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ENVIRONMENTALLY SUSTAINABLE DEVELOPMENT AGRICULTURAL RESEARCH AND EXTENSION GROUP SERIES Banana Improvement ProjectReport No. 1

BananaImprovement ResearchChallenges and Opportunities

Edited by GabrielleJ Persley Pamela George

The World Bank Washington, D. C. Copyright © 1996 The International Bank for Reconstruction and Development/THE WORLD BANK 1818 H Street, N.W. Washington, D.C. 20433, U.S.A.

All rights reserved Manufactured in the United States of America

First printing October 1996

This report has been prepared by the staff of the World Bank. The judgments expressed do not necessarily reflect the views of the Board of Executive Directors or of the governments they represent.

Cover art by Tomoko Hirata.

When the text was written, Gabrielle J. Persley was manager of the Banana Improvement Project (BIP) at the World Bank; she is now a member of the BIP ScientificAdvisory Panel. Pamela George is the coordi- nator of the project at the Bank.

Libraryof Congress Cataloging-in-PublicationData

Banana improvement: research challenges and opportunities / Gabrielle J. Persley, Pamela George, editors. p. cm. - (Environmentally sustainable development agricultural research and extension group series. Banana Improvement Project report ; no. 1) Papers from a meeting held at Katholieke Universiteit Leuven, Feb. 1996. Includes bibliographical references (p. ). ISBN0-8213-3740-8 1. Bananas-Breeding-Congresses. 2. Bananas-Biotechnology- Congresses. 3. Bananas-Disease and pest resistance-Genetic aspects-Congresses. 4. Bananas-Research-Congresses. 5. Crop improvement-Research-Congresses. I. Persley, Gabrielle J. II. George, Pamela, 1948- . III. Series. SB379.B2B345 1996 634'.77223-dc2O 96-31057 CIP Contents

Foreword v

Contributors vii

Acronyms and Abbreviations ix

Chapter 1. Banana Research Needs and Opportunities Ivan W. Buddenhagen 1 Key Findings I Current Status of Banana Research 2 Perspectives on the Commercial Industry and Synopsis of Breeding/Biotechnology Needs and Opportunities 14 Major Researchable Topics 16

Chapter 2. Overview of the Banana Improvement Project GabrielleJ. Persley 21 Objectives 21 Commissioned Research 22

Chapter 3. Portfolio of Projects 25 Field and Laboratory Evaluation of Diploid Bananas for Their Use in Breeding ChristopheJenny 27 Field Cross Experiments to Clarify the Mechanism Governing the Inheritance of Black-Leaf- Streak-Resistant Characters in Banana FredericBakry 27 Developing Improved Banana Varieties with Pest and Disease Resistance, Postharvest Superiority, and Maximum Export Potential N. KrishnanNair 28 Collection, Evaluation, and Characterization of Genetic Resources and Improved Banana Crop Plants in Viet Nam Do Nang Vinh 29 Banana Breeding in Brazil Sebastiaode Oliveirae Silva 30 Genetic Transformation and Non-Conventional Breeding Technology for Improvement of a Molecular Toolbox for Banana C. Arntzen and G. May 30

iii iv Contents

Novel Genes for Fungal Resistance and Postharvest Quality Charles Arntzen and Gregory May 31 The Development of Transgenic Bananas with Resistance to BBTV and BBMV James Dale 32 Genetic Transformation of Prototype Bananas for Black Sigatoka and Fusarium Resistance Rony Swennen and Laszlo Sagi 33 Genetic Engineering of Ethylene Biosynthesis in Bananas Shang-Fa Yang and Ning Li 33 Use of Biotechnology to Produce Transgenic Bananas That Are Resistant to BBTV Infection John S. Hu 34 Elimination of BSV from Improved Musa Germplasm and Related Studies on Transmission and Host Plant/Virus/Vector Interactions Jonathan H. Crouch 35 Variability and Relationships within Populations of Fusarium oxysporum f.sp. Cubense from Its Center of Origin Ken G. Pegg 36 Origin and Distribution of Fungicide-Resistant Strains of M. fijiensis in Banana Plantations in Costa Rica Ronald Romero-Calderon 37 Tolerance/Resistance of Bananas to Nematodes Roger Fogain 38 Identification of Nematode Resistance Sources in Banana and Plantain Dirk De Waele 38 Identification of Durable Pest and Disease Resistance Sources in Banana and Plantain Philip Rowe 39 Identification of Durable Nematode Resistance Sources in Banana and Plantain: Screening Methodologies Jorge Pinochet 39

Chapter 4. Summary and Recommendations GabrielleJ. Persley 41 Problem Identification: Where Are We Going? 41 Strategy: How Do We Get There? 42 Achievements: How Far Have We Got? 42 Future Directions: What Needs to Be Done? 43 Project Management: How Do We Do It? 44 Conclusion 45

References 47 Foreword

The Banana Improvement Project (BIP) is co-spon- general lack of connection between banana produc- sored by the Common Fund for Commodities (CFC), tion and handling at the commercial level and any the FAO Inter-governmental Group on Bananas public sector research programs. (FAO/IGB), and the World Bank. The World Bank The volume also presents the proceedings of the acts as Project Executing Agency on behalf of the three First Annual Scientific Meeting. The portfolio of re- co-sponsors and is responsible for the management search projects commissioned by the BIP is described of the BIP, including contractual arrangements and in Chapter 3, with a brief summary of research pro- financial activities. This volume is the first of a series gress as reported at the first Scientific Meeting of the reporting on the BIP. BIP, held at the Katholeike Universiteit Leuven The Common Fund for Commodities has provided (KUL), Belgium, in February 1996. Summary of dis- approximately US$3.5 million over five years to spon- cussions and recommendations from the KUL meet- sor specific research activities through BIP. The pro- ing is included as Chapter 4. ject has two important objectives: first, to develop, The research reports are most encouraging, and through research, improved banana varieties suitable over the five-year life of the project, we look forward for the international market; and, second, to improve to new and innovative results from the seventeen pest and disease control practices to reduce pesticide commissioned projects being undertaken worldwide. use and its harmful environmental impact. The This report is the result of inputs by many people. main research themes are: breeding and genetic The scientists who attended the 1996 Leuven meeting resources, biotechnology, disease control, and nema- are thanked for their reports on individual projects, tode control. which appear in abbreviated form here. One of the first initiatives sponsored by the project Reginald MacIntyre assisted with the editing, and was a review of banana and plantain improvement his valuable assistance is acknowledged. undertaken by Professor Ivan W. Buddenhagen in We hope this publication will prove useful to ba- 1994/95. This edited report is included as Chapter 1. nana researchers and to the many commercial pro- The review was based on a series of visits to banana ducers worldwide. research programs worldwide. It focused not only on research activities under way and planned, but also on innovation that might be fostered. The relevance of the programs to real world problems and opportu- nities was also assessed. These latter two points were Michel Petit considered important because of the 70-year record of Director lack of success in breeding a disease-resistant dessert Agricultural Research & Extension Group banana for the commercial trades and because of the (ESDAR)

v

Contributors

Arntzen, Charles. President, Boyce Thompson Insti- May, Gregory. Boyce Thompson Institute for Plant tute for Plant Research Inc., Tower Road, Ithaca, Research Inc., Tower Road, Ithaca, New York, NY New York, NY 14853-1801, USA 14853-1801, USA Bakry, Frederic. CIRAD/FLHOR, Station de Neuf- Nair, N. Krishnan. Associate Director of Research, chateau, Saint Marie, 97130 Capesterre Belle Eau, Regional Agricultural Research Station, Pattambi, Guadeloupe Kerala, India 679306 Buddenhagen, Ivan W. Professor Emeritus, Univer- de Oliveira e Silva, Sebastiao. EMBRAPA/CNPMF, sity of California, Davis, California, USA Rua Embrapa Cx Postal, 44.380-000 Cruz das Almas, Crouch, Jonathan H. Plantain and Banana Improve- Bahia, Brazil ment Program, International Institute of Tropical Pegg, Ken G. Plant Protection Unit, Department of Agriculture, High Rainfall Station, PMB 008, Nchia- Primary Industries, Meiers Road, Indooroopilly Eleme, Telga, Onne, Rivers State, Nigeria 4068 Qld., Australia Dale, James. Director, Centre for Molecular Biotech- Persley, Gabrielle J. AUSTRADE, Level 13,145 Eagle nology, School of Life Sciences, Queensland Univer- Street, Brisbane 4000 Qld, Australia sity of Technology, 2 George Street, Brisbane, Qld. Pinochet, Jorge. Deparamento de Pataologia Vegetal 4000, Australa (IRTA), 08348 Cabrils, Barcelona, Spain Do Nang, Vinh. Vice-Director, Institute of Agricul- Romero-Calderon, Ronald. Research Director, tural Genetics, Tu Liem, Hanoi, Viet Nam CORBANA, P.O. Box 6504-1000, San Jose, Costa Fogain, Roger. Head of Nematology Laboratory, Cen- Rica tre Regional Bananiers et Plantains (CRBP), Douala, Rowe, Philip. Fundacion Hondurena de Investigacion Cameroon Agricola (FHIA), Apartado Postal 2067, San Pedro Hu, John. Department of Plant Pathology, University of Sula, Honduras Hawaii, 3190 Maile Way, Honolulu, H 96822, USA Sagi, Laszlo. Laboratory of Tropical Crop Husbandry, Jenny, Christophe. CIRAD/FLHOR, Station de Neuf- Katholeike Universiteit Leuven, Kardinaal Mer- chateau, Saint Marie, 97130 Capesterre Belle Eau, cielaan 92, B-3001Heverlee, Belgium Guadeloupe Swennen, Rony. Laboratory of Tropical Crop Hus- Li,Ning. Department of Biology, Hong Kong Univer- bandry, Katholeike Universiteit Leuven, Kardinaal sity of Science and Technology, Clear Water Bay, Mercielaan 92, B-3001Heverlee, Belgium Kowloon, Hong Kong

vii viii Contributors

De Waele, Dirk. Laboratorium Tropische Planten- Yan, Shang-Fa. Department of Biology, Hong Kong teelt, Katholeike Universiteit Leuven, Kardinaal University of Science and Technology, Clear Water Mercielaan 92, B-3001 Heverlee, Belgium Bay, Kowloon, Hong Kong Acronyms and Abbreviations

ACIAR Australian Centre for International FOC Fusarium oxysporum f.sp. cubense Agricultural Research FRAC Fungicide Resistance Action Committee ACORBAT Asociation para la Cooperacion en Inves- IAEA Intemational Atomic Energy Agency tigaciones Bananeras en el Caribe (Austria) y en America Tropical (Costa Rica) IDRC International Development Research AUSTRADE Australian Trade Commission Centre (Canada) BADC Belgian Administration for Development HTA International Institute of Tropical Cooperation Agriculture (Nigeria) BBMV Banana bract mosaic virus INIBAP International Network for the BBTV Banana bunchy top virus Improvement of Banana and BIOTROP Biotechnology for Tropical Plant Plantain Improvement (France) IRTA Institut de Recerca i Technologia BPI Bureau of Plant Industry (Philippines) Agroelimenteries BSV Banana streak virus KUL Katholeike Universiteit, Leuven BTIBoyce Thompson Institute (USA) MARDI Malaysian Agricultural Research and CABIIntemational CAB Development Institute CABI CAB International NRI Natural Resources Institute (U.K.) CFC/FAO/WB Common Fund for Conmmodities/ PCR Polymerase chain reachon Food and Agriculture Organization/ Pc The World Bank QDPI Queensland Department of Primary CIRAD Centre de cooperation international Industries (Australia) en recheche agronomique pour QUT Queensland University of Technology le developpement (France) (Australia) CMV Cucumber mosaic virus TARS Tropical Agriculture Research Service CNPMF Centro Nacional de Pesquisa de (Puerto Rico) Mandioca e Fruiticultura TBRI Taiwan Banana Research Institute (EMBRAPA) UH University of Hawaii CORBANA Corporacion Bananera Nacional UPLB University of the Philippines, de Costa Rica Los Bafios CRBP Centre regional bananiers et plantains USAID United States Agency for International (Cameroon) Development DSIR Department of Scientific and Industrial USDA United States Department of Research (N.Z.) Agriculture EMBRAPA Empresa Brasileira de Pesquisa VCG Vegetative compatibility groups Agropecuaria VVOB Flemish Association for Development, FHIA Fundacion Hondurena de Investigacion Cooperation, and Technical Agricola (Honduras) Assistance FLHOR Departement des productions fruitieres WINBAN Windward Islands Banana Growers et horticoles (CIRAD) Association

ix

1 Banana ResearchNeeds and Opportunities

Ivan W. Buddenhagen

A review of banana research needs, banana improve- sistance to black Sigatoka, and they are moder- ment programs, and banana research laboratories ately more productive than their triploid female was conducted during 1994/95. The review was un- parent. Their relative fruit quality is still under dertaken on the initiative of the World Bank in its test. The major reason these targets have been capacity as executing agency for a Banana Improve- reached is that the best triploid clones of these ment Project (BIP) being co-sponsored by the Com- banana types are only mediocre in their agro- mon Fund for Commodities, the FAO Inter-Govern- nomic characters and in productivity. Thus, cross- mental Group on Bananas, and the World Bank ing with a vigorous resistant diploid parent (CFC/FAO/WB). This chapter contains highlights of improves both resistance and productivity. the review and information on the ongoing breeding 3. A really good substitute for the best sweet/acid and research programs at various locations visited. It banana, silk (Brazilian Mica), that also corrects its also describes research needs and opportunities. great weakness of Fusariumwilt susceptibility has New, innovative breeding approaches combined not yet been obtained. with advances in plant biotechnology offer really new 4. In general, the traditional approach for the above dimensions to banana improvement. Careful chan- breeding targets (3Xby 2Xto obtain a 4X)has gone neling of funds to precisely targeted research objec- about as far as it will go, and although useful tives with the right programs will lead to new, useful clones can be expected of acid dessert and various knowledge and to new profitable products. cooking types, major improvements above what has already been obtained should not be expected. Key Findings 5. No improvement on commercial Cavendish has been obtained by the traditional breeding ap- The key findings of the review are: proach, nor should it be expected. Seventy years of crossing a short Gros Michel commercial ba- 1. There are four active banana breeding programs nana with a resistant diploid to obtain a resistant where actual crossing (pollinating) is carried out tetraploid competitive with the more productive (in Brazil, Guadeloupe, Honduras, and Nigeria). In best Cavendish clones have been unsuccessful. most cases the targets are better plantains, cooking For decades, little interest, support, or effort has bananas, or sweet/acid dessert bananas. For these been devoted in this direction. objectives, the traditional approach of pollinating 6. Somaclonal and mutation breeding approaches to the major better triploid clones with a resistant improving Cavendish have been taken by the diploid parent to obtain a tetraploid is still used. International Atomic Energy Agency (IAEA) in 2. Better plantains, better cooking bananas, and bet- Vienna, with collaborators in developing coun- ter sweet/ acid dessert bananas (like "Prata") have tries; and by the Taiwan Banana Research Institute been obtained. These have good or moderate re- (TBRI).The IAEA program was targeted mainly

1 2 BananaImprovement: Research Challenges and Opportunities

at producing a black Sigatoka-resistant Caven- development that does not fail due to potential dish, but appropriate mass screening was not unexpected susceptibilities will require a better developed. The second program (involving so- multidisciplinary effort. This is also true for the maclonal variation only) was for obtaining a somaclonal/mutation programs. Cavendish variant resistant to the type of Fusarium 11. Better linkage of current efforts on banana re- race 4 attacking Cavendish in Taiwan. Moderate search and banana improvement with industry success (i.e., moderate resistance) has been ob- and with the extensive Musa research in laborato- tained for the Fusarium problem in Taiwan. ries in industrial countries is needed for further 7. Innovative approaches to breeding in the tradi- rapid progress and for efficient investment of re- tional mode (crossing/pollinating) have been at- search funds. tempted only by the CIRAD group in Guadeloupe during the last eight years. By using an approach Current Status of Banana Research to resynthesize new triploids from the best parthe- nocarpic diploids, considerable progress has been Latin America and the Caribbean made and new, interesting triploids should soon be forthcoming. The easier target is sweet/acid Honduras-FHIA desserts (AAB), but with more research and effort, a better Cavendish type is a definite possibility. The oldest active banana breeding program exists at This approach could be followed by any group if La Lima, Honduras, under a Honduran foundation, it so desired. FHIA. This organization was created in 1984 to work 8. Modern biotechnology has progressed suffi- on traditional and nontraditional export crops under ciently to impact on banana improvement if direc- a cooperative agreement between the Government of tion and support are applied; and if proper linkage Honduras, United Brands, and the U.S. Agency for to breeding programs, to industry's needs, and to International Development (USAID). In effect, the basic research were established. Transformation formerly extensive banana research program and re- methods are being developed at the Boyce search facilities of the United Fruit Company were Thompson Institute (USA) and the Katholeike given to this new organization. By 1984, the com- Universiteit Leuven (KUL), Belgium, for use in pany's banana research program had already dwin- obtaining resistance, initially to banana bunchy dled greatly but still retained one banana breeder and top virus (BBTV). These methods can be extended the extensive Musa genetic resources from a banana to introducing genes that will regulate ripening breeding program begun by the company in 1959. It characteristics. Solving the major needs of resis- is important to recognize, however, that the major tance to black Sigatoka, to Fusarium, and to nema- funds and staffing at FHIA are on crops other than todes through biotechnology appears more bananas, and that USAID did not support research on remote. Progress will require more basic research. commercial bananas. Additionally, the national na- 9. Recent attempts to commercialize Cavendish in ture of the organization tended to cut off banana Malaysia and Indonesia (Sumatra and Java) have activities and connections with the rest of Latin Amer- revealed how susceptible Cavendish clones are to ica. Thus, the program has been severely constrained the aggressive Fusarium strains existing there, pre- in funds, staffing, and scope for many years. Efforts sumably near the center of origin of the Musa/ from both within FHIA and from without to remove Fusarium pathosystem. This new development re- these constraints have been only partially successful. veals that the Cavendish banana trades elsewhere In a review of FHIA prepared for USAID in April are able to exist only because, by luck, these aggres- 1994, the problems and potentials of the banana re- sive strains were not moved to those regions with search program and how they fit within FHIA are the original planting stock. More research on the barely touched. banana pathosystems in the Southeast Asian area The shift from a USAID-supported organization to is a must, to avert further disasters similar to the a fee-based Honduran research service organization movement of black Sigatoka from the Asia/Pacific opens new problems for the banana program of region to America and Africa, and to enable ba- FHIA. The banana businesses in Honduras and Cen- nanas to be commercialized more widely in Asia. tral and South America do not look to FHIA as a 10. Both the traditional breeding programs and the source of banana research service. Thus, appreciating innovative breeding program in Guadeloupe suf- these structural problems and the historical back- fer from insufficient plant pathology and nematol- ground is important to any assessment of progress ogy research input. Real progress in varietal and future possibilities. The final key point in this BananaResearch Needs and Opportunities 3 regard is that the original purpose of the company hybrids are also grown. The disease develops better investment in banana breeding-to produce a resis- at this location. The biotechnologist planned to track tant commercial banana-was essentially abandoned resistant/susceptibility genes to black Sigatoka and by the time of the creation of FHIA. The concern Fusarium by analyzing F2 families from a cross of then became one of obtaining types of resistant advanced diploids available at FHIA, in collaboration bananas/plantains useful to smallholders in various with others. parts of the world who would not use pesticides. Thus, the targets were shifted to cooking bananas, Guadeloupe-CIRAD plantains, and to a replacement for the disease- susceptible acid-dessert bananas of Brazil. A banana breeding program is headquartered at the In the draft 5-year plan of FHIA 1994-98, the specific CIRAD-FLHOR Station de Neufchateau in Guade- breeding objectives listed are for: (1) plantains for loupe. The program is closely linked with other Latin America and West Africa; (2) a cooking banana French scientists at CIRAD, Montpellier, for more for Latin America, Asia/Pacific, and West Africa; basic research backup. This program is relatively new (3) a cooking banana for East Africa; (4) a sweet-acid but, by pursuing innovative breeding strategies, has silk-type dessert for Brazil and Australia; and (5) a made rapid progress. Earlier work included cytotax- black Sigatoka/nematode-resistant export banana onomic and evolutionary research. hybrid to be used in Latin America and the Asia/ The station is small, located at 250 m elevation, and Pacific region. This last objective, if attained, would receives a sea breeze so the location is not excessively have a major impact on the banana trade. hot. The existing laboratory is small yet it enables Attaining the first objective (resistant and higher- isozyme work and chromosome counting. A doctoral yielding plantains) could result in increased trade student is working on protoplast and anther culture. (over the small current amount) in plantains, both A new laboratory building is under construction. fresh and processed. FHIA has already produced a A recently received flow cytometer measures light candidate plantain for objective 1 (FHIA 21), a candi- absorption of very small particles (nuclei), reveal- date cooking banana for objectives 2 and 3 (FHIA 3), ing the ploidy of banana preparations (but not yet and a candidate acid-sweet banana for objective 4 aneuploids). (FHIA 1). These are vigorous, disease-resistant In additon to two breeders, there are five other tetraploids with considerable potential to replace scientists at the station, responsible for island agri- their parents. FHIA 1 has been released commercially cultural research and who may work part-time on in Australia as "Goldfinger," and is considered a bananas as needed (pathologist, pedologist, physiolo- potentially commercial banana to replace Fusarium- gist, entomologist, and agronomist). They also have a susceptible "ladyfinger" (an acid-dessert clone). The project with an INRA-station entomologist, in col- cold resistance of Goldfinger enhances its potential in laboration with the breeders, working on screening cool areas of Southern Queensland and Northern for weevil resistance. New South Wales. Present is a well-characterized, modest-sized Musa FHIA's approach to obtain a resistant banana to collection, much from the old CIRAD collection in competitively replace Cavendish, through conven- Njombe, Cameroon, plus material from recent collect- tional breeding based on pollinating a short Gros ing expeditions. By having close connection with Michel to obtain a tetraploid, seems as remote today French scientists in Cameroon, accurate and rapid as it did in 1960 when the company program started. assessment of parents and products for resistance to In 1994 FHIA has increased its technical staff by black Sigatoka is carried out. Only yellow Sigatoka is adding a pathologist and a biotechnologist (the latter present now in Guadeloupe. Since Moko disease is of whom has since left). In addition, the Natural Re- not present, they are able to leave the male buds, a sources Institute in the UK placed a postharvest tech- help in banana taxonomy. nologist at FHIA (funded through April 1995 by Although the program is breeding for all the fruit ODA) to assess postharvest performance of FHIA types (dessert AAA, AAB; AAB plantains and ABB hybrids. This postharvest work is of high quality. The cooking bananas), the targets of dessert AAA and pathologist established field plots for screening AABs are currently receiving most emphasis. against Fusarium races 1 and 2, and only now is it The key innovation in strategy that makes the work realized that FHIA 2 is susceptible to Fusarium. Addi- so interesting and the probability of success high, is tionally, a field plot has been established about 30 that they are attempting to resynthesize triploids de miles away (towards Puerto Cortez) for screening novo, by crossing doubled-diploids with diploids. By diploids against black Sigatoka and where the new having evolutionary analysis, it is possible to do this 4 BananaImprovement: Research Challenges and Opportunities

