitro banana accessions with different levels of ploidy. Mixoploidy was detected in triploid (3x) accessions with chromosomal regions being diploid (2x) and tetraploid (4x) and in diploid accessions (2x) where large deletions resulted in partial haploidy (1x). Such abnormal molecular karyotypes can potentially explain phenotypic aberrations observed in off type material. With the affordable cost of Next Generation Sequencing (NGS) technologies and the release of the presented bioinformatic pipeline, we aim to promote the application of this methodology as a routine operation for genebank management as an important step to monitor the genetic integrity of distributed material. Moreover, genebank users can be also empowered to apply the methodology and check the molecular karyotype of the ordered material.

Keywords Aneuploidy · Banana · Chromosomal variation · Musa spp. · Somaclonal variants · Genebanks · NGS

#### 1 Introduction

Somaclonal variation describes random cellular changes in plants regenerated through tissue culture. It occurs in certain crops that undergo micropropagation, and has been recorded in different explant sources, from leaves and shoots, to

© The Author(s) 2022

C. Breton (\*) · A. Cenci · J. Sardos · R. Chase · M. Ruas · M. Rouard · N. Roux (\*) Alliance Bioversity International-CIAT, Parc Scientifique Agropolis II, Montpellier, France e-mail: c.breton@cgiar.org; n.roux@cgiar.org

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_9

meristems and embryos. Banana (Musa spp.) is a clonal crop that can be conserved and multiplied in vitro. Somaclonal variations have been observed in banana after prolonged periods of in vitro culture and after intensive multiplication phases, both resulting in increased rates of subculturing for a given clone. Although somaclonal variation can result in advantageous mutations that can be useful for the genetic improvement of banana, it is undesirable in the context of micropropagation and plant conservation. This type of variation indeed is a problem for genebank managers, whose objectives are to maintain the genetic integrity of their collections for subsequent research and breeding purposes, thus preserving genetic resources for future generations.

The International Musa Germplasm Transit Centre (ITC), managed by the Alliance of Bioversity-CIAT and hosted at the Katholieke Universiteit Leuven in Belgium, is the world's largest collection of banana germplasm with more than 1600 accessions of cultivated and wild species of banana (Ruas et al. 2017; de Langhe et al. 2018). It ensures the long-term conservation of a wide banana genepool and supports germplasm distribution all over the world. Due to the vegetative mode of propagation, banana accessions are kept in vitro under slow-growth conditions and regenerated through tissue culture. Although stress during the in vitro process is minimized by optimized multiplication and growing conditions, somaclonal variations have been observed. To avoid the conservation, and distribution, of material that holds such variations, and therefore ensure the maintenance of the genetic integrity of the germplasm, the ITC has developed the Field Verification exercise. This exercise aims at monitoring the genetic integrity of its banana accessions and combines evidence from morphological and molecular characterization to determine genetic integrity. To do so, plantlets that were maintained in vitro for more than 10 years are sent to the Musa Genotyping Centre (MGC) and to a partner field collection, USDA-ARS. In the lab, leaves are analyzed by flow cytometry and SSR markers, while in the field, plants are grown, characterized and photo documented. Once all results are obtained after a year or two, a panel of taxonomists check the morphological and molecular data and compare them to known reference information for each accession. Given that plants that have undergone somaclonal variations are expected to change in morphology, from changes in color to obvious growth issues, the panel of experts then assesses whether the accession is True-To-Type (TTT) or Off-Type (OT) (Chase et al. 2014). A major limitation of this process is the amount of time that is necessary to grow the plants and document them. It is also cumbersome to request the availability and expertise of key experts on a voluntary basis.

To fasten the process, the development of early screening methods is therefore of great interest for the community. To overcome the plant phenotyping bottleneck, investigation of modifications at the genome level has been targeted (Sahijram et al. 2003; Oh et al. 2007). The molecular basis of somaclonal variation is not precisely known, but both genetic and epigenetic mechanisms have been proposed (Kaeppler et al. 2000). Somaclonal variants in plants can be the result of various types of mutations such point mutations, gene duplication, transposable elements activation or large chromosomal rearrangements (in number and structure) (Bairu et al. 2011).

Current advances in Next Generation Sequencing (NGS) technologies and associated Genotyping-by-Sequencing methods allow the generation of high-throughput genetic markers for a large number of samples in a fast and cost-effective way. These datasets can therefore be used to study many events, including large chromosomal variations as recently reported in wheat and barley (Keilwagen et al. 2019). Such chromosome changes (e.g. aneuploidy) have also recently been reported in bananas (Cenci et al. 2019, 2021; Baurens et al. 2019, 2020). Although not all these variations may be the cause of off-type phenotypes, likely due to polyploidy that can mitigate them, it remains a change in DNA integrity of the plant that should be flagged, as well as a good reason for accession regeneration from backup or reintroduction with new material. These large chromosomal variations will be the focus of this chapter.

The protocol described here provides an early detection method for large chromosomal indels applied to material which can be obtained at ITC. This method could be used as a routine operation to check the genetic integrity of germplasm conserved in vitro in genebanks, and also in tissue culture laboratories. It can also be used with other methods for the quick testing of variations in mutants.

#### 2 Materials

#### 2.1 Plant Material

Passport data of accessions held at the ITC are published on the Musa Germplasm Information System (MGIS; www.crop-diversity.org). Available material from the ITC collection can be ordered at no cost for research, education and breeding purposes using a straightforward workflow (Fig. 9.1).

The MGIS website offers several options to search for germplasm that presents specific criteria such as taxonomy, country of origin, and it is also possible to identify accessions that have been evaluated for pest and diseases resistance, biotic or abiotic stresses and genome composition. To do so:


MGIS permits users to create a list of accessions that, once registered and if the material is available for distribution, can be ordered online.



Fig. 9.1 Workflow to order banana samples from the International Musa Germplasm Transit Centre (ITC) via the Musa Germplasm Information System (MGIS)


The Musa Online Ordering System (MOOS) is a three-step process which generates at the end of the process a Standard Material Transfer Agreement (SMTA) in PDF format that is automatically sent to the curator of the ITC collection for preparation of the material.

	- (a) Signature: the document should be printed and signed by a person authorized to sign on behalf of the organization then sent to ITC for counter signature.
	- (b) Shrink-wrap: by accepting the parcel, the Recipient is accepting the terms of the SMTA attached.
	- (c) Clickwrap: you accept the SMTA online by clicking. It works the same way as when you order goods or services from other internet web sites.

Here we recommend selecting lyophilized leaf tissue which is the fastest way to receive it if your interest is DNA extraction for GBS or RADseq. Fresh leaves, that can be obtained by growing rooted plantlets, are recommended for whole genome sequencing.

11. Select the level of payment (article 6.7 or 6.11).

Although the material is distributed free of charge, several options exist to Enhance Benefit-Sharing if done for commercial purposes.


#### 2.2 Data Generation

To detect changes in chromosome ploidy, SNP ratio changes need to be monitored along the chromosomes. It is therefore necessary to choose a technology that will generate high-throughput SNP-based markers for genome-wide marker discovery. Technologies based on restriction enzyme-mediated genome complexity reduction such as Genotyping by Sequencing (GBS) (Elshire et al. 2011), Restriction Site Associated DNA Sequencing (RADseq) (Davey et al. 2010), Diversity Arrays Technology Sequencing (DArTseq™) (Kilian et al. 2012) have advantages and disadvantages (Table 9.1) and are all appropriate to detect aneuploidy events. The choice of the technology can be influenced by existing datasets, in-house facilities or affordable solutions offered by service providers. Therefore, we don't provide a protocol for data generation but list the main points related to the main methods previously tested for such analyses.

