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INIBAP’s MandateThe International Network for the Improvement of Banana and Plantain (INIBAP) wasestablished in 1984 and has its headquarters in Montpellier, France. INIBAP is anautonomous and nonprofit intergovernmental organization whose aim is to increase theproduction of banana and plantain on smallholdings by:– initiating, encouraging, supporting, conducting and coordinating research aimed at

improving the production of banana and plantain, with particular reference to thepeople of developing countries;

– strengthening regional and national programs concerned with improved and disease-free banana and plantain genetic material, and facilitating the interchange of suchmaterial by assisting in the establishment and analysis of regional and global trialsof new and improved cultivars;

– coordinating and supporting training programs for developing-country techniciansand scientists.

Planning for the creation of INIBAP began in 1981 in Ibadan with a resolution passed ata conference of the International Association for Research on Plantain and Bananas.INIBAP is a member of the Consultative Group on International Agricultural Research(CGIAR).

© INIBAP 1993Bât. 7, Parc Scientifique Agropolis34397 Montpellier Cedex 5, France

Biotechnology Applicationsfor Banana and Plantain

Improvement

Proceedings of the Workshop on

held in San José, Costa Rica27-31 January 1992

INTERNATIONAL NETWORK FOR THEIMPROVEMENT OF BANANA AND PLANTAIN

The opinions in this publication are those of the authors and not necessarily those ofINIBAP.

Acknowledgments

INIBAP is grateful to all participants, who came from all over the world, for theircontributions to the achievement of the Workshop’s objectives. Likewise, INIBAP isgrateful to:• The Centro Agronómico de Investigación y Enseñanza (CATIE), especially to

JV Escalant and the staff of the Biotechnology Unit, for hosting and handling the firstday of Workshop sessions.

• The Corporación Bananera Nacional de Costa Rica (CORBANA) for its cooperation inthe local arrangements and for its gracious hospitality.

• The Dutch Ministry of Foreign Affairs, Special Programme on Biotechnology andDevelopment Cooperation, for sponsoring the production of these proceedings.

• B Wills and RD Huggan who enthusiastically undertook the task of editing theproceedings, and to F Malafosse for her dedication in preparing the necessarydocuments for the editors.

• R Jaramillo, Coordinator of the INIBAP Network for Latin America and the Caribbean,who included even his family in the organization and administration of this event.

• USAID for its participation in the organization of the meeting.

Editorial note

In some papers references have been submitted without complete publishing data. Theymay therefore lack article titles, the full names of journals, and/or the place ofpublication and the publisher. Unavoidable delays in publication have also led to theomission of full publishing data about some items listed as “in press.” Should readershave difficulty in identifying particular references, staff at INIBAP will be glad to assist.

Contents Page

IntroductionE De Langhe (Director, INIBAP): Opening Speech 7R Tarté (Director General, CATIE): Introductory Remarks 9I Buddenhagen: Whence and Whither Banana Research and Development? 12

Part 1: Nonconventional Strategies for Producing Banana and Plantain Resistant to Pathogens and Pests

Genetic Maps

D González de León, S FauréGenetic Mapping of the Banana Diploid Genome 29

Transformation

F Bakry, R Haïcour, JP Horry, R Megia, L Rossignol Applications of Biotechnologies to Banana Breeding 52

Identifying and Generating Resistance

C Rivera, P Ramírez, R PereiraPreliminary Characterization of Viruses Infecting Banana in Costa Rica 63

CM Fauquet, RN BeachyStatus of Coat Protein-Mediated Resistance and its Potential Application for Banana Viruses 69

J Dale, T Burns, S Oehlschlager, M Karan, R HardingBanana Bunchy Top Virus 85

GA Strobel, AA Stierle, R Upadhyay, J Hershenhorn, G MolinaThe Phytotoxins of Mycosphaerella fijiensis 93

RC PloetzMolecular Approaches to Identifying Fusarium Wilt Resistance 104

D De WaelePotential of Gene Transfer for Engineering Resistance against Nematode Attack 116

N von Mende, P Burrows, J BridgeMolecular Aspects of Resistance to Nematodes 125

Cross-Pollination Breeding

PR Rowe, FE RosalesBreeding Cooking Bananas for areas with Marginal Growing Conditions 128

Part 2: In-Vitro Strategies for MusaGermplasm Handling

O AriasCommercial Micropropagation of Banana 139

FJ Novak, H Brunner, R Afza, R Morpurgo, RK Upadhyay, M Van Duren, M Sacchi, J Sitti Hawa, A Khatri; G Kahl, D Kaemmer, J Ramser, K WeisingImprovement of Musa through Biotechnology and Mutation Breeding 143

M Perea-DallosContribution to the study of Banana Anther Culture 159

MR Söndahl, C NoriegaBioreactor Micropropagation Technology for Rapid Commercialization Opportunities in Plantation Crops 163

Genetic ImprovementD Vuylsteke, R Swennen

Genetic Improvemement of Plantains 169JV Escalant, C Teisson

Somatic Embryogenesis and Cell Suspensions in Musa 177H Leblanc, JV Escalant

Induced Parthenogenesis to Obtain Haploid Plants in Musa 181W Parrott

Cell-Culture Techniques 183

Somaclonal VariationFX Côte, X Perrier, C Teisson

Somaclonal Variation in Musa sp. 192LA Withers

Early Detection of Somaclonal Variation 200

Part 3: Technology TransferJA Chambers, JI Cohen

Donor Assistance in Plant Biotechnology for the Developing World 211

Part 4: Recommendations(Working Group 1) Nonconventional Strategies for Producing Banana

and Plantain Resistant to Pathogens and Pests 225(Working Group 2) In-Vitro Strategies for Musa 235(Working Group 3) Technology Transfer 236

Annexes1. Acronyms and Abbreviations 2472. Participants 249

Introduction

7

Opening Speech

E De Langhe

Ladies and Gentlemen, this Musa Biotechnology Workshop has been made possiblewith the collaboration and input of several persons and institutes. It is a real pleasurefor me to explain their roles.

Dr Rodrigo Tarté, Director of CATIE, has been continually and consistentlyencouraging his Center in developing biotechnology research, and was most supportiveof the initiative to have the workshop take place in Costa Rica. CATIE is the RegionalBase of INIBAP in Latin America and the Caribbean, and INIBAP also considers CATIEas an excellent center for the transfer of new technology into the region.

The United States Agency for International Development (USAID) providedsubstantial financial support, allowing the participation of many specialists from theAmerican continent. Moreover, it allowed Dr Judy Chambers to collaborate actively inthe development of the workshop’s concept and technical organization.

The Technical Centre for Agriculture and Rural Cooperation (CTA) of the EECsupports the participation of our colleagues from Africa and from Asia. It will alsofinance the publication of the proceedings.

Dr Ramiro Jaramillo, our Regional Coordinator for Latin American countries, has,during the last 3 weeks, devoted most of his time to the numerous details regarding thepractical organization of the workshop, and the fax-telex line with headquarters,especially with Ms Susan Fauré of our secretariat, was “red-hot” at times. His base ofoperations, during the last few days, was at San José airport, rather than at CATIE.

Bob Huggan, our Editor, feels quite comfortable, having already received the text ofabout half of the presentations, and will no doubt be consulting you in the furtherpreparation of the proceedings. His long-standing professional experience is a guaranteeof the quality of that publication.

Dr Ivan Buddenhagen has agreed to make the general introduction to the scientificcontributions, and I have no doubt that he will do this in his typically provocative andneuron-stimulating style.

Our Germplasm Research Coordinator, Hugues Tezenas du Montcel, and our CropProtection Research Coordinator, David Jones, are here and are of course readilyavailable to provide any technical information you may wish to ask for.

INIBAP attaches particular importance to the recommendations of this workshopand on their follow-up within the Network. The background paper, which you havealready received, explains what we expect from this event, and I will, by the end of thepresentations, elaborate on how INIBAP expects to be helpful as a Network.

E De Langhe

Director, INIBAP

I am confident that the workshop will be productive in several senses. A good manyscientific contacts will be made — which is part of INIBAP’s task, to encourage informalnetworking. If the workshop formulates guidelines regarding INIBAP’s future role inMusa biotechnology, then its output will be maximal.

8 Opening Speech

Introductory Remarks

R Tarté

On behalf of the Tropical Agricultural Research and Training Center (CATIE), I wantto extend the warmest welcome to all participants in this extremely important workshop.

As you all know, CATIE has been the host for INIBAP’s regional coordination forLatin America and the Caribbean. We have, together, undertaken several importantactivities with the purpose of improving the production of bananas and plantains in theregion. Those activities are oriented towards coordinating the research efforts conductedby many institutions committed to this goal at a global level. The search for newtechnologies, the exchange of information, and the performance of joint collaborativeactivities require an institutional complementarity that must avoid the unnecessaryduplication of efforts.

Such complementarity and collaboration are regarded by CATIE as the mostimportant elements of its strategic development. They are also, I believe, the essence ofa networking mechanism like that promoted by INIBAP. And complementarity andcollaboration are the only ways of facing today’s challenges of agricultural research anddevelopment: the challenge of increasing productivity while maintaining the naturalresource base — the challenge of making agriculture sustainable, not only ecologically,but also economically, socially, and culturally.

Such challenges perhaps comprise a greater challenge than the one faced by thosewhose research led to the Green Revolution some 30 years ago.

Commodity and disciplinary research has evolved over many years intomultidisciplinary farm research, and today into what we call multidimensional research.Multidimensional because the search for agricultural development that is sustainablemust take into account the different dimensions involved in an integrated way: theecological, the economic, the social, the cultural, the policy, and the institutionaldimensions. And those require not only multidisciplinary research, but also integratedmulti-institutional action.

Multidimensional research, as we understand it in CATIE, can be regarded asresearch focused with an ecoregional approach. In other words, one that takes intoaccount the integrated management of natural resources at a regional level along withthe development of sustainable production systems.

It is along these lines that the new agricultural research for the 1990s must bedesigned. It is along these lines that our efforts will give rise to the new era of

9R Tarté

Director General, CATIE

modernization of agriculture that is demanded as we approach the beginning of the 21stcentury: the era of modernization with sustainability, equity, and quality of life.

Now, as the need for a new way of conducting agricultural research is outlined, wheredo we stand and where do we want to go in research on bananas and plantains?

In this part of the world, banana production is regarded as a very modern operationbecause of the high technological level required to assure the highest productivity andthe best quality fruit for our export markets. But it is also the most contaminating andecologically damaging of all agricultural operations. All of the technology used in bananaproduction to control major pests and diseases and to protect the fruit that is exported,is based on the heavy use of chemicals. In the last 20 or 30 years the major technologicalchanges that have occurred in the protection of bananas for export against thethreatening diseases, are changes in the kind of pesticide or fungicide used. Bananatechnology has depended on applications of chemicals, with new brands of productsappearing with relative frequency, and pesticide trials have become major appliedresearch activities. This also accounts for the very high cost of banana production.

While this is true for bananas, plantain growers, who are usually small farmersoperating on a small scale, are demanding low-cost technologies to improve theirproduction and to control the severe incidence of diseases affecting their crops,particularly black Sigatoka.

There is obvious need for a reorientation of banana and plantain research, a need tointroduce the new concept of plantain research, and a need to introduce the newconcept of modernization with sustainability that must guide all agricultural researchtoday. This is now urgent, particularly in the light of the extensive expansion of thebanana-growing area in Costa Rica and other Central American countries. For all thosereasons, we must maximize our creativity and imagination by planning our researchactivities through a major, unselfish cooperative effort among our scientists,policymakers, and farmers.

There are many opportunities for transforming research on bananas and plantains.Such opportunities must be found in the more fundamental areas related to thebiological and behavioral aspects of the major pests and diseases, such as black Sigatokaand nematodes, in order to help identify integrated pest management methods withemphasis on biological, genetic, and cultural methods of control rather than onchemicals. But perhaps the most challenging opportunities are in the field ofbiotechnology. CATIE has taken up such a challenge by designating biotechnology as themain research activity of the plantain working group that was set up 3 years ago. Withthe support of INIBAP, this research involves micropropagation, somatic embryogenesisand cell suspensions, haplomethods, somaclonal variation, and medium- and long-termgermplasm conservation.

The use of biotechnology for the improvement of bananas and plantains will, nodoubt, contribute to the modernization of their production on a sustainable basis,provided that efforts are directed towards the reduction of environmental hazards andproduction costs and to the development of new production systems for small farmers. Isincerely hope that the creativity needed, enhanced through worldwide collaboration in

10 Introductory Remarks

the field of biotechnology, will emerge as a very important outcome of this workshop. Butwe must also bear in mind that a multidimensional research approach must includeinteraction with other disciplines in the biological, economic, and social sciences as wellas with those related to policy, services, and marketing environments. I am sure CATIEand INIBAP will remain committed to this goal, and will find new and effective ways ofcoordinating this type of research.

Let me give you, once again, our warmest welcome and best wishes for a verysuccessful meeting.

11R Tarté

12 Whence and Whither Banana Research and Development?

Whence and Whither BananaResearch and Development?

IW Buddenhagen

PrefaceThis paper is dedicated to RH Stover and NW Simmonds — the two men who havecontributed the most to banana research both directly, and indirectly by influencingothers. Through thick and thin of political, bureaucratic, and funding problems theyhave remained steadfast to scientific enquiry and useful accomplishment on thiswonderful crop over their long careers.

IntroductionI view my task at this workshop as providing a historical setting of banana research andbanana problems to give perspective to the modern biotechnologist who wishes to applywonderful new techniques to this ancient crop. I use “banana” generically, to cover alsoplantains and various cooking bananas: in short, to mean Musa. Banana research hashad a fascinating but chequered history. Much has been learned; but the inability todevelop a good long-term organizational structure and support system formultidisciplinary teamwork research has slowed progress.

But it is important to realize how different the tropics were even only 50 years ago.French, British, Dutch, and Belgian empires dominated much of the tropics, except forLatin America which had thrown off the feudal Iberian yoke before anyone had evolvedthe concept of applied agricultural science. The Spanish empire fragmented into manysmall and a few larger tropical countries whose elites often lived in the highlands andhad no interest in lowland (banana) agriculture. (And little enough interest inagriculture per se except as a taxable resource.) Much later, as science became appliedto agriculture, the Latins and the Filipinos became influenced — and educated —directly or indirectly by the United States.

But to back up a bit, “modern science” evolved in Europe, mostly in northern Europe,in spite of an abortive try long before in Greece and elsewhere. Science that reveals anunderstanding of biological phenomena and of plants, which could be applied toagriculture, is very recent — say 150 years or so — and it was related to the unique

Department of Agronomy and Range Science, University of California, Davis, CA 95616, USA

north-European creation of the “industrial revolution”. Both activities were rapidlyadopted and amplified in the USA, with its early strong north-European heritage andconnections. Also, where new societies were formed of British people — in Canada, NewZealand, and Australia. They were also adopted soon by the Japanese.

But in the tropics, the ancient cultures, rich in many ways but poor in others,remained largely outside the advances of biological science and the industrial revolution.Neither science nor agricultural science evolved in any tropical country. What did finallydevelop was the application of evolving agricultural science through the variousEuropean colonial services to the crops of interest to the home country — oil palm,cocoa, coffee, cotton, various spices, tobacco, sugarcane, and rubber.

The Early Days: 1900 to the Second World War(WW2)

The Americans (USA) started the banana trade without any science, just buying,shipping, and selling bananas to fruit-hungry Americans. The British became interestedthrough the potential of bananas for their Caribbean Islands, and the British market, andthey went at it with typical British-botany bias — doing the first good work on the crop intaxonomy and cytogenetics. They even initiated a breeding program. This Britishinitiative, beginning in the 1920s, was the first real flowering of banana research.

The American banana companies (especially United Fruit Company) were soonconfronted with the dying of their one and only “shipping” banana, Gros Michel, by whatbecame known as Panama disease (Fusarium wilt) and they, too, started an abortivebreeding program — in Panama in the late 1920s — and hired the great GermanFusarium expert O Reinking to look at the problem. He went to Asia to collect resistantbananas and collected the eventual replacement for Gros Michel, in a yard in Saigon in1926. The great depression caused the abandonment of the company breeding program,but Sigatoka — the great leaf blight of bananas — finally reached the Americas in the1930s from its Asian homeland, causing a major shakedown of the Latin Americanbanana trades.

The French developed the agronomy of the crop on their hilly islands of Martiniqueand Guadeloupe and, much later, serendipitously discovered the effectiveness of oil sprayfor Sigatoka control.

Local country agricultural departments and agricultural universities developed slowlyin the poor Latin American countries and banana research hardly developed. The idea, ifconsidered at all, was that the banana companies should do banana research. Thus, thecompanies and the Latin American peasant farmer, growing plantains or bananas, wereleft to their own devices.

Since the USA had no colonial service and no tropical empire and little publicinterest in tropical agriculture, their potential agricultural research skills had littlepublic-sector outlet. Their three tropical territories — Puerto Rico, Hawaii, thePhilippines — were not seen as potential banana producers and, although the USDA

13IW Buddenhagen

established agricultural research positions in these territories and agriculturaluniversities were also established, no long-term commitment was made to conductresearch on bananas. There was no visionary in the USDA on tropical agriculture, letalone on bananas. There was no-one to link private and public sector research interestsand, indeed, the US attitude was and still largely is — let the companies do their ownresearch. Thus, the poor tropical peasant farmer remained short-changed, as did tropicalagriculture in general, including the large economic sectors dependent upon the bananatrade.

Of the three American tropical territories, Hawaii became the most westernized inagricultural tradition through American company developments of sugarcane andpineapple (not bananas), and very successful research organizations were set up onthese crops, across company interests, serving all companies contributing to theHawaiian Sugar Planters’ Association, and the Pineapple Research Institute. Later, bothUSDA and university input to these research organizations developed. Such good modelswere never copied for bananas by the American companies operating in Latin America(or later in the Philippines), even though several were the same companies that profitedfrom the pineapple and sugarcane research groups. Thus, no collective large researchgroup was ever formed for bananas, and no link with USDA or universities wasestablished. (As an aside, the C4 photosynthetic pathway was first discovered byscientists at the Hawaiian Sugar Planters’ Association.)

Back in the homeland of Musa, even less was done. The Dutch established much goodtropical agriculture work in their “Indies” (now Indonesia) but they hardly touchedbananas nor educated the local people in agricultural science. The heartland of Musaevolution was held by the French (Indochina) and the British (Malaya/Borneo/Burma),but in these regions essentially nothing was done on bananas. Although the Americansestablished a big collection in the Philippines by 1915, little else was done. Apparently,the colonial powers saw bananas in these regions as something nice to eat, andappreciated their diversity in local dishes, but basically accepted bananas as part of thebackground of the native cultures, which were exotic, complex, and left alone. Newoutside crops were seen as exploitable — oil palm, rubber, tobacco, etc., and thesereceived all the nascent tropical research.

Back in Africa, the Germans, French, and Belgians did just enough to try out the cropand cover its local agronomy.

It was in the Caribbean and Central American countries where the industry waspioneered before the 1st World War where initial successes led to large investments, andpractical problem-solving and development activities on a big scale. Making acommercial success of bananas meant exploiting the north American and Europeanmarkets, and it required trustable suppliers of quality fruit, new shipping and handlingpractices, good and fast communications, diversified geographic sources, and fertile,well-drained soils. The “banana business” required organization and efficientengineering, shipping, and marketing. Engineers and shippers ran the business, notagriculturalists. Everyone did the necessary local agronomy, but the key stimulus tobanana research came with the early appearance of Panama disease, both in


northeastern South America, Panama, and Central America and then in the Caribbeanand far-away Australia. This devastating and intractable Fusarium wilt was notunderstood, was spread everywhere in planting stock, and was fought in variousunsuccessful ways.

The Post-WW2 Roller-Coaster to the Early 1970sSince the American banana companies (especially the United Fruit Company) were theprimary victims of Fusarium wilt, they invested in various attempts at finding a solution.It was their “company” problem. They hired plant pathologists but their key approachwas escape — move to new lands. As escape to new lands became more and more costly,both economically and politically, “the Company” invested more heavily in research. Anew breeding program was initiated in Honduras in about 1960 and this, combined with alast-ditch switch to Fusarium-resistant Cavendish, stimulated the second great floweringof banana research. Multidisciplinary research on postharvest diseases and physiologywas established, as well as research on resistance, breeding, and cytogenetics, epide-miology, etc. New non-banana men at the top of the research management ladder openedup the narrowness developed under the old banana hands. Consultants from differentfields were hired and many new things happened. This flowering began in about 1958,grew more expansive (and more resented) in the mid-1960s and gradually withered withpolitical unrest, antitrust action, and the buying out of the United Fruit Company by firstone group and then another. Banana overproduction reduced prices and profits; newmanagement at the very top viewed bananas as a commodity like wheat, and saw littlecommitment to long-term tropical agriculture, to tropical-country development, or toinvesting in the future through research. The other big banana companies also were“acquired” by conglomerates, and even Del Monte is now only a subsidiary of a big,almost bankrupt, British conglomerate.