in a logical fashion to obtain maximum heterozygos- ratooning, and resistant to yellow Sigatoka. It is ity. It is also possible, by this approach, to obtain named Pioneira and is not resistant to Fusarium. abundant seed expressing many new genotypes. Tetraploid "Silk" has finally been produced and is Heritability and parental values may then be ob- now being tested. The program lacks sufficient pa- tained to establish heterotic groupings and even cy- thology back-up for the breeding program. clical inbreeding and recombination. Thus, breeding of bananas can be like for any other crop, making it Jamaica much less a "long-shot gamble." There is no history of Gros Michel on the island (the The breeding program in Jamaica is the oldest in exist- cultivar grown is the Cavendish type called Poyo) and ence, dating back to 1924. Considerable fundamental thus there is no history of Panama disease (Fusarium banana cytogenetics was done in Trinidad and then wilt), although the pathologist had found Fusarium breeding was established in Jamaica. Many improved wilt on the mountain slope where it is cold, on Poyo. synthetic diploids were developed as parents, and Also, Mysore ( = P. ceylan) had no banana streak virus various tetraploids, based on Gros Michel types, were (BSV)symptoms, unlike everywhere else, and it had produced. A few of these tetraploids have been tested indexed negative for BSV. The plantains on the is- beyond Jamaica, but none have been commercialized. lands probably have BSV, just as those in Central The original breeding objective was for resistance America. to Fusarium wilt. Later, objectives of resistance to The strategy and approach underway at Guade- yellow Sigatoka and to the lesion nematode Rado- loupe should be expanded and started elsewhere in a pholus were added. Lack of funds severely curtails an collaborative fashion to: (a) produce more doubled active breeding program at present. Nevertheless, diploids and analyze them; (b) obtain more wild and many good synthetic diploids are still held in the parthenocarpic diploids through collection and collection and limited efforts are made to cross these prospection; (c) try harder to obtain 2n x ln protoplast with Gros Michel-derived tetraploids. or cellular fusion and regeneration; and (d) have a much greater pathology (and entomology) involve- Africa ment with breeding. This work is probably best fos- tered through collaboration with other organizations Nigeria and Uganda-IITA or individuals. Pathology is a weak link in this and other banana improvement programs. There is great The International Institute of Tropical Agriculture opportunity at this station for students and others to (IITA)conducts research on plantains at its substation visit, collaborate with, and contribute to this program. at Onne in southern Nigeria. An initial small program on plantain agronomy, collection, and tissue culture Brazil-EMBRAPA/CNPMF was expanded in 1987-88to include breeding. Within a span of about five years many new and improved Comments on the Brazilian breeding program are plantains were produced. These have moderate resis- included here, on the basis of Shepherd et al. (1994), tance to black Sigatoka, which is the major disease rather than a site visit, since it is the other major threat in the region, and some are better agronomi- existing banana breeding program. Approximately cally. With the recent hiring of a breeder/geneticist 12 years have been spent in Brazil breeding for a better experienced in polyploid breeding, considerable pro- AAB-type dessert banana ("Silk" and "Apple" types). gress has been made very recently in Musa genetics. Two key disease problems are Fusarium wilt, espe- In May 1994 the previous head of the program cially on "Silk," and yellow Sigatoka, with the threat moved to a new IITA station based in Uganda, to join of black Sigatoka. The agronomy of existing clones is IITA entomology and nematology scientists and to poor and thus even moderate improvement of resis- consolidate a major effort on cooking bananas for East tance, if it went with a more productive plant, would Africa. Thus IITA will have two different Musa pro- be useful. This approach has been followed with the grams, one targeted at plantains for West and Central strategy of pollinating the better AAB clones with a Africa and one on cooking bananas in the East African diploid to obtain an AAAB tetraploid. This classical highlands. These programs will be loosely linked but approach has limitations (Stover and Buddenhagen are administered separately. In East Africa there will 1986),but for a target where current clones are gener- be one banana program in which breeding, pathol- ally poor agronomically, it is a reasonable approach. ogy, entomology, and nematology are administra- This program has already released a good tasting tively under a single program director. At Onne, Prata-type tetraploid, based on Lidi, that is fast- however, the "plantain improvement program" BananaResearch Needs and Opportunities 5

(breeding) is separate from the "plantain health pro- sufficiently poor to cause the dwindling and death of gram," reflecting a recent structural shift in the parent much Musa botanical germplasm. Moreover, even the institute, IITA, with a directorate of crop improve- best Musa lines generally grow poorly and are re- ment and a separate directorate of plant health. These duced in stature. Thus, judging progeny performance are institute divisions, and there is no directorate of at Onne is unrepresentative of better soils. Selections research. Nevertheless, the total effort on bananas there are likely to be too tall for cultivation in Central and plantains at IITA is impressive due to the dedica- and South America and their agronomic characters tion and dynamism of staff and the concern for the can only be poorly judged at Onne. The poor growth needs of all of tropical Africa. has been recognized for many years, hence the com- The plantain and banana improvement program mon comment that plantains are only good on the (PBIP) at Onne has one core position for breeding/ refuse heaps around houses. Examination of the roots genetics and one for tissue culture/biotechnology. In shows high frequency of death and branching and addition, a temporary International Development Re- rebranching. This is unusual for Musa, and although search Centre (IDRC)-funded postdoctoral position root-knot nematodes cause root tip stunting and currently adds work on postharvest evaluation and branching, it appears to be more than that. It is possible product utilization. The plant health management that aluminum toxicity (high aluminum saturation) is division has one core pathology position assigned to a major cause of root tip stunting and death, leading plantains at Onne. In East Africa that division also has to poor growth and the melting away of the clones. a core entomologist and a soft money nematology Aluminum toxicity inhibits mitosis and thus stops post assigned to bananas. root elongation in plants. This is probably combined In addition, at IITA headquarters in Ibadan, there with nematodes and associated root-rotting fungi not are good virology laboratories, screenhouse facilities, normally important. Given the strong soil research biotechnology facilities and staff who could supervise background of IITA and the original agronomic bias virology, entomology, or related research on plan- of the plantain program, it is not clear why no one has tains. This has been started to some degree already, addressed the possible toxicity problem and other, with links already established with advanced US and obviously serious, root problems. There is little avail- UEKlabs on virology and on molecular genetics. able information or past publications even indicating The goal of the IITA West African program is to the aluminum status, nor studies of the root problem. obtain better, disease-resistant plantains; that of the This is a major problem biasing the evaluation of both East African program will be better cooking/beer parents and progeny in the breeding program and it bananas for the highlands. Although these are the needs addressing. Also, although the Onne soil ap- goals, there is sufficient scientific staffing, curiosity, pears well drained, temporary waterlogging may oc- and drive to create many specific topics for research cur at shallow depth where roots should be. Recent projects to contribute new knowledge. In this respect, work in Western Australia reveals how stressed ba- these programs can be built upon to produce results nanas are by even temporary waterlogging. useful to other programs having different product The second major problem is the gradual realiza- goals. New knowledge may be generated especially tion that banana streak virus is present at Onne and on Musa genetics that could be used elsewhere on that it has been inadvertently transmitted into the banana streak virus, and possibly on black Sigatoka progeny, via seed or pollen transmission. For the and nematodes. breeding program to be successful, one must be able The location at Onne has proved ideal for obtaining to distribute the products and they must be pathogen- many seeds when the right plantain is pollinated. This free. Already, healthy appearing but putatively in- has been a great advantage in obtaining black Siga- fected progeny have been distributed to various toka-resistant, improved plantains. How much of this locations. This is also true for some of the FHIA high-seed production is due to climate/soil factors selections emanating from La Lima, Honduras. This and how much is due to the availability of diverse is a major problem requiring immediate action, good plantain germplasm, some of which is highly seed- research, and solution. This is new and it is important. prone, is not known since definitive genotype by Once again, the old adage: new varieties will create environment (GxE) experiments on seed set have not new problems. been conducted. There is an IRDC-supported postharvest and utili- The Onne location has two major negative charac- zation project at IITA connected with breeding that ters beyond the remoteness from IITA headquarters, has contributed greatly to knowledge of postharvest laboratories, and staffing. The first is that the soil is characteristics of the bred clones. 6 BananaImprovement: Research Challenges and Opportunities

Asia/Pacific Thus, this location and collection offer ample oppor- tunity for an overall expansion of focused banana Australia research with international scope.

North Queensland. Most banana production is in South Queensland.Banana research at and near Bris- northern Queensland yet banana research is concen- bane in various organizations is considerable, with an trated in southern Queensland in or near Brisbane at excellent connection between practical problems and the Queensland Department of Primary Industries both applied and basic research. This is probably due (QDPI) in Indooroopilly, the Maroochy Agricultural to the QDPI connection with the banana growers Research Station, Nambour, Queensland University problems, through extension agents and field plant of Technology (QUT), and at the University of pathologists and entomologists on the one hand, and Queensland (UQ), and the Cooperative Research their connection with universities and doctoral thesis Centre for Tropical Plant Pathology (CRCTPP). research on the other. In addition, an Australian Cen- There is also a QDPI research station in northern tre for International Agricultural Research (ACIAR) Queensland on 49 ha of land (South Johnstone Re- banana improvement project, begun in 1986, broad- search Station), 10 km south of Innisfail, where ba- ened the scope beyond Queensland by adding an nana research is conducted. Research at the station international dimension to the work. and surrounding areas covers bananas, papaya, other The most extensive work has been done on Fusar- tropical fruits, pastures, beef, and land conservation. ium wilt by QDPI scientists, to understand the distri- Some technology work is carried out at Innisfail. bution and origin of the races and vegetative Although the land area for bananas is currently compatibility groups (VCG) that occur in Australia limited, the South Johnstone station is well situated; (see various publications by Pegg, Moore, and Bent- nearby land could be leased, and the climate is much ley). This work has led to the rational hypothesis that more tropical and suitable for typical tropical banana Fusariumwas brought into Australia and distributed production than the areas south of Brisbane. There are with ladyfinger (AAB), and that some of the strains plans to expand the facilities at South Johnstone and later attacked Cavendish planted in the Fusarium- to place more banana research there. holes. These strains have been designated as race 4. There is a well-maintained collection of some 250 Later work has shown a different Fusariumhad been lines (Daniells 1995),and strong staff interest in pur- brought into Western Australia on windbreak ba- suing breeding and niche banana development. nanas, and that under waterlogging stress this appar- Daniells is also testing several somaclonal variants of ent race 1 strain can attack Cavendish. "Williams" from QDPI Maroochy Horticultural Re- This and earlier work on cold stress in southern search Station, several FHIA hybrids and many of the Queensland has led to interesting research on "stress" best parthenocarpic "local bananas" of Southeast as it affects susceptibility. This latter work continues Asian countries. Data are taken on yellow Sigatoka at the Maroochy Horticultural Research Station and reaction. Fusariumraces 1 and 2 occur in the area, but has expanded to add chlorophyll fluorescence meas- not race 4. Lesion nematodes also occur, as does scab urement to assess cold tolerance of germplasm in moth. A plant pathologist is conducting research on general. A temperature gradient panel has been de- screening fungicides, and on leaf spot monitoring and vised for this. Also root boxes have been made for root forecasting. studies, useful to study disease development under Some Australian scientists are interested in breed- different conditions. ing, some have pollinated many clones with FHIA The international Fusarium work has led to the diploid 3362, and FHIA 1 and obtained seeds and continued discovery of new Fusariumstrains in differ- plants. There is no strong support for a breeding ent parts of Southeast Asia, to the realization of the program at present. Their Mysore has banana streak complexity of strains in the center of origin of the virus (BSV)and 4X progeny from it also carry BSV, pathosystem and to the hypothesis of a biphyletic proving its seed transmissibility. Although there is origin of Fusariumoxysporum f.sp. cubense (Foc).It is some connection with the somaclonal variation work clear, on analysis, that the type of microanalysis of at the Maroochy Horticultural Research Station, there strains in Australia has not been carried out anywhere seems insufficient connection with the extensive pa- else. This is sorely needed in Southeast Asia to under- thology research at various Brisbane locations. Also, stand the pathosystem and to enable judgment of the this more tropical location offers ideal opportunity threat to new efforts to commercialize Cavendish, or for GxE studies on agronomy, flavor, fruit characters other clones, in places such as Malaysia, Sumatra, and as well as on disease reaction and climate effects. Sulawesi. BananaResearch Needs and Opportunities 7

It should be understood that the more basic work its pests and pathogens, and with the presence of a has found stimulus from practical findings from large commercial crop base and research system, Aus- QDPI, leading to new questions and their pursuit tralia is in a position to contribute more internation- through doctoral theses partially directed by re- ally to useful knowledge on bananas. If Australia searchers at QUT and at the University of Queens- should elect to add the key missing element-a breed- land. These linkages have been ideal for all concerned, ing program linked to modern biotechnology-it and extend to virus research as well. could produce desirable products in short order. Extensive basic research on BBTV has been con- ducted at QUT, leading to advanced knowledge of New Zealand pathosystem evolution, and viral structure and genes. Through recent collaboration with American and The Department of Scientific and Industrial Research European researchers, Cavendish is being trans- (DSIR) has been semi-privatized and the Mt. Albert formed with a BBTV replicase and also with a ri- Research Centre in Auckland is part of the Horticul- bozyme. These plants should soon be ready for field ture and Food Research Institute of New Zealand testing and appropriate permits need to be obtained. Ltd., where research was conducted on the black Si- This work should be pursued in collaboration with gatoka pathogen in conjunction with an ACIAR pro- the Philippines where BBTV is so serious. Of consid- ject. This work was connected with Tonga, Samoa, erable interest is their recent virus structural work and Cook Islands where the banana export trade had indicating two major strains, an "Asian" strain and a been more or less eliminated by a combination of "Pacific" strain of BBTV. Thus, it will be possible to factors, the main one being black Sigatoka. trace the routes of distribution of the virus to all DSIR holds the world's largest collection of banana distant locations. The original host/vector still re- Mycosphaerella strains, some 800 isolates in all, all mains to be discovered. lyophilized and sitting on the shelf. Also available are Work at QDPI on diagnostics has led to a workable records of this work, which consisted of glasshouse monoclonal antibody test for BBTV, which has been testing of isolates on leaves of a set of differential commercialized. However, in the Philippines it was varieties tested in the juvenile stage, and observations considered, in some blind tests, to be inaccurate. on the breakdown of resistance in the Pacific Islands Thus, more follow-through on its reliability is needed. and elsewhere (see Fullerton, various publications). QDPI researchers are working on diagnostics for The key points needing to be resolved are to judge BBMV, serious in the Philippines. A CMV diagnostic probability of resistance breakdown and of recombi- test is already available, works across strains, and is nation of virulence genes. These cannot be answered commercially available from a company in France. because the appropriate research has not been carried Some work has also been carried out on BSV that is out. Also, proper comparisons of juvenile leaf reac- present in the QDPI banana collection on Mysore and tion to inoculum and field performance of adult in some plantains from IITA. Also, interesting data plants have not been made. are available from a black Sigatoka trial planted in Although many researchers are applying various Western Samoa, which indicates that their Mysore molecular techniques to pathogen characterization (called Misiluke) is infected with BSV and that field and to analysis of diversity, the connection with field spread occurred to some of the test entries but not to reality is less clear. Part of the problem is that viru- the much more abundant Cavendish border plants. lence comparisons of diverse strains cannot be carried At the Maroochy Horticultural Research Station, in out in a banana-growing country in the field, and addition to the stress physiology research, there is a artificial challenge in a glasshouse does not simulate tissue-culture collection of Musa germplasm and con- well the natural pattern of inoculation, and potted siderable work is being done on tissue culture, so- plants do not grow normally. maclonal and mutational variation. This work could Part of the problem also is that no one is connected be expanded usefully if it were more closely involved closely with a breeding program where breeding and with clear objectives of a breeding program. genetics are practiced. Additionally, conventional Largely due to the presence of Fusariurm race 4, mycology and plant pathology are no longer in vogue Australia has received several FHIA hybrids, as so that sexual crossing of different pathotypes re- well as material from other breeding programs, for mains undone. Also left undone is analysis of the evaluation. different stages of pathogenesis wherein resistance Although considerable research is underway, genes could act to interfere with the pathogen and many key questions remain unanswered. With the contribute to the eventual resistance/susceptibility proximity to the center of origin of Musa and most of rating. Much creative mycology/genetics and resis- 8 BananaImprovement: Research Challenges and Opportunities tance genetics, based on homozygous diploids that are free of Fusarium wilt, even highly susceptible have been well analyzed, remains to be done. Pearson clones. Around Palopo, in central Sulawesi, Fusarium (from New Zealand) et al. (1983) analyzed black Siga- has been found in the cooking banana (P. manurung) toka reactions in the collections of Papua New and this has proved to be caused by a different strain Guinea, giving us the first published accounts of the than the aggressive strain in Sumatra. reaction of this unique indigenous germplasm. On the island of New Guinea, no Fusarium wilt is known, even in the extensive evolutionary center of Indonesia indigenous diploids. The situation on the large island of Borneo is unknown. As in Malaysia, bananas are being commercialized for It is not clear that any research useful to a nascent export for the first time. Cavendish is being planted banana industry is being conducted. The two Musa on a few sites, in dense stands allowing the pathogens collections in Java have no relevance, but apparently and pests present in this region of coevolution to a better collection exists in central Java and the gov- reveal their epidemic potential for the first time (the ernment is to collect and establish a planting at only exception is "blood disease" on Sulawesi, which, Sukamandi experiment station on Javas north coast. as a probable new introduction, became epidemic on One of the few practicing banana taxonomists in the "local bananas" in the early 1900s). world is located at Bogor. Also, a few researchers are Thus, in Southern Sumatra, within two years of located at various universities in Java, who do study planting Cavendish (cultivar Valery), Fusarium wilt some aspect of bananas. Thus, some expertise exists in has become epidemic. Also, the yellow Sigatoka origi- Java, which, if properly focused and connected, could nally present has been replaced by black Sigatoka on contribute useful knowledge on bananas. So far as is the Valery. Blood disease has become epidemic as a known, no breeding program exists or is planned. new introduction in the southern coastal areas around Lampung city in cooking bananas only. Malaysia Corky scab thrips are sufficiently damaging to neces- sitate bud injection of insecticide throughout any For the first time in the Malaysian peninsula, Cavendish plantings destined for export. Cavendish bananas are being commercialized, with In a smaller Cavendish plantation in central Suma- the establishment of modern small plantations. These tra (Grand Nain), Fusarium wilt is also rampant, but are as a rotation crop after old oil palm. The planta- the Sigatoka is still yellow Sigatoka. As in Malaysia, tions are severely affected by Fusarium wilt. The plan- Cladosporium speckle is potentially severe. Bud injec- tations were established with no concern for this dis- tion with insecticide is also practiced. Company tech- ease since it was believed that only race 1 was present. nical staff (usually Filipino) carry out technical This belief was not based on any analysis of the com- services to control the various diseases and pests, mon occurrence of Fusarium in many local clones. using experience from the banana plantations in Min- In general, bananas have received little research in danao. This is inadequate for the unexpected Fusar- this country, where they originated and where many ium problem, apparently caused by a different and of their pathosystems evolved. Local agriculture at the more Cavendish-aggressive strain. Research is not village level provided all the fruit needed for local under way to answer questions of origin, dissemina- markets, and the village production was left to its own tion, and containment. The meristems used in these devices, with research going to rubber, oil palm, and Sumatra plantings come from either Malaysia, Singa- later to rice. This is now changing due to the emer- pore, or Java. As in Malaysia, wild bananas occur gence of the intractible Fusarium problem and to the abundantly in Sumatra and their role in pathosystem desire to commercialize some of the local clones. How- evolution needs elucidation. Large areas in Sumatra ever, although some academic research on bananas were only recently taken out of jungle. exists at universities and at the Malaysian Agricultural In eastern Java, a recent attempt to commercialize Research and Development Institute (MARDI), there both Cavendish and P. berangan has also met serious is little connection with commercial development. Fusarium wilt problems, and again the origin is nei- MARDI and others have been local cooperators for ther understood nor questioned. IAEA's mutation breeding program and one can see A Cavendish plantation recently started on the is- plots of P. rastali treated with neutrons at MARDI; and land of Halmahera (east of Sulawesi) has had a few various clones, including Cavendish, which have re- cases of Fusarium wilt. Whether or not these were ceived gamma radiation at United Plantations, some caused by an aggressive strain is not known. Obser- two hours north of Kuala Lumpur. The objective is vations in Sulawesi indicate that most local bananas Fusariuni wilt resistance. Recently, a Cavendish clone BananaResearch Needs and Opportiunities 9