#### 3 Methods

The workflow described in Figs. 9.2 and 9.3 shows the different steps involved in the analysis. It is composed of two main processes. The aim of the first part is to map data DNA from DArTseq, GBS, RADseq and RNA from RNAseq onto a reference genome sequence (Musa acuminata genome (D'Hont et al. 2012; Martin et al. 2016), or M. balbisiana genome (Wang et al. 2019)), and perform a SNP calling. The second part of the pipeline uses the VCF file obtained from SNP calling to determine the genomic structure of the accessions and define their molecular karyotype in order to reveal possible ploidy change.


Table 9.1 NGS methods to generate high levels of polymorphism that can be used by the protocol

Step 1: Read cleaning, mapping, variant calling


Step 2: Merge data, Use VcfHunter to establish the molecular karyotype


The preprocessing pipeline presented in Fig. 9.2 shows the different steps that are compatible with four different sequencing technologies as listed in the previous section. The pipeline has to be done sample per sample according to the best practice of the software GATK (McKenna et al. 2010) used for the SNP calling part.

Fig. 9.2 Schematic overview of the bioinformatics workflow for SNP calling

Step 1: Read cleaning, mapping, variant calling

This pre-processing step performs the cleaning data to the SNP calling via the read mapping and should be performed on each individual in order to produce a single VCF by sample (to be combined in Step 2).

Read Quality Check

• Quality reads: Control the quality of the raw Fastq file with FASTQC

Description: In order to verify the quality of data reads, FastQC allows to check the quality score of each base and the presence or absence of the adaptor used to build the library. Adaptor depends on the sequencing technology used. https://www. bioinformatics.babraham.ac.uk/projects/fastqc/

• Trim low-quality base with Cutadapt

Description: According to the result of FastQC, Cutadapt (Martin 2011) trims low quality ends and removes adapters (Illumina). Website: http://cutadapt.readthedocs.io/en/stable/guide.html

Fixed Parameters


Fig. 9.3 SNP ratio calculation and visualization. Reads are mapped to the reference genome. For a given DNA base position, multiple reads will be aligned for a global coverage. SNP detected are assigned to a different color (corresponding to different genomes for hybrid species). SNP Frequency is calculated at each site (e.g. 0.5 ¼ half of the reads display this allele) and then plotted on a graph according to their physical position along the chromosome. Variation of SNP frequency combined with SNP coverage along the chromosome indicate chromosome segment with ploidy change


The tool generates a trimmed fastq file (\*\_cutadapt.fastq.gz) files for each accession.

#### Mapping Reads on the Reference

Description: Align reads on a reference genome (e.g. Musa acuminata 'DH Pahang'), with BWA (Li and Durbin 2010) for DNA, and STAR (Dobin et al. 2013) for RNA.

DNA Data: Map with BWA with default parameters with BWA-MEM.

Different types of genomic data such as DArTSeq, GBS and RADseq can be used (Fig. 9.2).

The tool generates a sam (\*\_.sam) files for each accession.

Website: http://bio-bwa.sourceforge.net/

RNA Data: Mapping with STAR in 2-pass mode.

Description: In the 2-pass mapping job, STAR will map the reads twice. In the first pass, the novel junctions will be detected and inserted into the genome indices. In the second pass, all reads will be re-mapped using annotated (from the GTF file given by the user) and novel (detected in the first pass) junctions. While this procedure doubles the run-time, it significantly increases sensitivity to novel splice junctions. In the absence of annotations, this option is strongly recommended.

The tool generates a folder for each accession, (names filled in column 3 "genome\_name") filled in the configuration file, which contained the SAM files (converted in BAM file) of aligned reads and a .final.out file of mapping statistics for each library. In addition, a (--prefix) folder containing a mapping statistics file (- prefix + mapping.tab) for all accession is generated. Website: https://github.com/alexdobin/STAR

#### Variant Discovery

• Add read group and (accession name from the fq.gz filename) sort BAM with Picard Tools

Description: This step replaces the reads Groups which describe the reads mapped on the reference, the sequencing technology, samples names, and library number are added.

ID <sup>¼</sup> Read Group identifier (e.g. FLOWCELL1.LANE1).

PU ¼ Platform Unit (e.g. FLOWCELL1.LANE1.UNIT1).

SM ¼ Sample (e.g. DAD).

PL ¼ Platform technology used to produce the read (e.g. ILLUMINA).

LB <sup>¼</sup> DNA library identifier (e.g. LIB-DAD-1).

Website: https://broadinstitute.github.io/picard/

The tool generates a bam (\*\_rmdup.bam) file for each accession with the RG (Read Group) modified.

• Mark duplicate reads and index BAM with MarkDuplicates from PicardTools

Description: PCR duplicate removal, where PCR duplicates arise from multiple PCR products from the same template molecule binding on the flow cell. These are removed because they can lead to false positive variant calls. Sort the BAM file and mark the duplicated reads.

Website: https://broadinstitute.github.io/picard/

The tool generate a bam (\*\_rmdup.bam) files for each accession with duplicated reads removed. In addition, a file named (--prefix + rmdup\*stat.tab) file containing duplicate statistics for each accession was generated in the (--prefix) folder.

• Index BAM with Samtools

Description: This step reorders the bam file according to the genome index position.

Website: http://samtools.sourceforge.net/

The tool generates a reordered (\*\_reorder.bam) and bai (\*\_reorder.bai) files for each accession.

• Split 'N CIGAR' reads with SplitNCigarReads from GATK

Description: Splits reads that contain Ns in their cigar string (e.g. spanning splicing events in RNAseq data). Identifies all N cigar elements and creates k+1 new reads (where k is the number of N cigar elements). The first read includes the bases that are to the left of the first N element, while the part of the read that is to the right of the N (including the Ns) is hard clipped and so on for the rest of the new reads. Used for post-processing RNA reads aligned against the full reference.

Website: https://gatk.broadinstitute.org/hc/en-us/articles/360036727811- SplitNCigarReads

The tool generates a split and trimmed (on splicing sites) bam (\*\_trim.bam) and bai (\*\_trim.bai) files for each accession.

• Realign indels with IndelRealigners from GATK (2 steps)

Description: The mapper BWA has some difficulties to manage the alignment close to the Indel. The step is not necessary with HaplotypeCaller but is necessary with UnifiedGenotyper. The tool generates a bam (\*\_realigned.bam) and bai (\*\_realigned.bai) files realigned around indel for each accession. This step is done with the GATK version 3.8.

Website: https://github.com/broadinstitute/gatk-docs/blob/master/gatk3-tutorials/ (howto)\_Perform\_local\_realignment\_around\_indels.md

The tool generates a bam (\*\_realigned.bam) and bai (\*\_realigned.bai) files realigned around indel for each accession.

• Create a VCF file with HaplotypeCaller from GATK

Description: The HaplotypeCaller is able to call SNPs and indels simultaneously via local de-novo assembly of haplotypes in an active region. Whenever the program encounters a region showing variation, it discards the existing mapping information and completely reassembles the reads in that region. This allows the HaplotypeCaller to be more accurate when calling regions that are traditionally difficult to call.

Parameters:


```
```

https://gatk.broadinstitute.org/hc/en-us/articles/360036712151-HaplotypeCaller

Note: All these steps can be performed separately or with workflows reported in the literature (e.g. Toggle(Monat et al. 2015; Tranchant-Dubreuil et al. 2018)) or using the scripts we made available on GitHub at https://github.com/CathyBreton/ Genomic\_Evolution, which follow all the steps.

The tools are developed in Perl, bash, Python3, Java and work on the Linux system and require:


Description: Get one FASTQ file ready for SNP calling per accession from raw sequence data (fastq.gz files).

USAGE:

<Technique>\_<Type\_of\_data>\_fastq\_to\_vcf\_job\_array\_Total\_GATK4.pl -r ref. fasta -x fq.gz -cu accession

Parameters:




Step 2: Molecular Karyotyping and Ploidy Change Detection

Based on known sequence variability, SNP variants can be assigned to the ancestral genomes in order to plot the genome allele coverage ratio and to calculate the normalized site coverage along chromosomes as described in (Baurens et al. 2019). This method can detect chromosome changes such as homoeologous exchanges (Cenci et al. 2021) but also is powerful enough to detect ploidy variation along the chromosomes as illustrated in Fig. 9.3.