Early on, the British considered the banana wilt problem as a colonial problemaffecting their prosperous Jamaican colony, not as a company problem. Thus theirapproach was to have the problem shouldered by the Imperial College of TropicalAgriculture in Trinidad and by the Jamaican Government Department of Agriculture. Theprewar breeding and research programs were resurrected after WW2, with new collectionexpeditions and further research in botany and cytogenetics. But by 1960 all work wasshifted to Jamaica under the Banana Board Research Department and the main “spring”of inspiration from the Imperial College dried up. But the important legacy from thissource came to us through two superb books by NW Simmonds, published in 1959 and1962. Following Jamaican independence, support to the Banana Board organizationdwindled and little research is supported there today. But the key point is that the earlybanana breeding effort sparked the basic cytogenetic studies of bananas and much of thebasic work on bananas. Much credit was due to the liberal policies of the ImperialCollege of Tropical Agriculture, which encouraged the development of long-term basicresearch programs. But that is past, and nowhere else did such an attitude prevail, nordoes it fully exist today anywhere.

15IW Buddenhagen

The French only gradually recovered an effective involvement with their tropicalcolonies after the war and the agitation of independence movements. But in theCaribbean, their islands remained a part of France and, early-on, they converted to wilt-resistant Cavendish. This stimulated a continued intensification of research throughtheir federal (public) overseas fruit research organization (IRFA), headquartered inGuadeloupe and Montpellier, France. (Like the British, the French viewed bananas as anational commitment, not as a company problem.) Work centered around plantprotection, nutrition, general agronomy, and shipping problems. Their early growing ofCavendish and their good research on “efficiency” enabled them to avoid beingconcerned with a breeding program until quite recently.

Historical Forces Driving Banana Breeding andOther Banana ResearchAlthough Fusarium wilt was the driving force that led to the first (Trinidad) and second(United Fruit/Honduras) flowering of banana research and breeding programs, it lost itspunch with economic success of the conversion of company bananas from Gros Michel toresistant Cavendish clones. Banana research had not flourished anywhere else, even inthe presence of Fusarium wilt, in regions not participating in export markets or wherelocal people had a diversity of clones only some of which were susceptible. There waslittle stimulus to start new, or continue old, breeding programs once Cavendish removedthe Fusarium threat. Those areas converting first had the least need to do bananaresearch except for adaptive agronomy. However, once the United Fruit Companyswitched to Cavendish, the push was for high yields and high quality and, with the newvariety, new problems, as always, appeared. Thus, the second banana research floweringin Honduras (1958-75) grew and continued, but with different objectives — moreefficient control of Sigatoka, nematode, and postharvest fruit disease and insects. Also,more efficient nutrition and agronomy and fruit quality control. Others had to play catch-up, be subsidized (as for the French and British markets), or wither away.

A temporary spark for research and breeding was kindled by the appearance of blackSigatoka in Honduras in 1972 and its subsequent spread as far as Colombia. Soon,however, a combination of good epidemiology research and new systemic fungicidesreduced control costs so that, once again, the spark for supporting resistance breedingdwindled. The pathogen has not yet got to Jamaica, and this absence combined withpolitical changes destabilized research support and the breeding program therecollapsed.

By 1975, interest in a new bred-variety also decreased in the United Fruit Company.Partly to blame was the lack of success from decades of investment in breeding. But theproblem had other roots. The Company breeding program had developed as theBritish/Caribbean program was waning, but there was hardly any connection with thisexperienced program. Discussions were held and attempts made to obtain germplasm,but no agreement could be reached. The Company went at it alone, hiring new


inexperienced people, and never utilizing the British talents directly. But they hiredgood people and mounted three very successful collection expeditions to Asia and tookevery lead from Simmonds’ excellent books. Enough money and talent led to the bestmultidisciplinary effort ever mounted on bananas, to many new research findings, to thelargest well-classified germplasm collection, to the development of the best male hybridsand to the best tetraploids for a dessert banana. But there were several key problems:the breeding target kept changing as practical Company agronomists switched from GrosMichel to Valery and then to Grande Naine. This shift to ever-shorter and higher-yieldingcultivars made for a shifting and much more difficult breeding target. Pathology researchand new chemicals leading to cheaper control of Sigatoka, and then of black Sigatoka ledto less interest by growers and managers in any new untested (and possiblyuntrustworthy) cultivar. Moreover, Company management became convinced that anynew cultivar would be stolen and used by others and the logical question — whysubsidize everybody else? — was asked. (The logical answer — get together on fundingwith the other companies and enlist public funds from governments with internationalinterests for the common good — was, unfortunately, never approached.)

Furthermore, in the 1970s, various Latin American exporting countries formed theirown private or public organizations dealing with export, shipping, policy, appliedresearch, and with self-financing through box taxes. These activities, plus strikes andlabor movements, led to a decrease of transnational involvement in land ownership andbanana growing and less interest in “Company” investment in research and in breeding.

Other Initiatives Since the Mid-1970s

Musa and the CGIARThe CGIAR is the “overseeing body” of the international agricultural research centers(IARCs) that grew out of original initiatives by the Rockefeller Foundation. The firstcenters were concerned with cereals but, with the formation of CIAT in the 1960s, theclonal crop cassava became a research target. I was working on bananas in CentralAmerica at the time and was interviewed by the organizers of CIAT for a position on rice.My suggestion that Musa should be an important research target and that they were astaple food was met with skepticism and lack of interest.

The formation of IITA in Nigeria as the CGIAR center for the lowland humid tropicsadded more clonal crops to the CGIAR system in about 1972 (Dioscorea yams, cocoyams,sweet potatoes). In addition, plantains were mentioned as a possible crop, but noadvocate came forward to develop a research program on them. Bananas, of course, werenot mentioned. (Bananas, for the CGIAR in general, and for donors who live far from thetropics, have a negative connotation because they can be a commercial crop from whichprofit can be derived by big companies. Their true nature and pervasive importance inlocal economies are little appreciated.) However, by 1976 a meeting was held at IITA toform an International Association for Research on Plantain and Other Cooking Bananas.

17IW Buddenhagen

IITA’s charter was revised and “plantains” as a crop was made more clear. IITA’s initialwork was minor and was all on plantain agronomy in poor delta soils. Management,board, and donors saw no reason to finance scientists to work on plantains full-time, noron a breeding program, nor on diseases; and dessert bananas were never included as acrop.

Thus, there was a CGIAR center in the country of greatest plantain production in theworld (Colombia), one in the Philippines, at a humid lowland site ideal for Musaresearch and in a center of origin of Musa (IRRI), and one dedicated to the lowlandhumid tropics but located in a non-Musa seasonally-dry site (IITA, Ibadan). Musa wasignored by the first two and was given only cursory treatment at IITA until about 1983. Itwas not until 1985 that IITA hired a full-time scientist for plantains; and breeding workwas begun only in 1987 and a breeder hired only in December 1991. No-one had had thevision in the IARCs nor in the CGIAR or its Technical Advisory Committee (TAC) tomount a study on the importance, problems, and research needs of Musa worldwide, andto go after solutions in a logical manner. Meanwhile the key banana research center andonly viable breeding program (United Fruit Company in Honduras) was dying by default.

The Brazilian initiativeAbout this time (1981), EMBRAPA of Brazil decided to go after solving their long-standing problem of Fusarium wilt and to preclude any problem from a future invasionof black Sigatoka for their very different AAB Brazilian dessert bananas — throughbreeding. They very sensibly dealt with the senior breeder (K Shepherd) in the waningJamaican breeding program. Shepherd, who inherited all the old Britishbreeding/genetics knowledge and germplasm, and who knew what to do, set up anoperation in Bahia and went after a Fusarium-wilt resistant dessert AAB type. Thisbreeding target was both “new” and much easier than trying to beat the best Cavendish,since the existing dessert AABs have low yields and poor agronomy. By dealing withCosta Rica for field screening of good progeny against black Sigatoka, they were able toobtain resistance to this pathogen also. Brazil has recently released a new desserttetraploid (AAB characters) from this program. Anyone familiar with the variability ofFusarium may wonder how good the resistance will be, and it is not yet clear to anoutsider how well the work of the program is continuing and how much multidisciplinaryresearch is involved. Nevertheless, this EMBRAPA initiative was useful to help getbanana breeding off dead-center.

The United Fruit Co./FAO/USAID/FHIAMeanwhile, back in Central America, the 1970s were fraught with problems of antitrust,new Latin exporting groups, tariffs, overproduction, hurricanes, buy-outs, and takeovers.Not a good climate for research, and only a few scientists remained in the United FruitCompany’s research group at La Lima. The advent and spread of black Sigatoka in 1972-80 gave a new brief spark to breeding but, by 1983, the Company even gave away its 24-year-old breeding program.


Actually, the idea was good — to create an “Agricultural Research Foundation” withthe Company facilities, which could work on many crops to benefit Honduras at large.USAID and FAO were alerted to this opportunity, and they responded in different ways. Aman with vision at FAO Rome (A Bozzini) decided the Musa germplasm and breedingprogram were too precious to let die. FAO is not a research-granting agency, but specialfunds were made available for 6 months (later renewed for another 6 months), tomaintain the germplasm and PR Rowe’s breeding program at survival level. Also — andthis is a key point — FAO sent a lawyer to help draft the FHIA charter, who insisted thatthe Musa part of the “Foundation” would be internationalized and not be just aHonduran activity. This created two great opportunities.

1. The breeding program could be internationalized and broadened in scope and inclientele. It could approach world needs without the past company narrowness.Moreover, while under FAO, the germplasm could become available to the worldcommunity.

2. The breeding program and Musa research overall could become the group forreceipt of international monies from international donors of the CGIAR and others forsolving any Musa problem, anywhere, that would be amenable to a breeding solution.

Unfortunately, neither great opportunity was realized, although some progress wasmade. The FAO connection was a short bridging one before a USAID commitment of 10years to FHIA was finalized. But this AID connection came out of the Latin Americandesk and had no connection with the part of AID that partially funds the CGIAR system.Moreover, they were not interested in supporting research on the banana export cropand they pushed for work on other crops. Musa was not really separated administrativelywithin FHIA or in the minds of the US donors or their program reviewers. Theconnection with the international agricultural research system was never really made.Musa research was hardly expanded, nor did it become multidisciplinary and fullyrounded.

Other forces were at work to undermine the great opportunities. 1. The new Latin American marketing and exporting unions and other banana or

research-related organizations could not grasp that a really good noncompany breedingprogram would be beneficial for themselves, as well as others. They could not separate FHIALa Lima from the “Company” La Lima, and the “Company” carried too much negative stigma.

2. IDRC in Canada, in looking around for a new “networking” project, seemed willing toput money into Musa unconnected to the CGIAR system, and listened to their Latin contactswho considered that the Honduras program should not be backed. (But IDRC did put moneydirectly into FHIA in support of the small breeding program.)

3. The French and Belgians wanted to establish their own “international” Musa organi-zation, mainly based on their African experience and the connection with the growinginternationalization of IITA’s small plantain program, and IDRC Canada was also interested.

4. IITA leadership could not visualize the world needs or opportunities on Musa, a cropconsidered to be just one more of its too-many-crops roster. Thus, the stage for the formationof INIBAP was set.

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The Taiwan situationMeanwhile, at about this time (1983), Fusarium wilt had become so common on the“resistant” Cavendish clones growing in Taiwan that a solution was needed. This“breakdown” of Cavendish resistance, starting in the late 1960s was also a red flag of fearto the tropical banana trades. It helped fuel the bare maintenance of the Honduranbreeding program. It stimulated research because it was gradually realized that “new”Fusarium races or strains could attack resistant Cavendish also in subtropical Australia,South Africa, and the Canaries. Even Cavendish in tropical Philippines was attacked, butat a low level.

The solution in Taiwan was to work out an economical replanting rotational systemwith paddy rice. The key was cheap, efficient raising of clean planting stock throughmeristem tissue culture. This provided the possibility for detecting somaclonal variantsfrom large populations — and just maybe, resistant ones. The dividend from a straightcommercialized laboratory propagation system, of a Cavendish resistant to theseresistance-breaking strains, would be great indeed. Most variants have poor agronomiccharacters, even if they appear to have short-term resistance. However, some goodvariants have been identified which also appear to have some resistance (Hwang, Ko1987; and personal information). This program appears not to have been broadened byattempting mutation treatments or in-vitro selection. These possible approaches weretried for a short time in the nearby Philippines by Del Monte Corporation, an effortabandoned in 1983 (Epp 1987).

IAEA, ViennaThe International Atomic Energy Agency in Vienna has a breeding and genetics sectionwhich conducts and sponsors mutation breeding activities in many developing countries.This long-standing joint FAO/IAEA division is funded by the United Nations, and amongits activities is a banana mutation/somaclonal laboratory at Seibersdorf near Vienna.Somaclonal material of potentially mutated variants is sent to any cooperator interestedin testing it. Somaclonal projects on Musa have been backed in several countries,including Costa Rica. Although mutation treatments may be given, in-vitro selection withfungal-product pressures have not yet been well worked out. However, interest has beensufficiently strong to generate research on somatic embryogenesis (a natural formutation breeding and now for biotechnology initiatives). Success in somaticembryogenesis has been reported (also at Louvain) and we should be brought up to dateat this workshop. It seems that IAEA is a logical place to add modern biotechnologyefforts to its existing older biotechnology efforts of mutation, applied to cropimprovement. They wish to do so.

The unique Australian experience (old and new)Over the years the pragmatic Australians gradually expanded their banana production(mostly in Queensland) to satisfy Australian demand. Long ago bunchy top virus wasaccidentally brought from Fiji and this prompted research and quarantine activities.


Banana research developed much as it does on crops in the USA — with farmersorganizing growers’ groups and pushing for public-sector help through extension andresearch — centered at both the State Agricultural Department (QDPI) at localagricultural universities and at the Department of Agriculture in New South Wales.

I think this unique situation for bananas was due to the very nature of society andagricultural development there. It is the only place where banana farmers and workerswere not part of a peasantry separate from a ruling elite, but were part and parcel of alocally developing economy which became self-supporting largely through a marriage ofindependent enterprise and local government concerned with local development ofeducation, industry, and farming, and a tradition of self-help through research, extension,and education. The farmers and farm workers were just as good as anyone else, andanyone might end up as an elected representative or with a PhD in agriculture. Researchwas done as was locally needed, mostly on agronomy, but also some good physiology andpathology have been done over the years. Banana researchers have long-term careers,though on a small scale, because funding and horizons largely reflect the problems ofbananas in Queensland. A major push to broaden the Queensland scope was provided byACIAR (Australian Centre for International Agricultural Research) in developing aninternational banana project in 1985, focused on international concerns of blackSigatoka, bunchy top virus, and breakdown of Fusarium-wilt resistance in Cavendish.The project was ostensibly focused on the “Pacific Islands” for the internationaldimension, with the first two problems. But the latter two problems were alsoQueensland problems, so a base for research was already ongoing. Adding ACIAR moniesalso gave a “germplasm diversity” dimension and the project became a focus for theIBPGR Musa collection from New Guinea, and a base for evaluation. Thus, researchbecame much broadened and deepened so that, now, monoclonals and a DNA probe forbunchy top virus exist, and work on tissue-culture variation and mutation, as well asbasic work on Fusarium variability, is carried out. The final pay-off would be for directinvolvement in a breeding program, but this most important and interesting aspect didnot quite get off the ground.

FAO and IBPGR

FAO continued its interest in Musa following its brief banana breeding rescue initiative,but mostly in the pure plant protection area. Reviews of black Sigatoka and of bananaresearch in South America were made (1984), but the lack of mechanisms for follow-through action by this nonresearch organization more or less invalidated this effort. In1990, FAO pushed an initiative to hold a meeting (jointly with INIBAP) to help resolvethe conflicting reports of what causes banana bunchy top. Also, they have teamed up withIBPGR to write a series of guidelines on the safe international movement of germplasm,one of which covers Musa. It is, of course, important to have “safe” movement ofgermplasm but, like many quarantine initiatives, one can always use them for delaying orinhibiting germplasm movement to one’s own advantage. The Musa guidelines are, in myview, overly restrictive, overly centralized, and yet still unsafe, and they have tended to

21IW Buddenhagen

inhibit or delay exchange of material among researchers — the more-apparent ready-availability and safety of which would have accelerated cooperative research.

FAO has been pressured by some of its developing-country members to help them notto be left out in the race to capitalize on modern biotechnology. FAO has recently hired aplant biotechnology officer to manage FAO’s incipient activities in this area. Also, astrategy paper has been developed that plots a course of action which attempts tobalance any new biotechnology initiatives with the old crop improvement projects andextensive country knowledge of FAO staff. It remains to be seen how efficient an FAO rolein brokering useful activities in this area can be. Without substantial expansion, FAO mayfind it difficult to become a viable player in this complex area.

At FAO, but completely unrelated to the Crop Production and Protection Division, ishoused an International Commodity Group which concerns itself with helping the bigagricultural commodities of international trade. Their concern is stabilization of pricesthrough various mechanisms. Banana is one of their commodities and, over the last 5years, they have decided to try to apply the reserve funds from banana trade levies tolowering production costs by backing research projects. Much discussion over divisionand use of these funds is under way. Theoretically, some could go to useful biotechnology.

IBPGR, earlier part of FAO, is now essentially an autonomous (and newly renamed)center within the CGIAR. Musa is just one of over 200 crops they are concerned with, andhow active a role they will take in being involved or catalyzing funding for collection,analysis, and evaluation of Musa remains to be seen. They were involved in a small way inhelping to catalyze the recent collecting expeditions to New Guinea, with ACIAR andAIDAB funding. What role they should play, since they are not directly involved withbanana breeding programs which would use and should study the germplasm, needscareful consideration.

Recent EventsThe original meetings which later catalyzed the formation of INIBAP were held at IITA inNigeria in July 1981 and later at IDRC in Ottawa in July 1982. At the first meetingJamaica was heavily represented, two IITA staff were present, and PR Rowe of SIATSA(now FHIA), as well as E De Langhe, now Director of INIBAP; the only potential donorpresent was IDRC. Since breeders were present, one suggestion was to perform inducedmutation as well as expand Dr Rowe’s small project in breeding AAB plantains. A strongsuggestion was made to reactivate the Jamaican program for breeding plantain andcooking bananas. The reason given for all this was only the threat of black Sigatoka —but no pathologist attended the meeting! By this time black Sigatoka had arrived inAfrica. Since black Sigatoka attacks plantains, Musa became more acceptable to receiveinternational public funds. There was no mention of a “network”, nor of “coordination”.

At the second meeting in Ottawa the pitch was that (a) “... urgent action is needed toestablish an informal international network linking national plantain and bananaresearch organizations ... so that disease-resistant planting material can be madeavailable as soon as possible.” And (b) “An important aspect of this network would be


the strengthening of the two existing banana breeding programs, in Honduras and inJamaica.” In retrospect, which gives the wisdom of hindsight, these conclusions andsuggestions seem terribly naive. Now, 10 years later, one may ask how many farmers aregrowing the “disease-resistant planting material [to] be made available as soon aspossible”. Also, one may question if the subsequently created INIBAP network hasindirectly resulted in the diversion of potential funds away from the key need ofexpanded, well-rounded breeding programs.

Also, 10 years later, the Jamaican breeding program is defunct; but, unrelated to theINIBAP network, three new breeding programs have been established, in disparatelocations (Brazil, Guadeloupe, and Nigeria). Moreover, two of these (Brazil and IITA)seem well on the way to solving, with minimal funding, the major problems of the AABdessert and plantain groups. The United Fruit breeding program is now that of FHIA, stillunder PR Rowe, still underfunded, but it is coming up with potential solutions for theeastern African cooking bananas and for ABBs in general, through a new breeding thrustfor these objectives. Separate from the network, the French have pushed hard to expandMusa research under CIRAD-IRFA and have started a breeding program andconsiderable basic research. It is hoped we will be updated on all these new activities atthis workshop.

INIBAP has done various useful things, in organizing meetings such as this one, incontributing to meetings of others, in communications of various sorts including theabstracting service, in sponsoring tours and consultancies. And on very limited researchseed-money disbursement arrangements. Very recently INIBAP has organized global andregional testing of a few new potential cultivars bred by FHIA and IITA. By assuming thisas an INIBAP activity, the active breeding programs tend to be less involved directly withpotential clientele and with analytical appraisal of environment/genotype interactions.Direct involvement by an active multidisciplinary crop improvement team of scientists inboth of these activities is very useful for any breeding program, to provide feedbackinsights. Where testing is separated from these scientists, the testing tends to become anend in itself, as has been seen in the massive international testing of rice and maize.Local research suffers.