has been released from this program as the new vari- no possibility of Fusariuni contamination in the mer- ety Novaria (to honor Dr Novak from IAEA), which is istems (but this needs checking), contamination in the reputed to have better flavor and be "earlier" than its nursery is quite possible. By not using a pasteurized parent Grand Nain. (This is the first release anywhere planting mix in the polybags, nor paying attention to of a mutated banana.) the purity of irrigation water, possible nursery con- At MARDI (25 km south of Kuala Lumpur), there tamination with both nematodes and Fusarium may is a good collection of local wild and parthenocarpic invalidate the great advantage of "disease-free" mer- diploids, and of some Thai material, plus local istems. This potential contamination either at the triploids. This collection has been replanted recently tissue-culture site, or at the site for planting, may be and is under good agronomic practices. However, a partial explanation for the rampant Fusarium wilt. "good agronomy" extends to spraying with Benlate Obviously research on this is urgently needed. monthly and deleafing, thus, work on assessing resis- A major opportunity for research on the wild ba- tance to leaf diseases cannot be carried out. It appears nana pathosystems exists in Malaysia, where wild to be planted on Fusarium-free soil, even though a acuminata diploid-seeded bananas appear abun- Fusarium-laden plot exists about 300 m away (with dantly, especially in hilly areas and near village settle- irradiated P. rastali dying of Fusarium wilt). The col- ments where local clones have been domesticated and lection could be useful if these deficiencies were ad- are diseased. The wild bananas appear healthy, but dressed, and if a linkage to a breeding program could what might be on or in their roots and on older leaves be developed. Analysis of the collection by patholo- is completely unknown. The biology and ecology of gists and by an active breeder would be very useful. these wild bananas appear to be complete unknowns. A well-run Cavendish plantation near Johore Yet they are among the key parents used in breeding Bahru (close to Singapore) was severely affected by programs. Fusarium wilt. This "new" plantation was planted on The absence of any banana breeding program in the land previously in oil palm, with laboratory-grown center of origin, combined with the non-exposure of meristems. No local or wild bananas were present. most people in existing banana breeding and research Thus, the origin of the Fusarium remains a mystery, programs in the Americas and in Africa (breeders, and whether the treatment practices used are increas- pathologists, and others) to the diversity of Musa and ing or decreasing natural spread remain unknown. its wild and domesticated pathosystems in Asia, are Good technical management exists, including good major lacunae that contribute to our relatively poor laboratories. Thus, a real potential exists for in-depth progress in banana breeding and to our poor manage- research to understand the Fusarium pathosystem. ment of banana problems. Management (Ulu Tiram Estate) would be willing to cooperate on such research, to include providing a Philippines "hot spot" for Fusarium screening, if a project were developed under the Banana Improvement Project. In Mindanao there are both industry researchers (Del In the north, at United Plantations (mainly oil Monte) and scientists from the Bureau of Plant Indus- palm), attempts to grow bananas are also met with a try (BPI) who work on banana problems. Fusarium wilt problem. This organization, linked The Del Monte operations are impressive both in through the University of Malaysia to IAEA, Vienna, technical activities and management of the diseases would be willing to cooperate on research through and the overall agronomy and handling of bananas. the Banana Improvement Project. An excellent labo- They have five plant pathologists and an entomolo- ratory exists, but the land is quite swampy for ba- gist. They do practical problem-solving activities in nanas. Consequent waterlogging makes for bacterial the management of black Sigatoka, Fusarium wilt, heart rot, poor roots, and confounds Fusarium wilt Moko disease, BBTV, and BBMV, and of nematodes screening. Severe Cladosporium leafspot also occurs in and "freckle," a leaf disease (Phyllostictina musarum) this humid location. Farther north at Penang, capable they consider harder to control than black Sigatoka as pathologists are working in the laboratory with Fusar- it is not affected by systemic fungicides. ium. Thus, the stage is set for doing some useful The plantations have excellent Sigatoka control. research on this important problem, provided a fo- The climate appears ideal for black Sigatoka and local cused project can be developed. bananas have plenty of it, yet the plantations have the Well-run commercial tissue culture laboratories ex- cleanest leaves of any location. The companies appar- ist in various locations in Malaysia (and in Singa- ently share information on control methods and use pore), which produce and export banana meristems, a different spray regime from those used in Central omamentals, and palm. Although there appears to be America. They use an integration of the youngest leaf 10 BananaImprovement: Research Challenges and Opportunities spotted and the amount of spotting to indicate when America and Sulawesi. Thus, much more needs to be to spray (essentially the Stover and Dickson method). done to determine if Bugtok disease was here before They do not use spore trapping nor an approach of the importation of Cavendish seed from Honduras in allowing abundant infection then hitting with TiltTM. the early 1960s. Thus, the origin of these diseases Also they never followed TiltTM with TiltTM (as oc- remains unresolved. curred in Central America) so they consider that there The Davao Musa collection, located adjacent to the has been no buildup of resistance to this fungicide. BPI headquarters, is poorly maintained and plants They refused from the start to follow the recommen- grow poorly (low N). Cavendish had only moderate dations of FRAC (Fungicide Resistance Action Com- black Sigatoka. Surprisingly, there was more resis- mittee) of the fungicide-producing companies. tance in clones from Malaysia and Thailand than from Their approach and management of Sigatoka con- Papua New Guinea. There are clear differences in trol is obviously working better than that in Central resistance to freckle. One case of BBTV and two of America. Comparisons of pathogen virulence and BBMV were obvious. The long-term usefulness of this climates are in order, versus fungicide regimes and collection is questionable. application methods, to clarify the true causes of the At Del Monte headquarters there were several in- superior control. No one is proposing to acually do teresting Cavendish clones, including Americani from such research. Cameroon. One clone called Caventan is like Lakatan Del Monte has eleven separate local producer farms (AAA) yet the fruit is said to taste like Cavendish. Is and Fusariumnwilt occurs in only four of them, at a rate this related to Cavendish? Del Monte may be willing of around 6,000 cases per year. Long-term trend data to conduct a field trial on agronomy and field spread are needed plus analysis of replanting losses. It would of any BBTV transformants. Thus, linkage is possible be of great interest to know the extent of VCG variabil- with industry via properly developed projects, the ity in the Panama disease within and among these four results of which would be mutualy beneficial. Espe- locations. Amazingly, given the great diversity of the cially useful is the industry experience and insight, fusaria from the Philippines, there was almost no and research staff. The Philippines would be inter- Fusarium wilt evident, not even in local bananas. Even ested in a niche banana. As an example, they have in Luzon around Los Bafios and in Cavite province in recently promoted a new fresh fruit pineapple which the extensive Sefnorita Farms (Pisang mas), there was is now in great demand in Japan. no Panama disease. Thus, microanalysis of Panama BPI management are interested in banana prob- disease in the Cavendish and in the major Philippine lems, and would welcome more support for banana clones is in order, as has been done in Australia. research. Discussions were held with BPI and NRI Both for Sigatoka and for Panama disease, and personnel about collaboration on BBTV and on BBMV also for Moko disease (described below), many ques- in Mindanao, involving also Australian virologists. tions can be asked which could be fruitfully investi- Potential linkage with field epidemiology and in test- gated and yet they are not, because of lack of contact ing BBTV-transformed bananas was also covered. between the Del Monte group and outside researchers The Institute of Plant Breeding, UPLB, proposes to concerned with the broader view of pathosystem analy- do research involving somaclonal and tissue culture sis. The Del Monte group must do troubleshooting work on BBTV resistance. They do have good tissue activities but they could contribute much more if there culture capability, but this route to BBTV resistance is were linkages with key researchers from outside univer- remote compared with other accomplishments on sities or other organizations. Such linkages could be transformation elsewhere. followed through on Moko, BBTV, and Fusarium. Postharvest research was discussed with staff at Moko symptoms were observed in two isolated their facilities at the Postharvest Horticulture Research small farms of Lakatan, the most favored dessert ba- and Training Center, UPLB. The facilities are ade- nana (an AAA = P. berangan in Malaysia). There were quate, and of special interest was the work on Sefiorita also Bugtok cases in Cardabalooking exactly like blood banana (the popular Pisang mas) wherein ripening was disease in Sulawesi or SFR insect-transmitted Moko inhibited within plastic bags containing a small sachet in Latin America. Except for doctoral student work at of a "natural product," which they plan to patent. the University of the Philippines in Los Banos (UPLB) These researchers would be potentially good collabo- in 1994 to compare at the molecular level the Bugtok rators if a postharvest project is developed. pathogen with the Cavendish Moko pathogen, no Pseudomonas solanacearumresearch (Bugtok and Moko other appropriate field work is being done on the diseases) was covered with representatives of the Na- disease in Cardaba, in Cavendish and Lakatan, nor in tional Institutes of Biotechnology and Applied Microbi- comparison with the different Moko strains in Latin ology, UPLB. They are completing work on molecular BananaResearch Needs and Opportunities 11 comparison of Bugtok and Moko isolates from Min- At Seibersdorf, work was conducted on Musa DNA danao. The proper link to field pathology and patho- size, tissue culture, chromosome doubling, and system analysis, and comparison to Moko in Latin ploidy monitoring. Facilities include greenhouses for American and with blood disease in Sulawesi, remain growing bananas. Some work on Fusarium culture as important questions and problems for the future. products as selective agents has also been conducted. The Musa collection in Los Bafios contains 52 entries. One stimulus for future collaboration should be the There is more BBMV and BBTV than in the Davao recognition that there are no proprietary rights to any collection and black cross disease occurs on Cardaba germplasm or techniques generated by IAEA. This (the location is very humid). Apparently, two wild needs to be appreciated by the potential users. bananas occur in the hills but they die out in the collection. One, Saging tnatsing, is rare but it has a Belgium-KULeuven pendulous bunch. A diseased condition not seen be- fore was on some ABBs, in which the plant appeared Belgium supports banana research mainly through vigorous and symptomless but the bunch was small, a center at the Katholeike Universiteit Leuven shriveled and necrotic, without internal symptoms. (KUL) in tropical agriculture. The major tropical con- There is considerable production of Senorita ba- nection is with the IITA plantain program at Onne, nana, a most favored local variety (P. rnas), in the Nigeria, where there has been an early and long- Cavite district. It is a small, rather weak AA diploid standing connection with Belgian support. There are grown on scattered farms, mostly quite derelict and now at least 12 staff/students working in Musa re- with abundant BBTV. Their leaf spot appears to be search at KUL. yellow Sigatoka, also moderately severe. Most farms A tissue-culture germplasm collection of Musa appear to use no inputs and weeds are rampant. In exists, with some 1,100 accessions, comprising most spite of this, roadside markets are full of fruit, hang- of the world's collected germplasm. This collection is ing ripe on the stem. Obviously Sefiorita should be well maintained, and experimentation continues transformed with BBTV resistance. on improving culture methodology. They propagate In conclusion, Mindanao in the Philippines is a materials for clients worldwide on request, and dis- major exporter of Cavendish and the industry is seminate those previously cleared by the virology well managed with good technical services and good unit at CIRAD, Montpellier, operating through rapport with the BPI staff. Almost no connection exists INIBAP. Research on cryopreservation (under liquid between industry and research at UPLB. Many disease N) is also being conducted, and appears promising. problems still exist, especially for local production Quality work is conducted on embryogenesis with where inputs are limited and farming is haphazard. embryogenic cell suspension (similar to work in Unresolved questions on pathosystems, even in the Montpellier, but they do not use the periodic "liquid commercial plantations, remain. More collaboration wash" system). Also, they recently started work with and synergy are possible if international connections Grand Nain. Research on protoplast culture is also con- were established through clearly focused projects. ducted, using an innovative nurse-culture technique. This is connected with research on transformation of Europe protoplasts. Actual molecular work is minimal, with emphasis on cellular and protoplast manipulation. Austria-IAEA Research on antifungal-proteins (AFPs) for fungal disease control is conducted in a nearby laboratory of IAEA-FAO have been conducting mutation breeding genetics. They are connected with the company and related backup research on Musa for many years. "Zeneca" on this AFP work, and the company holds This has operated through laboratory research at patents on the small peptides comprising the AFPs. Seibersdorf, and through linkage projects managed These peptides are much more effective in vitro out of IAEA,Vienna. These projects have developed against fungal pathogens of bananas than are the in various tropical countries, where putatively mu- earlier-found chitinases and lectins. tated germplasm could be tested. A publication sum- marizing this research is in press. France-CIRAD, Monttpellier There are ongoing IAEA cooperative research pro- jects to improve Fusarium resistance, black Sigatoka CIRAD is a very large French research organization resistance, and agronomic characters with several with worldwide operations in the tropics, but which countries, involving both Grand Nain and local ba- is run out of Montpellier. There are 1,800 staff, of nanas such as P. berangan. which 900 are senior and 400 of the latter are posted 12 BananaImprovement: Research Challenges and Opportunities overseas. The budget is derived from French federal For black Sigatoka, much work has been done (and monies, from industry, and from the EEC and other published) and work on pathogen variation, early multinational sources. inoculation methods, host/parasite interaction, etc., Since the banana-producing Caribbean islands of continues. Work on crossing isolates with different Martinique and Guadeloupe are part of France, they pathogenicities is needed and is possible at this loca- receive EEC money for banana research and develop- tion, which, however, is somewhat cut off from the ment. This situation dominates the CIRAD approach needs of a breeding program. to banana research and research management and strategy. Also, the ex-French tropical African coun- Spain/Canary Islands tries qualify within the ACP-protected markets for Europe, hence the French emphasis on banana re- There is an intensive and viable banana industry of search and development for that region. Cavendish in the Canary Islands, which are exported CIRAD has used INIBAP as a major source of infor- to Spain. The growers are organized and there is mation and contacts to the banana world, which has grower group support for research and extension, enabled the development of the present strategy and which is arranged as in Australia, with good links programs. between growers, extension and research-the latter CIRAD's tropical banana breeding centers are in also linked to researchers in Spain. They have a Guadeloupe and at CRBP in Cameroon, with re- unique cold subtropical climate for bananas and have searchers also in New Caledonia in the South Pacific. built good links to researchers on bananas in Israel, In addition, CIRAD staff are posted in various Latin South Africa, and Australia. They also share with two American countries, and in various scattered labora- of these locations the presence of race 4 Fusarium, and tories, which enable strong linkages also with Brazil have links to both NRI nematologists and with East and Cuba. CIRAD also provides technical services to Africa on nematodes, since P. goodeyi, the highland a commercial banana operation in the Philippines. African banana nematode, is present in the Canary Basic research to back these operations and connec- Islands. tions is headquartered at Montpellier, where re- An experienced person (Jorge Pinochet) on nema- searchers are in a position to interact with colleagues tode germplasm assessment for resistance in bananas who apply the same disciplines to other tropical is with the Spanish research system in IRTA near crops, such as rubber, coffee, cacao, coconuts, rice, Barcelona. They have major banana nematodes and maize, sorghum, etc. Research linkages exist with are interested in further germplasm screening, with other research centers, mainly in Europe. both field plots in the Canary Islands and laboratory The research emphasis at Montpellier centers work at IRTA. The growers (and their contacts in around tissue culture, somatic embryogenesis, and Israel) have what they consider superior clones for transformation. In the molecular laboratory, work is cold tolerance, derived from somaclonal variants, and conducted on mapping in various tropical crops, in- they are in communication with other subtropical cluding bananas, where doctoral students produce locations on this need. theses research, each building on previous work. Sev- There is also backing from Madrid to develop a enty-seven markers exist now and there is a target of subtropical fruit research center in La Palma, cen- 200. Targets are markers for A and B genomes, agro- tered around bananas, which is also supported by nomic characters, and disease resistance. Work is tar- the local government. This is also one of the very geted toward differentiating AA subspecies and few locations where there is serious, consistent triploid/diploid relationships. damage to bananas from Meloidogyne, which would For banana "plant protection," CIRAD has some 20 provide a location for screening potential parental staff. The main thrust is on black Sigatoka, viruses, germplasm for resistance to this nematode (and for and nematodes. tolerance to cold at the same time). Finally, the The virus work also covers the responsibility of Canary Islands growers have unique access to the indexing Musa material for INIBAP's distribution Spanish market, which has generated sufficient in- system, which is a sensitive area within the banana terest by the multinational companies for one com- research community. Of considerable interest was a pany to have located a technical expert on the collection of BBTV-inoculated Grand Nain plants ex- Islands. pressing very different symptoms with virus isolates Therefore, the Canary Islands offer a unique from different regions. This was surprising, and thus subtropical location, close to Europe, for research this virus symptom expression variation should re- on subtropical problems and potentials: cold toler- ceive rigorous and careful research. ance, race 4 Fusarium, and key nematodes Praty- BananaResearch Needs and Opportunities 13

lenchusgoodeyi, P. coffea, and Meloidogyne; also, on dustry involvement would also be useful. Especially agronomy at the commercial level for any collabo- deficient is the lack of Fusarium cultures and mi- rative GxE research. croanalysis of different sites in the old Panama dis- This is essentially a European banana operation ease areas. and it falls under the EU and other European sup- port organizations for research and development, Puerto Rico and the results would relate to the southern Medi- terranean. Puerto Rico has a US$50 million plantain and banana production industry for a population of about 3.5 United Kingdom million people. This industry has expanded mark- edly, and became profitable due to the recent avail- CAB International researchers representing the In- ability of good lowland soils in the south as the ternational Institute of Parasitology, the Interna- sugarcane industry receded. tional Mycological Institute, and the International There is a university, partly supported by US funds, Institute of Biological Control discussed past and with campuses near San Juan (Rio Piedras) and at proposed work on Fusarium and Mycosphaerella Mayaguez, which is ideally situated for research and stressing East Africa connections; nematodes, espe- training in tropical agriculture. However, a lack of cially P. goodeyi, in East Africa and advisory work strategic planning and an absence of a mission in in Cameroon, and on coevolution and dissemina- tropical agriculture from the Federal Government, tion; and the potential of "biological pesticides," have resulted in underuse of this potential great stressing connections with IITA/Benin and with resource for Latin America, and for banana research IITA in East Africa. These groups rely on interna- and training. This has occurred in spite of a long- tional tropical work and have all been involved term United States Department of Agriculture with banana research, mainly with ODA funding, (USDA) input into the island, and a current USDA but also from other sources. As in Australia, they Tropical Agriculture Research Service (TARS) Station rely heavily on graduate student thesis work, at Mayaguez. guided by experienced researchers. In the central north of the island, a 125-hectare The Natural Resources Institute (NRI) has had University Experiment Station exists where banana many banana projects on pathogens, especially black research is conducted and where a germplasm collec- Sigatoka, as well as postharvest, and they wish to tion of 54 entries is well maintained. The area has continue their involvement, which has been most rolling hills, high rainfall, low soil pH, magnesium strongly linked to Africa. Their connection with ac- deficiency, and considerable soil heterogeneity. Nev- tual breeding programs has been quite limited. ertheless, it is a traditional plantain-gowing region. Most work is on agronomy for local small-scale United States of America grower problems. Plantains "run down" quickly and need replanting, and most germplasm entries ap- Florida-University of Florida, Homestead peared virus free, but a few did not. Some germplasm entries looked interesting agronomically and these Techniques are being pursued to have a reliable seed- apparently are not present elsewhere. ling test for resistance to Fusarium wilt and for char- The good plantain producers receive US$7.50/box acterization of the pathogen at the molecular level. of plantains (US$6.50 for bananas) for the local mar- Ploetz and his colleagues have good connections with ket. The new plantain varieties of FHIA and IITA FHIA, but limited travel funds. The type of field should be tested at the USDA center on the west pathogen behavior studies (survival under central coast for agronomic performance in this high Cavendish) and variability in Latin America remain technology system. At present only a "plant crop" is as uninvestigated important topics. There is a need taken, due to reduced productivity of ratoons (osten- for carefully guided doctoral thesis work in these sibly nematodes and weevils but without research, it areas, and this is not happening. A much stronger is impossible to know). New plantains are not avail- connection between this group and Musa germplasm able to Puerto Rico in any reasonable time frame resistance assessment in cooperation with both through the cumbersome international delivery sys- pathogen and host genetics is a need that should be tem for Musa germplasm. Puerto Rico is one of the addressed, especially since the Latin American ba- best existing locations to experiment with commercial nana trade locations are nearby and it was Fusarium production of new plantains and to conduct good that created their greatest problem in the past. In- research on plantain decline. 14 BananaImprovement: Research Challenges and Opportnnities

The US variety collection of Musa is being trans- Perspectives on the Commercial Industry ferred from Miami to Mayaguez. The present collec- and Synopsis of Breeding/Biotechnology tion there is not well cared for, but could be assessed Needs and Opportunities for "resistances." Additionally, BSV symptoms were seen in several entries, but not in others. However, no The international export banana trade is based almost historic data are available on sources or origins of the exclusively on the efficient production of very similar collection entries, precluding hypothesis testing of clones of Cavendish. These triploid AAA clones are pathogen sources. agronomically excellent, highly stable across environ- ments, and produce fruit of high quality that is ac- Texas cepted as "the banana" by the purchasing public in most temperate countries. The scope of the molecular work on bananas at Texas Cavendish is susceptible to various pathogens, fun- A&M University is impressive. A simple, repeatable gal, viral, bacterial, and to several nematodes and transformation methodology has been worked out. insect pests. The relative importance of the pathogen/ Using a combination of particle gun and Agrobacterium pest problems varies from region to region, and even they have transformed small shoots of Grand Nain locally. with constructs potentially conferring BBTV resis- The industry in general maintains efficient produc- tance. They have a complete cDNA genome library of tion through the use of fungicides, nematicides, and Grand Nain, and are working to extract ripening genes insecticides (which constitute a considerable part of from banana peel and pulp, which they will use to the cost of production, both directly, and through downregulate ethylene biosynthesis. Their interest in costs of technical services, applied research, and ex- bananas lies in the idea of having the appropriate plant tension). The pesticides also pollute the environment produce vaccines to human viruses. The facilities are and, to some extent, negatively affect human and superb, and they welcome collaborators to bring their animal health. bananas and problems and work there using their The multinational banana companies are now techniques. As of September 1995, this group moved largely purchasing and shipping companies rather to the Boyce Thompson Institute for Plant Research, than plantation-owning and banana-producing com- Inc., affiliated with Cornell University, Ithaca, N.Y. panies. The companies do provide the "technical services" to national producers on disease and insect Other Locations control, based on either past research, limited current applied experimentation, or on trial and error. In a There are quite a few locations where banana research few areas these technical services are developed also is carried out that are not discussed here. Banana through growers associations and/or box taxes and researchers in many Latin American countries, in some governmental or state input. India, Southeast Asia, the Pacific, and Africa exist, These company technical services are in good com- trying to solve local problems and develop new munication and interaction with the input suppliers, knowledge. The reader may refer to INIBAP for lists the chemical companies. Although their objectives of banana researchers and for literature bibliog- differ, there is mutual understanding amongst the raphies and abstracts. The Asociation para la Coope- companies of their interdependence and of the need racion en Investigaciones Bananeras en el Caribe y en to use the best technical advances in the judicious use America Tropical (Costa Rica) (ACORBAT) and vari- of pesticides; the use of best pesticides; the develop- ous journals may also be useful sources. ment of new pesticides; and of the negative environ- The intent here is to focus on the major exisiting mental and public relations effects of pesticides. Their breeding programs and on basic research, including differing objectives, however, should be clearly un- biotechnology, that could be developed to contribute derstood. On the one hand for banana companies and to breeding better disease-resistant cultivars. In addi- their suppliers: (a) adequate disease/pest control and tion, there is a focus on more in-depth efforts on high production of a quality product with as little pathosystem evolution in the center of origin, so that pesticide application as possible to reduce costs of both resistance breeding and disease management inputs; and, on the other hand, for the chemical com- could be more rational. There is clearly a need for panies, (b) maximum sales and profit from the sale of research that goes beyond putting out the routine pesticides for use on bananas. It is the competitive- fires in existing banana plantings. There are great ness within each company group that makes the sys- opportunities to make useful contributions. tem work generally well. BananaResearch Needs and Opportunities 15