The method has been developed within the VCFHunter software (Baurens et al. 2019) and can be used with the following procedure (Fig. 9.4).

• Merge datasets

Script name: CombineVcf.pl.

Description: The script merges and prepares the final VCF file, this step combines multiple VCF and performs pre-filtering using GATK. The samples to analyze are combined to the reference samples to allow allele assignation. Reference samples are representative genotypes that are relevant to the identification of your samples (i.e. acuminata or balbisiana without admixture). Whenever necessary, such SNP datasets can be downloaded on MGIS (https://www.crop-diversity.org/mgis/gigwa with RADseq\_ABB\_AB datasets) via GIGWA (Cenci et al. 2021; Sempéré et al. 2019).

USAGE: perl CombineVcf.pl -r reference\_fasta -p output\_prefix -x extension\_file\_to\_treat.

Fig. 9.4 Pipeline to determine the genome structure with VCFHunter

Parameters:



Website: https://github.com/CathyBreton/Genomic\_Evolution

• Filter SNP dataset

Script name: vcfFilter.1.0.py

Description: Filter VCF file based on most common parameters such as the coverage, missing data, MAF (minor allele frequency). The tool keeps bi-allelic sites and removes mono-allelic, tri-allelic, tetra-allelic sites.

USAGE: python3 vcfFilter.1.0.py --vcf file.prefiltered.vcf --prefix file.filtered -- MinCov 8 --MaxCov 200 --MinAl 3 --MinFreq 0.05 --nMiss 50 --names All\_names. tab --RmAlAlt 1:3:4:5:6:7:8:9:10 --RmType SnpCluster.

Website: https://github.com/SouthGreenPlatform/VcfHunter/blob/master/tutorial\_ DnaSeqVariantCalling.md

Parameters:


• Split VCF by chromosome

Description: Generate a VCF file for each chromosome with VcfTools (Danecek et al. 2011), in order to obtain a representation (chromosome Painting) of the SNP position along each chromosome.

USAGE: vcftools --vcf file\_filtered.vcf --chr chr01 --recode --out batchall\_filt\_chr01.

Parameters:




• Generate molecular karyotype

Description: The program allows to perform a chromosome painting for all chromosomes of a given accession.

Script name: vcf2allPropAndCov.py.

USAGE: python3 vcf2allPropAndCov.py --conf <chromosomes.conf> --origin <origin.conf> --acc <sample\_name> --ploidy 2

Parameters:





The tool generates the 4 following files:


Website: https://github.com/SouthGreenPlatform/VcfHunter/blob/master/tutorial\_ ChromosomePainting.md

Use Cases: Application to Accessions at ITC

Fig. 9.5 Molecular karyotype of an ABB banana cultivar. On the left, SNP ratio distribution along the chromosomes. Each SNP is illustrated by a dot and assigned to a genome by a color (A ¼ green, B ¼ red). The red arrows with values (0.33/0.5/0.67) refer to the SNP frequency ratio. On the right, read coverage at SNP positions along the chromosomes. Heterozygous SNP frequency distribution around 0.5 and lower SNP coverage along the whole chromosome 8 indicate a BB pattern (loss of chromosome 8A) in 'Dole' (ITC0767)

This section describes several examples of samples across the banana taxonomy that were processed by our method and allowed the detection of large chromosome variations.

Chromosomal changes in allotriploids (AAB, ABB).

Comprehensive characterization of the ABB samples has been conducted on ITC materials, revealing the genome structure or molecular karyotypes of most of the existing taxonomic subgroups (Cenci et al. 2021). Using VCFHunter on RADseq data, we were able to uncover patterns of chromosome segment recombinations between A and B genomes for most of the accessions. Among them, one displayed a clear case of chromosome number change as illustrated on Fig. 9.5. For this genotype belonging to the Bluggoe subgroup, no SNP variant was assigned to the A subgenome (in green) along the whole chromosome 8. Most of the B variants (in red) were located at the top of the diagram (value ¼ 1) with an unimodal distribution for residual B genome heterozygosity around 0.5 (instead of 0.33/0.67 expected in the presence of three B chromosome 8). Moreover, the diagram on the right shows a lower SNP density coverage in comparison to all the other chromosomes, showing that chromosome 8 was diploid (2x) for this accession. This pattern observed here is due to the loss of the A genome version of chromosome 8. For comparison, chromosome 4 exhibits two regions with irregular patterns compared to

Fig. 9.6 Patterns of chromosome loss and gain in Musa ABB (A <sup>¼</sup> blue, B <sup>¼</sup> red, read coverage ¼ black). (a) Pattern of 'Simili Radjah' (ITC0123) chromosome 5, showing frequency of A and B variants (y axes) around 0.5 and lower SNP coverage on the second arm of one B chromosome, indicates AB pattern (loss of one B chromosome 5 second arm). (b) Pattern of 'INIVIT PB-2003' (ITC1600) chromosome 10, containing an interstitial region showing frequency of A and B variants (y axes) around 0.5 and higher read coverage indicates AABB pattern (duplication of A chromosome interstitial region)

expectation for an ABB. One is located on the first arm of the chromosome and the second one is placed on the distal part of the second arm. However, in both regions, allelic frequencies for heterozygous sites are consistent with triploidy (0.33/0.67) and no distortion of the coverage density is observed.

Two additional examples of aneuploidy detection in ABB are illustrated in Fig. 9.6. In the accession 'Simili Radjah' (ITC0123, ABB), the loss of the second arm in one of the B chromosomes of chromosome 5 can be inferred by the SNP frequency at 0.5 for both A and B assigned SNP and by the lower SNP density coverage compared to the first arm (Fig. 9.6a). In the accession 'INIVIT PF-2003' (ITC1600, ABB), in the second arm of chromosome 10, an interstitial region appears to have SNP frequency at 0.5. Since the SNP density coverage in this region is higher than in the remaining chromosome having 1A and 2B chromosomes, the duplication of the interstitial region in chromosome A was deducted, being the SNP ratio in this region 2A:2B.

Finally, other examples were also detected in AAB Plantain cultivars (Fig. 9.7). We observed in 'Nzumoigne' (ITC0718), a SNP frequency of A and B alleles at 0.5 on chromosome 2 that was combined to lower SNP density coverage (compared to other chromosomes as exemplified with chromosome 3) (Fig. 9.7a). In 'Ihitisim' (ITC0121), multiple events were detected including a chromosome gain on chromosome 3 with SNP ratio of ~0.25 and ~ 0.75, supported by a higher SNP coverage and a partial loss at the beginning of the chromosome 4 (Fig. 9.7b).

Fig. 9.7 Patterns of chromosome loss and gain in Musa AAB Plantains (A <sup>¼</sup> green, B <sup>¼</sup> red). (a) Pattern of 'Nzumoigne' (ITC0718) chromosome 3 with regular pattern (variants A and B with frequency at 0.67 and 0.33 (y axis), respectively) compared to chromosome 2 having both ends with A and B variants at 0.5 frequency. The pattern indicates loss of both ends of one A chromosome 2. (b) A and B variant frequencies (y axes) in chromosome 3 (0.75 and 0.25, respectively) and read coverage higher in chromosome 3 than in chromosome 4 indicate the presence of an additional chromosome 3A in 'Ihitisim' (ITC0121)

Chromosomal changes in diploid banana accessions (AA).

The survey of more than 200 AA accessions with the same procedure revealed molecular karyotypes corresponding to chromosome arms with large interstitial or terminal deletions for a few individuals (Fig. 9.8).