INIBAP concentrates on networking and coordination of others, and this activity isfraught with pitfalls. Research is competitive. Funding is short and struggled for on anindividual basis. Researchers don’t like to be “coordinated”. There is a long legacy ofcountry, regional, institute, and commercial rivalries. The carrot held out, of smallhandouts of funds, is accepted by some, but often resented. So there are many problems.But the fault lies less with INIBAP than with the very conceptualization of theorganization by the original organizers and donors. Most of the original CGIAR centersperform networking and coordination functions of some sort, and even they find theseactivities difficult, not very cost-efficient, and often politically sensitive. But at least theyare operating from a research base. One may question the wisdom of the formation of anorganization to conduct “networking and coordination” when it has no research base,especially on a tropical crop direly needing both better breeding and a better researchbase. To headquarter the organization in a nontropical country, far from banana

23IW Buddenhagen

problems and from research programs (except for the nearby French laboratories) hasalso posed great difficulties.

TAC has finally realized the importance of bananas and plantains and in their 1990priority study lists them among the top 10 world commodities in value of production. Thismay have influenced the move whereby INIBAP was recently allowed to enter the CGIARsystem, as one of several new centers under that mantle. Unique among the crop centers,it still is constituted not to have a research base. And, like IITA, it does not really addressdessert bananas. Thus, the CGIAR continues to side-step the banana industry that is sovital to the economic well-being of quite a few countries.

While all this has been going on many new initiatives have occurred. Some of the newassociations in Latin America and the Caribbean, such as CORBANA, UPEB, and others,as well as the older WINBAN, carry out applied research and technical services of variouskinds. In addition to those mentioned above in Brazil, Australia, Taiwan, IAEA, throughFAO, IBPGR and at IITA, scientists scattered here and there, with a dedicated interest inbanana research, have plugged away at modern research on embryogenesis, onFusarium, on Mycosphaerella, on moko and blood diseases, on the banana weevil andbanana nematodes and on biotechnology applied to taxonomy and evolution. Many ofthem are here at this workshop, very valuably arranged by INIBAP. Many of thesescientists are distant from banana lands, and they struggle for funds to keep a part-timeproject going. Few are really connected to a banana breeding program.

The FutureMuch new knowledge needs to be discovered that is useful for knowledge’s sake. Butwhere there are real problems and opportunities in the tropical banana world that canaffect the lives and livelihoods of millions, research should be applied to direct solutions.The solutions will come through breeding. And through developing the best possiblecompatible marriage of breeding and biotechnology. Both need to go forward together.And industry, international donors, foundations, and private donors should join togetherto construct rational, productive, useful and used research programs centered on severalfully developed breeding programs. More scientists at universities should be involved andmore PhD theses developed. There is much to accomplish. More and better research andcooperation is needed and less jockeying for political power and control.

There are many new players interested in the biotechnology of tropical crops:companies; national “overseas” organizations such as CIRAD in France, DGIS in theNetherlands, NRI in England; individuals and groups at universities; FAO/IAEA;“brokering” groups of various types; national agricultural systems in quite a fewcountries; and even the World Bank. The World Bank has appointed a biotechnologyspecialist to review and establish useful biotechnology projects connected to orappropriate for World Bank projects and interests. So there are enormous interest andpossibilities. The art and wisdom will be to add biotechnology where appropriate and insuch a way as not to deplete resources for both applied and basic research that is neededthat is not classifiable as biotechnology. For bananas, this is especially difficult since


fully-rounded multidisciplinary breeding programs are even yet not in existence nor wellsupported!

Biotechnology’s main opportunity will be a marriage with breeding and genetics ofbananas, to focus on the most difficult breeding target: a resistant Grande Naine orWilliams. These need transformation and appropriate genes for transformation. All theother banana classes should be bred conventionally since they are now proving so easy tobreed. Biotechnology may help as a breeding tool. There are other opportunities tocontribute to banana needs by a focus on enhancing potential biocontrol mechanisms ofanimal parasites. Whether the needs of postharvest physiology offer opportunities isuncertain. Genes are available to alter enzymes of the ripening process. Certainly themain investment should relate to solving the major biological pest and pathogenproblems.

Pathogen variability and host/pathogen coevolution have recently received newemphasis, but much more needs to be done, combining fieldwork and detailed collectingwith biotech-assisted analysis.

No fungal disease of any crop has yet fallen to the powerful techniques ofbiotechnology, so the challenge is very great. I see no reason why a banana pathogenshould not be the first — it is up to you, and others who wished to come to this workshopbut could not, to make it happen. It is also up to good leadership to catalyze goodcommunication between real problems, researchers, and those who make decisions onthe allocation of research funds. The model used for the biotechnology thrust of theRockefeller Foundation, which has had great success, is well worth considering.

Let this workshop be a turning point in banana research by catalyzing proper fundingof multidisciplinary breeding programs and enabling their flowering, with the aid ofbiotechnology, actually to solve the important problems. Sufficient appreciation of ourmistakes and the chequered history of banana research should enable a better course tobe charted for the future.

References and BibliographyBUDDENHAGEN IW. 1977. Resistance and vulnerability of tropical crops in relation to their evolution and

breeding. Annals New York Acad. Sci. 287:309-326.BUDDENHAGEN IW. 1987. Disease susceptibility and genetics in relation to breeding of bananas and plantains.

Pages 95-109 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds). ACIARProceedings no.21. Canberra, Australia: ACIAR.

BUDDENHAGEN IW. 1990. Banana breeding and Fusarium wilt. Pages 107-113 in Fusarium Wilt of Banana(Ploetz RC, ed.). St Paul, MN, USA: APS Press.

DHED’A D, DUMORTIER F, BARRIS B, VUYLSTEKE D, DE LANGHE E. 1991. Plant regeneration in cell suspensioncultures of the cooking banana cv. `Bluggoe’ (Musa spp. ABB group). Fruits 46:125-135.

EPP MD. 1987. Somaclonal variation in bananas: a case study with Fusarium wilt. Pages 140-150 in Bananaand Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds). ACIAR Proceedings no.21. Canberra,Australia: ACIAR.

FAWCETT W. 1913. The Banana. London.HWANG SC, KO WH. 1987. Somaclonal variation of bananas and screening for resistance to Fusarium wilt.

Pages 151-156 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds). ACIARProceedings 21, Canberra.

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KERVÉGANT D. 1935. Le Bananier et son Exploitation. Paris.KRIKORIAN AD, CRONAUER SS. 1984. Aseptic culture techniques for plantain improvement. Economic Botany

38:322-331.KRIKORIAN AD, SCOTT ME, CRONAUER-MITRA SS, SMITH DL. 1990. Musa callus and cell culture: strategies,

achievements and directions. Pages 9-23 in: In-Vitro Mutation Breeding of Bananas and Plantains I.Report of the First Research Coordination Meeting for the FAO/IAEA, May 29 - June 2, 1989. TechnicalDocument IAEA 312.D2.RC.411. Vienna, Austria: International Atomic Energy Agency.

PERSLEY GJ (ed.). 1990. Agricultural Biotechnology: Opportunities for International Development. Wallingford,Oxon, UK: CAB International.

PERSLEY GJ. 1990. Beyond Mendel’s Garden: Biotechnology in the Service of World Agriculture. Wallingford,Oxon, UK: CAB International.

PERSLEY GJ, De Langhe EA (eds). 1987. Banana and Plantain Breeding Strategies. ACIAR Proceedings no.21.Canberra, Australia: ACIAR.

PLOETZ RC (ed.). 1990. Fusarium Wilt of Banana. St Paul, MN, USA: APS Press.QUISUMBING E. 1919. Studies of Philippine bananas. Agric. Rev. 12:1-73.REYNOLDS PK. 1927. The Banana. Boston and New York.RIOS PG. 1930. Cultivo del banana en Puerto Rico. Ins. Agr. Exp. Sta. Rio Piedras Bull. 36. 58 pp.ROWE PR. 1981. Breeding an “intractable” crop: bananas. Pages 66-83 in Genetic Engineering for Crop

Improvement (Rachie KO, Lyman JM, eds). New York, USA: The Rockefeller Foundation.SIMMONDS NW. 1959. Bananas. London, UK: Longman.SIMMONDS NW. 1962. The Evolution of the Bananas. London, UK: Longman.SIMMONDS NW. 1983. Plant breeding: the state of the art. Pages 5-25 in: Genetic Engineering of Plants: An

Agricultural Perspective (Kosuge T, Meredith CP, Hollaender A, eds). New York and London: PlenumPress.

STOVER RH. 1972. Banana, Plantain and Abaca Diseases. Kew, Surrey, UK: Commonwealth MycologicalInstitute.

STOVER RH, BUDDENHAGEN IW. 1986. Banana breeding: polyploidy, disease resistance and productivity. Fruits41:175-191.

STOVER RH, SIMMONDS NW. 1987. Bananas. London, UK: Longman.VALMAYOR RV. 1976. Plantains and bananas in Philippine agriculture. Fruits 31:661-663.WARD FS. 1930. Banana growing in Malaya and the presence of diseases. Malayan Agric. J. 18:63-70.


Part 1

NonconventionalStrategies for

Producing Banana andPlantain Resistant toPathogens and Pests

29D González de León, S Fauré

Genetic Mapping of the BananaDiploid Genome: toward anintegrated approach to the study ofthe Musa genome and the use ofmolecular marker technologies inMusa breeding

D González de León, S Fauré

“So many genes. So little time.” (Ed Coe, maize geneticist)

IntroductionLike so many crops of relative importance in human diets, bananas and plantains havenot been studied as much as they deserve. In particular, much of what is known abouttheir natural history, genetics, and evolution is limited to the contributions ofcomparatively few researchers since the beginning of the century. Thus, while many ofthe cytotaxonomic riddles of the genus Musa have been elegantly unravelled (Simmonds1962, and references thereof), there is almost no knowledge of the genetics of any of itstaxa, let alone of their population genetics and the minutiae of their reproductivebiology. Modern banana breeding has suffered substantially from this situation, andprogress has been rather slow in breeding clones that fulfill current breeding objectives,such as resistance to major diseases (Buddenhagen 1993; Rowe, Rosales 1993). Evenwhen some of the objectives have been attained, it is typical, if not unfortunate, that theagronomic characters of the new clones are not as good as the most widely growntriploid varieties. It seems therefore imperative that more knowledge be gained aboutthe banana genome in general, in order to facilitate its manipulation and thuscontribute to giving new impetus to current breeding practices and helping developnovel improvement strategies, especially at the diploid level.

Genome Manipulation and Genetic MappingOver the last 6-10 years, with the flourishing of plant molecular genetics, new tools havebeen developed and refined that hold enormous promise in what they can reveal about

CIRAD-BIOTROP, BP 5035, 34032 Montpellier Cedex 1, France

the genome of almost any organism. These tools provide new kinds of genetic markers,that is characters that show inheritable variation at the DNA level, rather than at themorphological or biochemical levels. These characters are called markers because weuse them to obtain, albeit indirectly, information about the genetics of other traits ofinterest in the organism under study.

Restriction fragment length polymorphisms (RFLPs), random amplified polymorphicDNAs (RAPDs or AP-PCR markers) and other derived or analogous methodologies thatgenerate molecular markers have been expertly reviewed by numerous authors (see forexample Beckmann and Soller 1986; Burr et al. 1983; Rafalski et al. 1991; Stuber 1992;Tanksley et al. 1989; Williams et al. 1990).

With these genome manipulation techniques it is relatively easy to identify specificchromosome regions that are important for a particular phenotype. Thus, geneticmapping of the genome of hitherto poorly studied species has become a feasible, almosttrivial endeavor, as long as some conditions are fulfilled such as the availability ofsuitable segregating populations. Once particular genomic regions have been identified,they can be transferred from germplasm to germplasm by conventional crossing. In theseprocedures, it is the markers themselves that may serve as selection criteria.

This sort of “marker-assisted selection” has been around since the early days ofmodern genetics (see for example Stuber 1992 for a brief review). The difference withtoday’s approaches is simply in the kind of markers then available. Only a handful ofcrops have benefited from such early markers. Take, for example, the case of one of thebest genetically characterized plants: after more than 50 years of intensive geneticinvestigations, the current genetic database for maize (Zea mays) consists of over 600individual loci identified by their visible phenotype (“naked eye polymorphisms” orNEPs) or by the analysis of their biochemical bases (Coe et al. 1988). Of these, some 400have been placed onto specific segments of the corn genetic map; using conventionalapproaches, an enormous effort would be required to gather all of these points into aunique, comprehensive map, simply because it is quite impossible to accumulate morethan a few of these mutations into individual breeding stocks. By contrast, in less than10 years, research in only three different public laboratories has resolved more than1000 new loci defined by DNA polymorphisms. These have been added to the variousexisting versions of the corn map (Burr et al. 1988; Helentjaris 1987; Hoisington, Coe1990). Simple mapping strategies based on DNA markers such as “interval mapping”(Lander, Botstein 1987), are now being used to construct the comprehensive corn map(Hoisington, Coe 1990). Anyway, extensive genetic maps constructed on the basis ofmorphological, isozyme, and some other phenotypic traits (e.g., physiological mutants,disease resistances) exist for very few crop plant species (e.g., tomato, pea, maize).

In the bananas, what little is known of the formal genetics of NEPs is limited to oneor perhaps two, not too recent papers (Simmonds 1953; Vakili 1968). However, the maizeexample given above serves at least one purpose: it gives us the reassurance that rapidprogress in dissecting the genetics of the bananas is within our grasp. In effect,molecular tools provide a potentially indefinite number of markers that can serve asselection criteria in the manipulation of the banana genome.

30 Genetic Mapping of the Banana Diploid Genome

The use of molecular markers to locate major genes and loci contributing to theexpression of a continuously varying character (i.e., quantitative trait loci or QTLs)affecting agriculturally important traits is rapidly becoming the primary approach forunderstanding the genetic basis of these traits and, more importantly in the case ofcrops such as the bananas, to help develop novel breeding strategies.

High-density molecular marker linkage maps have now been constructed for morethan 15 different plant species including Arabidopsis thaliana (Chang et al. 1988; Namet al. 1989), barley (Graner et al. 1991; Heun et al. 1991), Brassica spp. (Landry et al.1991; Slocum et al. 1990; Song et al. 1991), chili peppers (Tanksley et al. 1988), lentils(Havey, Muehlbauer 1989), lettuce (Landry et al. 1987), maize (Burr et al. 1988;Hoisington, Coe 1990), potato (Gebhardt et al. 1989), rice (McCouch et al. 1988), rye(Wang et al. 1991), soybeans (Keim et al. 1990), tomato (Bernatsky, Tanksley 1986), andwheat (Gale et al. 1990).

Some examples of the localization of agriculturally important traits relative togenetic markers are briefly described elsewhere in these proceedings (Ploetz 1993). Theultimate use of molecular markers is the detection and potential manipulation of lociaffecting quantitative characters; at least two convincing demonstrations of thereduction of QTLs to Mendelian factors have very recently been published for tomato andmaize. These should indicate what strategies to adopt for the genetic dissection of QTLsin other crops that are far more difficult to manipulate (Paterson et al. 1991; Paterson etal. 1988; Stuber et al. 1992).

In the next section we briefly review some aspects of Musa biology that should not beoverlooked if the use of diploid germplasm is to be increased in current and futurebreeding strategies.

The Banana Genome: Genetic Constraints andtheir ConsequencesThe typical seedless edible banana appears to be the product of two essentialevolutionary processes accompanying domestication (Simmonds 1962): parthenocarpy,that is the autonomous stimulus to pulp growth; and sterility, both genic female (andmale?) sterility and that arising from structural hybridity. A third, additional andsubsequent process was the formation of triploid, fully sterile cultivated forms arisingfrom the fusion of reduced and nonreduced gametes following nuclear restitution. Bothat the diploid and triploid levels, domestication has therefore resulted in an obligatoryform of vegetative propagation of sexually sterile clones that has ensured the long-termpreservation of relatively unchanged phenotypes.

It is not difficult, therefore, to understand that breeding new triploid cultivars ofbananas or plantains has not been an easy task and that success has been rare (Rowe1984; Stover, Simmonds 1987). The classical schemes involve, for example, crossing adiploid, usually a fertile wild one showing some desirable trait, onto a good triploidrecipient cultivar having good female restitution. Not much had been drawn from this


method (Rowe 1984), but some recent successes are reported in these proceedings(Vuylsteke 1993). Schemes of this sort are nonetheless hindered by intrinsic geneticconstraints. Insufficient knowledge about the genomic origins and composition of thebest triploid cultivars complicates even further the choice of the best diploid materialsfor desirable introgressions.

Other strategies, based on breeding better diploids using both cultivated clones andwild accessions, are becoming increasingly important, not to say inescapable (Bakry etal. 1990; Rowe 1987).

The use of M. acuminata diploids is far from trivial. Take the cultivated types, forexample: most of the accessions investigated have been found to be heterozygous for oneor several translocations and/or inversions (reviewed by Simmonds 1962; pers. com.Bakry et al., CIRAD), and thus tend to be at least fairly male-sterile; genic femalesterility may further complicate the use of these materials also as female parents.

In the case of the wild acuminata taxa, the situation seems superficially better sincethese are fertile, seed-bearing types having normal meiosis, that should be easy subjectsin hybridization schemes. But data have been accumulating since the first half of thiscentury that there exists considerable chromosome structural differentiation within thespecies (reviewed by Simmonds 1962). Thus, most currently recognized subspecies suchas banksii and burmannicoides, to name but two, produce hybrids that are partiallysterile as a result of chromosome structural differences between the parents (e.g.,Dodds, Simmonds 1948; pers. com. Shepherd 1987: ms. on translocations in Musaacuminata). Bananas are one among many examples of plants and animals whosecytogenetic structure is made more complex through extensive chromosomalrepatterning (Burnham 1956; Jones 1970; Stebbins 1971; White 1978). These includetaxa from genera such as Triticum spp. (Fominaya, Jouve 1985), Lens (Tadmor et al.1987), Gossypium (Menzel et al. 1978), Secale (Rees, Sun 1965), Capsicum (Gonzálezde León 1986), Helianthus (Chandler et al. 1986), Caenorhabditis (McKim et al. 1988),and Drosophila (Sharp, Hilliker 1989).

This phenomenon establishes natural reproductive barriers between acuminata wilddiploids, and is an obvious component of subspecies divergence and increased geneticdiversity of the species as a whole. It is important for breeders, because it can seriouslyhinder their efforts at recombining certain characteristics of wild materials beforecreating suitable cultivars.

In effect, there are at least three important genetic consequences of structuralhybridity. First, there is interchromosomal linkage resulting from the fact that thenonhomologous chromosomes involved in a reciprocal translocation, for example, willassociate during the first meiotic prophase to form multivalents (as opposed to normalbivalents) which behave as a single recombination unit or linkage group. Thus, genesnormally situated on different chromosomes in one of the parents, that is genes thatusually segregate independently, will show varying degrees of genetic linkage in theprogeny of structural hybrids. Secondly, there is reduced recombination of genes locatednear the break-points at the origin of the translocations: see Figure 1 (Burnham 1962;Carlson 1988; Rees 1961; Rees, Jones 1977; Rickards 1983); this is reflected by


reductions in the genetic distances between those loci. And thirdly, given that thedisjunction of multiple chromosome associations at meiotic metaphase usually results inan unbalanced distribution of interchanged chromosomal segments, some gametes willbe inviable and their gene combinations will not be represented in the progeny, i.e.,there is differential gametic viability. This is simply reflected in the progenies as a moreor less severe distortion of the expected segregation of genes carried on rearrangedchromosomes (e.g., Rees 1961; Sharp, Hilliker 1989).


Figure. 1. Diagrammatic representation of meiotic pairing in a hypothetical heterozygote fortwo reciprocal translocations involving three pairs of chromosomes.

Thus, both the probability of finding unwanted linkages and the strength of theselinkages may increase; concomitantly, the frequency of certain genotypic classes maydecrease; the overall effects could well interfere with the breeder’s efforts atrecombining and transferring desirable traits from wild and cultivated diploids,sometimes forcing them, even unwittingly, to attempt bridging crosses to circumventthese reproductive barriers or to avoid apparent linkage of undesirable traits.Conversely, translocations can also be used to the breeder’s advantage for the transfer orthe localization of particular gene loci (Beckett 1991; Jones, Rawls 1988; Weber 1989).

Either way, knowledge of the extent and distribution of structural rearrangementswould be an invaluable asset to expedite breeding strategies involving diploid bananas.This could be achieved through the combined investigations of meiotic behavior ofhybrids of interest to improvement programs (e.g., Bakry et al., CIRAD) and the searchfor specific genetic markers (RFLP or other) closely associated to the rearrangedsegments. In other plant families, different RFLP marker orders have been shown toexist within and between species or higher taxonomic orders. For example, the peppergenetic map is highly rearranged with respect to those of the tomato and the potato,while in the latter two, marker order is conserved (Bonierbale et al. 1990; Bonierbale etal. 1988; Tanksley et al. 1988). Within pepper (Capsicum) alone, there are largenumbers of structural differences between and within the five species that have givenrise to today’s cultivated types (González de León 1986; Pickersgill 1977). Mappingstructural differences will certainly help manage and perhaps take advantage of theirgenetic effects for breeding purposes. Such information will guide breeders as to thechoice of better progenitors while providing them with tools that can help them detectdesirable structural homozygotes that can more easily be used for further recombinationand introgression.