It should also be understood that neither group has ease control. These are mutually synergistic. In addi- any faith that outside research, conducted in temper- tion, the program should work with other donors and ate countries on bananas, on banana pathogens, on research groups for more effort on local agronomy. modelling, etc. has any relevance to their operations This latter is needed to counter the idea that "better or needs. Also, they understand well that outside varieties" to come out of its efforts are the "end all" research in the tropics-in government-backed, or of the needs of banana growers. GxE interaction will international-backed organizations-is scarce, is con- become a major problem for new varieties. Research ducted by people with little understanding or connec- on GxE interaction scarcely exists for bananas at the tion with the banana business, and that the research moment, and no one is anticipating this problem or targets lie outside the banana business. Hence the lack planning research on it. of communication and interaction and the presence of No existing banana breeding program is really go- misunderstanding and the "in limbo" nature of the ing after the target of a better Cavendish variety for outside banana research. Australia is the only excep- the industry, although a few token efforts are stated. tion and possibly the small banana operations in the This has been true for at least 20 years. The industry Canary Islands, Israel, and South Africa. It is here itself is probably spending more money on this objec- where the traditional links amongst state research tive, by collecting and testing agronomically any support, university research, and actual practiced ag- clones that are considered as "better" somewhere in riculture, with the necessary extension and grower the world. group organizations, exists on bananas as well as on The only real exceptions have been the efforts of many other commodities. IAEA based in Vienna, on "mutation breeding" and It is this lack of commercial-agriculture/client- those of the Banana Research Institute in Taiwan, on identification, combined with the intractability of the "somaclonal variation." Private links to the industry social and economic systems of the small peasant have been made for testing agronomically any prod- farmer and the sheer magnitude of their numbers and ucts selected. So far, nothing beats the best Caven- distance, that contribute to the dissatisfaction of dish. The key problem with both of these programs donors, research groups, and clients, to the some- was, and is, the lack of connection on pathology times poor quality of research, the lack of strategy screening for black Sigatoka resistance, which should and long-term research plans, and the poor research have been the key target (other than race 4 Fusarium support. resistance in Taiwan). So here, again, as seems com- To temper this realistic view of how things actually mon for breeding programs, inadequate handling of work, one should appreciate that the banana compa- pathology precludes success. The current status of the nies and their technical services are in a good position IAEA program needs review. to take and use anything economically useful that The existing breeding programs have the following might emanate from someone elses research. Thus objectives: there is an opportunity for a new research orientation (a) A sweet-acid dessert banana (Brazil- and output through the Common Fund for Com- EMBRAPA, FHIA, CIRAD) modities/FAO/World Bank initiative. It is also true (b) Plantains (FHIA, IITA, CIRAD) that "better varieties" for the peasant farmer is the C ' realistic investment for him/her and that these farm- (c) Cooking bananas (FHIA, IITA) ers will quickly adopt "better varieties" if they really (e) A substitute for Cavendish (FHIA) are better and if they become aware of their existence (f) New niche bananas 'CIRADi ) and availability. The key problem here is the cumber- (C . some delivery system with its politics and rivalries at Objectives (d) and (e) are clearly the main target local, national, and international levels. appropriate for CFC/FAO/WB funding. One could The other key problem of the peasant farmer not argue that objectives (a) and (f) are similar and that solvable by "better varieties" is that of bad agronomy. they fit also since agronomically productive niche Since agronomic problems are area-specific, local bananas could enter international trade. work is needed to improve agronomy locally. Objective (b) could be considered appropriate based on the idea of increased within-tropics trade Objectives of the Project and expansion of the small international market (mostly for Latin Americans in the U.S.); also, for The new CFC/FAO/WB Banana Improvement Pro- a processed market (chips). Objective (c) could also ject initiative has two primary objectives: (1) better fit here for this latter market or for other industrial varieties; and (2) more knowledge for improved dis- markets. 16 BananaImprovement: Research Challenges and Opportunities

Modem biotechnology could be applied to any (c) More input towards cellular/protoplast fusion of these objectives, and it could even add another methodology objective: (d) Amplification of crossing and triploid recovery gImprgediblediploids already (e) More research on heterotic groups and evolu- (g) Improve the existing tionl tolodfinlprgentor having large internal markets in Asian coun- tion to fmd progemtors tries (mabe even making them into tradeable (f) More characterization of clone and group fla- niche bananas). vor characters, of breeding value for flavor, even of seeded parents The greatest payoff by far for successful biotechnol- (g) More analysis of resistance/susceptibility char- ogy would be on objective (d) (a better Cavendish). acters of all potential parents, etc. This is also the least likely objective (along with ob- The objective of a replacement for Cavendish jective (e)-a substitute for Cavendish) to find success through conventional breeding. A major commitment thogcnvtialbedgohrtanyte through conventionalhere.Sueding.sAmajoud scom meay above route is considered so remote as to be unattain- should be made here. Success could spill off easily able. Thus, barring new evidence that would reverse onto the other objectives. this estimation, it would be difficult to justify support The major targets for biotechnology are, in priority a order: (a) black Sigatoka resistance; (b) resistance to lesion nematodes; (c) resistance to Fusarium race 4; In summary, the two key routes for support for (d) resistance to bunchy top and other viruses; and attaining a better Cavendish are: (e) resistance to leaf diseases speckle and freckle, and Through biotechnology of various types and to yellow Sigatoka. Also, very high amongst this list o Through research on de novo triploid resynthesis. should be improvement of ripening characteristics (down-regulating ethylene biosynthesis) since this could predictably lower ripes and turnings and reduce Major Researchable Topics crown rot as well as increasing "green life" and "shelf The following points are a synopsis of the major life." A useful but more distant biotechnological objec- Theifollowmgana resare a should be thessed, tive would be hormonal regulation of height and ra- topicsuin b.anarearcat suld bye Badrse tooning, keys to improved agronomy. Also useful but and would be appropriate for funding by the Banana distant would be the ability to change flavor charac- Improvement Project. teristics. Also very useful, if attainable, would be in- Patholog creased cold-tolerance, for subtropical production. Conventional breeding programs have already pro- Most banana breeding programs are deficient in pa- duced (and are producing more) new banana vanie- thology. This needs addressing. Whether each pro- ties for targets: (a) sweet-acid dessert; (b) plantains; (c) cooking bananas, but not for objectives (d) and (e), gram should do all relevant pathology or whether the better~Caeds oasutitefrt. Th 'ol topics could be shared, will require further discussion i.e., a better Cavendsh or a substtute for It. The only nd analysis. progress on objective (f) (new niche bananas) may be The recommendation is to address this complex considered the output on objective (a). Any product issue and ensure that pathology is properly covered from these objectives actually good enough to be in relation to banana breeding. grown could be improved further by biotechnology (for instance, by adding resistance to BBTV). That part of objective (d) (a new or better Caven- Fusanum dish) which is "a new Cavendish" is really a different objctie"btte fomCaendsh, sice hislater Much has been learned about Fusarium, but key enig- means mo a extin Cavendis han at"ew mas remain. Most important is that we still cannot Cavendish" is the resynthesis de novo of a Cavendish identify an isolate as having a certain "pathotype" (butnwith" reitne.Tis t argresyntetis is beingo atvem d (host range) through laboratory techniques, in spite (butwitheresistanch).This programf Cuatemopted of all the molecular work. We need pathotype mark- only by theers. The diversity of Fusarium in its presumed center This target is considered realistic and attainable and of origin in Asia is scarcely touched. Are new should be supported there as well as amplified else- Cavendish plantations in Sumatra, Sulawesi, and where. Support should take various forms: Malaysia, picking up the same or different pathogens (a) New collecting and analysis of germplasm and from whence have they come (what local clones? (b) More chromosome doubling of diploids what about their biology?). Now that we have clean BananaResearch Needs and Opporttinities 17 tissue-culture plantlets, biology of spread could be management, different biological control factors, dif- researched in the field at a microscale level. But we ferent pathogen strains, or to differences in climate? need to know for sure if the symptomless in vitro In a polycyclic epidemic disease such as the Siga- plantlets could ever be carrying Fusarium. toka diseases, there are many potential host genetic The considerable experience with Fusarium on Min- steps that could affect epidemic rate (infection fre- danao in Cavendish needs analysis, as does the diver- quency, time to streaks, to spots, to sporulation; sity of the pathogen. What about the biology of sporulation rate and duration, ascospore production, Fusarium under Cavendish in the old banana areas of and leaf firing characters). In relation to resistance, Latin America? What would FHIA 1 or any other new these have never been dissected. variety be challenged by? Failing pathotype markers, Are different resistant clones affecting the same or can anyone devise a simpler pathogenicity test? How different steps in this complex cycle? Young plant sure are we that when a new area is planted with reaction in glasshouse is amenable to challenge in one Cavendish and cases occur, that the pathogen is from place by many strains, but how well is this correlated a previously planted local banana growing at the site? with field reaction, and can this correlation be im- What do we really know about susceptibility of wild proved by manipulation of climate or inoculation types in South and Southeast Asia? (So far as I am dose? This is a critical point to enable linking labora- aware, no one has ever seen a case of Fusarium wilt in tory studies to field reality. How can spore produc- the wild.) In addition to trying to answer these ques- tion in vitro be improved to enable more reliable tions, we need a few good sites in Southeast Asia screening of breeding material? where different pathogens occur, where germplasm What is the field biology of pathogen populations can be given a good field test-all potential parents where diverse clones are growing together? Does that anyone might want to use. There should also be each host or host group perpetuate its own patho- at least one site in Africa. (There are two at FHIA in gen strain? Where plantains and Cavendish are grow- Honduras; one in Brazil at the breeding site-but the ing nearby in a region, are their pathogens the same? pathogens are not well characterized; and several in In the Philippines it appears that Pisang rmas is still Australia where the pathogens have been well char- perpetuating yellow Sigatoka while other clones acterized, but where many potential parents have not have black Sigatoka. In Africa it appears that the been tested.) On VCGs, what happens to pathogenic- black Sigatoka resistant hybrid FHIA 3362 does the ity when different pathotypes (races) within the same same, but not so for Pisang mas. These enigmas need VCG, anastomose? Many questions remain. analysis. The recommendation is to go after the answers, espe- As for Fusarium, much is known about Mycos- ciallywheretheywillaffectvulnerabilityofnewvarieties, phaerella at the molecular level that is not linked to or of old ones in new areas, and where they will affect field reality. Likewise, the key ammunition on durability of resistance. We therefore need to know host/parasite interaction is present in the field, either the genetics of resistance/susceptibility and of viru- in collections or in breeding locations where host lence/avirulence, and cold stress vulnerability in re- diversity exists (for yellow Sigatoka, in Guadeloupe lation to pathogen strain differences, needs clarification. and distant Australia-ideal for comparative stud- ies). Analysis on-site, with linkage to laboratory Mycosphaerella work, and comparisons between Asia, Latin America, and Africa, are needed. Finally, both GxE effects and Strains in the Pacific appear to break resistance of genetics of virulence and resistance need clarification. some key parents (and progeny) used in breeding. The role of toxins is poorly understood. Could bet- How important is this to Latin America and Africa? ter research on toxins and host-parasite biochemistry Since the black Sigatoka pathogen has a perfect stage, lead to more precise control chemicals or biotech- recombination of virulence genes could readily occur nological routes to control? in areas of pathogen diversity unless these genes are allelic. Virulence genetics needs studying by making Other Leaf Fungi crosses of strains with different pathogenicities. Are strains in commercial plantations in Latin There are two different "speckle" diseases in Asia and America, Asia, and Africa different in aggressive- a disease called freckle. The Cladosporium speckle seen ness? in virulence (host range) genes? in fungicide recently in Indonesia and Malaysia is severe on resistance levels? Is the better control of black Siga- P. berangan, and unsprayed stands of this clone may toka in the Philippines due to different fungicide be as severely diseased as when affected by black 18 BananaImprovement: Research Challenges and Opportunities

Sigatoka. Cavendish may be equally susceptible but Rapid testing of putative BBTV-resistant bananas, it is sprayed, so we don't know. soon to be developed, for immunity, tolerance, and Also, in relation to potential future fungicide use, tolremicity should be backed vigorously. Also, con- if we had resistance to black Sigatoka, how much structs with the Asian strain should be developed. The spraying would be needed to control speckle and key commercial diploids in Asia should be trans- freckle where these diseases now occur, and would formed and the key diploids breeders would use to they become widespread problems? Differential recreate, de novo, a new Cavendish and a new AAB fungicide applications now, in experimental trials, dessert banana. Better linkage should be fostered with might tell us. the Philippines, the Pacific, and possibly elsewhere in Note that clones differ widely in resistance to these Asia. diseases-and no one is considering this while select- ing Sigatoka-resistant parents. Also, what will hap- Banana Streak Virms pen to crown rot and fruit diseases without spray? This could be tested now with FHIA 1 plots. Because of its apparent seed-transmission character, this virus is currently blocking dissemination of new Nemnatodes varieties from IITA and is also a threat to new varie- ties emanating from FHIA. It therefore needs imme- Much is written about nematodes but no breeding diate attention in terms of understanding its transmis- program is currently evaluating either parents or sion through pollen and/or ovules, and of progeny for reaction to nematodes. Possibly this developing eradication therapy. Also, research is work could be centralized in a few locations in the needed on the possibility of symptomless carriers in tropics/subtropics where species differ and research the parents in use, and of its presence or absence in competence exists. the major clones in different regions. Where field If laboratory work is set up outside these areas it spread is evidently occurring, the biology of this would need careful linkage and funds for such link- spread needs to be understood. IITA is the logical age. Although much has been written about differ- place to support this research, which would be rele- ences in population aggressiveness, it is not clear that vant to breeding programs elsewhere. Appropriate different pathotypes occur. Collection in the centers of relevant linkage with badnavirus experts in temper- coevolution and appropriate testing on a diversity of ate countries is in order. At breeding sites where field host clones is needed to determine this. Meanwhile, nurseries are grown, it should be determined if these one only needs to work where aggressive strains are locations for vector transmission. Diagnostic operate. work is already underway. For Cavendish, resistance to Radopholus is the major need. Resistance to Pratylenchus coffea would also be Bract Mosaic Virus (and CMV) useful, and for the subtropics, resistance to Meloi- dogyne (as in the Canary Islands, where also P. goodeyi Bract mosaic virus occurs in the Philippines in com- is important as it is also in highland Africa). Root knot mercial plantations as well as in local bananas. It may nematodes were seen to be severe in tropical Sulawesi occur in India and elsewhere but little is known about on Cavendish, and thus in Asian regions where ba- it. Work on diagnostics is under way in Australia nanas are becoming commercialized there may be a (QDPI) and this needs support as well as field re- real need for resistance to such nematodes as well. For search on transmission and the sources in alternate plantains in Latin America, resistance to both Rado- hosts, and on field management. As for BSV and pholus and to Pratylenchus coffea would be essential. CMV, one may question the need for transformed Although some work is underway on biological con- resistance against these viruses for dessert bananas. trol, and more should be done on this, field payoff is Possibly a transformed ABB cooking banana for bract likely to be years away. mosaic would be useful. For bract mosaic, the field biology should be collaboratively studied in Mindanao Banana Bunchy Top Virus in the Philippines.

The excellent work in Australia at the molecular level Bacterial Wilt on this virus needs to continue and be applied in the field. The payoff will be bananas with trans- The diseases known as Bugtok in the Philippines, formed constructs for resistance that will provide "blood disease" in Indonesia, and "Moko" in Latin either immunity or tolremicity (low field spread). America and the Philippines are all caused by various BananaResearch Needs and Opportunities 19 strains of Pseudomonas solanacearum.Commercial plan- natural groupings leading to progenitor identities tations suffer from these diseases in the Philippines; and heterotic groupings; (iii) better characterization and in Latin America, where most recently a new of subspecies and clones, especially on resistances banana development in Venezuela was abandoned and agronomic characters; (iv) more tetraploidization due to Moko disease. Development of commercial of diploids; (v) cellular biology research of various plantations in blood disease areas of Indonesia is types, including cvtology, to go after fusion to obtain threatened by this disease, about which little is known. triploids and to manipulate chromosomal fragments Thus, there are sufficient unknowns about these and parthenocarpy; (vi) characterization of flavor of diseases, their epidemiology, and about the ecology potential parents, including flavor of seeded diploids; of their different specific pathogens, to merit research (vii) characterization of postharvest behavior of po- to understand their field behavior and their patho- tential parents; (viii) studies on ovule and pollen for- genic strain origins and relationships. With carefully mation targeted at obtaining better seed set with 4X focused research, both the damage and the threat of by 2X crosses. these diseases to commercial banana production, and to development in new areas where the pathogens Biotechnology/Molecular Biology occur, can be greatly lessened. Biotechnology work should be focused on the target Traditional Breeding of a better Cavendish. Success here would have the greatest payoff. Later, the techniques could be applied Traditional breeding works in improving the resistance to other banana classes. Various disease (and insect) and the agronomy of the best clones represented by resistances are the most obvious to manipulate common plantains and AAB dessert types (3X by 2X = through transformation (and also through cellular bi- 4X) because they are poor. The three breeding programs ology techniques). These have been discussed above. pursuing this approach have probably gone about as A second area to manipulate is ethylene biosynthesis far as they can, although various resistances could still or action. Downregulation in the fruit to increase green be improved. This traditional breeding per se does not and shelf life would be of considerable practical worth seem to merit CFC/FAO/WB funding, but specific col- and would probably result in less crown-rot and peel laborative projects could usefully be developed by and disease. Manipulation of ethylene in leaves and roots with the traditional breeding programs. Also, tradi- may well affect disease reactions. Although more dif- tional breeding programs could be supported for ficult, but valuable to be able to manipulate, would be development of improved diploids, if tlese are made the hormonally regulated key agronomic characters of availableto otlhers.This could be very useful for innova- height and ratooning rate, and of tolerance to stress, tive breeding programs and biotechnology groups. especially cold, as discussed below.