Fig. 9.8 Chromosome pattern of mutated chromosomes in cultivated AA diploid accessions. (a) Patterns of 'No.110' (AA, ITC0413) second arm terminal region of chromosome 5. Absence of heterozygous variants and lower read coverage indicates terminal deletion in one copy of second arm. (b) Patterns of 'Pahang' (AA, ITC0727) second arm interstitial region of chromosome 8. Absence of heterozygous variants and lower read coverage indicates interstitial deletion of a copy on second arm

#### 4 Notes


Acknowledgements This work was supported by the CGIAR Fund, and in particular by the CGIAR Research Program Roots, Tubers and Bananas.

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Part IV Low-cost In Vitro Methods for Banana Micropropagation

## Chapter 10 Low-Cost In Vitro Options for Banana Mutation Breeding

Babita Jhurree-Dussoruth

Abstract Mutation fixation of irradiated banana cultures is achieved through at least three generation advancements by in vitro subculturing. The in vitro culture is a technique which allows rapid multiplication of plantlets within a short time and which often relies highly on expensive inputs that are almost unaffordable in many developing countries. This chapter highlights some easily affordable options that can be adopted for in vitro propagation and weaning of tissue-cultured banana plantlets and other horticultural crops. The presented options provide resource restricted laboratories opportunities to coopperate with irradiation facilities for mutation induction. Thus, when applied to any locally selected banana variety, the low-cost in vitro methods allow an efficient mutagenesis process to improve local accessions. Low-cost alternatives adopted to carry out in vitro mutagenesis activities in the current FAO/IAEA project are presented, by using as baseline other cheaper options developed and adapted through a locally funded project (supported by the Mauritius Research and Innovation Council).

Keywords Tissue-culture · Low-cost · In vitro mutagenesis · Banana · Mutation breeding

#### 1 Introduction

Banana breeding of popular varieties through conventional methods is a long and tedious process, challenged by low female fertility, polyploidy and heterozygosity (Jenny et al. 2002; Ortiz 2013). Unlike seeded species, the classical cross-breeding for genetic improvement of the mostly sterile and seedless banana can last over several years. This has prompted many countries to have recourse to mutation breeding as an alternative method for the improvement of banana.

B. Jhurree-Dussoruth (\*)

Food and Agricultural Research and Extension Institute, Reduit, Mauritius e-mail: bdussoruth@farei.mu

<sup>©</sup> The Author(s) 2022

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_10

Earliest studies on the effect of radiation on plant development dates back to 1928 by Lewis Stadler (Stadler 1928; FAO/IAEA 2011) with groundwork on induced mutation breeding of banana starting in the early 1970's by De Guzman, Ubalde and Del Rosario (De Guzman et al. 1976). Since then, significant advancement has been achieved in mutation breeding of banana through use of physical mutagens (X- or gamma rays) or chemical mutagens (ethyl methanesulphonate, EMS) (FAO/IAEA 1995). Successful applications of mutation in genetic improvement of banana include the development of an early flowering mutant of banana cv. Grande Naine, named 'Novaria' by F. J. Novak (Mak et al. 1996) and an improved local variety named "Al Beely" banana (FAO/IAEA 2011). Furthermore, Jain et al. (2011) reported on ten banana mutants/variants that were identified as promising with improved traits such as earliness, improved bunch and fruit size, reduced plant height or tolerance to Fusarium wilt.

Countries facing important banana pest and disease pressures can have access to improved genotypes such as FHIA hybrids developed by the Honduran Foundation for Agricultural Research, however, many countries still opt to improve their popular locally adapted variety. Mauritius is one such country which embarked on mutation breeding of its popular banana varieties through the joint support of the FAO and IAEA. A first project aimed at inducing tolerance/resistance to Panama disease in a highly prized local dessert type banana, namely 'Gingeli' banana (AAB genome) from year 2004 to 2015 using gamma rays on in vitro cultures. In the absence of hotspots, the generated mutant lines were screened at greenhouse level and the surviving lines were multiplied for field testing. Till date, no promising line has been identified. In another FAO/IAEA funded project, mutagenic treatments were performed on three other popular varieties, the 'Mamoul' (ABB), 'Ollier' (AAA) and D. Cavendish banana (AAA) with aim to induce tolerance to Fusarium wilt race 1 (in 'Mamoul') and race 4 in the others.

In vitro mutation breeding is a powerful tool for induction of desired traits. However, the restricted access to irradiation and in vitro facilities remains among the main withdrawal factors for these countries to embark on banana breeding using plant biotechnology techniques. The Plant and Breeding Genetics laboratory of the FAO/IAEA in Seibersdorf, Austria is one such laboratory that supports developing countries by irradiating plant materials, free-of-charge. Similarly, Mauritius can also extend physical mutagenic treatments to neighboring countries.

In vitro culture methods are used in banana mutation breeding whereby emerging buds from the mutagenized cultures are isolated and brought back into culture, through a series of up to four subcultures (generations) (M1V0 to M1V4) which then allows the mutation to be fixed in the genome (in vitro mutagenesis) (Roux 2004). The in vitro tissue culture (TC) is often associated with high production costs as it relies on aseptic conditions, high quality chemicals, high-energy and expensive glassware which restrict its broad application in plant breeding. To make TC affordable without compromising the quality of the plants, several low-cost alternative components of tissue culture have been considered and adopted (Kodym and Zapata-Arias 2001; FAO/IAEA 2004; Ogero et al. 2012; Datta et al. 2017).

A minimum, recommended number of shoot tips (500–1000) can be irradiated and if managed in batches, the number of plants generated by the M1V4 population can be easily handled using the low-cost approach. The sections below highlight the low-budget approach of in vitro banana mutation breeding.

#### 1.1 Low-Budget In Vitro Options

#### 1.1.1 Infrastructure

A typical TC laboratory consists of rooms dedicated to each aspect of in vitro culture: A first room (usually the first entry point) is for the reception and handling of mother plants received from the field. The other rooms include the media preparation room, the transfer room with laminar flow cabinets which serve as an aseptic area for handling of in vitro cultures and a growth room with controlled temperature and light for growth of cultures. In a low-cost situation, a simple large room or two-rooms can be partitioned to accommodate the above areas while respecting the flow of each section and ensuring that the aseptic area is against the flow of air.

#### 1.1.2 Consumables and Equipment

Banana cultures are mainly established in Murashige and Skoog (MS media) or modified MS media supplemented with auxin and cytokinin. The media can be prepared by using powdered form of ready-made MS media or by mixing aliquots of four stock solutions of macronutrients, micronutrients, iron and vitamins (see Annex 10.1). The latter is more accessible and relatively cheaper for developing countries. High quality analytical grade chemicals are expensive and not a requisite for routine TC. On the other hand, commercial grade chemicals of lower purity can be easily procured from school suppliers that provide such chemicals as teaching materials and their use can lead to up to 70% savings (Dussoruth et al. unpublished). In a study, Gitonga et al. (2010) reduced the cost of producing tissue culture banana seedlings by 93.9% by using alternative nutrient sources. Similarly, Ogero et al. (2012) successfully realized cost reduction of over 80–95% by substituting conventional MS compounds by locally available fertilizers (for macro/micronutrients), over-the-counter vitamins (for vitamins) and seaweeds or agricultural plant hormones (for plant growth regulators, PGR's). However, Dussoruth et al. (unpublished) found that the quality of agricultural fertilizers had significant effect on in vitro plants. Aliquots of field fertilizers used to make the MS media did not dissolve well, leaving behind deposits of impurities that reduced the effective percentage of each element in the media. Moreover, the explants inoculated in these media died due to phytotoxicity. On the other hand, MS media made up with hydroponic salts did not cause phytotoxicity but significantly reduced the multiplication rate of banana plants. Thus, all substitutions that aim in cost reduction need to be fine-tuned as these can affect the quantity and quality of the plants.