First Elements of a Genetic Map of Musaacuminata: a glimpse at work in progress atCIRADThe first elements of an RFLP linkage map of the Musa acuminata genome have beenderived by screening two genomic libraries isolated from young banana leaves. Eachlibrary was developed through digestion of nuclear-enriched genomic DNA with one oftwo restriction enzymes (EcoRI and PstI). Fragments from 500 to 2000 bp were selected,cloned into a suitable plasmid, and transformed into a bacterial host. The libraries werescreened for repeated sequence inserts (Amson 1989; Fauré et al. 1991; Fauré et al.1992). Some of the results of the screening of a number of banana accessions withinserts from these libraries are given in Table 1. Separate samples of DNA of eachaccession were digested with one of two restriction enzymes (DraI and EcoRV). Apartfrom the ease of isolation of some 250 usable probes that reveal unique sequences indiploid bananas, the essential fact illustrated by these results is the high degree ofpolymorphism revealed by the probes; thus, most markers will segregate in the progenies


of most crosses. Furthermore, by using two more enzymes, between 60% and 98%polymorphism is revealed between any pair of accessions tried so far. This is comparableto some of the most polymorphic plant species reported in the literature.

At present, the mapping work is being conducted strictly within the framework ofCIRAD’s banana improvement program. Mapping populations are being developed inGuadeloupe (CIRAD-IRFA) in collaboration with F Bakry and JP Horry. The nature offour of these populations is outlined in Figure 2. Field evaluations of important


Table 1. Summary of the characteristics of the probe libraries (unpublished data available upto December 1991). The results correspond to the overall polymorphism revealed after restrictiondigestion of the DNA of each accession with two different enzymes (see also Fig.3).

Figure 2. Four of the mapping populations under current development and/or evaluation inCIRAD’s banana improvement program.

characters such as parthenocarpy and disease resistance will be made on all of thesepopulations. When possible, all other characters that show variation in the progeny willbe measured and their inheritance analyzed.

The first population available for study was the F2 of SF265 x banksii. This was easyto create since SF265 (AAcv) appears to be a derivative of ssp. banksii; for the samereason, however, it is less polymorphic than the others. In addition, the female parentwas found to be heterozygous at many loci thus reducing even further the number ofusable probes in the F2 progeny. Two examples of segregation profiles revealed by suchprobes are shown in Figure 3.

Up to December 1991 the linkage map developed using 82 F2 individuals of thispopulation comprises 27 RFLP loci and 5 isozyme loci. About a third of these markersshowed a significantly distorted segregation (data not shown).

Linkage analysis of the segregation data was performed using multipoint techniquesas implemented in the computer package Mapmaker V1.0 (Macintosh) (pers. com.Tingey et al., Dupont Co. 1990; Lander et al. 1987). The resulting provisional map isshown in Figure 4; 6 linkage groups with 2 to 5 loci each have been found so far. Twelveother markers segregated independently.

Our preliminary cytological investigations indicate that the wild banksii parent hasnormal male meiosis, that SF265 is heterozygous for a reciprocal interchange, and thatthe F1 hybrid plant that gave rise to the F2 is heterozygous for two such rearrangementsinvolving three pairs of nonhomologous chromosomes (a situation similar to thatdepicted in Figure 1). As was outlined in an earlier section, there is a strong probability


Figure 3. Typical RFLP segregation patterns observed among the F2 progeny of SF265 x banksii. A. Hybridization signal of probe pMaCIR1006 of the EcoRI library onto DNAdigested with DraI; B. Hybridization signal of probe pMaCIR27 of the PstI library onto DNAdigested with EcoRV.

that the distorted segregation ratios were due to differential gamete viability in the F1parent. This connection is being investigated at present. Additional geneticconsequences have already been discussed, notably some form of interchromosomallinkage between the rearranged chromosomes. In this cross, we could then expect todetect fewer than the 11 linkage groups corresponding to the basic number ofchromosomes in these diploids. Besides, not only should one of these groups be relativelylarger, but it should also contain all or some of the markers that showed distortedsegregation. The possibility of stronger linkage among these, owing to the likelyreduction in recombination near the break-points of the interchange meioticconfiguration, will be explored.

Our plan is to put between 60 and 100 markers on this map in 1992. These willinclude RFLP, RAPD, isozyme and perhaps a few morphological markers. With thepublication of the map toward the end of 1992, we shall endeavor to make freelyavailable all of the probes that will have been used in this or other maps based on otherpopulations (Fig.2).

The Suitability of Musa Plant Materials forMolecular Marker AnalysesThe feasibility of constructing a genetic map of the M. acuminata genome was, we think,well established in the previous section. In order to support this claim even further, andperhaps motivate potential or actual banana breeders and geneticists to take a deeper


Figure 4. First elements of a linkage map of the banana genome (Musa acuminata)constructed from unpublished data available up to December 1991.

interest in this kind of research, we rapidly review below what, in our experience, makesdiploid bananas suitable for molecular marker analyses.

Indeed, the obvious shortcomings of bananas as model genetic organisms, resulting fromtheir relatively long life cycle and enormous plant habit as compared with typical annualcrops, are outweighed by numerous advantages for genetic and molecular marker work.

Pollinations between numerous diploid accessions are fairly easy to perform at leastin one direction, even when the flowers are perfect, as in the case of M. acuminata ssp.banksii. Besides, the relatively slow sequential development of the female inflorescence,the long-term availability of large amounts of pollen from single individuals, and theclonal reproduction of each plant, all allow for self- and cross-pollinations to beperformed given enough planning. There are difficulties, of course, when differentdegrees of female and/or male sterility complicate the task, or when there is a need forrescuing embryos through tissue culture to ensure their survival; but none of theseobstacles is unique to or specially acute in the bananas. In favorable cases, very largeamounts of seed can be produced from a single cross.

Once the progeny of a given cross is established in the field, it essentially representsan eternal population, indefinitely available for further study and evaluation, givenenough maintenance. Not only that, it can also be cloned and thus replicated in one ormore different environments. The equivalent operation can be more difficult and costlyin the case of more widely studied annuals.

Leaf material is available in great, almost unlimited amounts from any young to adultindividual plant, at any time of the year, and is very easily harvested and stored.Extraction of fairly pure DNA in large quantities and for large numbers of samples iseasily and cheaply achieved. In our laboratory for instance, in 2 weeks, a single personcan extract, quantify, and check for quality enough DNA for a fair-sized project involving200 individuals to be analyzed for 40 probe-enzyme combinations (protocols, fromharvest to final results, are available from the authors).

The high polymorphism that has been revealed at the DNA level means that anessentially unlimited number of markers can be developed even for crosses betweenfairly related accessions (e.g., SF265 x banksii, etc., Table 1).

Finally, owing to the increased representation of wild diploids in the largercollections (CIRAD, Guadeloupe; Vakili 1968), it should be possible to establish a widevariety of segregating populations arising from crosses between them. Prior screening forpolymorphic loci will contribute to the genetic description of these materials well beforemuch else is known about them on other grounds.

Towards an Integrated Approach to the Study ofthe Musa GenomeIt has been suggested above that at least some of the genetic characteristics andconstraints of the diploid bananas can be dissected using molecular markers. Later, itwas argued that one of the means toward such a target is the construction of a genetic


map, and that this, in itself, is now a tangible reality. This reality was made possible bythe suitability of the bananas themselves as materials for genetic and molecular markermanipulations. It is therefore only natural to propose that molecular markers should beused to complete our picture of the banana genome in terms of its wealth of variationthroughout Musa and even beyond it. The program developed at CIRAD is an attempt atintegrating mapping and formal genetic studies with the investigation, description, andanalysis of molecular diversity in the two putative progenitor species of the ediblebananas and plantains. A synoptic diagram of this program, giving an approximatechronology for the start of various subprograms and final objectives, is given in Figure 5.The central concept in this scheme is that of genome “reconstruction”: it may well bepossible to reconstruct improved polyploid assemblages (AAA, AAB, ABB, AABB, etc.)from chosen recombined diploids; the building blocks would be provided by data on gene


Figure 5. A scheme for an integrated approach to the study of the Musa genome usingmolecular marker technologies: the approximate chronology of CIRAD’s current program isoutlined on the left.

orders found in the genus and their correlation with the presence or absence,throughout the genome, of particular alleles in specific combinations. Inherent to thishypothesis is the idea that the genome architecture that will be revealed in today’sbananas still reflects the ancestral contributions to existing domesticates.

Current diversity data, whether at the morphological or genetic levels, give sufficientsupport to our working hypothesis. The classic classificatory scheme, whose mainelements were the morphological recognition of the bispecific origins of bananas andplantains coupled with knowledge of chromosome numbers (Simmonds, Shepherd 1955),has been, in essence, supported by recent studies using various kinds of genetic orbiochemical markers. Thus, isozyme markers (Horry 1989; Jarret, Litz 1986a,b),flavonoids (Horry, Jay 1988), and chloroplast DNA markers (Gawel, Jarret 1991a,b), allsupport the concept of bispecific origins. Indeed, diagnostic isozymes and chloroplastDNA bands have been described. But perhaps the more pertinent data in the presentcontext is that the isozyme, flavonoid and, more recently, the data obtained from theanalysis of length variation in the intergenic gene spacer of banana ribosomal genes(rDNA) (Lanaud et al. 1992), all confirm earlier beliefs that the diversity of thecultivated types represents a fraction of that encountered at the wild, diploid level.

The use of mapped loci as markers for diversity studies will help strengthen thecurrent biosystematic treatment of the genus, by providing data on numerouschromosome segments well distributed over the entire genome. Moreover, it will bepossible to verify the existence of linkage disequilibria fixed in present sterile, clonaledible types.

In order to build this integrated approach into the study of the banana genome onsolid ground, it is essential that the mapping effort itself rests on a structured,coordinated plan that it is hoped will involve many participants. The followingparagraphs suggest some ideas as to what ingredients such a plan should include.

An integrative core map of Musa and beyond: somesuggestions and propositions

1. Development of reference mapping populations A concerted effort, possibly coordinated through INIBAP, for the development andestablishment of multiple reference mapping populations (RMPs) should be madewithin the framework of collaborative efforts between existing field and laboratoryfacilities; some guidelines for prospective developers of such populations are given in theappendix to this paper. Moreover, some or all of these populations should be multipliedfor multisite evaluations after quarantine clearing. Tests of the behavior of thesepopulations under various biotic and abiotic stresses would then be simplified and eachbreeding program would be able to obtain data pertaining to their specific improvementobjectives during all or some specific growing cycles.

2. Construction and use of a “core” mapIt is proposed that initial efforts should concentrate on the construction of a basicreference or “core” map to be used for coordinating all mapping efforts and applications.


Recently developed for maize (Gardiner et al.: pers. com. EHJ Coe), the idea of a coremap consists in identifying a standardized set of RFLP markers that would be more orless evenly spaced in the genetic map(s) developed from the RMPs. The probes forrevealing such loci should give strong, simple-to-read signals, and their relative distancesshould be such that their relative order be at least 1000 times more probable than anyother order for the same probes. Other probes revealing loci within shorter distances ofthe core markers will simply be put into the intervals or “bins” defined by the latter. Inother words, depending on the size of the starting mapping population, the core map willhave a defined density (see the appendix). As more information is obtained from othercrosses and experiments, some of the bins defined by core markers can be furthersubdivided into smaller segments. Also, as more information about structuralrearrangements becomes available, different bins will “contain” break-points markingspecific translocations or inversions. The core map will slowly evolve into an integrativeone (see below).

The main advantage of a core map is that it simplifies data-sharing between researchgroups and should expedite the integration of segregation data obtained from RMPs andother, more specific, populations. Eventually, any genetic trait would be localized to asmall region of the genome by using a limited number of agreed-upon markers.Furthermore, the bins themselves should serve as the indexing basis of a mappingdatabase system (Gardiner et al.).

3. From a core map to an “integrative” map and beyond

Eventually the map should be “integrative” in at least two ways.

First, it should aim at representing the plethora of structural rearrangements thathave accompanied genome evolution and domestication in Musa (see above). This willnot only clarify phylogenetic relationships between taxa (especially in conjunction withmolecular systematic data), it will also help guide crossing strategies so as to avoidunwanted linkages and favor specific genic recombinations.

Secondly, the integrative map should combine all kinds of markers at themorphological, biochemical, and DNA levels. Each marker type has its own intrinsicadvantages and limitations, and they all should be used to answer specific questions inspecific ways.

Molecular genetic markers should include those based on anonymous RFLP probessuch as those described in the previous section, new marker types such as RAPDs, andcloned genes, of which there are more than 100 now available that have been isolatedfrom other species. These can reveal RFLPs corresponding to putatively functional geneswhen used as heterologous probes.

In the case of PCR-based methods such as RAPDs that have a few intrinsicdisadvantages, such as the detection of different loci using the same oligonucleotide(acting as a “probe” in this case) on different populations (Williams et al. 1991),researchers would be encouraged to isolate the polymorphic DNA bands and use them asRFLP probes on some or all of the RMPs (Williams et al. 1990).


Conversely, sequencing of the ends of existing probes should help develop PCR-baseddetection systems for the fine genetic analysis of specific loci or for increasing theprobability of finding a polymorphism when this has not been possible through othermeans. In effect, the sequenced ends can be used as primers for amplification of thesequence corresponding to the original probe, and the product can be digested by one ormore restriction enzymes to reveal differences between otherwise monomorphicaccessions at that locus (Williams et al. 1991).

Such methods could conceivably evolve into +/- detection systems that rely on visibleproducts that can be measured directly in the test-tube used for DNA amplification,(e.g., Higuchi et al. 1992).

Progressively, a map should emerge that combines all types of available markers andtherefore becomes neutral with respect to technical approaches.

While the minute chromosomes found in Musa have resisted attempts at establishingadequate karyological descriptions, their low number (2n=2x=22) and the rather smallsize of the genome as a whole (Arumuganathan, Earle 1991, our own unpublished data,Novak 1993) could help motivate the construction of a library of very large banananuclear DNA fragments cloned into yeast artificial chromosomes (YACs: see for exampleGanal et al. 1989; Garza et al. 1989; Guzmán, Ecker 1988). The sequence-tagged sitesgenerated upon partial sequencing of RFLP probes could serve as a starting point forrapidly locating these “long-distance” probes (i.e., several hundred kilobases) on thecore map. The eventual construction of physical maps of the banana genome, i.e., theordering of YAC clones that cover most, if not all, the genome would then be achievedand would open up new options such as map-based cloning of banana genes (e.g.,Tanksley et al. 1989).

4. Coordination of mapping activities: a mapping information database and clonebank

The scant present research on banana genetics certainly needs a substantial boost;meanwhile, it is all the more important to optimize what little is being done around theworld. As more laboratories start applying molecular technologies, it seems desirable tostart coordinating their activities as soon as possible. One way of achieving this would beto set up a mapping information database and a clone (probe) bank that would promotesharing of tools and information, and help define genetic nomenclatural rules right fromthe start. Initially, INIBAP could help set up a probe transfer and exchange system tofacilitate and promote the use of the best probes that are already available.

ConclusionsDespite their major importance as a staple starch source and their significant place inthe export market under their dessert forms, bananas and plantains still remain on themargin of modern genetics research and its applications to breeding practices.

On account of their highly polymorphic nature and their ease of handling formolecular marker studies, the bananas are an easy target of such studies.


Moreover, with the increasingly important role that wild diploid clones play in theimprovement of the crop, and the complex biological problems that these diversematerials pose to breeders and geneticists alike, it seems logical, if not imperative, toincrease our investigations of their genetics and reproductive biology. We have arguedthat genetic mapping provides powerful tools to achieve these goals, and havedemonstrated that it is feasible. Further progress in the genetic dissection of Musa spp.should benefit from an approach that would integrate cytogenetic, mapping, and geneticdiversity data into a coherent description of the Musa genome.

Thus, far from being an end in itself, mapping is a means toward a betterunderstanding of genome evolution, organization, and function; it provides a set ofpowerful handlers — or genetic markers — that allow extensive genetic manipulations.Genetic markers should quickly enhance and strengthen our knowledge of genomediversity and organization of the diploid bananas and their ancestral contributions totriploid cultivars; in gaining such knowledge, breeding practices will have more rationalbases and, eventually, we shall be in a better position to assess how and for whatspecific purposes marker technologies and other molecular tools can have a direct andcost-effective impact on banana and plantain improvement.

AcknowledgmentsJL Noyer’s expert technical support is greatly appreciated. Our special thanks to F Bakryand JP Horry and their team in Guadeloupe, to J Ganry and H Tezenas du Montcel,without whose dedication and support little would have been possible. This research isbeing supported in part by a grant from the Commission of European Communities’ STD2program (TS 2A-0094-F(SD)), and a doctoral studentship (S Fauré) from the FrenchMinistry of Research and Technology.

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PATERSON AH, DAMON S, HEWITT JD, ZAMIR D, RABINOWITCH HD, LINCOLN SE, LANDER ES, TANKSLEY SD. 1991.Mendelian factors underlying quantitative traits in tomato: comparison across species, generations, andenvironments. Genetics 127:181-197.

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Appendix: Mapping Populations — a fewguidelines for prospective developers(by M Lorieux, D González de León, CIRAD, France)

The purpose of these brief guidelines is to orient prospective developers of populationsfor genetic studies and mapping. A few theoretical considerations are discussed and aselected bibliography is suggested for further reading.

Construction of a genetic map: marker density, populationsizes, and precision of estimated genetic distances

Population types of practical interest to banana geneticsIdeally, the type and size of a population chosen for mapping characters of interest shouldallow an optimal estimation of the recombination frequencies between segregating


markers and the traits themselves. Practical considerations may suggest specialpopulation types, such as recombinant inbreds. In the case of the bananas, populationsare potentially eternal and therefore one should aim at generating F2 progeny populationsgiven that these are the most informative when using markers such as RFLPs (seebelow). However, in many cases, the production of F2 populations may not be possiblebecause it is impractical to obtain entirely homozygous diploid lines, unless these alreadyexist (e.g., banksii accessions). Figure A(1) shows the normal crossing scheme forobtaining various types of segregating populations from two homozygous parents. Thiswas illustrated in Figure 2 of the main article by the cross between the wild diploid clonesCalcutta 4 and banksii.

Other population types, such as those shown on Figure A, may be easier to obtain inthe short term. Although these populations may not be optimal for mapping, the relativeloss of information at a given population size may not be significant for certainapplications (Gebhardt et al. 1989; Ritter et al. 1990). For instance, in order to speed upthe mapping of structural differences within and between Musa taxa, a number ofprogeny populations could be developed from crosses or selfings within structural groups;maps could then be constructed using markers already localized in a reference map: onlytwo markers per chromosome arm (about 44 in all) should help detect major changes ingene order. Then, more intensive mapping in and around that linkage region would bepursued with additional probes to get a better estimate of break-point location.

The precision of estimated genetic distances

The precision of estimations of recombination frequencies or genetic distances, dependson three interrelated factors: the size of the population, the types of markers to bemapped, and the density of these markers (and therefore the genetic size of the genome).The more individuals are genotyped, i.e., the more information is available, the moreprecise is the estimation. This precision can be expressed as the standard error sr of therecombination frequency r. Allard (1956) provided formulae and tables to calculate sr inthe case of pair-wise segregation data (two-point analysis), for different population andmarker types. The latter fall into two categories according to their mode of segregation:codominant markers (e.g., isozymes or RFLPs) and dominant markers (e.g., RAPDs andmany morphological single gene mutations).

Codominant markers (isozymes, RFLPs). When markers are codominant, bothhomozygous and heterozygous individuals are detected. Thus, each individual is fullyinformative, and the estimation of the recombination frequency attains its maximalprecision. Marker order is inferred from recombination fractions between two, three, ormore loci taken simultaneously. It is therefore desirable that the standard errors be verysmall in comparison with the value of r itself. This is illustrated in Figure B for markershaving a recombination frequency of about 20%: for example, an F2 of 50 individualsshould suffice to keep sr < 5% when using codominant markers. By contrast, about 28more individuals from a backcross population are needed to achieve the same precision.


Dominant markers (RAPDs). In the case of dominant markers, the estimation of therecombination frequencies is less precise than with codominant markers unless thepopulation is a backcross, because in an F2 homozygous and heterozygous individualscannot be distinguished. Moreover, the precision of this estimation depends on the phaseof the alleles at each of the two loci in the F1 parent (see Figure B for explanations).

Analysis of dominant and codominant markers simultaneously. When bothcodominant (e.g., RFLPs) and dominant (e.g., RAPDs) markers segregate in the samepopulation, the precision of the estimation of their recombination frequency isintermediate between the two preceding cases.