Triploid Resynthesis Agronomic Characters

Triploid resynthesis to obtain a new Cavendish, a A commercial banana must be short and must cycle superiorAABdessert,andothernichebananas,merits rapidly with good ratooning. Little is known about top priority for support. These objectives and the basic the genetics or the physiology of these characters. and applied research required for a rapid successful Research in this area is needed to support breeding attainment of the objectives should be supported. This programs. It is clear that cellular biology techniques will require organizing and commissioning such re- can change these characteristics (somaclonal vari- search, and this should occur in at least two locations. ation), but it would be most advantageous to be able One definite location is Guadeloupe, where the to convert an otherwise ideal but too-tall clone into CIRAD scientists have already made good progress. one of the right height, at will. Fruit and bunch mor- Hopefully, the techniques can be shared and devel- phology and geotropv are other characters important oped elsewhere, closer to the center of origin of Musa, to acceptability that are little understood genetically as amplification and diversity of output will occur. or physiologicallv. Research backup is required in several areas: (i) prospection, collection, and analysis inisitu of more Genotype by Environment Interactionl Musa, including wild species, while simultaneously Cold Tolerance analyzing the pathosystem locally, and collecting the pathogens; (ii) better taxonomic work on Musa, both The wide environmental stabilitv of Cavendish is traditional and molecular, focused on describing unusual. It is already clear from the behavior of dip- 20 Baania Inllproveiilent:Research Clallenigesand Opportunities loid parents and of FHIA 1 that their amplitude of of transmitting their characteristics to progeny is al- adaptation for both agronomic characters and for fruit most unresearched. flavor, etc. is much narrower than Cavendish. FHIA 1 There is another aspect of postharvest research that is much superior in the cooler part of Queensland than needs development and exploitation, especially in in the tropical part, where it is unacceptable. Some expectation of the emergence of highly productive FHIA diploids "melt away" in cooler Queensland. ABB cooking bananas and of plantains. What "value Breeders have largely ignored any potential GxE added" products can be made from them to commer- interaction possibilities for both parents and progeny. cialize them and even reach international trade? Also, In Taiwan it is recognized that winter fruit is es- with the enormous tonnage of reject Cavendish ba- teemed on the market and that their summer fruit is nanas that high quality standards dictate, what can be of poorer quality and only equals tropical fruit, and produced from them that is economic? Industry has thus they cannot compete. So for both productivity, looked at this question, but it is considered that the growth, cycling, and fruit characters including flavor, issue needs a new look. These points reveal that there there are GxE interactions, almost totally unexplored are three quite different areas of postharvest research by banana researchers. This great deficiency needs that could be developed. addressing. Especially important would be to have a few sites in the subtropics, e.g., Southern Queensland InsectsIMites or the Canary Islands, where all potential parents and progenies emanating from breeding programs could Various insects and mites damage bananas, and the be evaluated and compared with their performance amount of damage and pesticides used varies from in a few tropical locations. This should include both place to place. The industry generally controls insects agronomy and fruit characters. well and has backed away from the former practice of Results would reveal parents with wide amplitude abundant use of insecticides, allowing natural bio- and parents with cold tolerance. One could then breed control factors to operate. However, the need for bananas better adapted to cool climates. Likewise one blemish-free fruit dictates bud injection or other treat- could determine parents producing better fruit in hot ment for scab moth control in the Australasian region climates. where this insect occurs. Also, generally, bunch-cover Such experiments could even be carried out on plastic bags are impregnated with a pesticide to keep existing Cavendish clones to see if some are really down various species that blemish fruit. The so-called better in cooler climates (or in warmer ones). Like- weevil- borer, Cosmopolites sordiduis,is the pest receiv- wise, deficiencies in parents in use would be revealed, ing most outside research. Plantations generally do and they could be discarded before investing years in not treat for this insect. The amount of damage in their use. The industry should be interested in partici- "local" bananas varies from place to place but the pest pating in this GxE research. may be overrated. Research towards improved biocontrol is con- Postharvest Research ducted on a small scale and it would be worthwhile to intensify research towards this end. The industry The industry knows well the postharvest perform- could do more on IPM in commercial plantings. Since ance of Cavendish, and has invested millions in regu- there are constructs already in use for transformation lating that performance to be within narrow limits. for resistance to certain insect groups, it might be of They will not be amenable to adopting a new banana interest to experiment with them in bananas. How- with inferior, or even different, performance. A ever, this possibility should be reviewed further to breeding program paying insufficient attention to make a better judgment on the pros and cons, before both flavor and postharvest characteristics of parents proceeding. is bound to fail, because the industry will not change There are differences in susceptibility to weevil- for a resistant clone whose fruit character has been borers, and presumably to other insects, but this has lessened. There are many other obstacles: finger drop, been little studied. In relation to breeding, this area short green life, short shelf life, soft fruit, bruisable merits more work. Also, as mentioned under viruses fruit, etc. Much more characterization of potential and bacterial wilt, more research on vectors should be parents is in order. Their breeding behavior in terms carried out. 2 Overview of the Banana Improvement Project

Gabrielle J. Persley

Bananas and plantains play an important role in the of great importance and potential. This philosophy nutritional and economic status of millions of families underpinned the establishment of the Banana Im- in tropical countries of Africa, Asia, Latin America, provement Project for the benefit of the millions of and Oceania. Although dessert bananas are mainly small producers in developing countries. exported, plantains and cooking bananas are impor- To address the serious disease problems confront- tant sources of food. Only 10 percent of the bananas ing the export banana industry, the Common Fund produced are exported. However, banana is one of for Commodities (CFC), the FAO Inter-Governien- the five most important crops of export interest to tal Group on Bananas (FAO/IGB), and the World developing countries. Annual exports have doubled Bank agreed in 1993 to establish a new 5-year project in the past 25 to 30 years to the present level of about on banana improvement. The World Bank agreed to 9 million tons. Demand has also risen markedly in the act as the Project Executing Agency (PEA) at the re- major consuming industrial countries, which ac- quest of the CFC and the FAO/IGB. The project com- counts for 90 percent of total world imports. menced in January 1994. Dessert bananas and plantains (both Musa genus) are susceptible to disease. The export banana crop is Objectives especially vulnerable since it depends on a narrow genetic base. Presently the industry depends mainly BIP has two main objectives: on clones of a single variety "Cavendish," which is under attack by the highly damaging black Sigatoka . To develop and evaluate a range of improved ba- disease, as well as new races of Ftusariurn wilt. The nana varieties with export potential, incorporating costs of production have increased substantially due increased productivity and durable disease resis- to the need for increased fungicide applications tance through conventional and nonconventional against black Sigatoka disease. However, pesticides breeding techniques. cause environmental damage and are too expensive . To develop more efficient and integrated disease for many small producers. management practices, especially for black Siga- No suitable altemative to Cavendish in terms of toka disease. productivity, quality, and market acceptability exists to date. Disease-resistant varieties are the only viable The research undertaken to address the two objec- option for the many smallholders growing bananas tives of the project has been commissioned from pro- and plantains as staple foods for local consumption. posals selected from leading research institutes Since research on one Musa type is directly relevant worldwide, on the basis of competitive research and beneficial to all other types, the spillover effects grants. Scientists from over 200 national, regional, of research on dessert bananas, cooking bananas, and and intemational research institutes were invited to plantains for both local consumption and export are submit proposals. The Project Executing Agency estab-

21 22 BanianaIniprovemtienit: Research ChallenLgesanid Opportunities lished an internationally competitive research grants * The epidemiology and ecology of black Sigatoka scheme in 1994. This was an innovative approach disease, as a basis for better management of the dis- agreed to by the cosponsors, and aimed at attracting ease, through reducing the frequency of fungicidal high-quality scientists worldwide to participate in the applications while still maintaining effective control. program and contribute towards its objectives. A Scientific Advisory Panel was convened by the Initial Project Portfolio World Bank in 1994, to advise on the research propos- als received. The panel consisted of several eminenit The seventeeni currently contracted commissioned re- scientists, with expertise in plant breeding, molecular search projects are listed in Table 3.1 and are briefly biology, and pathology, as well as knowledge of Musa described in Chapter 3. Project leaders have submit- research needs. An FAO scientist also participated in ted detailed work programs and objectives, which the meetings of the panel. The panel met with repre- give their key tasks and time frames. This information sentatives of industry and the development assis- will assist the cosponsors in monitoring the research. tance community to discuss their views on research In association with the Second Annual Meeting of priorities, prior to the Panel's consideration of the the BIP Cosponsors, a meeting of principal investiga- individual research proposals. tors of the commissioned research projects was held The panel assessed all the research proposals re- at the Katholeike Universiteit Leuven (KUL), Bel- ceived, in terms of their relevance to the Project objec- gium. The meeting also included some members of tives, as well as their scientific quality and likely the Scientific Advisory Panel and other experts to chance of success. The panel recommended approxi- assist in the review of the quality and relevance of the mately 20 proposals for possible funding from over research. The meeting reviewed progress to date, 100 proposals received. helped build synergies amongst existing activities, Research progress by the commissioned research and identified gaps requiring further attention. teams is summarized later in this report (Chapter 3). The most important research highlights are sum- marized below, for the key thematic areas of banana Commissioned Research breeding, Mutsa germplasm collection and evaluation, biotechnology, and disease control. Details are con- Research Strategy tained in the individual progress reports containied in Chapter 3. The research strategy being pursued by BIP, through its portfolio of commissioned research, is to foster the Banana Breeding development of new banana varieties with export potential and to reduce excessive pesticide use, The objective of the diploid evaluation project being through two approaches: conducted by the Centre de cooperation international en recherche agronomique pour le developpement New banianiavarieties, developed tlhrouigh (CIRAD) in Guadeloupe is to evaluate about 200 dip- loids to assess their potential usefulness as parents in * Musa germplasm collection, evaluation, and en- banana breeding programs. Field evaluation for agro- hancement to generate new varieties with higher nomic characteristics of some 70 diploids has been yields and improved pest and disease resistance. initiated in Guadeloupe. Arrangements are being * Biotechnology-based breeding to genetically mod- made for the diploids to be evaluated also in New ify presently cultivated clones to enhance key char- Caledonia for black Sigatoka resistance, in Australia acteristics, such as resistance to selected fungal and for Fuisariun,iiwilt resistance, and WINBAN in the viral diseases and delayed ripening to extend shelf Caribbean for yellow Sigatoka resistance. life. A second project also being implemented by CIRAD in Guadeloupe and Cameroon will focus on the inheri- Imnprovedpest and disease control practices, throuiglha tance of black Sigatoka (black leaf streak) resistance in better uinderstanditngof bananas. The objective is to identify the most promis- ing sources of r esistance for use in breeding programs. * Sources of resistance genes and variation in the Screening using existing techniques has started in major pathogens, causing black Sigatoka and Cameroon, complemented by studies in Guadeloupe, Fusariunmwilt, to enable more accurate and rapid to identify resistance genes by molecular markers to screening of potentially resistant varieties and pa- expedite breeding using new technologies in conjunc- rental material for use in breeding programs. tion with established breeding practices. Overviewof the BananaImprovement Project 23

The status of the banana and plantain breeding by the International Plant Genetic Resources Insti- program at FHIA, Honduras, was appraised during tute/International Network for the Improvement of 1995. A mission sponsored by the World Bank and the Banana and Plantain (IPGRI/INIBAP) and UNDP, United Nations Development Programme (UNDP) and focus on the agronomic and molecular charac- was undertaken to the Fundacion Hondurena de In- terization of the Musa collections in Viet Nam. vestigacion Agricola (FHIA), Honduras, in May 1995, by Dr. L. Sequeira from the BIP Scientific Advisory Safe Exchange of Musa Gernplasm Panel, Dr. G. Wilson from the FAO/IGB Technical Advisory Committee, and Dr. G. J. Persley, the BIP Safe international movement of Musa germplasm is Project Manager. Its purpose was to consult with threatened by a number of virus diseases. One of the FHIA scientists and management on ways to involve newly reported viruses is banana streak badnavirus FHIA more closely in the banana breeding activities (BSV), which is widespread in Africa and also occurs being sponsored by BIP. Although potentially useful on other continents. The lack of rapid and reliable banana and plantain types have been bred at FHIA indexing methods and procedures to remove BSV over the past 30 years, the first new banana variety from Musa germplasm and the inadequate under- (Goldfinger) was just released commercially in 1995. standing of the distribution and extent of damage Its release has stimulated new interest in the potential caused by BSV is a major impediment to Musa for breeding additional banana varieties, especially germplasm exchange worldwide. Research at the In- if conventional breeding can be linked actively to ternational Institute of Tropical Agriculture (IITA) is modern biotechnology. For example, this could be concentrating on developing improved diagnostic done through the use of molecular mapping tech- tests for BSV and improved therapeutic methods to niques to accelerate breeding, and the introduction of develop virus-free material. These techniques will be novel genes for disease resistance or postharvest of value to allbanana/plantain programs worldwide. quality into varieties with other desirable agronomic They will also facilitate the wider distribution of im- characteristics. proved banana and plantain germplasm by IITA for its agronomic evaluation and use, both within Africa Musa Germplasm Collection and Evaluation and on other continents.

Musa germplasm collection and its evaluation has Banana Biotechnology been initiated in India and Viet Nam, both centers of diversity of dessert bananas where the range of types Methods for banana transformation and regeneration has not been fully collected and evaluated for their have been developed by participating laboratories in commercial potential. the banana biotechnology consortium initiated by BIP The Viet Nam Institute of Agricultural Genetics (May et al. 1995, Sagi et al. 1995). These methods will hosted a workshop and field visit in October 1995 enable, for the first time, the introduction of poten- related to its BIP-sponsored research, and concerning tially useful genes into existing varieties such as banana production, biotechnology, and biodiversity. Cavendish, to improve characteristics such as pest The purpose of that workshop was to discuss the and disease resistance and postharvest quality, in- details of the proposed research with scientists within cluding improved shelf life. Viet Nam and with some outside experts to establish Scientists from the laboratories participating in the collaboration with other relevant institutes in Viet biotechnology consortium [Boyce Thompson Insti- Nam and to identify training needs. tute (BTI), Katholeike Universiteit Leuven (KUL), and The 50 participants were mainly from within Viet the Queensland University of Technology (QUT)], Nam, and a network of banana researchers has been met in Leuven in June 1995 to review progress to date established within the country. Collaborative re- and to plan future collaborative research. Current search has been established between the Institute of effort is concentrated on ensuring the reproducibility Agricultural Genetics and the research stations con- of the banana transformation and regeneration proto- taining the two largest Musa collections in Viet Nam, cols in different laboratories, and in identifying and Phuho Centre for Fruit Research in North Viet Nam, effectively introducing agronomically useful genes and Long Dinh Fruit Research Centre in South Viet into banana varieties for evaluation. Nam. The three institutes have agreed to collaborate The genetic engineering techniques are being ex- in the evaluation and use of collected materials. This changed and evaluated initially amongst the three will enable the BIP-sponsored research to comple- participating laboratories in the consortium and are ment earlier Musa collections in Viet Nam sponsored also available to other interested scientists. A molecu- 24 BananaImprovement: Research Challenges and Opportunities lar "toolbox" is being developed which will be made Fusarium wilt. It would also be advantageous for available to other laboratories. Initial training of sci- these materials to be screened in Indonesia and Ma- entists from producing countries in the use of the new laysia against the locally occurring and damaging molecular techniques was undertaken in 1995 by the Fusarium strains. consortium members. A genomic library of banana has also been prepared at BTI, and useful genes and Sigatoka Disease promoters identified and made available to collabo- rators interested in isolating banana genes. The emergence of fungicide resistance in the black Banana biotechnology research being supported by Sigatoka pathogen in Costa Rica has been a cause of BIP also concerns the identification, introduction, and concern there and elsewhere in Latin America. It is evaluation of potentially useful genes. Genes are reducing the efficiency of disease control leading to presently being introduced into various Musa types more pesticide applications, with less effect. BIP- to enhance resistance to BBTV. Additional genes to supported research at Corporacion Bananera Na- enhance resistance to fungal diseases, especially black cional de Costa Rica (CORBANA), Costa Rica, in- Sigatoka and Fusarium wilt, are being sought for volves development of a greenhouse screening evaluation. method for black Sigatoka disease, examining the The rapid developments in banana biotechnology epidemiology of the disease and the development of during 1995, which have been stimulated in part by resistance in the pathogen, with the view to develop- BIP's initiation of the banana biotechnology consor- ing improved disease management. The research is tium, are producing useful results. There is a need for jointly supported by BIP and the Costa Rican banana the wider application of modern molecular biology to industry, through CORBANA. It is being conducted banana and plantain improvement, especially in the in collaboration with three of the major banana ex- areas of the development of enabling technologies, porting companies in Costa Rica (Dole, Del Monte, the identification of potentially useful genes for dis- and Geest), each of whom are also contributing ease resistance, and the development of molecular through the conduct of experiments on their farms. maps to accelerate breeding. Nematode Resistance Fusarium Wilt (Panama Disease) Given the environmentally toxic effects of nemati- The presence of a tropical race of the Fusarium wilt cides from banana plantations, the incorporation of pathogen able to attack Cavendish types of bananas nematode resistance in any potential new varieties is in Asia has been confirmed from several sources. This very important. A BIP-initiated research consortium race is causing severe losses in new banana planta- has been established amongst Musa breeding pro- tions, especially in Indonesia and Malaysia. A full grams in FHIA in Honduras, IITA in Uganda and understanding of Fusarium wilt variability in Asia Nigeria, and nematologists at KUL, Belgium; and the and the identification of potential new sources of Institut de Recerca i Tecnologia Agroelimenteries/ resistance are highly desirable. BIP has commis- Centro de Investigacion y Tecnologia Agraria (IRTA/ sioned research led by the Queensland Department CITA), Spain. KUL is the convener of the consortium. of Primary Industries (QDPI) to collect and charac- The objective is to develop more effective and effi- terize isolates of Fusarium oxysporum f.sp. cubense (Foc) cient screening of new banana and plantain varieties in the center of origin of Musa spp. in Asia. Two for their tolerance to nematodes. This consortium missions collected isolates in Southeast Asia in 1995. represents the first substantial input of nematologists These isolates are being characterized, by biochemical into Musa breeding programs. BIP's initial support for and molecular means, to determine the pathogenic the nematode/breeding work has catalyzed the iden- variability of the Fusarium wilt pathogen. Arrange- tification of substantial cofinancing funds from devel- ments are being made to screen potential new varie- opment agencies in Belgium (BADC and VVOB) that ties and parental breeding material from Musa breed- will be financing most of the field work at FHIA, ing programs of FHIA in Honduras and CIRAD, Honduras, and IITA, by locating nematologists at Guadeloupe, in Australia against the new race 4 of each site. 3 Portfolioof Projects

Table 3.1. Banana Improvement Project: Commissioned Research Projects Title Leadagency Collaborators Breeding and Genetic Resources 1. Field and laboratory evaluation of diploid CIRAD-FLHOR,Guadeloupe QDPI, Australia; FHIA, Honduras; bananas for their use in breeding IITA, Nigeria; IAEA, Austria 2. Field cross experiments to clarify the mecha- CIRAD-FLHOR,Guadeloupe BIOTROP,France nism governing the inheritance of Black- Leaf-Streak-resistantcharacters in banana 3. Developing improved varieties with pest Kerala Agricultural University, Viet Nam; Brazil and disease resistance, postharvest supe- India riority, and maximum export potential 4. Collection, evaluation, and characterization Ministry of Agriculture and Food, Inst. of Plant Protection, Viet Nam, of genetic resources and improved banana Viet Nam Phuho Centre for Fruit Research, crop plants in Viet Nam Long Dinh Fruit Research Center 5. Banana breeding in Brazil EMBRAPA,Brazil USP-CENA,CATIE, Costa Rica; FHIA, Honduras; INIBAP, France; CIRAD-FLHOR,Guadeloupe Biotechnology BiotechnologyConsortium 7. Genetic transformation and non-conven- Boyce Thompson Institute Queensland University of tional breeding technology for improve- for Plant Research, USA Technology, Australia; Katholeike ment of a molecular toolbox for banana Universiteit Leuven, Belgium BiotechnologyConsortiuim 8. Novel genes for fungal resistance and post- Boyce Thompson Institute MSU-DOEPlant Research Lab.; harvest quality for Plant Research, USA FHIA, Honduras

BiotechnologyConsortium 9. The development of transgenic bananas Queensland University of UPLB,Los Baiios, BPI Davao, with resistance to BBTVand BBMV Technology, Australia Philippines; Hanoi Agricultural University, Viet Nam; University of Hawaii, USA (Tablecontinues on thefollowingpage.)

25 26 Banana Improvement: Research Challenges and Opportunities

Table 3.1. (continued) Title Lead agency Collaborators

Biotechnology Consortium 10. Genetic transformation of prototype Katholeike University Leuven, Queensland University of bananas for black Sigatoka and Fusariutm Belgium Technology, Australia; Boyce resistance Thompson Institute, USA 11. Genetic engineering of ethylene Hong Kong University of Science Boyce Thompson Institute; biosynthesis in bananas and Technology CIVESTAV, Mexico; National Taiwan Univ.; FHIA, Honduras; Hainan Province, China 12. Use of biotechnology to produce University of Hawaii, USA Queensland University of transgenic bananas that are resistant to Technology, Australia; Hawaii BBTVinfection Banana Industry Association, USA; Boyce Thomspon Institute, USA Pest and Disease Control: Diseases 14. Elimination of BSV from improved Musa IITA, Nigeria Univ. of Minnesota, USA; John germplasm and related studies on Innes Institute, UK; INIBAP, France transmission and host plant/virus/vector interactions 15. Variability and relationships within Queensland Dept. of Primary Univ of Florida, USA; Institute of Fusarium oxysporum f.sp. cubense from Industries, (QDPI) Australia Plant Protection, Viet Nam; its centre of origin MARDI; University of Malaya, Malaysia 17. Origin and distribution of fungicide- CORBANA, Costa Rica Univ. of Hawaii; North Carolina resistant strain of M. fijiensis in banana State Univ., USA; FHIA, Honduras plantations in Costa Rica Pest and Disease Control: Nematodes 18. Tolerance/resistance of banana to CRBP, Cameroon IITA, Nigeria; FHIA, Honduras; nematodes CIRAD-FLHOR,Guadeloupe; KUL, Belgium Nematode Consortium 19. Identification of nematode resistance Katholeike Universiteit Leuven IRTA/CITA, Spain; FHIA, sources in banana and plantain Honduras; IITA, Nigeria; and Uganda Nematode Consortium 20. Identification of pest and disease FHIA, Honduras KUL, Belgium; IRTA/CITA, Spain; resistance sources in banana and plantain IITA, Nigeria; and Uganda Nematode Consortium 21. Development of techniques for the IRTA/CITA, Spain KUL, Belgium; FHIA, Honduras; identification of nematode resistance IITA, Nigeria; and Uganda sources in banana and plantain: screening methodologies Portfolio of Project 27