Gelling agents and high-grade sucrose constitute the most expensive components of the culture media (Sahu and Sahu 2013). The gelling agents account for over 70% of media costs while the media chemicals contribute to 5–15% of production costs (Prakash 1993). Dussoruth et al. (unpublished) also noted that when both highquality chemicals and analytical grade sucrose are used for MS media, sucrose becomes the largest components of MS media (63–78%) and this is reduced to about 3–8% when replaced by household sugarcane-based sugar (see Table 10.1). On the other hand, when commercial grade chemicals and household sugar are used, the gelling agents account to approximately 70–80% of media cost (Dussoruth et al. unpublished). As the media chemicals (nutrients and PGR's) contribute to only about 10–20% of overall cost of 1 L media (see Table 10.1), a significant cost reduction in media is possible mainly by substituting the expensive high-quality gelling agents and analytical sucrose by cheaper commercial substitutes. It is now standard practice by many laboratories to use commercial sugar in media preparation.

Banana cultures can be generated in solid, semi-solid or liquid media. The liquid media allows a more rapid proliferation (Ahloowalia and Prakash 2004) but relies on expensive orbital shaker at around 60–80 rpm for media aeration to prevent asphyxia of the explants. The orbital shaker can, however, be replaced by a locally mounted shaker. In the absence of a shaker, the cultures can be multiplied in stationary liquid media by using supports as interface (matrix), which serve as an intermediate layer between the liquid and explants. Cheaper matrices such as marbles (beads), paper shreds, glass wool, filter paper bridges, cotton fibers among others were used by several researchers as reported by Prakash et al. (2004) and Datta et al. (2017). Dussoruth et al. (unpublished) assessed the effectiveness of sugarcane based bagasse, cotton wool, paper shreds, tissue-paper, marbles (see Fig. 10.1) for micropropagation of banana and African violet (an ornamental, Saintpaulia ionantha). Tissue-paper and cotton wool were as effective as phytagel or agar and agitated liquid, but the disadvantage was that explants, including the roots, were strongly attached to the matrix. The size of the marbles are important because large spaces among the beads can lead to drowning of explants.

As shakers/agitators add to production costs, many laboratories opt for semi-solid or solid media by using gelling agents which contribute to the viscosity of the

Table 10.1 Percentage cost contribution of each component in MS media depending on quality of chemicals and quality of gelling agents used


Fig. 10.1 Use of low-cost physical support matrices for growth of cultures. The matrices (from left: cotton, cotton, tea paper, marble, liquid-agitated, bagasse) and the banana cultures, 4 weeks after subculture

medium. The solidified media acts as an interface allowing the explants to be seated in the media while uptaking necessary nutrients for growth and development. Conventional gelling agents are agar, agarose, and gellan gum (marketed under trade names such as phytagel or gelrite) (Prakash et al. 2004). The gellan gums produce transparent media, allow easy detection of contamination and are of higher quality than agar, however they are often expensive. In Mauritius, the gelrite and phytagel cost around 1200–1600 €/kg and 500–700 €/kg respectively while agar costs 90–400 €/kg, depending on the purity. To reduce the cost of media, cheaper sources of phytagel can be mixed with agar. Agar is the most widely used gelling agent, since it is usually unnecessary for high purity agar in large-scale micropropagation (Prakash et al. 2004). Extensive research has been done into cheaper alternatives to replace agar and gellan gums or in combination with other gelling agents (Puchooa et al. 1999; Wilson and Tenkouano 2020 and as compiled by Prakash et al. 2004; Datta et al. 2017).

Dussoruth et al. (unpublished) tested household tapioca pearls (locally called as 'sagoo'), rice flour, cornstarch and arrowroot as alternative gelling agents, of which rice and tapioca were least suitable. Arrowroot gel led to improved plant development compared to agar. However, the 100% cornstarch-based or arrowroot-based media is firm and the opaque white to grayish-white color make difficult to detect any contamination (see Fig. 10.2). Addition of agar (1:1 ratio) softened the media while allowing explant growth and development. To allow detection of contamination during the initial stages, it is advisable to avoid the opaque media and use mainly agar as gelling agent. An agar or phytagel-based medium can be cooked in a microwave to produce a homogeneous medium prior to dispensing in culture jars, while the household gelling agents need to be carefully cooked in a pan on a stove with continuous stirring, to avoid lumps.

Fig. 10.2 Effects of different low-cost gelling agents and physical matrices. Effect of (from left to the right) agar, arrow root, cornstarch, arrowroot plus agar and cornstarch plus agar on banana shoot proliferation. Addition of agar to the low-cost gelling agents improved the structure of the media

The protocol in 'Methodology' section describes the banana micropropagation on modified MS media solidified with agar-cornflour (1:1) and agar-arrowroot (1:1). Growth of cultures was comparable to those grown in agar-based media (see Fig. 10.2). Cost of cornstarch and arrowroot was around 4 €/kg which is only 0.1% of the cost of agar. As 80–100 g of the cornstarch and arrowroot is used per litre of MS media compared to 2.5 g/l phytagel and 8.0 g/l agar, this represented a respective 60% and 80% savings over agar and phytagel.

Water is another main component which is used to make stock solutions and the culture media. Conventionally, distilled, doubled distilled or de-ionized water is used (Ahloowalia and Prakash 2004). These are expensive and can further significantly increase the cost of production if they are operated using electricity. As reported by Ahloowalia and Prakash (2004), several researchers used cheaper options such as operating distillatory with gas or altogether replacing distilled/ deionized water with cheaper alternatives like tap, rain or bottled water. Experiments using distilled/deionized, tap, bottled and household-filtered water showed that cultures were successfully produced using filtered water (see Fig. 10.3). Filtered water was then adopted as the most economic source for preparation of both media and stock solutions used in this project. The unit cost of a home-scale filtration unit ranged from € 50 to € 125, depending on brand and quality compared to about € 5000 for a deionizer/distillatory unit leading to a cost reduction of over 95%.

Big commercial laboratories use high quality glassware that tend to be costly and fragile. Similarly, chemicals or stock solutions which are accurately measured during media preparation using expensive equipment can be approximate to the

Fig. 10.3 Effect of five water sources (from left to the right tap, bottled, household-scale filtered, deionized and double-distilled) used in media preparation. Performance of banana cultures in media made using filtered water was comparable to those from deionized water

nearest amount. Over 90% cost savings can be made by using easily available, hardy and cheap wares such as syringes (for dispensing on aliquots of 1 ml to 50 ml) or plastic cylinders for larger volumes. These can easily be procured over the counter from school supply shops or medical stores. Access to culture vessels, namely 'Magenta' commonly used for TC may not be easily found in developing countries, alternatively recycled jam jars can be used. Cheap polypropylene (PP) lids can be used to replace the metal caps with added advantage that they do not rust and allow light to reach the cultures. Additionally, cling film or sterile PP plastic films can also be placed around the mouth of the jar and held tight using rubber bands (see Fig. 10.4). This method is not recommended, as wrapping the cover/film around the mouth of the jar takes more time than closing with a lid. It however, remains an option for those not having access to required lids. The Guangdong Academy of Agricultural Sciences (GAAS) uses a special PP pouch (see Fig. 10.4) for growing cultures both under natural and artificial LED lights.

While most activities can be easily handled using cheaper alternatives, some steps such as pH testing, require a closer monitoring. Some laboratories used pH indicator paper as low-cost option, however this method is not very accurate (Ahloowalia and Prakash 2004) because slight changes in pH are not easily detected through the indicator paper. Alternatively, portable hand-held cheaper pH meters can be used and they cost about 10% of analytical high accuracy pH meters. The expensive magnetic stirrers (€ 300 to € 900) can equally be substituted by simple ones at less than € 50.