Figure A. Types of populations that can be developed for genetic studies in the bananas andplantains. 1. Generation of F2 and backcross progenies from homozygous parents (some wilddiploids). BC progenies can be helpful in the analysis of dominant/recessive characters and ofsome nucleocytoplasmic interactions. 2. A “pseudo-F2” is obtained by self-fertilization of aheterozygous individual. The difficulty here is that the parental genotypes are unknown andneither is the phase of the markers (see Fig.B). 3. “Pseudo-backcross” obtained from parentshaving common alleles or not. 4. When mostly heterozygous parents are used, havingcommon alleles or not, certain loci will be informative in an “F1” but uninformative in anotherone; a strategy would consist in developing several complementary “F1”s and F2s.

Marker density and order, and the “core” mapThe concept of the “core” map has been defined in the main paper. Here we shall touchon some statistical aspects.

The relative order of core markers should be established with a high probability at agiven density and for a specific population size. There are several methods fordetermining marker order. The most powerful takes into account all the recombinationevents that have occurred in a linkage group in all the population (“multipoint linkageanalysis”: Lander et al. 1987). Here we shall illustrate the relationship between thedensity of the markers given by their mean distance (Dm), the population size, and theprecision of estimated r values, using simple two-point analysis.

Assuming a normal distribution of the values of r, there is a 99% probability that theestimation of r is within the interval [r-2.58sr, r+2.58sr]. Such confidence intervals aroundthe recombination frequencies should not overlap, so as to have a high probability that


Figure B. Standard error (sr) of the recombination frequency (r) as a function of thepopulation size (n), when the recombination frequency between two loci is 20%. sr decreasesexponentially as the population size increases, with different rates according to the type ofpopulation. BC: backcross population, codominant or dominant markers; F2 2 codo: F2population, two codominant loci. F2 2 domC: F2 population, two loci dominant and incoupling, i.e., the two dominant alleles are on the same chromosome of F1 parent. F2 2 domR:F2 population, two loci dominant and in repulsion, i.e., the two dominant alleles are each onone of two homologous chromosomes of F1 parent. F2 1codo 1dom: F2 population, one locuscodominant , the other dominant.

the relative sequence of the markers is true. This means that the mean distance betweenmarkers Dm should not have a confidence interval larger than Dm/2, i.e., 2.58sr ≤ Dm/2 fora 99% probability, or 1.96sr ≤ Dm/2 for a 95% probability. For example, given an F2population of n=80 individuals mapped with codominant markers, we have to find, inFigure C, a value of r (= Dm) for which sr ≤ r/(2*2.58). We then find that for Dm= 0.2, sr =0.036, so we have sr ≤ Dm/(2*2.58) =0.0388. Thus, a core map with an average distancebetween markers of 20 cM can be constructed with an F2 population of 80 individuals, forcodominant markers, and with 99% confidence intervals for the estimations of therecombination values. If the genome has a total length of, say, 2000 cM, then about 100regularly distributed markers would be needed to approximate a usable core map.

Suggested referencesALLARD RW. 1956. Formulas and tables to facilitate the calculation of recombination values in heredity.

Hilgardia 24: 235-278.BURR B, BURR FA. 1991. Recombinant inbreds for molecular mapping in maize: theoretical and practical

considerations. TIG 7:55-60.LANDER ES, GREEN P, ABRAHAMSON J, BARLOW A, DALY MJ, LINCOLN SE, NEWBURG L. 1987. Mapmaker: an interactive

computer package for constructing primary genetic linkage maps of experimental and natural populations.Genomics 1:174-181.


Figure C. Standard error (sr) of the recombination frequency (r) as a function of thepopulation size (n), when the recombination frequency between two loci takes a range ofdifferent values (inset). These curves were constructed for an F2 population mapped withcodominant markers.

RITTER E, GEBHARDT C, SALAMINI F. 1990. Estimation of recombination frequencies and construction of RFLPlinkage maps in plants from crosses between heterozygous parents. Genetics 125:645-654.

SNAPE JW. 1988. The detection and estimation of linkage using doubled haploid or single seed descentpopulations. Theor. Appl. Genet. 76:125-128.

TANKSLEY SD, YOUNG ND, PATERSON AH, BONIERBALE MW. 1989. RFLP mapping in plant breeding: new tools for anold science. BioTechonology 7:257-264.

TANKSLEY SD, MILLER J, PATERSON A, BERNATSKY R. 1988. Molecular mapping of plant chromosomes. Pages 157-173 in Chromosome Structure and Function: impact of new concepts (Gustafson JP, Appels R, eds). NewYork, USA: Plenum Press.

WILLIAMS JGK, KUBELIK AR, LIVAK KJ, RAFALSKI JA, TINGEY SV. 1990. DNA polymorphisms amplified by arbitraryprimers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535.

Mapping QTLsWhen a quantitative trait is observed in a mapping population, it is possible to establishcorrelations between the “phenotypic values” of the trait for each individual and thecorresponding genotypes for all segregating markers in the population. Obviously, themore genotypic classes there are, the more precise will be the analysis of the trait.Codominant markers such as RFLPs will therefore be more powerful than dominantmarkers in F2 progeny populations. Large populations may be needed for dissectingcharacters under the control of many genes. The power of different methods for detectinggenomic segments underlying quantitative trait loci (QTLs) has been discussed by variousauthors:ASINS MJ, CARBONELL EA. 1988. Detection of linkage between restriction fragment length polymorphism

markers and quantitative traits. Theor. Appl. Genet. 76:623-626.KNAPP SJ. 1991. Using molecular markers to map multiple quantitative trait loci: models for backcross,

recombinant inbred, and doubled haploid progeny. Theor. Appl. Genet 81:333-338.LANDER ES, BOTSTEIN D. 1989. Mapping mendelian factors underlying quantitative traits using RFLP linkage

maps. Genetics 121:185-199.PATERSON AH, DAMON S, HEWITT JD, ZAMIR D, RABINOWITCH HD, LINCOLN SE, LANDER ES, TANKSLEY SD. 1991.

Mendelian factors underlying quantitative traits in tomato: comparison across species, generations, andenvironments. Genetics 127:181-197.

STUBER CW, LINCOLN SE, WOLFF DW, HELENTJARIS T, LANDER ES. 1992. Identification of genetic factorscontributing to heterosis in a hybrid from two elite maize inbred lines using molecular markers. Genetics132:823-839.


52 Applications of Biotechnologies to Banana Breeding

Applications of Biotechnologies toBanana Breeding: haplogenesis,plant regeneration fromprotoplasts, and transformation

F Bakry1, R Haïcour2, JP Horry1, R Megia2, L Rossignol2

Introduction

Creation of resistant varieties is necessary for banana cultivationIn comparison with other food crops such as cereals, little is known about bananagenetics, especially as regards the determinism of main characters such as parthenocarpyor female sterility, which are a requirement for the production of edible seedless bananas(Dessaw 1988).

Despite 60 years’ work in the field of hybridization, all cultivated varieties are stillnatural and issued from clonal selection (Rowe 1984). Due to disease extension inbananas the creation of new cultivars or the improvement of already-existing bananasbecomes necessary. The main diseases are black leaf streak due to Mycosphaerellafijiensis Morelet, Panama disease due to Fusarium oxysporum Schlecht f. cubense (E.F.Smith) Snyd. & Hams race 1, with some new pathogenic strains, moko disease due to Pseudomonas solanacearum E.F. Smith race 2, banana bunchy top virus (BBTV),cucumber mosaic virus (CMV), weevils, nematodes, etc.

The restricted genetic base of cultivated clones makes banana growing a particularyfragile activity because of the rapid extension of new strains of diseases. In addition, thethreat of pest and disease attack is increased by monoculture and the lack of soil rotation(Delvaux et al. 1990).

For many diseases — Panama, moko, BBTV, CMV — no chemical treatment exists, anddestruction of the affected plants is the only method of control. For the others, the cost ofchemical treatments is increasing as more virulent strains appear, or even prohibitive forsmall farmers in developing countries, or else damaging to the environment. In particular,this is obvious for black leaf streak which has expanded through South America and Africa(De Langhe 1987; Bakry et al. 1989).1Station de Neufchâteau, CIRAD-IRFA, 97130 Sainte Marie, Guadeloupe, FWI; 2Laboratoire de Morphogenèsevégétale expérimentale, Université Paris-Sud XI, Bât.360, 91405 Orsay Cedex, France

Banana breeding programs supported by the EECSince 1982 CIRAD-IRFA has developed a program of banana breeding supported by theEEC. The results obtained during programs STD1 and STD2 improved our knowledge of thestructure of the Musa genus and gave a better understanding of the mechanisms ofsterility in Musa (CIRAD-IRFA, Neufchateau Station, Guadeloupe, FWI) (Dessauw 1988),as well as the resistance to Cercospora spp. (CIRAD-IRFA Phytopathology Laboratory,Montpellier, France; CRBP Cameroon) (Carlier, Mourichon 1990; Fouré et al. 1990).Progress has also been achieved in the genetic mapping of diploid bananas, which willprovide better genetic control of agronomic traits (CIRAD-BIOTROP, Montpellier, France)(Fauré et al. 1991). In addition, progress has been obtained in in-vitro culture, especiallyregarding vegetative propagation and regeneration (in-vitro culture laboratories at KULeuven, Belgium; CIRAD-IFRA/CATIE, Costa Rica; CIRAD-BIOTROP, Montpellier, France;Paris XI University, Orsay, France) (Banerjee et al. 1987; Sannasgala 1989; Escalant,Teisson 1988, 1989; Harran 1991; Megia et al. 1992).

The aim of the STD3 program, which has just been agreed by the EEC, is to identifynew banana cultivars suitable for export and new plantains for local use resistant to ortolerant of black leaf streak, which is the main threat to banana production. In addition,good productivity, structure, and organoleptic quality of fruits have to be maintained, aswell as parthenocarpy and sterility.

For that purpose six different projects will be developed by the groups involved in theprevious STD1 and STD2 programs, as follows.

1. Genetic resources: collection and conservation of clones selected on the basis ofmorphotaxonomic and genetic evaluation, to permit the creation of databases that can bemade available for breeders.

2. Genetic studies and diploid breeding: to control sterility, to understand the geneticcoding for agronomic traits and resistance characters, and to create new lines for triploids.

3. Agronomic evaluation of diploids and newly created varieties.4. Varietal improvement through 3x cv x 2x → 4x cv crosses.5. Nonconventional varietal improvement by using protoplasts and genetic engineering.6. Studies on the local distribution and genetic variability of Mycosphaerella fijiensis

in relation with the host.This paper describes original data obtained during the STD2 program by three groups

that are now involved in the STD3 program in the field of biotechnology: - from Paris XIUniversity, Orsay, France; CIRAD-IRFA, Neufchâteau Station, Guadeloupe, FWI; andCATIE, Costa Rica.

The Strategy Chosen by CIRAD-IRFA and itsCollaboratorsThis strategy, as agreed for the STD3 program, is focused on two main topics.

1. The correction of defects in well-established cultivars, such as Cavendish (AAA) orPlantain (AAB), in order to acquire a specific resistance, for example.

53F Bakry, R Haïcour, JP Horry, R Megia, L Rossignol

2. The creation of new synthetic hybrids, which will be either sweet bananas or cookingbananas, according to the selection criteria chosen.

A combination of conventional breeding methods with new techniques in geneticengineering is necessary to achieve these objectives.

ResultsMost of the natural edible parthenocarpic varieties of bananas are heterozygous, unliketheir wild relatives. This implies a complicated inheritance pattern because the observedcharacters rely both on simple or polygenic traits (additive values) and on improvedperformance gained from poly-allelism (heterosis) at multiple loci (epistasis).

Beside the genetic aspects, structural heterozygoty (exchanges, inversions) can alsopartly explain the sterility of edible diploid bananas.

Thus the most rational use of diploid varieties is the obtention of more fertile purelines in which the gene potential will be highly inheritable and can produce F1 hybridsusable in banana breeding. It is therefore necessary to obtain homozygotic bananas inorder to create controlled F1 hybrids suitable for our breeding program.

Haplo-diploidization techniques, already applied in some monocotyledon programs, area valuable model for the creation of new pure lines in bananas. This is why we started ahaplomethod program with banana using immature pollen, i.e., in-vitro androgenesis onthe one hand, and as unfecunded oospheres (i.e., induced parthenogenesis) on the other.

Haplomethods

Androgenesis

Recent successful results were obtained in Guadeloupe with anther culture of wild Musaacuminata ssp. burmannica cv Long Tavoy (Fig.1).

A creamy white callus (1 mm diameter) emerging through the dehiscence split wasisolated and subcultured. After transfer to a shoot-initiating medium, vegetative budsdeveloped on the callus. Plantlets rooted when isolated, and were transferred to a basalmedium (BM). About 20 weak plants were weaned, but 10 only survived in the greenhouse,showing more or less variegated leaves.

Chromosome counts carried out on root tips showed four plants to be diploid(2n=2x=22) and the others are probably diploid too, as suggested by their identical form.

The microsporial origin of these four plants was proved by isoenzymes studies: themother plant presents a three-band pattern heterozygous locus with dimeric enzymes(Got. B and Est. A), whereas there is a one-band pattern homozygous locus with theregenerated plants.

Induced parthenogenesis

The method currently developed at CATIE consists of the use of irradiated pollen (bygamma ray) to stimulate, after pollination, the embryo development of the oosphere



Figure 1. The different stages of anther culture, material, and culture conditions.

without fertilization, to obtain, in the seedlings, haploid or spontaneously doubled haploidplants.

Pollen from M. acuminata ssp. burmannicoides and M. balbisiana cv Tani wasirradiated with different gamma radiation doses (3-100 krad, 60cobalt source), and thenused to pollinate female flowers of M. acuminata ssp. burmannicoides.

Observations with a fluorescent microscope revealed that the germinating tube growsto reach the ovule at any tested dose of pollen irradiation, but only the treatmentsbetween 0-10 krad permitted embryo development. Moreover, endosperm-vacant seedswere found in all doses used, but more markedly at those above 8 krad.

Embryo rescue by in-vitro transfer permitted the development of whole plantsoriginating from hybridization with pollen irradiated with 3, 5, 7, 8, and 10-krad treatments.

Karyotypes carried out on root tips showed the plants evaluated to be mainly diploid,although two weak plants were found to be haploid (one with 5-krad and the other with 7-krad treatments).

Discussion

These preliminary results obtained by our group for the first time show that bothtechniques described above (androgenesis and induced parthenogenesis) are applicableto banana. The techniques make possible a reduction in the cost and duration ofexperiments (at least eight generations are necessary to reach homozygosity by self-fertilization (Fig.2). Additionally, in-vitro androgenesis is the only technique available forobtaining homozygotic plants in some clones from 2x parthenocarpic clones as 2n, whichare usually female-sterile, with weak male fertility.

Finally, these techniques enable us to obtain new kinds of plant material such ashaploid callus and haploid cell cultures, which create new perspectives in the field ofsomatic cell fusions or transformations.

Protoplast regeneration and transformationFor this purpose the group of nonconventional banana breeders in Orsay has developedspecial protoplast techniques. These are of considerable interest in the production ofnonconventional hybrids, and for electroporation transformation.

At this time the main difficulty with banana protoplasts is the low potential ofprotoplasts to divide and to differentiate into a whole plant. To overcome this problem theOrsay group has focused its work on the culture and differentiation of protoplasts. Bakry(1984), followed by Da Silva Conceicâo (1989), observed low reactivity in bananaprotoplasts, and Da Silva Conceicâo reported that few divisions could be obtained, andonly with embryogenic material.

Working with the CIRAD-BIOTROP CIV laboratory (CIRAD-IRFA, Montpellier, France),which kindly provided appropriate plant material (embryogenic suspension of diploidMusa acuminata ssp. burmannica cv Long Tavoy obtained by Escalant [Escalant, Teisson1989]), the Orsay team, using a feeder cell technique, succeeded in obtaining continuousdivisions from protoplasts to microcolonies, up to the callus stage.

This kind of experimentation, however, is limited by the long- term embryogenicculture used as cell suspension, which does not allow us to obtain embryos and wholeplants. So we have used other plant material with better potential. Through suitablecollaboration with the Laboratory of Tropical Crop Husbandry (at KU Leuven, Belgium),which has proven experience in somatic embryogenesis (Dhed’a et al. 1991), we have beenable to improve our results significantly.

We cultivated thin layers (called scalps) from proliferating buds from shoot meristemsof a Bluggoe clone (ABB). They provide highly morphogenic material (with highlyembryogenic cell suspension) from which we have been able to obtain protoplasts. Whencultured, these protoplasts differentiated into embryos and later in whole plants.



These last results are of considerable interest regarding the use of biotechnologies inCIRAD’s genetic program.

In addition, unpublished original data have been obtained by the Orsay group. Using aparticle gun, expression of a foreign inserted gene (GUS) in the diploid cell culture ofbananas has been obtained for the first time. This result opens up promising potentials inthe transformation of this plant.

Figure 2. Comparison between the conventional pedigree method and in-vitro androgenesis.

Conclusions and ProspectsThe techniques described above are useful for CIRAD-IRFA’s work on banana improvement(Bakry et al. 1990), in which the hybridization strategy is focused on diploid plantsynthesis. Special attention is also being given to the improvement of wild clones andedible cultivars (Fig.3).


Figure 3. The diploid-triploid approach to banana breeding at CIRAD-IRFA.

First, however, we have to improve some diploid populations, targeting diseaseresistance and agronomic and organoleptic traits. For this purpose bulk selection followedby genetic mixing will be done in recurrent selection. From these improved populations,pure lines will be chosen and intercrossed to obtain heterozygous, interpopulation, diploidhybrids F1. To obtain homozygotic plants, the classical scheme consists in self-repeatedpollinations. At present, first-generation plants are observed and CIRAD-IRFA is workingon in-vitro haplogenesis in order to obtain homozygotic plants more rapidly.

Obtention of triploid varieties, which is the ultimate objective, may be achieved inthree different ways.

1. By crossing a diploid F1 hybrid (restitution of the diploid female F1 hybrid genome in2x female gametes) with another homozygotic male diploid (x male gametes). Howeverthis procedure is uncertain and uncontrolled.

2. By crossing a tetraploid plant (from an F1 diploid clone, coupled with colchicinetreatment) with another homozygotic improved diploid. The success of this schemedepends on the meiotic behavior of the tetraploid. The best gametes, containing geneticpotential of the F1 diploid parent, will be obtained from the allotetraploid AABB structure.

3. By using biotechnologies: fusion of 2x F1 protoplasts with a haploid protoplastproduced from anther culture via haploid embryogenic callus and haploid cell suspension,like those obtained with other crops (Sun et al. 1989). These 3x somatic hybrids will bescreened using flow-cytometry apparatus, for example. The advantage of this scheme isthat the F1 diploid genetic structure of the 2x parent is always preserved.

Our new results in the field of banana cell transformation open up new perspectives inthe breeding of such edible diploid or triploid bananas as Cavendish. It is necessary toobtain transferable DNA, whether it comes from banana or not, and to choose withprecision the best plant target, tissue, or cell type.

A particle gun would be used to transform all plant material with morphogenicpotential. Electroporation would also be used on suitable protoplasts.

Many techniques are already used daily with success in our program. In the field ofhybridization, electrophoresis reveals the similarity between clones or the degree ofheterozygous differentiation; and cytogenetics permits the control of interspecificity ofhybrids. In-vitro culture will strengthen our program with vegetative propagation, zygoticimmature embryo culture, callogenesis, somatic embryogenesis, and polyploidization totetraploid level with meristem diploid clones treated with colchicine.

And finally, haplogenesis, all protoplast techniques, and genetic engineering enable usto anticipate what challenges lie ahead for us in our work.

Acknowledgments

This research has been undertaken with financial support from CIRAD-IRFA via EECfunding of DG 12 (Contract no.TS ZA-0094-F). We also thank C Teisson and researchworkers in the Laboratory of Tropical Crop Husbandry for their invaluable assistance.


ReferencesBAKRY F. 1984. Choix du matériel à utiliser pour l’isolement de protoplastes de bananiers (Musa spp.),

Musacées. Fruits 39:449-452.BAKRY F, HORRY JP, TEZENAS DU MONTCEL H, GANRY J. 1989. L’amélioration génétique des bananiers à

l’IRFA/CIRAD. Fruits (numéro spécial): 25-40.BANERJEE N, SCHOOFS J, HOLLEVOET S, DUMORTIER F, DE LANGHE E. 1987. Aspects and prospects of somatic

enbrogenesis in Musa AAB, cv. Bluggoe. Acta Hort. 212:727-729.CARLIER J, MOURICHON X. 1990. Polymorphisme de fragments de restriction (RFLP) chez les Mycosphaerella ssp.,

pathogène des bananiers et plantains. IIème Congrès Société Française de Phythopathologie, Montpellier,November 1990.

DA SILVA CONCEICÂO A. 1989. Isolement et culture de protoplastes de bananiers (Musa sp.): étude de diversfacteurs. Orsay, Paris: DEA Génétique et Sélection Animale et Végétale, option Végétale UPS. 14pp.

DESSAW D. 1988. Etude des facteurs de stérilité du bananier (Musa ssp.) et des relations cytotaxonomiques entreM. acuminata Colla et M. balbisiana Colla. Parts 1,2,3. Fruits 43:539-558;615-638; 685-700.