1. Field and Laboratory Evaluation of Diploid sion, these data will be specified with cycle duration Bananas for Their Use in Breeding and fruit quality. A morphotaxonomic description will be the last step before laboratory characterization Lead institution: CIRAD-FLHOR, Guadeloupe using molecular markers. Principal investigator: Christophe Jenny 2. Field Cross Experiments to Clarify Banana breeding programs traditionally rely on the the Mechanism Governing the Inheritance use of diploid clones, regardless of the end-product of Black-Leaf-Streak-Resistant Characters to be created (tetraploid or triploid, local consump- in Banana tion, or export bananas). Intensive breeding efforts can only be invested upon a relatively small number Lead institution: CIRAD-FLHOR, Guadelouipe of accessions at any one time. Thus, a rational basis is Principal investigator: Frederic Bakry needed to identify the material of greatest potential, both within the natural populations as well as Screening for resistance against Black Leaf Streak amongst the intermediate breeding products (diploid (BLS) leads to a range of bananas from highly suscep- hybrids), for their use for further breeding. tible to resistant. Two different forms of resistance have been described. With the first one, reported as Objective highly resistant (HR), symptom development is blocked at stage 1 or 2 and there is no sexual or asexual The main objective of the project is to obtain agro- sporulation. With the second one, which is called nomic and molecular evaluation data on landrace Partial Resistance (PR), the disease develops slowly diploid cultivars and on bred hybrid diploids. Pre- from stage 1 to necrosis, leaving a large number of liminary evaluation data will include information on functional leaves at harvest for a long time. There is basic agronomic characters such as cycle duration, asexual and sexual sporulation. plant height, ratooning, bunch characteristics (shape, The genetic determinism of inheritance of these weight, number of hands and fruits), fruit charac- forms of resistance is unclear. HR phenotype reaction teristics (shape, length, taste), and resistance to Black is suspected to be based on a monogenic or oligo- Leaf Streak disease. genevic vertical host-pathogen interaction, and there- Basic agronomic data will be recorded for about 200 fore liable to be broken down due to the spread of diploids, including 85 acuminata cultivars. Evaluation different virulent strains in the pathogenic popula- for resistance to M.fijiensis will be evaluated on clones tions. PR phenotype reaction expressed on a continu- (hybrids and natural landraces) selected among them ous scale is a quantitative resistance and may rely on for good agronomic qualities. a polygenic, horizontal host-pathogen interaction, Two locations will be used: Guadeloupe (French and should consequently be durable facing the evo- West Indies) and New Caledonia (Melanesia). CIRAD- lution of fungus populations. FLHOR in Guadeloupe has a large collection of dip- Thus, the integration of durable resistance in culti- loid clones (wild and cultivars) and many crosses and vated export bananas requires basic studies on the hybrids. Most of the main diseases are absent, allow- genetic determinism on both forms of resistance. This ing for a good expression of agronomic value. New knowledge on the inheritance of both types of resis- Caledonia is of great interest for the selection of clones tance is necessary for conventional breeding as well and hybrids for disease resistance, as very aggressive as for the biotechnology/transformation strategy to strains of M. fijiensis have been identified there. The ensure durable resistance to the fungal pathogens. diploids will also be evaluated in Australia for their This study will lead to the identification of the resistance to Fusarium wilt disease. number of genes involved for resistance supported by mendelian determinism and the number of quantita- Research Progress tive trait loci (QTL) involved. These results will also lead to the molecular estimation and identification of During the first six months, it has been possible to allelic variability for genes conferring resistance to confirm the ploidy level of 74 assessions of the CIRAD- BLS in bananas. FLHOR collection, using the flow cytometry method. The project will benefit breeders in the implemen- Characterization of the clones has begun with ag- tation of strategies using various sources of resistance ronomic features. The first harvest data showed great to BLS. It will lead to the identification of the banana variability among these assessions. For each asses- genome regions coding for resistances. In the future, 28 BananaImprovement: Research Challenges and Opportunities these loci could be isolated and integrated in plasmids 3. Developing Improved Banana Varieties for transformation purposes for the production of with Pest and Disease Resistance, genetically transformed, resistant plants. Postharvest Superiority, and Maximum Export Potential Research Progress Lead itnstitution:Kerala Agricultuiral University, India Crosses have been conducted between three wild ho- Principal investigator: N. Krishnan Nair mozygous Musa acuminata clones displaying different types of reaction against BLS disease (S for suscepti- In spite of the fact that India ranks second in banana ble, PR, and RP). The selected clones are diploids, production among the various banana-producing fertiles, and homozygous in order to avoid most of countries in the world, the contribution to the world the segregation distortion due to the population export market is negligible. The largest area under ploidy or size and to facilitate interpretation. The Fl banana is in Kerala, but the productivity is very low, and F2 progenies derived from the three crosses HR x which is attributed to the intercropping system of S, PR x S, and PR x HR are being established at CRBP cultivation coupled with the prevalance of diseases in Cameroon. All field evaluations for resistance to such as BBTV and other pest and disease problems. BLS and for segregating morphological characters In this project, cultivation of bananas will be carried will be conducted at CRBP. out in a newly established Banana Research Unit in During the first six months, the selected parents Kerala, a humid tropical region of India. were intercrossed. The Fis have been planted after in This project will link traditional breeding with vitro germination of the three seed populations were modem molecular technology aimed at improving obtained. Initial results suggest that HR form of resis- the fruit quality, bunch weight, and plant height to tance is dominant over the susceptible form. There- enhance the export potential of the crop. Musa species fore, introgression of the HR form of resistance into will be collected from all available types both from susceptible clone will be easier; at the diploid level, it India and other countries. The morphological, quan- only needs to be present at a heterozygous state. This titative, and quality characteristics will be studied to result will be confirmed by the study of the F2 progeny ascertain the influence of the genomic constitution of issued from the selfing of one Fl. the clones, the extent of genetic diversity, and the In the next phase of the project, the study of the two relative contribution of various characters toward other F1 progenies will give information on the reac- genetic diversity. Clones displaying desirable tion of the PR phenotype of resistance toward the traits will be selected for the breeding program susceptible and the HR phenotypes. The F2 popula- and will be further improved using techniques of bio- tion issued from the HR x S cross will be duplicated technology. in vitro. After planting in two different locations, ob- Spontaneous mutations in the AAB group have servations for BLS resistance will be undertaken. By been reported from India, which is the original home the end of the project, it will be possible to estimate of this group. The clone is grown in vastly different the number of genes involved in each form of resis- agroclimatic areas of India and is therefore extremely tance by studying the F2 populations. The main genes variable, with six natural mutants/ecotypes of the involved will be localized on the genome by molecu- variant Palayankodan reported. These clones have lar markers. shown variation in fruit yield, pest resistance, and in In the next phase of the project, molecular tech- fruit quality. Clones of Palayankodan, Njalipoovan, niques will become available at CIRAD-FLHOR in and another naturally occurring type, Nendran, will Guadeloupe. Two types of markers will be devel- be collected, conserved, and analyzed. Superior geno- oped: microsatellites using the PCR technique and types will be isolated and tested under different agro- genomic probes using the RFLP technique. The mark- climatic situations in Kerala. ers mapped at CIRAD-GERDAT will be used and The most popular cultivar, Nendran, will be im- tested to identify polymorphisms between the three proved by reducing height and duration, increasing selected parents. By the end of the project, two F2 yield, and tolerance to pests and diseases, especially populations will be characterized with molecular Sigatoka. Improvement of Palayankodan will be markers. The F2 clones issued from the HR x S and PR aimed at reduction of fruit acidity, and Njalipoovan x S crosses will allow the molecular localization of the will be improved for stature, fruit size, and yield. main loci involved in the two forms of resistance to This latter variety is of importance as it is a shade- BLS disease. The molecular markers identified will tolerant variety suitable for the intercropping system then be used by breeders to assist selection. with perennial trees used in many home gardens. Portfolioof Project 29

The project aims to breed a variety that will meet tion of virus diseases. The new varieties must be the heavy demands of internal and export markets; introduced into large-scale production in regions and display a high yield and quality with reduced with suitable soil and climate conditions. potential for damage in storage and transit for the export market. Research Progress

4. Collection, Evaluation, and Characterization A Workshop on "Banana Production, Biotechnology, of Genetic Resources and Improved Banana and Biodiversity" and field trip, funded by the Gov- Crop Plants in Viet Nam emient of Vietnam and the CFC/FAO/World Bank project, and organized in Hanoi in October 1995, con- Lead institution: Institute of Agricultural Genetics, tributed greatly to the future orientation of banana Viet Nani research and banana research cooperation. Some sug- Principal investigator: Do Nang Vinh gestions on future research activity in our project were presented there by Dr. Ivan Buddenhagen and Viet Nam is one of the centers of origin of MAusa spp., Dr. Nader Vakili for discussion at the workshop. We and has many species, varieties, and clones. Many of highly appreciated this input. these are highly productive and disease resistant or Field trips have been organized to evaluate banana have other special qualities. This project combines production problems and to collect banana genetic collection and evaluation of Musa germplasm with resources. A total of 95 new banana assessions have the identification of new banana varieties with export been collected from different locations. potential. Agreement has been reached between the Institute Banana is the most important fruit in Viet Nam, of Agricultural Genetics, the Phuho Center for Fruit ranking first in importance among fruit exports and Research (North Viet Nam), and the Long Dinh Fruit providing income for small-scale farmers and retail- Research Center (South Viet Nam) on cooperation in ers. The banana export trade is primarily based on evaluation and mutual use of the collected material local varieties of Cavendish cultivars, which origi- for breeding purposes. nated in Vietnam. A significant part of banana produc- The field collection in HaiHung province is neces- tion is consumed locally, contributing significantly to sary to duplicate the collection of banana in Viet Nam, the Vietnamese diet and nutrition. to avoid losing gerrnplasm. The HaiHung station will The former Soviet Union and countries of Eastern provide the materials and facilities for banana breed- Europe were once the main importers of Vietnamese ing and genetic research. The collected materials have bananas, but currently the quantities required are not been planted at the station. available. Most Vietnamese bananas are exported to For future breeding research, a cytogenetic study is China. Improvement in both quality and uniformity required to provide information on ploidy levels of of product is required to enable Viet Nam to export the genotypes. The fertility and sterility of the pollen to other countries. The Government of Viet Nam has in relation to ploidy level of cultivars or wild species recently made a decision to increase fruit exports to have been established. A method for chromosome achieve a return of US$100 million annually by the observation in banana has been developed. Chromo- year 2000. Bananas are a first priority as a fruit export. some numbers of nine local banana varieties have Banana is replacing rice as a crop on 10 percent of been identified. the area currently under rice cultivation, adding to the Major fungus and virus diseases have been identi- 90,000 hectares already under banana. The current fied. Three virus diseases (BBTV, BSV, and BBMV) banana crop is very low in export standards, and in have been identified. The most important fungus dis- some provinces only 20 percent of the crop meets ease pathogens are: Leptosphaeria musanumn (leaf fun- standards required. The farmers are therefore return- gus), Mycosphaerella innsicola, and M.fijiensis. ing to rice which is more profitable. There is therefore They cause losses mainly in the genomic group an urgent need to find and propagate more produc- AAA/AA. Fusariuri wilt is the most dangerous fun- tive and uniform cultivars of export quality banana. gus, causing severe losses in Chuoi xiem/Chuoitay or We plan to give priority to the collection of banana klue Namwa, AAB dessert banana. germplasm in vivo and in vitro, systematize it botani- In variety improvement work, the callus culture of cally and cytologically, evaluate the germnplasm for banana variety Chuoi tieu trung, Cavendish greup, productivity and disease resistance, and to select va- was established by culture of young growing points rieties with export quality. Techniques need to be of banana inflorescences. The formation of somatic devised for their micropropagation and for elimina- embryos and plant regeneration were achieved. 30 BananaImprovement: Research Challenges and Opportunities

In Viet Nam, different banana clones exist in the ing low nutritional requirements; and to develop tech- same cultivar. Among the Cavendish group, different niques for in vitro fertilization and for propagation with clones of semi-dwarf Chuoi tieu cultivar express reduced somaclonal variation (lower than 5 percent). differences in yield, fruit quality, and disease resis- tance. In Chuoi cau, a cultivar with high fruit qual- 7. Genetic Transformation and Non-Conventional ity, there are two clones with differences in fruit Breeding Technology for Improvement of a skin thickness, in storage duration, and in flavor. The Molecular Toolbox for Banana evolution of banana in Viet Nam has resulted in many useful natural mutations. Field evaluation is valu- Consortium memtber:Boyce Thompson Institute able for selection of good quality fruit and disease for Plant Research, USA resistance. Queensland University of Technology, Australia Katholeike University Leuven, Belgium 5. Banana Breeding in Brazil Banana Biotechnology Consortium - I Lead institution: Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA-CNPMF), Brazil The application of molecular approaches to crop im- Principal investigator: Sebastiao de Oliveira e Silva provement has now been investigated in detail for several major crops, so that a paradigm for the proc- The focus of the project is to obtain more productive ess of technology generation and utilization is avail- varieties resistant or tolerant to the main phytosani- able. It is necessary to have the core technologies that tary problems that affect Brazilian banana produc- are broadly applicable to the desirable cultivars of the tion. This should result in a reduction of environ- crop capable of: (a) producing large numbers of trans- mental hazards as well as production costs, and in an formed plants for detailed analysis in a short time enhancement of sustainable production in the crop. period; (b) using one or several candidate genes of One of the biggest problems of the banana crop in importance to the crop; and (c) widely adaptable for Brazil is lack of high-yield varieties with adequate use by molecular biologists and crop breeders. We height and resistance to pests and diseases. We now describe the steps that will be necessary to generate have Panama disease, yellow Sigatoka, Moko disease, the required core technologies needed for a compre- nematodes, and rhizome weevil borer. Black Siga- hensive Musa molecular improvement program. toka, if accidentally introduced into Brazil, may cause Agrobacteriurn-based banana transformation is a serious problems to the banana crop. rapid and widely applicable strategy for introduction Several trials will be conducted at EMBRAPA/ of novel genes into Musa spp. This system has been CNPMF, in Cruz das Almas-BA, to develop banana demonstrated through analyses of "model" genes in- varieties resistant to yellow and black Sigatoka dis- troduced into Dwarf Cavendish. Our goal is to apply eases, Panama disease, Moko disease, nematodes, this technology to broad-scale crop improvement; to and rhizome weevil borer. The varieties should do so will require optimizing the transformation with show adequate height, a shorter period from plant- multiple cultivars, and regulating the activity of ing to harvest, and high yield. We hope to accom- introduced genes to achieve desired physiological plish this by crossing improved diploids with changes. Our objective, therefore, is to create the cultivated triploids. "molecular toolbox" which will be necessary to Additional objectives include evaluation and selec- achieve this goal. tion of promising varieties in several ecosystems; iden- The research will generate the following "molecu- tification of promising genotypes with low fertilizer lar reagents" and information relating to their opti- requirements; comparison of fruit quality of promis- mal use: ing hybrids with that of their parents; development of techniques for in vitro fertilization and propagation 1. A collection of Agrobacterium strains for use in with reduced risk of somaclonal variation. evaluating transformation efficiency in diverse We expect to produce and evaluate 10,000 diploid Musa cultivars. hybrids and 1,000 tetraploid hybrids in order to obtain 2. Multiple vectors containing different selectable 10 superior diploids and five tetraploid cultivars resis- marker genes plus suitable cloning sites for addi- tant to pests and diseases, with fruit quality and yield tional genes of potential value for banana im- better than cultivars belonging to the Prata type; to provement. identify five new banana varieties showing superior 3. Regulatory DNA elements for use in driving gene characteristics; to identify at least two varieties show- expression in transgenic banana (including an Portfolioof Project 31

evaluation of their activity and patterns of expres- We believe that the creation of a "molecular tool- sion). box" will be achieved most cost effectively through 4. Genomic DNA libraries for banana. the consortium. The "molecular toolbox" will be 5. cDNA libraries from various banana tissues. developed for the reliable and efficient genetic trans- 6. A "molecular toolbox" that can be made available formation of banana using, inter alia, an Agrobacteriurn- to scientists in developing countries as a means to mediated gene transfer system, including vectors for demonstrate banana genetic transformation and different gene introduction strategies, and genetic li- optimize its use for local cultivars. braries for isolation and manipulation of genetic ele- ments from banana to create economically useful The cooperative effort, through a banana biotech- phenotypes. The enabling technologies will be made nology consortium, to create a "molecular toolbox" widely available to collaborating laboratories in de- for banana transformation will allow the rapid ad- veloping countries. vancement of the core technologies needed for this To create transgenic banana plants with altered approach to banana improvement. This collaborative ethylene metabolism, we will extend and utilize the effort will speed the dissemination of information "molecular toolbox" by characterizing genes in ba- about successful aspects of the new technologies and nana whose expression is modulated by fungal biological materials that are time-consuming to create pathogen stress and fruit ripening. In these studies, and verify for biological value. To ensure that there is we propose cloning and characterizing the genes con- free exchange of information concerning progress in trolling ethylene metabolism in banana and charac- molecular improvement, an Internet "bulletin board" terizing their pattern of expression and those of other will be established that can be used to monitor re- "pathogenesis-related" gene families during black search advances and to pose questions concerning Sigatoka and Fusariurtn infection of susceptible and methodologies and techniques. resistant cultivars and during the ripening of banana Research progress by the banana biotechnology fruit. From these studies, potential sites of control can consortium members, and molecular tools, are de- be manipulated by modification of gene expression in scribed in the next three projects. transgenic banana and creation of new prototype cul- tivars designed for disease resistance and/or with 8. Novel Genes for Fungal Resistance and altered ripening properties. Evaluation of new Postharvest Quality germplasm will be done through collaboration with researchers experienced in the evaluation of disease Lead institution: Boyce Thompson Institutefor resistance. Plant Research, USA The outcome of this project will have three compo- Principal investigator. Charles Arntzen and Greg May nents: (1) a reliable and efficient genetic transforma- tion system for banana; (2) a detailed characterization Banana Biotechnology Consortium -2 of genes that are expressed in fungal pathogen- stressed leaves, roots, and ripening fruit; and (3) This project forms part of a consortium with Dr. Rony transgenic bananas in which the regulation of gene Swennen at the Katholeike Universiteit Leuven, and expression has been modified, thereby allowing Dr. James Dale of the Queensland University of Tech- evaluation of novel means to create disease resistance nology, Australia, to create and utilize the "molecular and improved postharvest fruit quality. toolbox." We plan to clone and characterize the genes controlling ethylene metabolism in banana, and char- Research Progress acterize their pattern of expression, and those of other "pathogenesis-related" gene families during black Agrobacterium strains LBA 4404, EHA 101, and EHA Sigatoka and Fusarium infection of susceptible and 105 were selected for comparison. These have been resistant cultivars and during the ripening of banana used by the USA group to evaluate transformation of fruit. From these studies, potential sites of control can Grand Nain. The EHA strains did not appear more be manipulated by modification of gene expression efficient in the transformation of Grand Nain than the in transgenic bananas and create new prototype standard strain LBA 4404. cultivars designed for disease resistance and/or We tested the Agrobacterium-mediated transforma- with altered ripening properties. Evaluation of tion protocol using ten clones of Musa with different new germplasm will be done through collaboration ploidy and genomic composition. Gus histochemical with researchers experienced in the evaluation of assays showed Gus activity in all clones subjected to resistance. transformation. This confirmed that A. tumefasciens 32 BananaImprovement: Research Challenges and Opportutnities is able to colonize Musa and that it can be used with transformation cassettes for resistance; and (3) trans- Agrobacterium-based vectors for foreign gene transfer forming bananas initially with a single resistance into bananas and plantains. These findings are an transgene and ultimately multiple resistance trans- important part of determining the broad utility of the genes, screening transformants for resistance in the "molecular toolbox" for multiple banana cultivars; greenhouse and field. the data are needed to provide assurance that the Viruses are a major limiting factor for banana. system will have wide applicability. BBTV is the most important of these viruses. BBMV, Agrobacterium-based vectors containing genes for already a major problem in the Philippines, has the kanamycin, basta, and hygromycin resistance have potential to become economically important outside been obtained or constructed. Continued efforts will the Philippines. BBTV is widespread in the Asia/ be made to add additional genes of value for transfor- Pacific region and in some parts of Africa; it is not mation protocol development, or with desired useful present in South and Central America. However, genes by all participants in the Consortium. The im- BBTV is still moving and is therefore a potential threat portance of these results is that we are assured that to regions that are currently free of the virus. there will be several mechanisms to introduce genes There is no known resistance to BBTV or BBMV in sequentially into banana, thereby giving confidence Musa and there are no current programs to develop that multiple traits can be conferred, as is expected to resistance by conventional breeding. BBTV has been be necessary for fungal resistance. controlled, but not eradicated, in Australia using cDNA libraries of expressed genes from various strict phytosanitary strategies. This approach has not stages of ripening fruit have also been prepared. been successful elsewhere. There have been no con- Ethylene-responsive genes have been identified and certed attempts to control BBMV in the Philippines. characterized; this work continues in collaboration It is proposed to develop transgenic resistance to with the researchers at CINVESTAV in Irapuato, these two viruses using "pathogen-derived resis- Mexico, who have also received a cDNA library. A tance genes," a strategy that has proved successful genomic library of banana (Grand Nain) is now main- for other plant viruses. QUT has been working with tained at Cornell University in Ithaca, and is available BBTV for more than ten years and with BBMV for to other interested scientists. two years. QUT now have available six components We have identified genes that are up-regulated of the BBTV genome from which to design resis- during fruit ripening and are now comparing five tance genes and have commenced this work in non-coding regulatory sequences of corresponding collaboration with Boyce Thompson Institute for genes isolated from genomic libraries. Research is Plant Research. It will be necessary to further charac- also under way to identify the gene, and its corre- terize the BBMV genome before resistance genes can sponding promoter, that encodes the dominant pro- be designed. QUT will be collaborating closely with tein found in green fruit. These results will be our first BTI and KUL on transformation of and transgene detailed study of comparisons of gene control in ba- expression in bananas. nana and other crop species. The outcomes and benefits of this project include: (1) BBTV- and BBMV-resistant banana cultivars 10. The Development of Transgenic Bananas (based on Cavendish cultivars) will be available to with Resistance to BBTV and BBMV control these two viruses in regions where they occur. This should result in increased yield in both planta- Lead institution: Quieensland University of tions and small holdings; (2) a contingency strategy Technology,Australia for other regions if and when these viruses arrive; Principal investigator: James Dale (3) technology for the expression of single and multi- ple genes in bananas; (4) a model system for the Banana Biotechnology Consortium -3 practical testing of transgenic bananas and disease resistance; and (5) strategies for virus resistance that The aim of this project is to controlbananabunchy top may be used for other Musa viruses. virus (BBTV) and banana bract mosaic virus (BBMV) through the development of multivirus resistant Research Progress transgenic bananas. This will be achieved by: (1) de- veloping transformation cassettes for BBTV resis- Four different BBTV resistance cassettes have been tance based on current and emerging knowledge for constructed each in binary vectors such that they can stable resistance to all strains; (2) characterizing the be used for both microprojectile mediated and Agro- BBMV genome and from this information developing bacterium-mediated transformation. Each construct Portfolioof Project 33 contains the NPT 11 gene driven by the Nos promoter Research Progress with the virus resistance contructs all driven by the CaMV 35S promoter. The four constructs are full We published the first protocol for the production length replicase gene, mutated replicase gene, trun- of transgenic banana plants using optimized particle cated replicase gene, and a ribozyme designed bombardment equipment and conditions, and embryo- against the replicase gene. While it was orginally genic cell suspensions of cv. Bluggoe (ABB) (Sagi et al. envisaged that these constructs would form the basis 1995a, b, c). We worked on the genetic transformation of this resistance project, other constructs based on of a wide range of banana cultivars (AAA, AAB, ABB) other BBTV genes, other promoters, and resistance with transient and stable expression of the gusA re- strategies will be developed. porter gene in new embryogenic cell suspensions. The complete coat protein gene of BBMV has been Comparative analysis was carried out on transient cloned and sequenced from BBMV isolates derived expression levels of heterologous promoters in ba- from the Philippines and India. It appears that there nana, using promoter-gusA constructs bombarded or is about 4 percent sequence difference between these electroporated into embryogenic cells or protoplasts, geographically separated isolates, Further, more than respectively. Polyubiquitin and actin genes were iso- 70 percent of the replicase (NIb) has been cloned and lated from a genomic library of cv. Grand Nain sequenced. This task is ahead of schedule and has (AAA), and we worked on the preparation of a cDNA been expanded to include different geographical iso- library from leaves of cv. Grand Nain. These results lates of BBMV. offer opportunities for banana genetic manipulation At QUT, three transformation protocols will be and isolation of genes for practical applications. investigated: microprojectile bombarded embryo- Our future research activities will include: initia- genic cell suspensions, Agrobacterium mediated trans- tion of transformation experiments using Agrobac- formation of banana meristems, and transformation teriunmtumefaciens on banana meristematic tissues; of embryogenic callus derived from male flowers. We characterization of the isolated polyubiquitin and ac- are currently culturing the male flower explants and tin genes from banana; and analysis of tissue-specific are well along with generating embryogenic cell sus- gene expression in banana. pensions of Bluggoe. 11. Genetic Engineering of Ethylene 10. Genetic Transformation of Prototype Biosynthesis in Bananas Bananas for Black Sigatoka and Fusarium Resistance Lead institution: Hong Kong University of Science & Technology, Hong Kong Lead institution: Katholeike Universiteit Leuven Principal investigators:SHANG-FA Yan and Ning Li (KUL), Belgiurm Principal investigators: Rony Swennen and Laszlo Sagi The plant hormone ethylene is produced by the banana plant and fruit and plays an important role in disease Banana Biotechnology Consortium -4 susceptibility and fruit ripening. Ethylene produc- tion is induced by pathogen attacks resulting in accel- The goal of the proposed research is to create and erated leaf senescence and yellowing. Ethylene can utilize a broadly applicable system for the noncon- also cause premature ripening of the fruit, resulting ventional breeding of superior banana germplasm. in reduced storage life and poor quality of the fruit. The project has a strategic objective of creating ena- 1-Aminosyclopropane-1-carboxylic acid (ACC) syn- bling technologies that will facilitate the genetic trans- thase and ACC oxidase, which catalyze the last two formation process. steps in the pathway of ethylene biosynthesis, partici- The objective will be achieved through a consor- pate in the regulation of ethylene biosynthesis. While tium program with BTI and QUT and by pursuing both ACC synthase and ACC oxidase genes have been further the KUL transformation strategy. The aim is cloned and characterized in a number of species, cloning to develop a "molecular toolbox" for the reliable and of banana genes has notbeen achieved. Recent advances efficient genetic transformation of banana. in genetic engineering of higher plants makes it possible The in vitro manipulation of banana is well devel- to use biotechnology to genetically engineer crop plants oped in the KUL laboratory, as is the establishment of with reduced endogenous ethylene production in fruits, regenerable embyronic cell suspensions (ECSs), and resulting in prolonged storage life. from this, the high frequency of plant regeneration Partial ACC synthase and ACC oxidase DNA frag- from isolated protoplasts. ments have been obtained by polymerase chain reac- 34 BananaImprovement: Research Challenges and Opportunities tion. We will obtain full sequences by screening a tial areas throughout the island of Oahu, indicating banana genomic library using these DNA fragments that additional control strategies and long-term solu- as probes. The expression of these genes will also be tions to the problem must be considered and studied. studied in ripening fruits and in leaves infected with Development of resistant varieties is the ideal way to Sigatoka and Fusarium pathogens. We will also se- control plant virus diseases because it is the most envi- quence the upstream regulatory region, the informa- ronrentally sound, economical, and effective way to tion from which could be useful for transformation achieve control. However, all cultivated bananas are experiments. Once transformed plant tissue has been natural selections originally found in the center of origin obtained, RNA will be prepared from the plantlets to of Musa in the Asia / Pacific region. Breeding efforts have determine the presence of the constitutively ex- been complicated by ploidy level differences and steril- pressed antisense transcript as well as the sense tran- ity in the commercial clones. Since bananas have not script using specific DNA as probes. been bred successfully, and all Musa species are sensi- Transgenic plants expressing the lowest ACC tive to BBTV infection, the use of unconventional meth- synthase/ACC oxidase activity, gene transcript, or ods for production of resistant cultivars for control of ethylene production rates will be screened. Plants will BBTV is warranted. Recent developments in biotech- be selected that display decreased levels of ACC nology have provided new opportunities to solve prac- and/or ethylene production rates, and the plant re- tical agricultural problems. Genetic engineering offers sponse to pathogen attack, shelf life, and over-ripen- new approaches to produce disease-resistant varieties. ing will be determined. Decreased leaf chlorosis is The biotechnology approach will add disease resis- expected, which will be measured by determining tance to the existing Cavendish bananas with mini- changes in chlorophyll content over time following a mal change in agronomic and quality characteristics. pathological infection (Mycosphaerella and Fusariumn Resistant bananas will provide an environmentally oxysporum), and the responses to these pathogens safe approach for control of BBTV, reduce use of will be examined. Changes in color, starch to sugar insecticides, and help small-scale banana growers to- conversion, and texture associated with ripening will wards a more sustainable method of production. be compared between wildtype and transgenic fruits to ascertain their postharvest behavior. The effects of Research Progress exogenous ethylene applied to the control and trans- genic fruits on ripening and fruit shelf life will also be Particle bombardment was used as a primary method studied. of wounding banana meristem tissue for Agrobacterium Successful completion of the project will result in infection. To determine the optimum bombard- increased shelf life, better quality of fruit, and in- ment conditions with our gene gun, which is a Bio-Rad creased resistance of the plant to pathogens, thus helium-driven PDS-1000/He system, experiments enhancing its export potential. were conducted on transient expression of - glucuro- nidase (GUS) gene in tobacco (Nicotiana tabacum Turk- 12. Use of Biotechnology to Produce ish) leaves and banana (Grand Nain) meristem under Transgenic Bananas That Are Resistant various conditions. The conditions tested in transient to BBTV Infection experiments were: two different microparticle types; two different particle sizes; and five different helium Lead instituition: University of Hazvaii at Manoa, USA pressures. Tissues were bombarded under various Principal investigator: John S. Hit conditions; they were then incubated under light at 27°C for 48 hours. GUS histochemical or flurogenic BBTV is the most devastating virus disease of bananas assays were conducted and individual blue spots were in many banana-producing areas of Asia, Africa, and counted as the result of transient expression. the South Pacific. In the 1920s this virus nearly destroyed Our preliminary results show that: (1) the level of the banana industry in southeast Queensland and north- GUS expression in tobacco is about 200 times higher em New South Wales in Australia. Its spread has caused than in banana; (2) among the microparticle types devastating problems to many countries, including the and sizes, 1.6 um gold particle gave higher transient South Pacific, India, Egypt, Viet Nam, and China. expression than others; and (3) comparing different Since 1989 the Hawaii Department of Agriculture helium pressures at 1,300 pounds per square inch (PSI) has been attempting to eradicate the virus, an ap- appeared to be the best in terms of level of GUS proach that worked well in Australia, but which expression. Therefore, 1.6 m gold particles and 1,300 appears insufficient for management of BBTV in PSI helium pressure were selected to use in further Hawaii. BBTV has been found in farms and residen- particle bombardment experiments. Portfolio of Project 35