An aseptic condition is a major requisite in any TC activity. Wares and media are best sterilized using autoclave, which can be costly. Commercial laboratories working with large volumes can afford autoclaves but this can be an expensive option for laboratories with small turn-over rates. Stericlave (portable medical pressure steam

Fig. 10.4 Options for culture vessels and covers. From left: jar covered with polypropylene (PP) cap, conical flask covered with an aluminium and jam jar with metal cap

Fig. 10.5 (a) Stericlave. (b) Pressure cooker. (c) Low-cost, glass hood

sterilizer) of above 40 l, which cost about 30% of a conventional autoclave is an affordable option (see Fig. 10.5a). Similarly a pressure cooker (20–25 l volume) which cost about 6–10% of an average autoclave can also be used (see Fig. 10.5b). Effective steam sterilization can be achieved through automatic setting of autoclaves at a pressure of 15 psi (1.05 kg/sq.cm) along with a temperature of 121 C for at least 30 min. On the other hand, the pressure cooker needs closer monitoring as overcooking can lead to caramelization of the medium (see 'Methods' section). Another point to consider is that a filled pressure cooker can be very heavy to lift and thus the height of the table, where the stove will be placed to heat the cooker, may need to be adjusted for easy handling. A pressure cooker is an easily available option for small laboratories where for example in a 20 l capacity pressure cooker, only about thirty 200–250 ml capacity culture (jam) jars can be stacked. As each jam jar is filled with about 30 ml medium, a total of only 1 l media can be sterilized at a time in a 20 l capacity cooker.

In vitro culture manipulation is done inside a laminar hood in a transfer room under aseptic conditions. Instead of using high purity ethanol, commercial ethanol can be equally used to clean working surface and a flame to sterilize the scalpels and forceps. In the absence of glass beads sterilizer, the sterilization can easily be done by dipping the working ends of the tools in alcohol and then immediately flaming them over an alcohol lamp or gas burner. A laminar flow cabinet allows handling of cultures in a sterile environment and the cheapest ones varies around 5000–6000 €. If the laboratory intends to undertake TC works for many years, then it is advisable to invest in a laminar hood. Otherwise, if the hood is needed only to allow generation advancement of the mutated population, a glass hood can be designed such that the opening is wide enough only to allow easy manual handling of the cultures. The glass hood can be cuboidal about 1 m wide, 40 cm deep and 60 cm high with an opening on one side (of about 30–40 cm from base) (see Fig. 10.5c). The hood should be placed against flow of air and surface sterilized regularly.

After placing the explant in the media, the jar is immediately closed with the cover to prevent microbial contamination. A layer of parafilm is often additionally wrapped around the base of the cover to ensure that the entry point for any microbes is blocked. The parafilm, however, is relatively expensive and can be easily substituted by equally effective food wrap (cling films), which thus allows a 90% savings over parafilm.

#### 1.1.3 Light and Energy

The cultures are conventionally placed on shelves illuminated with cool light fluorescent tubes (conventionally at 1200–2000 lux) (Ahloowalia and Prakash 2004) under a 16 h:8 h light/dark cycle at 25 <sup>2</sup> C. Artificial lighting of cultures accounts to nearly 60% production cost (Ahloowalia and Savangikar 2004). However, many in vitro growing plants can tolerate wide fluctuations with temperatures (Ahloowalia and Savangikar 2004) as high as 30 C and as low as 10 C with improved plant growth (Kodym et al. 2001). Research works have also been reported on the performance of in vitro cultures under natural light using solatube (Kodym et al. 2001), nethouse, light-emitting diodes (LEDs) (Datta et al. 2017), domelight or diffused light (Ahloowalia and Savangikar 2004) and sidewise lighting system rather than downward illumination of culture racks (Datta et al. 2017).

In this project, all cultures were grown under natural light from either a solatube (purchased at around 1200 €) (see Fig. 10.6) or from a 1 m<sup>2</sup> domelight made of superior quality acrylic (purchased at around 120 €), that were fixed on the roof of a 9 m<sup>2</sup> room. In a separate study, cultures were grown in a netcloth shed that was lined with plastic running from the roof to the sides (see Fig. 10.6), and also in a room, receiving diffused light through the windows.

Alternative culture growing conditions are summarized in Table 10.2. The solatube redirects the daylight through reflective tubing without heating the room and can illuminate an area of 3–5 m2 (Kodym et al. 2001) whereas the domelight can heat up the room, requiring the window to be opened for air flow.

Depending on the season, the number of buds produced per explant in diffused light was reduced or comparable to conventional controlled conditions. The growth

Fig. 10.6 Alternative sources of natural light that can be potentially used for micropropagation. Left image: Shed, lined up with plastic and netcloth, which can be rolled up depending on heat accumulation. Right image: Solatube directs diffused light into the room, without heating the room


Table 10.2 Comparison of radiation and temperature of the different environment in Mauritus

a Measured using LIG1050 lux meter

rate was also slower under diffused light. Conditions with the solatube were more stable, while the domelight's conditions were directly dependent on the weather. Proliferation rates were better in rooms lit by the solatube than by the domelight. While no electricity is involved in use of natural light, each system has its own advantages and constraints and need to be optimized prior to application (see Fig. 10.7).

In the shed, the rate of development was not uniform. Mortality was relatively high (50–80%) depending on the outside conditions. The afternoon sun, especially in summer, led to intense scorching due to hot microclimate within the jars. This can be reduced by covering the west side of shed with shade netting. The other problem

Fig. 10.7 Response of banana cultures under different growing conditions, from left to the right: control, diffused light (2 plants), shed. Those grown under diffused light were comparable to control, but with slower rate. In shed, mortality and scorching were high. Remaining explants developed but with rate of development depending on the season. Development of rooted plantlets was also possible under diffused light

associated with growing cultures in a shed was the accumulation of dusts which intensified risks of contamination when brought back in the transfer room. However, cultures at rooting stage can be grown in such conditions as the plants can be sent to nursery for hardening.

In a commercial micropropagation laboratory, where the volume and quality of planting materials to be produced are crucial, there is often little concern about using cheaper options. The above low-cost substitutes have been proposed for countries with restricted or no TC facilities so that they can proceed with in vitro mutagenesis to improve any local banana cultivar for which conventional breeding can be too lengthy or complex. The low-cost alternatives can as well be included in a routine TC where up to 80% cost reduction can be achieved (see Table 10.3).

#### 1.2 In Vitro Mutagenesis

In vitro methods as described by Vuylsteke (1998) and Lopez et al. (2017), can be applied to mass multiply the desired local accessions to generate enough explants that can then be sent to another laboratory for irradiation. This section describes only the physical mutagen (gamma rays) which was applied on excised banana shoot tips.


Table 10.3 Cost savings through adoption of low-cost substitutes of the TC components

In order to reduce risks of chimerism, the explants are trimmed to produce shoot-tips of about 2–3 mm length.

After irradiation, the mutation is fixed in the genome by carrying out three subcultures (the whole process is referred to as generation advancement). In order to dissolve potential chimeric sectors during each subculture, longitudinal sections are made through the explant to select buds or propagules (see Fig. 10.8). The mother explant can be further subcultured to recover the main meristem mutation after homohiston formation.

In practice, however, it is often difficult to dissociate the buds from the mother explant. In such cases, routine subculturing/decapitating the main explant is practiced to promote shoot proliferation. After irradiation, the cultures are referred to as the M1V0 generation and the buds thus produced (after 4–6 weeks of incubation) are subcultured and make the population of M1V1 individuals and this process is repeated after each incubation period until the population of M1V3 is reached. The resultant M1V3 population is placed in regeneration medium (usually of half strength macronutrients and without any PGR) for the buds/shoot to regenerate into plantlets (or referred to as putative mutant line). These individual mutant lines might be clonal propagated for multiple traits screening, particularly abiotic or biotic stress which usually uses destructive methods. Moreover, the development of multiple clones per individual is also important as avoid loss of the mutant lines during the weaning of ex vitro plantlets.