DE LANGHE E. 1987. Necesidas de una estrategia internacional para al mejoramiento genetica del Banana y delPlantano. Pages 181-199 in Memoria de la Reunion Regional de INIBAP para America Latina y el Caribe,San José.

DELVAUX B, PERRIER X, GUYOT P. 1990. Diagnostic de la fertilité de systèmes culturaux intensifs en bananeraies àla Martinique. Fruits 45:223-236.

DHED’A D, DUMORTIER F, PANIS B, VUYLSTEKE D, DE LANGHE E. 1991. Plant regeneration in cell suspension culturesof the cooking banana cv “Bluggoe” (Musa ssp. AAB Group). Fruits 46:125-135.

ESCALANT JV,TEISSON C. 1988. Embryogenèse somatique chez Musa sp. C.R. Acad. Sci. Paris, série III 306:277-281.ESCALANT JV, TEISSON C. 1989. Somatic embryogenesis and plants from immature zygotic embryos of species

Musa acuminata and Musa balbisiana. Plant Cell Reports 7:665-668.FAURÉ S, CARREEL F, AMSON C, AUBERT G, HORRY JP, GONZÁLEZ DE LEÓN D, LANAUD C. 1991. RFLP analysis in the

bananas (Musa ssp.). Eucarpia Symposium on Genetic Manipulation in Plant Breeding: MolecularBiology/Breeding Interface, Tarragona, Spain.

FOURÉ E, MOULIOM PEFOURA A, MOURICHON X. 1990. Etude de la sensibilité variétale des bananiers et des plantainsà Mycosphaerella fijiensis Morelet au Cameroun. Caractérisation de la résistance au champ de bananiersappartenant à divers groupes génétiques. Fruits 45:339-345.

HARRAN S. 1991. Etude de cals inflorescentiels de deux cultivars de bananiers (901 et GN) et des plantes qui ensont issues. Ph.D. thesis, Uuniversité de Paris-Sud XI, Orsay, France. 110 pp.

MEGIA R, HAÏCOUR R, ROSSIGNOL L. 1992. Callus formation from banana (Musa sp.) protoplasts. Plant BreedingReviews 7:135-155.

ROWE PR. 1984. Breeding bananas and plantains. Plant Breeding Reviews 7:135-155.SANNASGALA K. 1989. In vitro somatic embrogenesis in Musa. Ph.D. thesis. KU Leuven, Heverlee, Belgium.SUN CS, PRIOLI LM, SÖNDAHL MR. 1989. Regeneration of haploid and dihaploid plants from protoplasts of

supersweet (sh2sh2) corn. Plant Cell Reports 8:313-316.

Further Information on Protoplast Regenerationand Transformation in Musa(by R Haïcour, L Rossignol)

IntroductionA combination of conventional breeding programs with new methods in genetic engineeringis necessary for banana improvement. These new strategies must be interpreted ascomplements to and not replacements for conventional breeding.


This is why at the Laboratoire de Morphogenèse végétale expérimentale, Université deParis-Sud XI, we decided to develop protoplast techniques for use in the banana breedingprogram.

Protoplasts, as single-cell systems, are particularly appropriate for applications ofplant genetic engineering such as somatic hybridization and transformation by protoplastfusion and electroporation, respectively. Protoplasts appear to be tools of special interestin banana and plantain improvement.

Protoplasts: obtention, division, and regenerationThe first banana protoplasts were isolated in our group in 1983 by Bakry. Then Cronauerand Krikorian (1984-1986), Chen (1985), Chaput in our laboratory (1986), and Da SilvaConceicâo (1989) also obtained protoplasts from different explants of banana.

But, up to now (1993), the main difficulty with banana protoplasts has been their lowpotential to divide. The very low frequency of first and second divisions of bananaprotoplasts cultured in sea plaque agarose has been reported, but only when embryogeniccells were used (Da Silva Conceicâo, 1989).

However, sustained protoplast division and differentiation in plants are a prerequire-ment for using protoplasts in a breeding program. To overcome this problem, we decided towork with the most reactive material, i.e., with embryogenic material. So we usedembryogenic cells of an aged diploid suspension of Musa acuminata ssp. burmannica cvLong Tavoy (AA), kindly provided in 1987 by JV Escalant (CIRAD, Montpellier, France) andmaintained in our laboratory by weekly subcultures.

To stimulate protoplast division, we tried to use feeder cells. Cell suspensions fromthree species, Lolium multiflorum, Triticum monococcum, and Zea mays were tested.The best results were obtained with Lolium feeder cells, with a high density of protoplastsplated above the feeder cell layer and separated from the nurse cells by a physical barrier(a double nylon mesh).

The nurse cells in the feeder layers proved to be critical in supporting the sustainedprotoplast divisions, the formation of microcolonies, and subsequent growth up to thecallus stage.

After 3 months of culture, protoplast-derived calli of banana died, even though thenurse cells were still growing. Conversely, calli continued to grow when subcultured onfresh nurse medium.

Cytohistological sections showed that the cells of protoplast-derived calli retained the embryogenic characters previously observed in the cell suspensions used as a source of protoplasts: particularly a large nucleus, important reserves, and a thick cell wall.Moreover, morphological and cytological study revealed that cells of banana were differentfrom those of Lolium and the protoplast-derived calli obtained had all the characteristicsof banana cells. These latter were smaller in size than Lolium cells and had a densercytoplasm. Chromosomes were larger in Lolium than in dividing cells of banana.

In order to confirm that calli developed above the nylon filters derived from bananaprotoplasts and not from the contamination of nurse cells, such calli were examined forisoenzymes. Phosphoglucomutase (PGM) and alcohol deshydrogenase (ADH) were found


to distinguish banana from Lolium cells. Since the zymogram of the calli located above thenylon filters showed an identical pattern to that of banana cells, it was concluded thatthey really derived from banana protoplasts.

But this technique was still limited by a lack of regeneration due probably to the long-term embryogenic culture used as cell suspension. At present (1993), this suspension doesnot allow us to obtain morphogenesis with calli derived from protoplasts (Megia et al.1992). So we have used other plant material with better potential. Collaboration with theLaboratory of Tropical Crop Husbandry (at KU Leuven, Belgium), which is experienced insomatic embryogenesis, permitted us significantly to improve our results.

Referring to data from Dhed’a et al. (1991), we cultivated thin layers (scalps) fromproliferating shoot-tip cultures of a cooking banana (ABB), subgroup Bluggoe cv Matavia.They provided a good embryogenic cell suspension from which we have been able to obtainprotoplasts. When cultured on medium with feeder cells, protoplasts divided anddifferentiated into globular embryos and later in whole plants (pers. com. Megia et al.).These original results obtained by the Orsay group are of particular interest in relation tothe use of biotechnologies in the CIRAD genetic program.

First results with cell transformationAn unpublished original result has just been obtained (1993) by the Orsay team using apowder gun, as described by Zunbrunn et al. (1989).

Fresh diploid cell suspension culture of M. acuminata ssp. burmannica cv Long Tavoywere shot with tungsten particles of 0.5-1.0 µm diameter coated with a DNA solution.

Plasmid DNA derived from PI BI 221, containing the Ca MV 35S promoter, Gus codingregion, and Nos terminator was used as a chimeric gene introduced in banana cells.Histochemical Gus assay was performed with X glu substrate, and then we obtained for thefirst time the expression of a foreign-inserted gene in banana.

This preliminary result shows the feasibility of transformation in this important crop.

ReferenceZUNBRUNN G, SCHNEIDER M, ROCHAIX JD. 1989. A simple particle gun for DNA mediated cell transformation.

Technique A. Journal of Methods in Cell and Molecular Biology 1: 204-216.


63C Rivera, P Ramírez, R Pereira

Preliminary Characterization ofViruses Infecting Banana in Costa Rica

C Rivera, P Ramírez, R Pereira

IntroductionCosta Rica, the second largest worldwide exporter of bananas, has retained this positionby increasing yields through better methods of production and the recuperation ofcultivated areas (Informe UPEB 1990).

Some viral diseases are a real threat for the production of bananas (Dale et al. 1986).Different symptoms have been observed in commercial banana fields in Costa Rica thatcould be associated with viral infection. These symptoms are “distorted candle leaf”, rugoseleaves with thickened and chlorotic veins, pockets of necrosis in the sheaths, mild or severechlorosis, chlorotic streaking or flecking, and bunched leaves at the apex of the plant.CORBANA (Corporación Bananera Nacional) and the University of Costa Rica have started acooperative research project to characterize the viral diseases that could be associated withthese different symptoms. Although only one virus disease has been reported in Costa Rica’splantations — cucumber mosaic virus (CMV), the type member of the Cucumovirus group(Brioso et al. 1986) — the possibility of finding other virus diseases is present.

The Cucumovirus is a small group of serologically interrelated viruses each of whichincludes numerous biological variants (Francki 1985). A large number of strains of CMVhave been recognized by host range and symptomatology (Kaper, Waterworth 1981),serological relationships (Devergne, Cardin 1973), peptide mapping of the viral coatprotein (Edward, Gonsalves 1983), physical and chemical properties (Lot, Kaper 1976),and nucleic acid hybridization (Gonda, Symons 1978; Piazolla et al. 1979; Owen, Palukaitis1988). Devergne and Cardin (1973) and Devergne et al. (1981) have grouped a largenumber of isolates of CMV on the basis of biological properties as well as precipitin andElisa testing into two main groups: ToRS and DTL. They have reported that there is a thirdserotype — CMVCo — that appears to be serologically distinct from both of these groups.

This paper describes the preliminary characterization of two CMV-related virusesisolated from banana by symptomatology, host range, serology, and electron microscopy,and reports the presence of two other virus-like particles in commercial bananaplantations in Costa Rica.

Centro de Investigación en Biología Celular y Molecular, Universidad de Costa Rica, San José, Costa Rica

Materials and Methods

Preliminary analysis by Elisa of the different symptomatologiesIn 1990, a survey was made in several banana fields, in which plants were selected thatpresented different symptomatologies frequently observed. Leaf samples were collectedfrom 39 different plants. Sheath samples were collected only from those plants showingpockets of necrosis. All samples were analyzed by DAS-Elisa using the CMVer and CMVvidiagnosis kit from AGDIA and the Sanofi CMV of banana diagnosis kit. The Sanofi kit is made of polyclonal antibodies produced against the three main serotypes of CMV, known as DTL, ToRS, and Co. The AGDIA CMVvi kit reacts with some strains of serotype DTL and the AGDIA CMVcr kit reacts with some strains of serotype ToRS.The Elisa test was donefollowing the protocolsrecommended by themanufacturer.

One plant from eachof two different symptomprototypes (Types 1 and2) commonly observed inthe field (Fig.1), pre-senting also very differentresults by Elisa testing(Table 1), were selectedfor further analysis. Bothselected plants were of cvGrande Naine. Type 1 wasreproduced by rhizome,while Type 2 was repro-duced by tissue culture.Type 1 showed bunchedleaves at the apex of the plant, leaf distortion(Fig.1,A), pockets ofnecrosis in the sheaths(Fig.1,B), and distortedfruit (Fig.1,C). Type 2showed “distorted candle”,rugose leaves withthickened and chloroticveins (Fig.1,D). Type 2 did not show pockets of necrosis in the sheaths.

64 Preliminary Characterization of Viruses Infecting Banana in Costa Rica

Figure 1. Symptoms associated with banana collected in CostaRica. A: Bunched leaves. B: Necrotic pockets in the sheaths.C: Distorted fruits. D: Distorted candle—rugose leaves withthickened and chlorotic veins.

Virus purificationVirus was partially purified from banana Types 1 and 2 by a modification of the methoddescribed by Palukaitis (pers. com.). Infected leaf tissue, minus midrib, was finelychopped and ground in liquid nitrogen and homogenized in 1.5 vol (w/v) of 0.5 Msodium-citrate buffer, pH 6.5, containing 5mM EDTA and thioglycolic acid 0.5% and 1.5vol (v/v) of chloroform. After a cycle of differential centrifugation (15 000 g for 10 min,186 500 g for 90 min) the pellet was resuspended overnight at 4°C in 5 mM sodium-borate buffer, pH 9, containing 5mM EDTA and 2% Triton X-100. The suspension wasclarified by low-speed centrifugation (7000 g for 10 min) and the virus was precipitatedfrom clarified extract by ultracentrifugation (218 000 g for 60 min) through a 10% (w/v)sucrose cushion prepared on 5 mM sodium-borate buffer, pH 9, containing 2% (v/v)Triton X-100, 5 mM EDTA. The virus preparation was resuspended in 1 mL of the samebuffer.

Electron microscopyPartially purified virus preparations of Types 1 and 2 were negative-strained with 1%uranyl acetate, pH 5, on Formvar-Carbon coated grids (400 mesh), and observed in an H-7000 Hitachi electron microscope.

Host range symptomatologyPartially purified virus preparations from banana Type 1 and crude sap of banana Type2 were mechanically inoculated into healthy plants of Curcurbita pepo, Cucumissativus, and Nicotiana tabacco cv Samsun. Inoculations were made by rubbingcarborundum-dusted cotyledons and expanding first leaves. The inocula were preparedin cold 5-mM sodium-borate buffer, pH 9, containing 5 mM EDTA, 2% Triton X-100. Theleaves were washed with distilled water after inoculation and were observed forsymptoms.

Results

Table 1. Elisa test results of the two selected types.

Absorbance 405 nm

Elisa Type 1 X + 3s Type 2 X + 3shealthy healthy

CMVvi 0.265 0.047 0.049 0.014CMVcr 0.203 0.054 0.036 0.022CMV Sanofi 0.134 0.038 0.003 0.005

To establish limits for the negative and positive values the upper negative limit was taken as the absorbancemean of the healthy values (X) plus 3s (standard deviation) (P =0.003).



Host range symptomatology

Symptoms induced by Types 1 and 2 were compared on zucchini, cucumber, and tobacco(Table 2). Figure 3 shows some of the most important symptoms observed.

Table 2. Host range symptomatology.

Hosts

Inoculum source Cucumber Zucchini Tobacco

Type 1 Mild, mosaic, Mild mosaic, Symptomlessrugose leaf rugose leaf

Type 2 Chlorotic local Mild mosaic, Symptomlesslesion, later rugose andsystemic rugose curling leafand curling leaf,enations

Figure 2. Virus particles associated with infected field bananas. A: Virus particles purified frombanana Type 1. B,C,D: Virus particles purified from banana Type 2. The bar represents 100 nm.

Electron microscopyIsometrical particles of about 29 nm with hollow centers were observed in the partialpurified preparation from banana Types 1 and 2 (Fig.2, A-B). Flexuous-filamentous andrigid-filamentous virus-like particles were also observed in banana Type 2 (Fig.2, C-D).


ConclusionsThe results obtained in this work suggest the presence of CMV associated with the Type 1plant. The symptoms observed in this type are similar to some of those reported for someCMV strains of banana (Frison, Putter 1989). The electron micrographs of partiallypurified virus preparation showed spherical particles of about 29 nm with hollow centerssimilar to those reported for CMV (Tolin 1977). The DAS-Elisa results confirmed the CMVpresence in Type 1. However, these results did not permit differentiation to be madebetween serological subgroups.

The electron microscopy of partially purified samples of plant Type 2 showed very fewisometrical viral particles similar to those found in the Type 1 plant. However, the DAS-Elisa results of these samples did not permit confirmation of the presence of CMV. By thestandards commonly used in virus-plant Elisa testing (X + 3s of the healthy controlsamples), our results would be rated positive. However, these values are very lowpositives or unusually high negatives. There exist very few test results with banana sapanalysis by DAS-Elisa commercial kits. It is necessary to analyze a bigger number ofhealthy samples and samples showing Type 2 symptoms in order to establish positive-negative thresholds.

The symptoms observed in zucchini and tobacco when they were inoculated withboth types of banana plants, were similar. Some differences were observed in cucumberplants inoculated with Types 1 and 2. Only Type 2 developed chlorotic local lesions in theinoculated leaves and enations on systemic infected leaves.

Figure 3. Symptomsobserved in indicator plantsinoculated separately withbanana Type 1 and Type 2.A: Local lesions induced incucumber by inoculationwith Type 2. B: Enation observed incucumber after inoculationwith Type 2. C: Chlorotic leaf veins andsevere mosaic observed incucumber by inoculationwith Type 2. D: Mild mosaic and leafcurling after theinoculation of zucchiniseparately with Type 1 andType 2.

The two other virus-like particles associated also with Type 2 have not yet beenanalyzed by serology.

It is necessary to complete the characterization of the banana CMV on the followingbasis: (a) biological properties, using a large variety of indicator plants; (b) serologicalproperties, determining the serological relationship with known CMV strains and othermembers of the Cucumovirus group, such as TAV and PSV; and (c) molecular properties,analyzing the ability to hybridize with eDNA from the members of the Cucumovirus group.

It is also important to start the characterization of the two other virus-like particlesassociated with the Type 2 plant.

ReferencesBRIOSO PST, RIVERA C, ARROYO T, RODRIGUEZ CM. 1986. Identificao e distribucao du virus do mosaico do pepino em

areas de cultivo de bananeira e platano na Costa Rica (America Central). Fitopatología Brasileira 11:392.DALE JL, PHILLIPS DA, PARRY JN. 1986. Double-stranded RNA in banana plants with bunchy top disease. J. Gen.

Virol. 67:371-375.DEVERGNE JC, CARDIN L. 1973, Contribution a l’étude du virus de la mosaïque du concombre (CMV). IV. Essai de

classification de plusieurs isolats sur la base de leur structure antigénique. Ann. Phytopathol. 5:409-430.DEVERGNE JC, CARDIN L, BURCKARD J, VAN REGENMORTEL MHV. 1981. Comparison of direct and indirect ELISA for

detecting antigenically related cucumovirus. J. Virol. Methods 3:193.EDWARDS MC, GONSALVES D. 1983. Grouping of seven biologically defined isolates of cucumber mosaic virus by

peptide mapping. Phytopatology 73:1177-1120.FRANCKI RIB. 1985. The viruses and their taxonomy. Pages 1-15 in The Plant Viruses, vol.1 (Francki RIB, ed.).

New York, USA: Plenum Press.FRISON EA, PUTTER CAJ (eds). 1989 FAO/IBPGR Technical guidelines for the safe movement of Musa germplasm.

Rome, Italy: Food and Agriculture Organization of the United Nations/International Board for Plant GeneticResources.

GONDA TJ, SYMONS RH. 1978. The use of hybridization analysis with complementary DNA to determine the RNAsequence homology between strains of plant viruses: its application to several strains of cucumoviruses.Virology 88:361-370.

Informe UPEB. 1990. Panamá 14:7-20.KAPER JM, WATERWORTH HE. 1981. Cucumoviruses. Pages 257-332 in Plant Virus Infections and Comparative

Diagnosis (Kurstak E, ed.). New York, USA: Elsevier/Holland Biomedical.LOT H, KAPER JM. 1976. Physical and chemical differentiation of three strains of cucumber mosaic virus and

peanut stunt virus, Virology 74:209-222.OWEN J, PALUKAITIS P. 1988. Characterization of cucumber mosaic virus. I. Molecular heterogeneity mapping of

RNA3 in eight CMV strains. Virology 166:495-502.PIAZZOLA P, DIAZ-RUIZ JR, KAPER JM. 1979. Nucleic acid homologies of eighteen cucumber mosaic virus isolates

determined by competition hybridization, J. Gen. Virol. 45:361-369.TOLIN SA. 1977. Cucumovirus group. Pages 303-309 in The Atlas of Insects and Plant Viruses, including

mycoplasma viruses and viroids. New York, USA: Academic Press.


69CM Fauquet, RN Beachy

Status of Coat Protein-MediatedResistance and its PotentialApplication for Banana Viruses

CM Fauquet1, RN Beachy2

IntroductionThe recent development of gene transfer technologies to plants has made it possible totransfer useful traits to a number of crop plants. Among the different possibilities, virusresistance is probably one of the most successful applications of plant geneticengineering. When used to complement current breeding programs, these technologieshave the potential to help control plant viruses and consequently to decrease the impactof plant viruses on crop productivity. During the past 6 years different molecularapproaches have been developed to attempt to control viruses, some of which are stillunder investigation in laboratories, while others have reached the field-testing level. Thispaper briefly reviews these different approaches, discussing the state of development, theefficacy, and the stability of each concept for controlling viruses by genetic engineering.Particular emphasis is placed on coat protein-mediated resistance (CPMR) because ofthe success of this strategy and because of the large resource of data and examples nowavailable derived from the use of this technique. The type of resistance obtained and thespectrum of protection produced is exposed, and the examples of viruses belonging tovirus groups the members of which are infecting bananas are particularly detailed inorder to evaluate the potential of application of the coat protein strategy for this crop.

Multiple Strategies for Controlling VirusesWith the increase of the knowledge of virus replication and virus genome organization,scientists are testing different approaches for controlling virus infection. Different viralgenes or sequences, inserted into the plant genome, interfere with the virus replication ina beneficial way for the infected plant.