We have transformed banana cvs. Williams and diseases has serious implications for the safe move- Grand Nain with the 19kDa gene using particle bom- ment of germplasm and for the adoption of improved bardment and Agrobacterium-mediated transforma- breeding material. tion systems. Forty transformation experiments have Banana and plantain are important food crops and been conducted. Our primary assay method for trans- sources of revenue for smallholder farmers in Africa. genic plants is polymerase chain reaction (PCR). Two Over 70 million Africans obtain more than 10 percent hundred and forty-nine and 209 plants were tested by of dietary calories from banana and/or plantain, and PCR for NPTII gene and 19kDa gene, respectively. the gross value of annual production ranks first Results from preliminary experiments showed that amongst food crops in the region. Black Sigatoka some of the banana plants were transformed. The disease, caused by the fungal pathogen Mycosphaerella potentially transformed banana plants will be further fijiensis, is the major constraint to banana and plantain tested by Southern hybridizations. In addition, N. production worldwide. The pathogen was acciden- tabacum Turkish tobacco was transformed as a model tally introduced into Africa about two decades ago system to rapidly check the transformation and gene and has spread rapidly through all production zones. expression results. Fifty-one kanamycin-resistant to- IITA has developed tetraploid plantain and cooking bacco plants were tested, and 100 percent of them banana hybrids with resistance to black Sigatoka and were ELISA positive for NPTII. Five out of 20 plants has an active breeding program involved in the fur- tested were positive in Southern blot hybridization ther improvement of material for other constraints. for the 19kDa gene. Currently, we are analyzing the Given the serious effects of black Sigatoka, it is of transformed tobacco plants in Northern blot hybridi- major importance that IITA's resistant material be zation experiments. evaluated by national programs. A BBTV DNA fragment carrying the 19kDa gene However, banana streak (BSV) is present in several was subcloned into expression vector pET24d and African countries and this has serious implications for transformed into E. coli BL21. After IPTG induction, the safe movement of germplasm and breeding mate- only pET 24d containing the 19kDa gene produced a rial across national boundaries. BSV is transmitted in distinct protein band with a size about 2lkDa, but not a semipersistent manner by the citrus mealybug from the control vector pET24d. The distinct protein Planococcus citri. Other species of mealybug are also band was isolated from 12 percent SDS-PAGE gel and believed to be vectors. However, the epidemiology of injected into a rabbit to produce antisera. The antisera BSV and the ecology of its vectors have not been is being characterized for detection of BBTV proteins studied in Africa. The role that insect vectors play in from BBTV-infected banana plants. If it works, it will relation to planting material in virus spread in the be used to analyze transgenic plants. field is not clear. Gene constructs have been subcloned from cloning There is a possibility that BSV is transmitted vector pBI525 into plant expression vector pBII21 and through pollen, embyro, endosperm, and suckers. In will be used in transform experiments. The transfor- order to reduce the virus spread and contamination mation system will be used to transform banana using of germplasm awaiting distribution to national pro- other BBTV gene constructs. The putative trans- grams, it is essential to determine if indeed virus formed banana and tobacco plants will be further transmission occurs through any of these means. characterized. This work will be carried out in close There is also an urgent need to develop therapeutic consultation with BTI and QUT, under the auspices methods for the elimination of BSV and other viruses of the banana biotechnology consortium. from germplasm awaiting international distribution. It is also crucial to identify the virus vectors and 14. Elimination of BSV from Improved Musa sources of resistance to the virus. Germplasm and Related Studies on The initial research is applied in nature and will Transmission and Host Plant!Virus/ provide a general protocol for the elimination of vi- Vector Interactions ruses from tissue culture material of banana and plantain. The other part of the project is a more stra- Lead institution: International Institute of Tropical tegic generation of knowledge that will greatly aid Agriculture (IITA), Nigeria future quarantine and breeding efforts. In these ways Principal investigator: Jonathan H. Crouch the project will be of direct benefit to both national programs and local producers. Banana viruses infect numerous genotypes of Musa This project will provide a protocol for the elimina- and occur in many banana- and plantain-producing tion of BSV from infected Musa genotypes. It will also countries around the world. The occurrence of virus result in better understanding of the epidemiology of 36 BananaImprovement: Research Challenges and Opportunities

BSV, the identity of its vectors, host plant/virus/vec- the Malaysian/Indonesian centre of Foc diversity tor relationships, and virus transmission and spread. could not be placed in existing vegetative compatibil- Interactive collaboration will be maintained with ity groups (VCGs). It is intended that phenetic and John Innes Centre (JIC) and the University of Minne- genetic procedures be used to reliably and unambigu- sota (UM), USA. ously distinguish an isolate of Foc and relate these where possible to pathogenicity. Research Progress Genetic and molecular studies with Asian isolates suggest that strains of Foc with race 4 virulence co- Research in progress includes: evolved with Musa acuminata clones in the Indo- Malaysia region, and that strains of Foc with races 1 * Identification of escapes from infection from tissue and 2 virulence coevolved with Musa balbisianaclones culture plantlets using PCR diagnostics; in the north of Asia. Eight of the sixteen VCGs de- . Development of a protocol for virus elimination scribed for Foc in Asia have been found, as well as four through meristem micro-dissection, chemother- new VCGs and many isolates of unknown VCG. This apy, thermo-therapy, or a combination of all amount of variability is not unexpected if the coevo- three; lution theory is correct due to great host diversity and . Laboratory and screenhouse tests to determine if the age of the pathosystem in the region. pollen transmission of BSV occurs through in vitro The urgency of work in Asia has been highlighted or in vivo seed germination and suckers; with the recent recovery of isolates in two VCG iso- • Laboratory and field tests to determine the identity lates from wilt-affected Cavendish cultivars growing of BSV vector(s); in Sumatra and Malaysia. Until recently, Fusarium • Field and laboratory experiments to study vector wilt in Cavendish cultivars has been considered to transmission in relation to natural dissemination be confined to the subtropics. If no predisposing fac- through vegetative material; and tors are present in Sumatra and Malaysia, then these . Field screening of local landraces, hybrids, and two VCGs may constitute a tropical race 4 which exotic germplasm for resistance to BSV. would have the capacity to threaten the world export trades. 15. Variability and Relationships within During collecting missions for coevolved patho- Populations of Fusarium Oxysporum f.sp. gens, it may be possible to locate and collect Musa Cubense from Its Center of Origin germplasm resistant to pathogen populations present in particular areas. This represents a potentially valu- Lead institution: Department of Primary Industries, able source of resistant germplasm for use in breeding Queensland, Australia programs. In addition to assessing their field per- Principal investigator: Ken G. Pegg formance, these accessions should be screened for resistance against different pathogen populations. The success of any plant breeding/selection program for disease resistance depends on a complete under- Research Progress standing of pathogen variability. Recent studies have shown Fusarium oxysporum f.sp. cubense (Foc) to be a Isolates in VCGs 0122, 01213, and 01216 are capable highly diverse pathogen. In the past, limited variabil- of attacking Cavendish in tropical areas, although ity at screening and selection sites has been one reason isolates in VCG 0122 appear to require host predispo- for the failure to breed or select universally resistant sition. Isolates in VCGs 0120, 0121, 0129, and 01211 cultivars to this pathogen. Thus an important step in attack Cavendish in subtropical production areas any plant improvement program for disease resis- where predisposition of some sort contributes to sus- tance is to understand as completely as possible ceptibility. Either cold winter temperatures or high pathogen variability and host/pathogen coevolution. water tables are involved. Isolates decimating The major aim is to assess variability in Foc in its Cavendish in Malaysia and Sumatra belong in the center of origin with Musa in the Asia/Pacific region. race 4 VCGs 01213 and 01216. As no predisposing Already over 100 isolates of Foc from Asia have factors have been identified in these countries, it is been collected and characterized. This work needs to apparent that Cavendish clones will not fare well in be expanded as very few isolates were available from these regions and export production with these clones Indonesia, India, and South China, and isolates from is unlikely to succeed. Outbreaks in these countries Myanmar, Viet Nam, and Cambodia were not repre- warrant further attention and should be of interest to sented in this study. Also many of the isolates from international breeding programs. Portfolioof Project 37

In a preliminary study to develop a reliable field pair of the following VCGs: 01213/01216, 0129/01211, screening method, 58 percent of Cavendish cv. Wil- 0124/0125 and 0129/01211-these VCGs also contain liams plants derived from tissue culture became in- cross compatible isolates and probably represent di- fected with Foc (VCGs 0120 and 0129), compared with verging populations. 2 percent of plants derived from conventional plant- ing materials (bits and suckers). In more comprehen- 17. Origin and Distribution of Fungicide- sive trials with Cavendish cv. Williams established in Resistant Strains of M. fijiensis in Banana a VCG 0120 and 0129 infested field, 54.5 percent of Plantations in Costa Rica plants derived from tissue culture; 17.9 percent of plants derived from bits; and 9.1 percent of plants Lead institution: Corporacion Bananera Nacional derived from bits established in pots showed corm de Costa Rica (CORBANA) invasion by Foc.When internal corm symptoms were Principal investigator: Ronald Romero-Calderon rated on the INIBAP 1-6 scale, where 1 = corm com- pletely clean, no fascular discoloration, and 6 = total The development of fungicide resistance in a patho- discoloration of vascular tissue, results were as fol- gen population may cause failures of disease control lows: tissue culture 2.7 (1.7); bits 1.2 (0.5); and bits and consequently serious crop losses. The use of an established in pots 1.1 (0.4). In another field test 28.6 effective fungicide could be restricted or abandoned percent of Cavendish cv. Williams plants established due to the buildup of resistance. Banana growers in from tissue culture were infected, whereas no FHIA Costa Rica are confronting a problem that limits their 01 (Goldfinger) plants were affected. In a third field ability to control black Sigatoka disease adequately. test, 9.4 percent of FHIA 01 (Goldfinger) plants de- Since 1992 this disease has caused an increase of about rived from tissue culture were affected, whereas no 100 percent in the cost of control, accompanied by FHIA 01 (Goldfinger) plants derived from bits and severe losses. The factors contributing to this problem suckers showed any corm discoloration. are: (1) the buildup of resistance to the fungicides With field screening, it is expected that the relative benomyl and propiconazole; (2) the lack of informa- resistance/susceptibility for clones will remain the tion on origin and distribution of the resistant strains same even though absolute levels of disease will be compared to the sensitive isolates; and (3) the absence higher for tissue culture vs. conventional planting of studies on the fitness and persistence of M. fijiensis material (bits and suckers). It is quite apparent that if isolates resistant to these fungicides. only a small percentage of plants of a given genotype This project will investigate the changes in the tem- are affected by Foc when tissue-cultured planting ma- poral distribution of M. fijiensisbenomyl- and propi- terial is used, the clone should not be discarded. conazole-resistant isolates, over a period of three During a collecting mission in Viet Nam, Fusarium years, under different strategies for use of propicona- wilt was found to be widespread, but was found zole and in the absence of applications of benomyl. attacking only Chuoi xiem (a Pisang Awak clone). Biological characteristics of benomyl- and propicona- Isolates characerized to date belong to the "inodora- zole-resistant isolates will also be studied with regard tum" group and VCGs 0124/25. This indicates that to their parasitic fitness (aggressiveness), conidial Foc in Viet Nam does not represent a threat to production in culture media, and their ability to form Cavendish cultivars, and provided suitable lands are fertile pseudothecia. The ratio of phenotypes in pro- available, an export industry based on these cultivars genies from crosses between propiconazole-resistant could be developed. and sensitive isolates of M. fijiensis should allow the Since Foc reproduces strictly by asexual means, determination of the inheritance of resistance for each VCGs represent clonal lineages within the species. It fungicide. Previous studies by the project's principal is important to understand the degree of variation investigator suggest a qualitiative expression of resis- that exists in VCGs and especially whether there is a tance to benomyl and a quantitiative expression correlation between VCG, fungal genotype deter- of propiconazole. A high proportion of isolates of mined by DNA fingerprint pattern, and pathotype. M. fijiensis has been found that are resistant to both The situation with Foc is complex. More than one benomyl and to propiconazole. genotype and pathotype may be present within a This project will relate changes in the dynamics of single VCG (e.g., race 1 and race 2 pathotypes occur sensitivity to fungicides, with changes in the parasitic in VCG 0124 and these two pathotypes can be differ- fitness of resistant isolates. This should allow the entiated by their genotype); and isolates in different design of better disease control strategies for coping VCGs may have the same genotype as determined by with resistance in fungal populations, thereby im- DNA fingerprint pattern (e.g., isolates within each proving the efficacy and longevity of systemic fungi- 38 BananaImprovement: Research Challenges and Opportunities cides. The results will be of relevance not only to nematode damage. Promising hybrids are being re- Costa Rica, where there is an epidemic of black Siga- leased from various breeding programs, and it is toka, but also to other banana-producing countries in important to determine the degree of susceptibility to Latin America at risk from the disease. nematodes of these new materials. Different degrees of susceptibility have been re- Research Progress ported both between cultivars and within the genomic group. It is believed that this is a tolerance Although no statistic analyses have been per- mechanism rather than resistance. This project will formed yet, the results of disease assessment did not look for cultivars with a degree of resistance/toler- indicate differences in the control of the disease be- ance to nematodes that can be recommended directly tween treatments. The results on sensitivity con- to farmers as substitutes for susceptible Cavendish firmed previous studies that indicated a significant clones and plantain cultivars common in West Africa. shift in sensitivity of M. fijiensis to propiconazole and benomyl in banana plantations in Costa Rica. It is still Research Progress too early to determine the effect of the triazole-free period on changes in the sensitivity of M. fijiensis to Studies conducted so far were made using suckers propiconazole. collected from the Musa germplasm in Njombe Benomyl resistance is very high, despite the nonu- (Cameroon). Suckers of approximately the same size tilization of this compound for several years in most were selected, roots and necroses on corm were re- banana farms in Costa Rica. This indicates no appar- moved and they were sterilized using hot water treat- ent cost on fitness in benomyl-resistant isolates. The ment (55°C for 20 minutes). They were then grown in results also suggest the involvement of fungicide re- pots of 10 liters previously filled with a sterilized sistance as one of the main causes for the unsatisfac- substrate made up on an equal mixture of volcanic tory control of black Sigatoka in Costa Rica. soil and coffee husk. Plants were allowed to grow for two weeks before inoculation. Each plant was inocu- 18. Tolerance/Resistance of Bananas lated with 1,000 R. similis from stock cultures main- to Nematodes tained on carrot discs at 280C. Plants were harvested two months after inoculation for nematode extrac- Lead institution: Centre Regional Bananiers tion. Roots were macerated and nematodes were ex- et Plantains (CRBP), Cameroon tracted using direct sieving technique. Damage was Principal investigator: Roger Fogain assessed using root lesion and corm lesion indices. Data were analyzed using SYSTAT 5.02. Nematode Bananas and plantains are an important staple food populations and RLI were Log (X=1) and angular for a significant proportion of the Cameroon popula- transformed, respectively, before analysis of variance tion, as well as being an important source of revenue was performed. Mean separation was done using for the country. Nematodes are one of the most im- LSD (least significant difference). portant groups of pests that attack banana. In Cameroon, two species of nematodes (Radopholus 19. Identification of Durable Nematode similis and Pratylenchus goodeyi) are very damaging. Resistance Sources in Banana and The former is the most important nematode in com- Plantain mercial plantations located in low-altitude areas, whereas the latter is dominant in smallholder planta- Lead institution: Katholeike Universiteit Leuven tions situated in highland areas with cool tempera- (KUL), Belgium tures. No control of nematodes is attempted in small- Principal investigator: Dirk de Waele holder plantations, whereas in commercial plantations the use of nematicides is one of the main Nematode Consortium -1 costs of production. Most of the cultivated bananas and plantains are Nematodes constitute an important limiting factor of susceptible to nematodes. Early screenings have banana and plantain production throughout the shown that sources of resistance to nematodes may world. Usually nematicides are used to control ba- exist in the diploids. Moreover, different degrees of nana nematodes. However, because of the high cost resistance/tolerance have been observed between and toxicity of these nematicides, the incorporation of cultivars, suggesting that some cultivars may tolerate nematode resistance in banana and plantain through Portfolioof Project 39 classical breeding is urgently needed. The lack of 20. Identification of Durable Pest and Disease nematode resistance sources and the lack of early, Resistance Sources in Banana and Plantain rapid, and reliable methods for nematode resis- tance screening are the major obstacles to incorpo- Lead institution: Fundacion Hondurena de rating durable nematode resistance in banana and Investigacion Agricola (FHIA), Honduras plantain. Principalinvestigator: Philip Rowe The objective of this project is to adapt or develop early, rapid, and reliable methods for nematode resis- Nematode Consortium -2 tance screening, and to screen banana and plantain genotypes for durable nematode resistance, in col- The objective of this project, which is the FHIA com- laboration with the existing classical breeding pro- ponent of the nematode consortium, is to adapt or grams for Musa improvement of FHIA in Honduras. develop early, rapid, and reliable methods for The project forms part of a consortium of four nema- nematode resistance screening; and to screen ba- tode projects. nana and plantain genotypes for durable nematode The project consists of three parts: (1) adaptation resistance, in collaboration with the existing classical and development of techniques for the establishment breeding programs for Musa improvement of FHIA in and maintenance of stock cultures of the most impor- Honduras. tant banana nematodes; (2) adaptation or develop- The project includes three parts: (1) adaptation and ment of soil-root bioassays for the early, rapid, and development of techniques for the establishment reliable determination of the reproductive and dam- and maintenance of stock cultures of the most impor- age potential of nematodes on banana and plantain; tant banana nematodes; (2) adaptation or develop- and (3) nematode screening of banana and plantain ment of soil-root bioassays for the early, rapid, and genotypes. reliable determination of the reproductive and dam- The project links three of the most important exist- age potential of nematodes on banana and plantain; ing classical banana and plantain breeding programs, and (3) nematode screening of banana and plantain i.e., those of FHIA in Honduras and of IITA in Nigeria genotypes. and Uganda, with each other and with the Laboratory The project links three of the most important exist- of Tropical Crop Husbandry of KUL, where the ing classical banana and plantain breeding programs, INIBAP Transit Centre and the world germplasm those of IITA in Nigeria and Uganda, and with the collection of banana and plantain is based, and with Laboratory of Tropical Crop Husbandry of KUL, IRTA, Spain. where the INIBAP Transit Centre and the world The major outputs of the project will be sources germplasm collection of banana and plantain is of nematode resistance for further breeding for based, and with IRTA, Spain. nematode resistance. Improved nematode manage- Major outputs of the project will be sources of ment on banana and plantain will benefit both the nematode resistance for further breeding for nema- producer and the environment in the developing tode resistance. Improved nematode management world through the increase of yields, the decrease will benefit producers and the environment through of production costs, and the lower application of increased yields, decreased production costs, and nematicides. lower application of nematicides.