#### 1.3 Weaning of Mutant Lines

Prior to screening the mutant lines for any traits of interest, such as to Fusarium wilt using the double-tray system or field tests, the rooted plantlets are hardened so that they adapt to the outside environment where the light intensity (4000–12,000 lux) is higher compared to growth rooms that are artificially illuminated (1200–2000 lux) and the relative humidity lower (40–80% v/s 98–100%) (Ahloowalia and Savangikar 2004). Weaning is preferably done gradually, starting with high humidity and partial shade which is then gradually decreased. Commercial laboratories rely on greenhouses with sections of (i) partial shading (70–80%) and intermittent misting, (ii) 40–50% shade with regular irrigation/overhead fine irrigation and (iii) 20% shade to full sunlight with irrigation. In order to support developing countries, an easy method of weaning is proposed to handle the mutant lines in batches.

The high humidity in the weaning stage is created by placing the plantlets on a table and covering them with a frame, lined with clear polyethylene sheeting, and regularly watering the plants (or mist using a spray bottle) (see Fig. 10.9). The partial shade is provided by covering the structure with shadecloth (70–80% shade). The plastic sheet is removed after about 2–3 weeks to allow the plants to adjust to ambient relative humidity. The hardening media should be freely draining and relatively rich. Jhurree-Dussoruth and Kallydin (2011) successfully hardened banana plantlets in media consisting of sterile soil and manure (see Fig. 10.9). The addition of perlite improves the porosity of the medium and is important mostly in the first stage hardening.

In this chapter, a low-budget TC and weaning procedure are presented to allow laboratories with limited resources to proceed with banana mutation breeding after an outsourced irradiation. Banana cultures in this project were irradiated using local facilities, but an additional section has been included to guide those who need to seek an international support for irradiation.

Fig. 10.9 Weaning and hardening stages. (a)&(b) Simple structures constructed out of metal framework, lined with polyethylene sheeting. During the hardening, the sheet covers the whole structure and is gradually lifted after 2–3 weeks, to allow plantlets to adapt to ambient humidity. (c) Testing of most appropriate potting media from locally available materials. Sterile mix of manure and soil (1:1) allowed rapid development of plants 4 weeks after weaning. (d) Fully acclimatized plants ready for transfer to the experimental plot

#### 2 Materials

#### 2.1 Preparation of Media


#### 2.2 Shoot-Tip Establishment


#### 2.3 Shoot Tip Multiplication/Generation Advancement


#### 2.4 Growing of Cultures


#### 2.5 Mutation Breeding/Generation Advancement


#### 2.6 Rooting


#### 2.7 Weaning


#### 3 Methods

#### 3.1 Preparation of Stock Solutions


#### 3.2 Preparation of Multiplication Medium

	- (a) Stericlave set at 121 C at a pressure of 15 psi for 20–25 min
	- (b) Pressure cooker as follows:
		- Place a perforated plate (on a stand, with gap of about 4–6 cm) inside cooker, add tap water just below the rim
		- Place jars on plate, cover and heat cooker on medium flame until first whistle
		- Leave on medium flame for 25–30 min after first whistle

#### 3.3 Preparation of Rooting Medium

1. Proceed as above (Sect. 3.2), except in step 1 use only 50 ml aliquot of Stock I, and in step 2 no plant growth regulator is added. The media are labelled as AC0 or AA0, respectively for the agar/cornflour and agar/arrow root gelling agents (see Note 2).

#### 3.4 Sterilization of Tools


#### 3.5 Sterilization of Working Surface

Prior to all works under laminar or hood, spray household alcohol to whole working table and side of hood with cotton.

#### 3.6 Establishing Banana Cultures

This section highlights steps from culture initiation until cultures reach stage for mutagenesis. Details can be obtained in Vuylsteke (1998).

#### 3.6.1 Culture Initiation


#### 3.6.2 Multiplication of Shoot-Tip Cultures


Fig. 10.10 Number of buds produced from explant after each subculture (4–6 weeks) in banana (var. Dwarf Cavendish, AAA) at different BAP levels. The explants entered the rapid phase of multiplication after the 4th subculture. Explants for irradiation were selected among the population of explants from the 4th or 5th subculture onwards

4. After repeated subculture of explants (often by 4th to 5th, depending on variety), the multiplication rate will increase rapidly, and explants will enter the log phase of multiplication (see Fig. 10.10). Select explants from these stages for irradiation.

#### 3.6.3 Shoot-Tip for Mutagenesis – Dispatch and Reception


Fig. 10.11 Sending banana cultures for irradiation to an international or regional facility. (a) Sealed culture box with banana shoot tips. (b) The seal should remain intact if arrangements have been made for irradiation without opening the culture vessel. (c) Culture vessels placed tightly in vertical position. (d) Box ready for dispatch

#### 3.7 Generation Advancement Under Low-Cost Conditions


#### 3.8 Regeneration of Mutant Lines


#### 3.9 Low-Cost Weaning

This stage concerns post-flask low-cost hardening of the in vitro derived plantlets.

	- (a) Hardening for field planting: Individually transfer plugged plants into planting pots (1.5–2 l) containing autoclaved potting mix (soil:manure:perlite: peat at 1:1:1:1 or soil:manure:perlite at 1:1:1). Keep plants under 50–40% shade for about 1 month and later at full sunlight or 30% shade until they reach a height of about 30 cm for field planting. Use a slow release to fertilise the plants, otherwise use a locally available complex fertiliser high in nitrogen at the rate of ½ teaspoon per pot to avoid phytotoxicity (at 5 weeks interval).
	- (b) For nursery level screening of potted plants: Transfer plugged plants in large potting boats with bottom tray to collect exudates. Use same potting media as used above. The plants can be used immediately for screening using an adapted low-cost modified double-tray system and applying same practices as highlighted in this book.

#### 4 Notes


If irradiation is done locally then the explants are gently placed horizontal on the media, so as maximise chance of irradiation. Sucrose can be excluded.



Table 10.4 Conditions associated with outsourcing for mutagenic treatments


Acknowledgements The author would like to acknowledge the Food and Agricultural Research and Extension Institute (FAREI) for technical support provided. Financial assistance was provided by the Mauritius Research and Innovation Council, the International Atomic Energy Agency (Joint FAO/IAEA) and FAREI is acknowledged. Author is also grateful for the support of Mrs. Hemlata Kallydin-Gaur and Mrs. Dheema Burthia for conducting all the laboratory activities.

#### Annexure


Annex 10.1 Composition per litre of the modified Murashige and Skoog (1962) and preparation of stock solution

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.

## Chapter 11 Protocol for Mass Propagation of Plants Using a Low-Cost Bioreactor

Affrida Abu Hassan, Norazlina Noordin, Zaiton Ahmad, Mustapha Akil, Faiz Ahmad, and Rusli Ibrahim

Abstract Conventional in vitro mass propagation methods are labour-intensive, costly and have a low degree of automation. Bioreactor or automated growth vessel systems using liquid media were developed to overcome these problems. The use of liquid instead of solid culture medium for plant micropropagation offers better access to medium components and scalability through automation. However, the cost of setting up a bioreactor system is one of its disadvantages as such systems are expensive with limited number of manufacturers. A low-cost bioreactor system was set up using recycled, low biodegradable plastic bottles. This low-cost bioreactor, based on temporary immersion principle, has proven to be effective as a vessel for rapid plant propagation. It is designed to reduce the production cost of plant micropropagation. This chapter explains the step-by-step methods for setting up a low-cost bioreactor for banana seedling production. This low-cost bioreactor system has the potential to be adapted for large scale in vitro cultivation of the plant seedlings.