1ORSTOM, Division of Plant Biology, MRC7, 10666 Torrey Pines Road, La Jolla, CA 92037, USA; 2TSRI Plant Division,MRC7, at the same address

Ribozyme strategyA novel approach to achieve virus resistance is the use of autocatalytic RNA-cleavingmolecules, also called “ribozymes” (Cech 1986). Viroid RNAs such as avocado sunblotchviroid and satellite RNAs such as the satellite of tobacco ring spot virus (TobRSV) havethe possibility of self-cleavage during replication (Buzayan et al. 1986; Forster, Symons1987; Hutchins et al. 1986; Prody et al. 1986). Cleavage is effective both on the positiveand negative strand of the RNA and is highly specific and associated with conservedsequence domains. Several studies have been conducted to determine the optimal in-vitroconditions of cleavage (Gerlach 1989; Haseloff, Gerlach 1988). Replicase genes of tobaccomosaic virus (TMV) and barley yellow dwarf virus (BYDV) encoding sequences bearingspecific virus cleavage sites have been integrated in transgenic plants and shouldgenerate sequence-specific endonuclease activities (Gerlach 1989), but no in-vivo resultshave been published yet.

Translation strategyBy translation strategy we refer to the strategies involving integration in the plantgenome of sequences generating complementary sequences to viral RNA that interferewith the translation of viral genes by hybridization of the coding sequence. It has beenreported that synthesis of complementary RNA (antisense RNA) can decrease theaccumulation of gene products in both procaryotes and eukaryotes (Ecker, Davis 1986;Green et al. 1986). It is likely that antisense RNAs anneal with sense RNAs to form adouble-strand complex which is rapidly degradated or which inhibits translation of theRNA. Several viral coat protein antisense constructs including TMV, cucumber mosaicvirus (CMV), and potato virus X (PVX) have been integrated into plants and tested forresistance to infection (Cuozzo et al. 1988; Hemenway et al. 1988; Powell et al. 1989). Inall these cases resistance has been reported against infection by the homologous virus butonly at low virus inoculum concentrations. Recently, a similar study done with the potatoleafroll virus (PLRV) (Kawchuk et al. 1991) proved to induce resistance to the virusinoculation of the same level as the CPMR.

Replication strategyOne of the first steps of the virus cycle is replication of the viral genome and it seemslogical that any approach to block this phase should be an efficient way to protect plants.Two approaches have been considered to block a virus infection, the antisense and thesense approaches.

Antisense approach of the replication strategyThis strategy attempts to block the replication of virus by hybridization of complementarysequences to the replicase viral gene or to sequences recognized by the replicase duringreplication. This strategy is at a preliminary stage of investigation but is presentedbecause of promising in-vitro results and the potential that it may be applicable whenother approaches fail. The complete antisense sequence of the replicase gene of thetomato golden mosaic virus (TGMV) was integrated into tobacco genome and several lines

70 Status of Coat Protein-Mediated Resistance and its Potential Application for Banana Viruses

were reported to exhibit a high level of resistance when challenged with differentconcentrations of TGMV (Lichtenstein, Buck 1990). This approach may be an interestingalternative but must be further tested before being considered as a useful strategy forpractical purposes. The last example concerns turnip yellow mosaic virus (TYMV) whereantisense sequences corresponding to the tRNA-like structure of the 3’ extremity of theTYMV RNA can strongly inhibit replicase activity (Cellier et al. 1990).

Sense approach of the replication strategy

Competitor RNA. A study using sense sequences comprising the tRNA-like structureof TYMV (see above) in order to compete with the similar viral sequences and therebydecrease virus replication activity has been conducted. In-vitro studies have shown suchcompetition (Morch et al. 1987) and in-vivo experiments show some level of resistance(Cellier et al. 1991). In contrast a similar approach used with the TMV seemed not toinduce any resistance (Powell et al. 1990).

Subgenomic DNA. Some viruses produce subgenomic molecules during virusinfection; for example several geminiviruses produce subgenomic DNA molecules of the Bcomponent. Insertion of one copy of such DNA of the African cassava mosaic virus(ACMV) into the tobacco genome reduced disease symptoms when the plants werechallenged with ACMV (Stanley et al. 1990). Symptom amelioration is associated with areduction in the level of viral DNA, including B DNA which is responsible for thesymptomatology, and the resistance is specific to ACMV.

Satellite RNA. Another approach taken to confer protection against viruses is tocause the expression of virus satellite (Sat) RNAs. Sat RNAs are associated with severalviruses and are dependent upon a helper virus for their replication and spread in theinfected plant. It has been reported that the presence of Sat RNAs in cucumber mosaicvirus (CMV)-infected tobacco reduces disease symptoms (Mossop, Francki 1979).Similarly, tobacco plants infected with a mixture of tobacco ring spot virus (TobRSV) andToBRV Sat caused amelioration of symptoms (Gerlach et al. 1986). Transgenic plants thatexpress these satellite sequences were shown to decrease symptoms and virus replication(Gerlach et al. 1987; Harrison et al. 1987; Jacquemond et al. 1988). However, when theplants expressing CMV Sat RNA are infected with a related but different cucumovirus,there is symptom amelioration but no decrease in virus replication. Recently, this strategyhas been applied to tomato (Tien et al. 1990; Tousch et al. 1990), and proven to beefficient for the reduction of symptoms both in greenhouse and field experiments (Tien etal. 1990). Not all satellite sequences provide symptom attenuation but sometimes theycan also cause necrosis; the sequences responsible for severe symptoms are reduced to afew nucleotides (Devic et al. 1990; Jaegle et al., 1993). There is a risk that amplifying asatellite in transgenic plants may result in some of the molecule reverting to a necroticform, causing dramatic symptoms when naturally infected by the helper virus. Thispossibility will greatly limit the utilization of the Sat RNA strategy unless further studiescan demonstrate a great stability of the system.

TMV replicase strategy. Lately, a new source of genetically engineered resistance hasbeen identified involving the transformation of plants with nonstructural viral genes.


Tobacco plants transformed with the TMV 54 kDa gene, which is derived from a portion ofthe replicase complex, are immune to extremely high concentrations of TMV virions orRNA (up to 500 µg mL-1) of the strain U1 (Golemboski et al. 1990). This immunity ishighly specific to the strain U1, or a mutant YS1/1 of TMV and susceptible to the otherstrains, including the related U2 strain of TMV. This approach is undertaken with severalviruses and preliminary results with the expression of the entire replicase gene of thepotato virus X are producing a high level of resistance into transgenic tobacco plants(Braun, pers. com.) and portions of the replicase gene of CMV as well (Anderson, pers.com.).

Status of the Coat Protein Strategy

Among the different strategies for controlling viruses by genetic engineering, the coatprotein strategy is currently the most promising. Many examples have been published andthe efficiency in terms of protection, stability, and specificity has been evaluated forseveral viruses. The type of resistance and the mechanisms of action of the CPMR havebeen investigated and much information is available. Finally, both laboratory experimentsand field tests with different crops have been conducted and the first commercial use ofthis type of resistance is scheduled for 1995.

Definition, concept, and production of coat protein-mediatedresistance (CPMR)

CPMR refers to the resistance to virus infection caused by expression of a coat protein(CP) gene in transgenic plants. The expression of a CP gene confers resistance to thevirus from which the CP gene was derived. Resistance is associated to the expression ofthe CP gene, and is stably inherited to subsequent generations.

The CP strategy is the expression by the plant of the viral CP gene integrated into theplant genome. Construction of the chimaeric gene should include the selection of anappropriate transcriptional promoter to cause the expression of the CP gene at sufficientlevels to produce disease resistance. Several different transcriptional promoters havebeen used and the promoter that has proven most effective is the CaMV 35S. Thispromoter leads to high levels of mRNA and protein in most of the plants in which it hasbeen tested. Furthermore, an enhanced 35S promoter, e35S, which is produced byduplication of an upstream regulatory sequence (Kay et al. 1987), causes higher levels ofgene expression and is now widely used. The coding region used for the gene is obtainedby deriving double-stranded DNA from the virus genome. When necessary, specificmutations of the gene may be needed to increase the translation of any mRNA followingthe consensus sequence rules described by others (Kozak 1988). The third part of thechimaeric genes is a sequence to confer transcript termination and polyadenylation.Little evidence has been published to date to indicate that a specific 3’ end is preferred intransgenic plants. The 3’ ends used for most chimaeric genes expressed in plants have


been taken from the T-DNA region of the Ti plasmid (Nelson et al. 1988; Powell et al.1986; Powell et al. 1990).

Assessing disease resistance involves inoculation with virus plants that express the CPgene (CP+) and those that do not (CP-). There is also increasing evidence thatnontranslated messengers might interfere with virus replication and, consequently,transgenic plants expressing the CP messengers without CP production should be used ascontrol. A precise estimation of the number of inserted copies of the chimaeric gene, thelevel of messenger expression, and the amount of CP produced are required for a valuableevaluation of the CPMR. A comparison in the numbers of sites of infection, the diseaseincidence, the development of disease symptoms, and the accumulation of virus aregenerally used to evaluate resistance.

In order to use populations of plants that are identical in age, growth rate, and size,R1 or successive generations of plants are used. Prior to the inoculation of seedlings withvirus, the segregation of the introduced gene is determined generally by animmunological reaction to detect the CP or by following the expression of a gene that isco-introduced with the CP gene.

Efficacy of coat protein-mediated resistance

The efficacy of CPMR is demonstrated by the number of examples where resistance hasbeen achieved, by the spectrum of specificity of protection, and also by the type ofresistance achieved.

Specificity of coat protein-mediated resistance

Since 1986, the date of the first publication describing the coat-protein strategy (Powellet al. 1986), there have been a number of reports of CPMR involving a variety of differentviruses and host plants (Beachy et al. 1990). A list of published and unpublished reportsis presented in Table 1. It includes viruses belonging to 13 different virus groups andhosts that belong to dicotyledons and monocotyledons. Most of the examples are fromnonenveloped ssRNA(+) viruses, but it seems that the morphology of the virus, the factthat the viral genome is divided or not, and the type of viral genome organization does notmatter for the obtention of resistance. There are positive examples of CPMR for twogroups of enveloped ssRNA(-) viruses: Tenuivirus and Tospovirus, but there is somequestion about the mode of action of the strategy because the CP mRNA could also act asantisense of the complementary strand of the CP. As for the DNA viruses, we haveinformation only about geminiviruses where the CPMR has been achieved: the tomatoyellow leaf curl virus from Thailand (TYLCV-Th) and the African cassava mosaic virus(ACMV); but in these cases the CP levels were extremely low and the protection limited(Rochester, pers. com.). There is only one example of the use of CPMR in monocotyledonswhere the CP expression is very high and the resistance to the virus evaluated by insecttransmission is also very high (Hayakawa et al. 1991). It is anticipated that moreexamples of CPMR for different virus groups will be achieved in the very near future,which will provide substantial information about the strategy.


Spectrum of coat protein-mediated resistance

The best TMV CP(+) tobacco lines were inoculated with members of different virusgroups including CMV, AlMV, PVX, and PVY (see Table 1), but there was no noticeableresistance against infection by any of the tested viruses (Anderson et al. 1989). Thoughthere is no protection for viruses belonging to other virus groups, there is growingevidence that the CPMR has a large spectrum of specificity within the group and that aparticular CP can provide resistance to more than the homologous virus.

A complete study on the spectrum of resistance of transgenic plants expressing a CPgene was carried out on tobamoviruses (Nejidat, Beachy 1990); CP(+) tobacco plant linesthat expressed the U1 TMV CP gene were inoculated with other tobamoviruses. Basedupon comparisons of amino acid sequences of CPs (Gibbs 1986), tobamoviruses havedegrees of relatedness to TMV ranging from 85 to 39%. Infection by TMV, ToMV, peppermild mosaic virus (PMMV), and tomato mild green mosaic virus (TMGMV) were inhibitedby 95-98%, Odotonglossum ring spot virus (ORSV) by 80-95%, ribgrass mosaic virus(RMV), and sunn hemp mosaic virus (SHMV) by 40-60% (Nejidat, Beachy 1990). Similarly,transgenic tomato plants expressing the CP of TMGMV were found resistant to TMV andToMV (Sturtevant et al. 1991). On the basis of these studies it was concluded that virusesthat are related to the CP of TMV by more than 60% are less able to infect the resistantlines than are less related tobamoviruses.

Results of experiments of CPMR in the Potyvirus group, particularly important sincemany economically important plant viruses belong to this virus group, demonstrated thatexpression of a particular CP gene can induce protection for several other potyviruses.The soybean mosaic virus (SMV) CP protected tobacco plants from infection by tobaccoetch virus (TEV) and potato virus Y (PVY) (Stark, Beachy 1989). Transgenic tobaccoplants expressing the CP of papaya ringspot virus (PRSV) were found resistant to PVY,PeMV and TEV (Ling et al. 1991). The CP gene sequences of these potyviruses arehomologous to the level of 65%.

In the case of tobraviruses the heterologous protection is also effective for viruseshaving about 60% homology (van Dun, Bol 1988). For cucumoviruses it has been provenfor several cases that the CPMR is extended to several strains among and across the twosubgroups of the Cucumovirus group (see below).

Multiple manifestations of coat protein-mediated resistance

Resistance to inoculation. In each of the examples of CPMR described to date,resistance is manifested by several features. First, there is a reduction in the numbers ofsites where infection occurs on inoculated leaves. Fewer starch lesions were producedafter inoculation with PVX on CP(+) tobacco plants than on CP(-) plants (Hemenway etal. 1988), and there are fewer chlorotic lesions caused by TMV infection on tobacco plantsthat express the TMV CP gene than on those that did not (Powell et al. 1986). Likewise,the numbers of necrotic local lesions caused by TMV infection on CP(+) Xanthi nctobacco local lesion were 95-98% lower than on CP(-) plants (Nelson et al. 1987). Theseexperiments indicate that the expression of a CP gene causes a reduction in the numberof sites where infection is established upon inoculation.


Table 1. Examples of coat protein-mediated resistance in transgenic plants.Virus group CP gene Transgenic plant Virus resistance ReferenceTobamovirus TMV tobacco TMV (Powell et al. 1986)

" " ToMV (Nelson et al. 1988)" " PMMV (Nejidat, Beachy 1990)." " TMGMV "" " HRSV "" " ORSV "" tomato TMV (Nelson et al. 1988)" " ToMV "

ToMV tomato ToMV (Sanders et al. 1990)TMGMV tobacco TMGMV (Sturtevant et al. 1991)

" tomato TMV "" " ToMV "

Tobravirus TRV tobacco TRV (van Dun, Bol 1988)" " PEBV "

Carlavirus PVM potato PVM (Wefels et al. 1990)PVS tobacco PVS (MacKenzie, Tremaine 1990)

potato PVS (MacKenzie et al. 1991)Potexvirus PVX tobacco PVX (Hemenway et al. 1988)

" potato PVX (Lawson et al. 1990)CCMV tobacco CCMV (Fauquet et al. 1991)

Potyvirus PVY potato PVY (Lawson et al. 1990)SMV tobacco PVY (Stark, Beachy 1989)

" " TEV "ZYMV tobacco ZYMV (Namba et al. 1990)WMV II tobacco WMV II "PRSV tobacco PVY (Ling et al. 1991)

" " TEV "" " PeMV "

Furovirus BNYVV beet (protoplast) BNYVV (Kallerhoff et al. 1990)AlMV group AlMV tobacco AlMV (Loesch-Fries et al. 1987)

" " " (Tumer et al. 1987)" " " (van Dun et al. 1987)" tomato " (Tumer et al. 1987)

Cucumovirus CMV-D tobacco CMV-D (Cuozzo et al. 1988)" " " (Nakayama et al. 1990)" tomato CMV-D (Cuozzo et al. 1988)

CMV-C tobacco CMV-C (Quemada et al. 1991)CMV-Chi "CMV-WL "

CMV-WL tobacco CMV-WL (Namba et al. 1991)CMV-C "CMV-Chi "

Ilarvirus TSV tobacco TSV (van Dun et al. 1988)Luteovirus PLRV potato PLRV (Tumer et al. 1990)

" " " (Kawchuk et al. 1990)Tenuivirus RSV rice RSV (Hayakawa et al. 1991)Tospovirus TSWV tobacco TSWV (MacKenzie, Ellis 1992)

" " " (Gielen et al. 1991)

Abbreviations: AlMV, alfalfa mosaic virus; BNYVV, beet necrotic yellow vein virus; CCMV, cassava commonmosaic virus; CMV, cucumber mosaic virus; ORSV, Odotonglossum ringspot virus; PEBV, pea early browningvirus; PLRV, potato leafroll virus; PMMV, pepper mild mosaic virus; PRSV, papaya ringspot virus; PVM, potatovirus M; PVS, potato virus S;.PVX, potato virus X; PVY, potato virus Y; RSV, rice stripe virus; SMV, soybeanmosaic virus; TEV, tobacco etch virus; TMGMV, tobacco mild green mosaic virus; TMV, tobacco mosaic virus;ToMV, tomato mosaic virus; TRV, tobacco rattle virus; TSV, tobacco streak virus; TSWV, tomato spotted wiltvirus; WMV II, water melon mosaic virus II; ZYMV, zucchini yellow mosaic virus.


Resistance to virus spread within the plant. The second manifestation of resistanceof CP-engineered plants is a reduced rate of systemic disease development throughoutthe CP(+) plants. Thus, if inoculation results in infection on the inoculated leaves, thelikelihood that the infection will become systemic is considerably lower in CP(+) plantsthan in CP(-) plants. Grafting studies in which a stem segment of a transgenic TMV(CP+) tobacco plant was inserted between the rootstock and apex of a wild-type tobacco,demonstrated that the (CP+) segment prevented the virus from moving to the upper partof the grafted plant. This experiment shows that the CP may play a role in the long-distance movement of the virus and, consequently, resistance has a component thataffects systemic spread of the infection, at least in the TMV-tobacco system (Wisniewskiet al. 1990).

Resistance to symptom expression. A third manifestation of resistance is a reducedrate of disease development on systemic hosts that are CP(+). In most of the examples ofCPMR, CP(+) plant lines were less likely to develop systemic disease symptoms thanthose that were CP(-). Several plant lines that expressed the PVX CP gene did notbecome severely infected when inoculated with high levels of virus (Hemenway et al.1988). Similar results were reported for CPMR against CMV (Cuozzo et al. 1988), TMV(Powell et al. 1986), PVY and TEV (Stark, Beachy 1989), and other viruses. On thecontrary, in other cases the transgenic plants that are becoming infected showed similarsymptoms to those of the control plants as, for example, the CMV in transgenic tobaccoplants (Quemada et al. 1991) and the cassava common mosaic virus (CCMV) in transgenicNicotiana benthamiana (Fauquet et al. 1991). In one transgenic line of tobacco withCMV the plants were asymptomatic but showed a normal level of virus replication(Quemada et al. 1991), suggesting that the CP might play a direct role in the suppressionof symptoms in addition or not to suppression of virus replication.

Resistance to virus multiplication. Another manifestation of resistance is loweraccumulation of virus in CP(+) compared with CP(-) plant lines. Elisa and semi-quantitative western blots have been used to quantify virus accumulation in inoculatedleaves and other plant parts in most examples of CPMR (Cuozzo et al. 1988; Hemenway etal. 1988; Lawson et al. 1990; Nelson et al. 1987; Powell et al. 1986). In certain examples ofresistance, plants accumulate no virus after inoculation (Hemenway et al. 1988; Lawsonet al. 1990), and can therefore be considered to be immune to infection under theconditions of the tests. In other cases, the virus accumulation in the infected transgenicplants is normal and comparable to control plants (Fauquet et al. 1991; Quemada et al.1991).

All resistance manifestations of CPMR can usually, but not always, be overcome byinoculating with relatively high concentrations of virus. A virus concentration of 10 µgmL-1 of TMV nearly breaks the CPMR to TMV in a system where 0.01 µg mL-1 causesdisease in CP(-) plants (Powell et al. 1986). Fifty µg mL-1 are needed to overcome CPresistance to AlMV, PVX, PVY and TEV (see Table 1) (Hemenway et al. 1988; Lawson et al.1990; Stark, Beachy 1989; Tumer et al. 1987) and 100 µg mL-1 of CCMV are required tobreak the CPMR in tobacco plants (Fauquet et al. 1991). Resistance is largely overcomeby inoculation with RNA rather than virions in many cases except the PVX CP(+) lines of


tobacco and potato (Hemenway et al. 1988; Lawson et al. 1990), and the CCMV lines oftobacco (Fauquet et al. 1991); this feature, shared by two members of the potexvirusgroup may reflect a property specific to viruses of this group.