Research Progress 21. Identification of Durable Nematode Resistance Sources in Banana and Work on the adaptation or development of tech- Plantain: Screening Methodologies niques for the establishment and maintenance of stock cultures of the most important banana nema- Lead institution: Institut de Recerca i Tecnologia todes has been initiated at KUL and IRTA. Agroalimentaries/Centro de Investigacion A preliminary list of the banana nematode popula- y TecnologiaAgraria (IRTA/CITA), Spain tions that will be collected and cultured has been Principal investigator: Dr. Jorge Pinochet made. Seven banana nematode populations are in culture at KUL and five banana nematode popula- Nematode Consortium -3 tions at IRTA. Several other nematode populations have been recently isolated but their successful estab- The objective of this project, which is the IRTA com- lishment is still unknown. ponent of the nematode consortium, is to adapt or 40 BananaImprovement: Research Challenges and Opportunities develop early, rapid, and reliable methods for nema- This project component also links three of the most tode resistance screening, to screen banana and plan- important existing classical banana and plantain tain genotypes for durable nematode resistance. This breeding programs, i.e., those of FHIA in Honduras will be done in collaboration with the existing classi- and of IITA in Nigeria and Uganda, with each other cal breeding programs for Musa improvement of IITA and with the Laboratory of Tropical Crop Husbandry in Africa and FHIA in Honduras. of KUL, where the INIBAP Transit Centre and the The project will follow similar approaches, includ- world germplasm collection of banana and plantain ing: (1) the adaptation and development of tech- is based. niques for the establishment and maintenance of The major outputs of the project will be sources of stock cultures of the most important banana nema- nematode resistance for further breeding for nema- todes; (2) the adaptation or development of soil-root tode resistance. Improved nematode management on bioassays for the early, rapid, and reliable determina- banana and plantain will benefit producers and the tion of the reproductive and damage potential of environment through increased yields, decreased nematodes on banana and plantain; and (3) the nema- production costs, and lower use of nematicides. tode screening of banana and plantain genotypes. 4 Summary and Recommendations

Gabrielle J. Persley

The first scientific meeting of the Banana Improve- Problem Identification: Where Are We Going? ment Project (BIP) was held at the Katholeike Univer- siteit Leuven (KUL), Belgium, on 27-28 February The key objectives are: 1996. An initial portfolio of 17 commissioned research . To identify new banana varieties with export po- projects, which address one or both of the above tential objectives, was initiated in 1995. The projects were . To develop better disease control practices in or- identified through an internationally competitive re- der to reduce pesticide use. search grants scheme, during which over 100 project proposals were received from research institutes These are challenging objectives in that few new ba- worldwide. These were appraised by a Scientific Ad- nana varieties havebeen bred overthe past 40 years, and visory Panel for their scientific quality and their rele- none are yet significant in the international trade of vance to the above objectives. dessertbananas.Allbananaandplantainvarietiesinthe Most of the projects came into operation during international trade are natural selections, and the vast 1995. Some were initiating their activities only in early majority are dessert bananas of the Cavendish type. 1996 after the completion of contracts and recruitment Why change? The question must be asked if there of staff. are new factors of sufficient importance to move the The purposes of the KUL meeting were: (1) to export industry towards new varieties. Historically review the scientific progress to date of the commis- there has been only one major varietal shift in export sioned research; (2) to identify future research pri- dessert bananas. This occurred in the 1950s when orities within the commissioned research portfolio; Cavendish replaced Gros Michel as the principal ex- (3) to identify important gaps not currently being portvariety from LatinAmerica and theCaribbean, due addressed; and (4) to foster collaboration amongst to the latter's extreme susceptibility to Fusarium wilt. the BIP-supported projects and with other interna- The current factors that are predisposing the export tionally supported banana and plantain research industry to consider varietal change are the increas- programs. ing threats from pests and diseases. The 50 participants included lead scientists from New developments include: the 17 BIP-commissioned research projects, repre- sentatives of the cosponsors and their technical advis- * The increasing cost of disease control, especially for ers, as well as representatives of other international black Sigatoka disease, which is requiring more Musa research programs, and some observers from frequent fungicide applications. Also, fungicide- the banana exporting industry. resistant strains of the pathogen are emerging.

41 42 BananaImprovement: Research Challenges and Opportunities

* Potential environmentaldamage caused by runoff of America, Europe, and Australia. In 1996, approxi- excess fungicides and nematicides into streams mately 200,000 ha will be planted to genetically engi- and rivers. Nematicides, which are especially toxic neered crop varieties in North America. These in waterways, are more widely used now, as include soybean, cotton, potato, with improved resis- Cavendish varieties are susceptible to nematodes, tance to insects and virus diseases, and tomatoes whereas Gros Michel was more resistant. Pesti- with extended shelf life. cides are also of increasing concern to consumers The successful application of transformation tech- as an environmental and human health risk. nology to banana will require: . New disease threats such as a tropical race of the Fusarium wilt pathogen (tropical race 4) that is able * Enabling technologies for transformation and re- to attack Cavendish types in Asia has been identi- generation fied in Indonesia and Malaysia, where it has de- . Identification, isolation and introduction of poten- stroyed some newly established Cavendish tially useful genes (e.g., for disease resistance) into plantations. existing varieties, such as Cavendish * New virus diseases are emerging. For example, * Field evaluation of novel varieties under appropri- banana streak virus has been found to be wide- ate regulatory requirements. spread in Africa. It persists through tissue culture, and thus is inhibiting international exchange of This strategy has particular promise with dessert Musa germplasm. bananas that are difficult to breed, since it is possible to introduce novel genes into a desirable background. Strategy: How Do We Get There? Potentially, the Cavendish types could be modified for a few key characteristics (especially Sigatoka re- Potential new varieties are being identified by a three- sistance), while retaining other desirable charac- pronged, integrated approach, based on: teristics. This is a long-term strategy, leading towards commercially viable cultivars as replacements for Germplasm Evaluation Cavendish.

Selection and evaluation of naturally occurring Achievements: How Far Have We Got? germplasm from the center of origin in the Asia/ Pacific region, for its commercial potential and for its The key developments from the commissioned re- potential usefulness in breeding programs. search portfolio in its first year of operation are:

Breeding of New Varieties Germplasm Evaluation

Using new tools, including molecular maps and other * Field and molecular characterization of 200 diploid techniques of molecular biology, to accelerate banana bananas for their potential as parents in breeding pro- breeding and to bring greater precision in selecting grams has been initiated by CIRAD in Guadeloupe; and breeding for key traits. These traits include: * Evaluation of approximately 100 naturally occur- ring accessions has been initiated in Viet Nam, * Black Sigatoka resistance including identifying the range of naturally occur- * Nematode tolerance ring Cavendish types in their center of origin. . Fusarium wilt resistance, including resistance to the . An indexing technique for banana streak (BSV) strains attacking Cavendish in Asia based on polymerase chain reaction (PCR) technol- • Agronomic characteristics (including productiv- ogy is being developed by IITA in Nigeria to facili- ity, fruit size, and postharvest characteristics). tate the safe interchange of Musa germplasm.

Genetically Engineer Banana Varieties Breedingfor Pest and Disease Resistance for a Few Key Characteristics Nematode Research Genetic transformation and regeneration of several plant species enables the introduction of specific * Nematode screening has been initiated to support genes to convey novel traits. These techniques were breeding programs in Cameroon (Centre regional first applied to plants in 1983. The first commercial bananiers et plantains-CRBP/CIRAD), Hondu- varieties are now appearing in the field in North ras (FHIA), and Nigeria and Uganda (IITA). Summary and Recommendations 43

* In Cameroon, a screening method has been de- These are being introduced into banana, as a model veloped and 84 accessions (mainly triploids) system to take through to field trials as transgenic have been screened, and some triploids resistant materials. to Radopholus similis, the major pathogen at low . New genes involved in fruit ripening have been altitudes, identified. identified, cloned, and sequenced. * Work in Belgium (KUL) and Spain (IRTA) is culti- . A genomic library of Grand Nain has been pre- vating selected nematode populations for use by pared by BTI and is being distributed to interested breeding programs worldwide and developing researchers. early screening techniques. * Other genes useful as promoters, terminators, * Three nematologists (supported by the Flemish and molecular markers in genetic engineering of ba- Development Agency-VVOB) will be joining the nana have been identified from the genetic library. breeding programs of FHIA Honduras and IITA in 1996 to initiate field screening of parental material Future Directions: What Needs to Be Done? and new hybrids. The priority areas identified by the participants at the Black Sigatoka (Black Leaf Streak) Resistance workshop were:

Crosses between resistant and susceptible lines have Modern Breeding Techniques been made by CIRAD in Guadeloupe to study the genetics of black Sigatoka (BLS) resistance. Molecular . The development of molecular maps to accelerate mapping is being initiated to locate markers and ulti- breeding by the use of molecular markers for the mately the genes for resistance in order to accelerate early identification of desirable traits. This would breeding for disease resistance. best be done through co-operation and sharing of responsibility and results amongst all Musa breed- Fusarium Wilt (Panama disease) ing programs, of which there is a small number and all are participants in BIP. * The diversity within the Fusarium wilt pathogen * The further development of different types of (Foc) has been classified through the use of new genomic libraries in which potentially useful genes molecular techniques (DNA fingerprinting) and can be identified and isolated. These genomic librar- vegetative compatibility groups (VCG). ies will also be shared amongst interested scientists * The Fusarium wilt pathogen appears to have co- to accelerate progress. evolved with Musa in Asia, from two ancestral populations. There are two distinct geographical Biotechnology groups, with races 1 and 2 widely distributed throughout Asia and the "tropical race 4" types * The newly reported banana transformation and regen- restricted to the equatorial belt through Indonesia eration systems need to be refined to develop one or and Malaysia. more systems that are applicable to a wide range of * Tissue-cultured plantlets are more susceptible to cultivars. These systems need to be efficient, repro- Fusarium wilt. ducible, and transferable to different laboratories. * A model system for banana transformation needs Biotechnology to be established, which demonstrates that a spe- cific gene can be inserted into a banana cultivar and * Banana transformation has been demonstrated for fundamentally expressed as a useful trait. Resis- the first time in several cultivars by KUL (Belgium) tance to virus diseases (BBTV and BBMV) is the and BTI (USA). most advanced trait at present. Transgenic ba- * Transformation via three different systems is pos- nanas with a novel trait need to be taken through sible and the relative merits and reproducibility of the regulatory process for environmental release, each system are being compared by their origina- perhaps in the USA and Australia initially, to es- tors at KUL and BTI, as well as the Queensland tablish a precedent for the safe environmental re- University of Technology (QUT), Australia, and lease of transgenic bananas. the University of Hawaii (UH), USA. * The next generation of potentially useful genes for * A range of virus resistance genes has been identi- insertion into banana needs to be identified, par- fied for banana bunchy top virus (BBTV) and ba- ticularly for conferring resistance to black Sigatoka nana bract mosaic virus (BBMV) by QUT and UH. disease and Fusarium wilt. 44 BananaImprovement: Research Challenges and Opportunities

Pest and Disease Control wide and involve a greater breadth of expertise to tackle problems of banana production. This process * Reliable earlyscreening techniques need to be devel- took the form of an internationally competitive re- oped urgently for the major pathogens (black Siga- search grants scheme where recommendations for toka, Fusariumwilt, and nematodes) to accelerate funding by a ScientificAdvisory Panel were based on: breeding when linked with the use of molecular (a) peer review of proposals for their scientific excel- markers. Ideally, multiple pathogen screening at a lence, and (b) their relevance to the BIP objectives of few sites would be desirable. new varieties with export potential and improved * A better understanding of variabilityin the Sigatoka disease control. complex is required (similar to that being under- taken for the Fusarium wilt pathogen) in order to Research Collaboration assist breeders in the development of new materi- als with durable resistance. In establishing the research portfolio, the World Bank . There needs to be multi-site evaluation of parents has encouraged creative partnerships amongst the and progenies from breeding programs world- scientists being supported by BIP and with other in- wide for their resistanceto the tropicaland subtropical ternational Musa research programs. Some linkages race4 of Fusariumwilt presently occurring in Asia were established at the proposal stage and this re- and Australia, respectively, as a precaution against sulted in the formation of a biotechnologyconsortium this race becoming more widespread. and a nematoderesearch consortium. • The apparent increased susceptibility of tissue- Other linkages were fostered by the holding of the cultured planitletsto Fusariumwilt, as compared to first scientific meeting within the first year of imple- adult plants, needs to be better understood. mentation of the research projects. This has enabled * Integrated managemient of banana diseases needs to early results to be shared and research collaboration be studied in order to incorporate host plant resis- established amongst scientists with similar interests. tance with other disease control components, Closer cooperation and integration amongst the Musa including biocontrol agents, to reduce pesticide breeding programs worldwide that are focusing on use. various types of banana and plantain important in different geographic regions would be desirable. The Project Management: How Do We Do It? Leuven meeting has been especially successful in fos- tering scientific collaboration amongst the key play- The Banana Improvement Project has a number of ers in banana research, and this should accelerate innovative features. Some of these are unusual in a progress towards shared objectives. project cosponsored by a number of multilateral de- velopment agencies. The innovative features in the Financial Resources design and implementation of the project are: The important issue in considering future financing Clear Problem Identification of banana research is to ensure that funds from differ- ent sources can address common objectives, with This includes the recognition that there are different agreed milestones to measure progress towards problems in different regions and the dynamic nature achieving these objectives. of the problems. Consultations with technical special- The Common Fund for Commodities (CFC) has ists from banana-producing countries and industry provided approximately US$3.5m over 5 years to have been especially helpful in this process. The pri- sponsor specific research activities through BIP. ority problems being addressed are the increased im- The establishment of BIP has also leveraged other portance of black Sigatoka disease, Fusariumwilt, and investments in banana research from diverse sources. nematodes. The Flemish Association for Development Coopera- tion and Technical Assistance (VVOB) and the Bel- Mobilizing Additional Scientific gium Development Agency (BADC) are providing and Financial Resources substantial funding to the nematode consortium, working with the breeding programs in Honduras, This is necessary to address the problems of dessert and East and West Africa. banana especially. Scientific resources are being mo- BIPalso works closely with the Consultative Group bilized through a science-driven process, designed to on International Agricultural Research (CGIAR)insti- attract the best available sources of expertise world- tutes involved in banana and plantain research, IITA Summary and Recommendations 45 in Africa, and IPGRI and its International Network for Conclusion the Improvement of Banana and Plantain (INIBAP). The CGIAR donors, including the World Bank, invest BIP has established itself as a virtual international cen- approximately US$3m annually in banana and plan- ter for banana research since its inception two years tain research, primarily because of their importance ago. It has mobilized a critical mass of scientific re- as staple foods. sources to tackle some of the key problems constrain- The present funding for BIP comes from public ing banana production. Some of the commissioned sector sources. The time may now be opportune to research projects have made significant progress dur- seek to establish some innovative public/private sec- ing their first year. However, breeding new banana tor partnerships in banana research where public in- varieties with export potential and improving disease stitutions and private companies can identify some management are difficult and long-term targets. Con- common objectives of mutual interest, and identify a tinued assessment of progress in the research portfo- modus operandi to jointly support research to resolve lio, commissioning additional research to fill the iden- the major problems of banana production. The World tified gaps in the portfolio, especially in relation to Bank undertook to continue discussions during 1996 black Sigatoka disease and banana breeding, and with national and multinational companies in the seeking the active financial and technical participa- banana industry to determine their interest in partici- tion of the banana industry in the effort are the impor- pating in the project. tant challenges ahead.

References

Daniells, J. 1995. "Illustrated Guide to the Identifica- Transformation of Banana and Plantain (Musa spp.) tion of Banana Varieties in the South Pacific." via Particle Bombardment." Bio/lTechnology13:481- ACIAR Monograph No. 33. Canberra. 485. May, G. D. et al. 1995. "Generation of Transgenic Sagi, L., S. Remy, B. Verelst, B. Panis, B. Cammue, G. Banana (Musa acuminata) plants via Agrobacterium- Volckaert, and R. Swennen. 1995. "Transient Gene mediated Transformation." BiofTechnology 13: 486- Expression in Transformed Banana (Musa, cv. Blug- 492. goe) Protoplasts and Embryogenic Cell Suspen- Pearson, M. H., P. B. Bull, and K. Shepherd. 1983. sions." Euphytica 85: 89-95. "Possible Sources of Resistance to Black Sigatoka in Sagi, L., S. Remy, B. Verelst, R. Swennen, and B. Panis. the Papua New Guinea Biological Foundation Ba- 1995. "Stable and Transient Genetic Transforma- nana Collection." Tropical Pest Management 29(4): tion of Banana (Musa spp.) Protoplasts and Cells." 303-308. In Bajaj, Y. P. S. (ed.), Biotechnology in Agriculture and Pegg, K. G., N. Y. Moore, and S. Sorensen. 1994. Forestry. Vol. 34. Plant Protoplasts and Genetic En- "Variability in Populations of Fusarium oxysporum gineering VI. Springer. Berlin-Heidelberg-New f.sp. cubense from the Asia/Pacific Region." In York-Tokyo. Jones, D. R. (ed.), "The Improvement and Testing Shepherd, K., J. L. L. Dantas, and S. de Oliveria e of Musa: A Global Partnership." Proceedings of the Silva. 1994. "Breeding Prata and Maca Cultivars First Global Conference of the International Musa for Brazil." In Jones, D. R. (ed.), "The Improve- Testing Program held at FHIA, Honduras, 27-30 ment and Testing of Musa: A Global Partner- April 1994. INIBAP, Parc Scientifique Agropolis, ship." Proceedings of the First Global Conference 34397 Montpellier, Cedex, France. of the International Musa Testing Program held Persley, G. J., and E. A. deLanghe (ed.). 1987. "Banana at FHIA, Honduras, 27-30 April 1994. INIBAP, and Plantain Breeding Strategies: Proceedings of an Parc Scientifique Agropolis, 34397 Montpellier, International Workshop held at Cairns, Australia, Cedex, France. 13-17 October 1986." ACIAR Proceedings No. 21. Stover, R. H., and I. W. Buddenhagen. 1986. "Banana Canberra. Breeding: Polyploidy, Disease Resistance, and Pro- Sagi, L., B. Panis, K. DeSmet, H. Schoofs, S. Remy, ductivity." Fruits 41: 175-191. R. Swennen, and B. P. A. Cammue. 1995. " Genetic

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