Keywords Low-cost bioreactor · Temporary immersion · Plant micropropagation · Seedling production

#### 1 Introduction

The conventional micropropagation technique requires regular sub-culturing, manual handling at various stages of the process (labour-intensive) and more shelf space that contributes to high running and labour cost. Scaled-up and automated systems are therefore desirable to reduce the amount of handling, increase multiplication rates, hence overcome and/or minimize production costs of the conventional

A. Abu Hassan (\*) · N. Noordin · Z. Ahmad · M. Akil · F. Ahmad · R. Ibrahim Agrotechnology and Biosciences Division, Malaysian Nuclear Agency, Ministry of Science, Technology and Innovation (MOSTI), Kajang, Selangor, Malaysia

e-mail: affrida@nuclearmalaysia.gov.my

J. Jankowicz-Cieslak, I. L. Ingelbrecht (eds.), Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, https://doi.org/10.1007/978-3-662-64915-2\_11

micropropagation technique as initially reported by Aitken-Christie et al. (1995). This can be achieved by using a bioreactor to scale up propagation. Bioreactors are usually described in a biochemical context as a self-contained, sterile environment which incorporates liquid nutrient or liquid/air inflow and outflow systems, designed for intensive culture and affording maximal opportunity for monitoring and control over micro environmental conditions (agitation, aeration, temperature, dissolved oxygen, pH etc). The use of bioreactors in controlled condition increases the multiplication rate and plant quality and has been proven as an efficient tool for rapid production of plant cells, tissue or organ culture and metabolites. The first reported use of bioreactor for micropropagation was in 1981 for Begonia propagation (Takayama and Misawa 1981). Since then, it has been widely used and proved applicable to many plant species including cassava (Golle et al. 2019), carnation (Marzieh et al. 2017), gerbera (Frómeta et al. 2017).

Various types of bioreactor systems, with different types and different sizes of vessels and agitation mechanisms (non-agitated, mechanical or pneumatically agitated) have been developed and used as described by Paek et al. (2005), Eibl et al. (2018) and Alireza et al. (2019). Among them, temporary immersion system (TIS) bioreactor is highly suitable for use in semi-automated micropropagation. This principle of temporary immersion was first tested by Harris et al. (1983) through alternate exposure and submergence of explants by tilting a flat-bottomed vessel to opposite direction using semi-automatic system. TIS bioreactor allows immersion of explants in medium for a specific duration at specified intervals, control of contamination, adequate nutrient and oxygen supply and mixing, relatively infrequent subculturing, ease of medium changes and limited shear damage. The temporary immersion of the plant with the media is a good technique to avoid damage, since long exposure can lead to physiological malformation which causes poor regeneration. In comparison with both, solid and liquid culture systems, TIS has technological and quantitative advantages such as higher multiplication rate and reduction of production cost (Etienne and Berthouly 2002). The use of TIS for large scale micropropagation produces better plant quality and higher multiplication rate (Ziv 2005). Examples of TIS bioreactor available today include BIT® twin-flasks system (Escolana et al. 1999), Reactor with Automatized Temporary Immersion (RITA®) (Alvard et al. 1993) and Bioreactor of Immersion by Bubbles (BIB®) (Soccol et al. 2008).

#### 1.1 Low-Cost Bioreactor System

Many established TIS bioreactor systems were patented and are quite costly, hence less preferable for large scale mass propagation. Option for a simpler and cheaper TIS bioreactor system was explored through the development of a TIS bioreactor prototype called BIO-TIS (Ibrahim 2017). BIO-TIS consists of two glass vessels, one for the in vitro shoots and the other for liquid culture media which is connected by silicone tubing that permits the flow of the liquid medium from one vessel to the other. It has been tested for mass propagation of horticultural crops such as: fruit trees (pineapple, banana), ornamental plants (orchids, chrysanthemums) and herbal plants (Eurycoma longifolia Jack, Labisia pumila and Stevia rebaudiana). In a study on pineapple propagation, the multiplication rate with BIO-TIS was found to be much higher in comparison to the established RITA® bioreactor (Ibrahim 2017).

Modification was done by replacing the glass bottles in BIO-TIS with recycled plastic bottles as an alternative for a cheaper setting up cost. A silicone cap with stainless steel tubing is fabricated for liquid nutrient or liquid/air inflow and outflow. This low-cost bioreactor is capable of supplying planting materials in large quantities for various plants, able to increase the multiplication rate of in vitro plantlets up to ten-fold (Ibrahim 2017; Mustapha et al. 2017), improve the quality of tissue culture plantlets by reducing vitrification and is environmentally friendly. This system can be used by the plant biotechnology industry and agro-industry to save on the production cost. Furthermore, recycling of plastic bottles helps to reduce issue of the disposal of unused material in landfills thus reduce environmental pollution from disposal of used plastic bottles.

#### 2 Materials

#### 2.1 Liquid Media


#### 2.2 Bioreactor System (Fig. 11.1)


#### 2.3 Culture Initiation

1. 30–40 day old in vitro shoots of banana measuring 1.5 cm with 2–4 leaves.

2. Forceps.

Fig. 11.1 (a) Recycled plastic bottles. (b) Modified silicone cap. (c) Silicone tube. (d) Air filter. (e) Air compressor pump. (f) Timer. (g) PVC pipe


#### 3 Methods

#### 3.1 Preparation of MS Medium


#### 3.2 Preparation of Bioreactor Set and Culture Initiation (See Note 3)


#### 3.3 Setting up Low-Cost Bioreactor

1. Connect the low-cost bioreactor set to the air compressor pump (Fig. 11.4). A number of low-cost bioreactors can be combined and run simultaneously as Fig. 11.2 A close view of the modified silicone cap that consists of a fabricated silicone cap, stainless steel tubes to allow media/air outlet and inlet, and silicone tube

Fig. 11.3 Explants are transferred into sterile plastic bottle under aseptic condition in the laminar air flow cabinet (left). A complete low-cost bioreactor ready for incubation (right)

shown in Fig. 11.5 and Fig. 11.6. For this protocol, the system was tested for a max of 4 sets merged together. However, the efficiency of the system must be revaluated if there is additional set added.


Fig. 11.4 Two bottles of the same size are used. Bottle 1 is filled with media and Bottle 2 is for explants

Fig. 11.5 Sets of low-cost bioreactor can be combined and run simultaneously

Fig. 11.6 Large-scale production of banana plantlets through low-cost bioreactor

#### 3.4 Harvesting


#### 4 Notes


Fig. 11.7 Operational principle of low-cost bioreactor system: (A) The liquid medium is located in Bottle 1 and explants in Bottle 2 (B) The air compressor pump 1 is run for 15 min and all medium flow from Bottle 1 to Bottle 2 (C) The explants are immersed into the liquid medium for 30 minutes (D) After immersion is complete, the air compressor pump 2 is run for 15 min to allow all medium flow back into Bottle 1

6. Due to the efficient gaseous exchange between plant tissue and gas phase inside the vessel, the yield obtained from this procedure can increase up to 10–15 old compared to solid media.

Acknowledgments Authors wish to thank Ms. Norhayati Irwan, Ms. Nashimatul Adadiah Yahya and Mr. Nor Hafiz Talib for their dedication and assistance. We would also like to thank Malaysian Nuclear Agency for their continuous support. This work was funded by the Ministry of Science, Technology and Innovation (MOSTI) of Malaysia under MOSTI Social Innovation Funding.

#### References


The opinions expressed in this chapter are those of the author(s) and do not necessarily reflect the views of the IAEA: International Atomic Energy Agency, its Board of Directors, or the countries they represent

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 3.0 IGO license (http://creativecommons.org/licenses/by/3.0/igo/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the IAEA: International Atomic Energy Agency, provide a link to the Creative Commons license and indicate if changes were made.

Any dispute related to the use of the works of the IAEA: International Atomic Energy Agency that cannot be settled amicably shall be submitted to arbitration pursuant to the UNCITRAL rules. The use of the IAEA: International Atomic Energy Agency's name for any purpose other than for attribution, and the use of the IAEA: International Atomic Energy Agency's logo, shall be subject to a separate written license agreement between the IAEA: International Atomic Energy Agency and the user and is not authorized as part of this CC-IGO license. Note that the link provided above includes additional terms and conditions of the license.

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.