The majority of evaluation of CPMR has been assessed by mechanical inoculation ofthe virus and, of course, the most important criterion for virus resistance is its evaluationin natural modes of contamination, i.e., in vegetative propagation and with the naturalvectors. Information related to these points is limited but significant. In the case of thedually engineered resistance against PVX and PVY in potatoes (Lawson et al. 1990), it hasnot been possible to recover in the potato tubers any of these two viruses. At least oneline of potato showed a good level of resistance through aphid inoculation of PVY. PLRV, amember of the Luteovirus group, is nonmechanically transmissible and the CP-engineered potato plants have all been challenged by using aphid inoculation (Kawchuket al. 1990; Tumer et al., 1991), and demonstrated some degree of resistance. CMV CP(+)transgenic tobacco plants have been challenged with viruliferous aphids and proven to beresistant as for mechanical inoculations (Quemada et al. 1991).

Stability of coat protein-mediated resistance

CPMR is, in most cases, monogenic and is inherited by the following generations, like anyother genetic trait. Consequently the genetic stability of this resistance gene is expectedto be the same as any other gene. The biological stability of such resistance can bequestioned, but no answer can be provided until it is used in natural conditions. One mayargue that a single-point mutation can change a vital amino acid in the expressed coatprotein and consequently alter the resistance, but the probability will not be greater thanfor any other monogenic resistance gene. Furthermore, we know that CPMR is effectivefor viruses differing in up to 40% in their CP sequences. In order to test the stability of thesystem, TMV CP(+) lines of tomato have been inoculated successively 15 times with thesame isolate of virus, with the idea of selecting TMV molecules able to overcome theprotection; however, no resistance breaking strain has yet been identified (White andBeachy, pers. com.).

Field experiments with coat protein-mediated resistant plants

There have been several field tests of virus-engineered resistant plants. Tomato plantsexpressing the TMV and ToMV CP genes have been tested in the field for several years,and potato plants expressing the PVX and PVY CP genes have also been tested in thefield.

Tobacco plants that expressed the CP gene of AlMV were field-tested in Wisconsin in1988 (Krahn, pers. com.). CP(+) plants developed disease more slowly or not at allcompared with CP(-) plants; symptom development was correlated with virusaccumulation. At 85 days after inoculation only 9% of the CP(+) plants had developed asystemic infection, while 93% of the CP(-) plants had systemic infections.

The first field test with TMV-resistant tomato plants, from cultivar VF36, wasconducted in 1987 (Nelson et al. 1988). CP(+) plants, mechanically inoculated with TMV,exhibited a delay in the development of disease symptoms or did not develop symptomscompared with the CP(-) plants. No more than 5% of the CP(+) plants developed disease


symptoms by fruit harvest, while 99% of the VF36 plants developed symptoms. Lack ofvisual symptoms was associated with a lack of virus accumulation. Fruit yields of theinfected VF36 plants decreased 26-35% compared with healthy plants, whereas yieldsfrom the CP(+) line were equal to those of uninoculated VF36 plants.

To determine if the TMV CP gene conferred protection against infection by fieldisolates of ToMV, tests were conducted in 1988 in Florida and Illinois. Progeny that werehomozygous for the TMV CP gene and VF36 CP(-) plants were challenged with a Floridaisolate of ToMV, Naples C, in Florida. The field test in Illinois was conducted to determineif expression of the TMV CP gene in tomato would protect it against a number of differentstrains of TMV and ToMV. The TMV CP gene conferred resistance against ToMV-Naples Cinfection under Florida field conditions and against two strains of TMV under Illinois fieldconditions. Only weak protection was conferred against infection by the ToMV strainsunder Illinois field conditions (Sanders et al. 1990).

To enhance protection against ToMV, plants were produced that expressed a CP genederived from ToMV-Naples C. These lines were evaluated under field conditions in Illinoisalong with the control tomato line UC82B, with lines expressing the TMV CP gene, andwith lines expressing both TMV and ToMV CP genes. The TMV CP(+) lines were resistantto TMV infection, as shown in the earlier field test; however, they were less resistant toinfection by ToMV-Naples C. The tomato lines expressing ToMV CP gene were highlyresistant to infection by ToMV-Naples C. Plants that expressed both TMV and ToMV CPgenes were equally well protected against TMV and ToMV.

Field tests were conducted with Russet Burbank potato plants expressing the CPgenes of PVX and PVY (Lawson et al. 1990). PVY causes significant yield depression inpotato and, in combination with PVX, PVY produces a severe disease called “rugosemosaic”. To determine if expression of PVX and PVY CP genes would protect potato plantsfrom the synergistic effects of PVX and PVY infection in the field, plants propagated fromCP(-) Russet Burbank and from plant lines expressing both CP genes were inoculatedwith both PVX and PVY and transplanted into the field (Kaniewski et al. 1990). Plantsfrom four CP(+) lines were significantly protected from infection by PVX. However, threeof the lines were not protected from infection by PVY when simultaneously inoculated byboth viruses. Plants of one line, however, were highly resistant to PVX and PVY, aspredicted from growth-chamber tests. Tuber yields at maturity in uninoculated plots werethe same for all the lines. In contrast, tuber yields of all inoculated lines were markedlyreduced, except the line resistant to both viruses, which was unaffected by virusinoculation.

For the last 3 years, field trials of cucurbits transformed with the CMV CP gene havebeen conducted with success (Gonzalves et al. 1991). In 1991, four transgenic lines of thecv Poinsett 76 were compared with resistant cv Marketmore 76; infection was allowed tooccur naturally by aphids using a low percentage of infected plants as initial virussources. After 4 months of growth, the transgenic plants performed much better than thecontrol plants; 8% of transgenic plants showed symptoms versus 98% for the control and8% for the resistant line.


Potential Use of Coat Protein-MediatedResistance for Banana VirusesThe choice of a particular strategy for controlling plant viruses by genetic engineeringdepends greatly on the types of virus chosen as a target; but, because of the apparentuniversality of the efficacy of CPMR and because of the strength of the resistance, coatprotein strategy should be first attempted.

Viruses infecting bananasViruses infecting banana are limited in number and still insufficiently described; however,the following have been identified :

Banana bunchy top DNA-virus (Dietzgen, Thomas 1991; Harding et al. 1991; Thomas,Dietzgen 1991; Wu, Su 1990). This virus has been isolated only in Australia, and it is notknown whether it is the causal agent of the banana bunchy top disease or a helper of thebanana bunchy top luteovirus. This virus with a genome of multipartite single-strandedDNA most probably belongs to a new group of viruses. The genome comprises a largenumber (seven or more) of monogenic components. It is somewhat similar to thegeminiviruses for which CPMR so far has not been extremely effective and, if the CPstrategy was poorly efficient with this virus, another strategy, like the sense or antisensestrategy for the replicase gene, should be considered.

Banana bunchy top luteovirus (Brunt et al. 1990). Luteoviruses are single-strandedRNA+ viruses with a monopartite genome. With the example of the PLRV, we know thatCPMR is an efficient strategy to create resistance by biotechnology, provided that the CPis engineered and the gene expression increased. Resistance to the virus through insect-vector inoculation has been demonstrated in potato and tobacco, and should therefore beeffective in banana with appropriate chimaeric gene constructs. If banana bunchy topdisease results from the effect of two viruses, the usefulness of CPMR would have to beinvestigated using one or the other CP or a combination of the two CPs.

Cucumber mosaic cucumovirus (Brunt et al. 1990). This virus is one of the mostcommonly used for CPMR and we know that the CP strategy is extremely effective with awide spectrum of protection (see below). In any project of banana transformation tocontrol viruses through biotechnology, the CMV CP gene should be used because it is animportant viral disease of banana and because it can be an internal marker for other viralgenes. We know that if CPMR cannot be achieved with CMV, the unique bananaenvironment should be taken into account to improve gene expression and efficacy ofCPMR.

Banana bract mosaic potyvirus (Dale, pers. com.). This virus has been recentlydiscovered in the Philippines where it is causing a lot of damage. There are manyexamples of CPMR for such viruses and the strategy could be applied directly to bananawith the appropriate cloning of the CP gene for this potyvirus. There are also indicationsthat different potyviruses in other places in the world infect banana: proper identificationis needed, but, with the wide spectrum of protection effected by CPMR, an overallprotection against banana potyviruses using one CP could be envisaged.


Banana streak badnavirus (Lockhart 1990). Badnaviruses are double-stranded DNAviruses for which we have not achieved any examples of CPMR. Studies with a virus of thesame group are under way with the rice tungro bacilliform virus, and should providevaluable information to extrapolate to the banana streak virus. This virus does not seemto be very widespread in the world and, therefore, is not an important economic target.

Summary of coat protein-mediated resistance for PLRVThe CPMR for PLRV has been achieved by two different groups which obtained similarresults (Kawchuk et al. 1991; Tumer et al. 1991). The CP of the PLRV is expressed at avery low level, at the limit of the detection (0.01%), but resistance to the virus replicationwas nevertheless effective. The transgenic plants were resistant to inoculation by thenatural aphid vector, even in the case where large numbers of insects were used. Therewas no relation between the amount of CP detected in the transgenic lines and the levelof resistance estimated in the same lines. Field trials have been successfully conducted,but the spectrum of specificity remains to be established. The CP expression has beenimproved by sequence protein engineering, removing the internal ORF, resynthesizing theCP coding sequence, and by expressing two chimaeric genes in the same transgenic plant.In these conditions up to 9% of the regenerated lines were highly resistant or immune(Cuozzo et al. 1988; Tumer et al. 1991). Furthermore, it has been proven that the CPantisense strategy is also effective against PLRV, providing an alternative to the CPMR(Kawchuk et al. 1991).

Summary of coat protein-mediated resistance for CMVCPMR for CMV has been achieved by three different groups which obtained similarresults in different plants (Ye et al. 1991; Cuozzo et al. 1988; Beachy, pers. com.). The CPof CMV was expressed at a very high level (0.6%), and resistance to the virusmultiplication was effective. The transgenic plants were resistant to mechanicalinoculation and/or inoculation with the natural aphid vector. There was no direct relationbetween the amount of CP detected in the transgenic lines and the level of resistanceestimated in the same lines, though the highest expressors were among the mostresistant lines, and no line was highly resistant without any CMV CP. Field trials oftransgenic cucumbers have been conducted successfully for 3 years, where the besttransgenic lines had less than 10% of contaminated plants after 4 months of survey.Studies have been conducted to evaluate the spectrum of specificity of the resistance, andit was shown that the plants were resistant to strains within one subgroup of CMV strainsand across the two subgroups of the cucumovirus group.

ConclusionThe last few years have provided a profusion of techniques to control viruses by geneticengineering. Most have demonstrated potential for conferring resistance to plants andconstitute future hopes for crop improvement. Several techniques have already been


applied in field experiments and have proven to be real sources of resistance to viruses,e.g., the satellite and the coat protein strategies. The ribozyme strategy is interestingbecause of its potential applicability to all viruses, and because of its high specificity, butit needs to be demonstrated under in-vivo conditions. The satellite strategy, though veryeffective, is strictly limited to viruses having satellites, which are very few in the plantvirus world, and its use is limited by the risk that mutations would accompany widespreaduse.

CPMR for controlling viruses is the safest, most efficient, and widely documented typeof engineered resistance. Several field experiments have been conducted with differentcrops and CPMR was shown effective in all cases. The spectrum of specificity of CPMR isbroader than in any other type of virus resistance (including natural resistance), becauseplants can be protected against viruses of the same group sharing up to 60% in their coatprotein sequence. Coat protein transgenic plants are resistant to high concentrations ofvirus inoculum, and even in some cases to RNA inoculum. Resistance against vectorinoculations and vegetative propagation have also been demonstrated. The resistancegenerated by the coat protein strategy is of a multiple type, reducing the number ofinfection sites, the symptoms, the virus multiplication and the long-distance spreadthrough plants. It seems that the banana is an excellent candidate for usingbiotechnology to control viruses. And because there is no natural source of resistance forany banana virus, because it is a vegetatively propagated crop, and because CPMR hasbeen used to describe most of the viruses of banana described to date, CPMR isapplicable and should be useful.

All the manifestations of CPMR can be the result of a single mechanism or of multipleeffects of the coat protein on different targets. Nevertheless, despite the number ofstudies conducted to understand the mechanisms of the action of the coat proteinstrategy, the question of how the coat protein confers resistance to engineered plantsremains unanswered.

AcknowledgmentsThis publication has been made possible with the participation of ORSTOM and TSRI.

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85J Dale, T Burns, S Oehlschlager, M Karan, R Harding

Banana Bunchy Top Virus: prospects for control throughbiotechnology

J Dale, T Burns, S Oehlschlager, M Karan, R Harding

IntroductionThe banana, as with most major food crops, is plagued by a number of virus diseases thatcan considerably reduce both the yield and economics of growing bananas. The mostimportant of these viruses is banana bunchy top virus (BBTV). This virus was firstrecorded in Fiji in 1879 and, since that time, has spread to many banana-producingcountries. Cucumber mosaic virus (also known as banana mosaic virus and infectiouschlorosis) infects bananas to a greater or lesser extent in most, if not all, banana-producing countries; banana streak virus, a mealybug-transmitted badnavirus, was firstrecorded in Morocco and appears to be confined to Africa; banana bract mosaic virus is aprobable potyvirus reported only from the Philippines. There are also two virus diseasesof abaca (Musa textalis) reported in the Philippines: abaca bunchy top, which appears tobe a strain of banana bunchy top virus and abaca mosaic, which is probably caused bysugarcane mosaic virus.

Considering the importance of both bananas and BBTV, there has been relatively littleresearch effort devoted to this virus. Consequently, there has been little informationpublished about BBTV until recently. There are probably a number of reasons for thisneglect including: (a) bananas are grown primarily in the tropics and subtropics whereasmost virologists and research funds are concentrated in the temperate regions; (b) thebanana is a difficult plant to work with from a virological aspect; (c) BBTV does not occurin Central or South America, the regions which supply nearly 90% of the world’s bananaexports and where most international banana funding has been directed; and (d) untilrelatively recently, there was no international organization to coordinate bananaresearch.

There have, however, been considerable advances in our knowledge of BBTV in thelast 4-5 years and, with the integration of biotechnology and BBTV research, there is nowa realistic prospect of developing resistant cultivars in the foreseeable future.

Centre for Molecular Biotechnology, Queensland University of Technology (QUT), 2434 Brisbane, 4001 Queensland,Australia

Banana Bunchy Top: the disease

The importance of the diseaseBBTV is considered the most economically damaging of the viruses that infect bananas. Itvirtually destroyed the banana industry in the southern Queensland and northern NewSouth Wales regions of Australia in the 1920s in probably the best documented outbreakof BBTV. Currently, the virus is a major limiting factor in the production of bananas inmany countries including the Philippines, China, and India. There is, of course, the ever-present danger of the inadvertent introduction of BBTV into Central and South America.

Symptoms of the diseaseThe symptoms of advanced infections in Musa spp. are quite distinctive. The leaves at theapex of the plant become narrower, dwarfed, and upright giving the top of the plant abunched appearance. The leaves also show marginal chlorosis. Infected plants may notproduce a bunch or the bunch does not emerge from the pseudostem depending on thetime the plant has been infected. The first symptoms of infection are less distinct but verycharacteristic. Dark green streaks develop on the petioles, midribs, and leaf veins. Thesestreaks are of varying length and the resulting dot-dash pattern is often referred to as a“morse code” pattern. Variation in the severity of symptoms has been reported and it ispossible that this is due to strain variation (H Su, pers. com.).

Geographical distributionThe disease was first recorded in Fiji in 1879 and, since this initial report, the disease hasbeen recorded in most banana-producing regions of the world including Australia, Asia(China, Indonesia, the Philippines, Vietnam, India, Pakistan, Taiwan), Africa (Egypt,Gabon, Burundi), Sri Lanka, and a number of the Pacific islands (Hawaii, Tonga, WesternSamoa, Guam).

There are a number of notable regions or countries where BBTV has not beenrecorded, particularly South and Central America, the Caribbean, Papua New Guinea, andThailand as well as many African countries.

BBTV is almost certainly continuing to be moved internationally; for instance, itseems that the virus has only recently reached Hawaii. The first report of the virus inAfrica was from Egypt in 1901. There was then a considerable time before reports of thevirus in central Africa (Burundi and Gabon); it appears that BBTV is now spreading incentral Africa. It is somewhat puzzling as to how countries such as Thailand have avoidedthe virus considering the close proximity to “infected” countries and the ease ofmovement of germplasm in the region.

Transmission of the diseaseThere are two reported mechanisms for the transmission of BBTV. The virus ispersistently transmitted by the black banana aphid, Pentalonia nigronervosa. There is

86 Banana Bunchy Top Virus: prospects for control through biotechnology

no information available as to whether the virus replicates in its aphid vector. This aphidhas been recorded in virtually all banana-growing countries.

The virus can also be transmitted through the vegetative parts of the banana plantincluding suckers and the corm. It has been demonstrated that the virus can readily passthrough the tissue-culture process.

The virus cannot be transmitted by mechanical (sap) inoculation.

Banana Bunchy Top: the virus

The search for the virusBanana bunchy top disease was always assumed to be caused by a virus and was classifiedas a possible luteovirus by Matthews (1982). The rationale for such a classification wasthe biological properties of the disease: it is transmitted by aphids in a persistent mannerbut not by mechanical inoculation; the symptoms of the disease included chlorosis andstunting; and the phloem of infected plants was damaged (Dale 1987). These are allprimary biological characteristics of luteoviruses and luteovirus infections.

Numerous attempts were made to purify virions from BBTV-infected plants usingprocedures successfully adopted for luteovirus purification but without significantsuccess. However, further evidence for the association of a luteovirus with the diseasecame from the reported occurrence of double-stranded RNAs in bananas infected withBBTV but not in healthy bananas (Dale et al. 1986). These dsRNAs were of similar size tothose reported in luteovirus-infected plants. These observations have been confirmed by anumber of other groups (B Kummert, pers. com.; H Su, pers. com.; M Iskra, pers. com.).

Small isometric virus-like particlesIn 1989, two groups reported the purification of virus-like particles (VLPs) from BBTV-infected plants. Iskra et al. (1989) obtained isometric particles about 28 nm in diameter,within the size range expected for a luteovirus; Su and Wu (1989) also obtained isometricparticles but these were 20-22 nm in diameter, which apparently contained single-stranded RNA approximately 2.0 x 106 in molecular weight and a single protein of 21 000molecular weight. Wu and Su (1990) classified these particles as belonging to a “small”luteovirus.

Harding et al. (1991) and Thomas and Dietzgen (1991) also reported the purificationof small isometric VLPs, 18-20 nm in diameter, from BBTV-infected plants. However,Harding et al. determined that these particles contained single-stranded DNA of about 1 kb. Thomas and Dietzgen confirmed this result. Both groups reported a single coatprotein of about 20 000 molecular weight. Subsequently, Harding et al. cloned a portion ofthis ssDNA and, by using this clone as a DNA probe, demonstrated that this 1 kb DNA waspresent only in BBTV-infected bananas, not healthy bananas, and was transmitted frominfected bananas to healthy bananas with the disease and the 18-20 nm VLPs. The DNAprobe hybridized with all BBTV-infected samples including samples obtained from


Australia, Burundi, Egypt, Gabon, Indonesia, the Philippines, and Taiwan, indicating thatat least one virus is common to all BBTV infections and that this virus contains ssDNA.

Antisera, both polyclonal and monoclonal, has been produced by Su and Wu (1989)and Thomas and Dietzgen (1991). These antisera react with isolates of BBTV from manydifferent regions confirming that a small isometric virus is intimately associated with thedisease.

The properties of the particles of BBTV reported by Harding et al. and Thomas andDietzgen preclude these particles as being virions of a luteovirus because luteoviruseshave virions of about 25 nm containing ssRNA and a coat protein subunit with a molecularweight of about 24 000. The properties are, however, most similar to the properties of theVLPs associated with subterranean clover stunt virus (SCSV) (Chu, Helms 1988). Theseparticles are isometric, 17-19 nm in diameter, and contain ssDNA (0.85-0.88 kb) and asingle protein about 19 000 in molecular weight. SCSV is also transmitted by aphids in apersistent manner and is not transmissible by sap.

Current status of research at QUTSince the publication of the association of ssDNA with the virions of BBTV (Harding et al.1991), our research has centered on the cloning sequencing and analysis of the BBTVgenome.

The original clone, pBT338, contained a 977 bp insert. The nucleotide sequence of thisinsert was determined. From this sequence, two adjacent oligonucleotide primers weresynthesized. In a polymerase chain reaction (PCR), these primers would be extendedaway from each other; thus, amplification would occur only if the molecule were circular.When these primers were used in a PCR reaction with BBTV ssDNA extracted frompurified virions, a 1.1 kb product was amplified. This demonstrated that the ssDNA fromBBTV virions is circular.

The PCR product, which should represent the complete sequence of this circle, wascloned into a “T-tailed” vector and the nucleotide sequence determined. Analysis of thissequence revealed the following.

• A sequence of 1111 bases.• A strong possible stem/loop structure which could represent the origin of

replication. The stem consisted of 11 base pairs. The loop, 10 nucleotides, contained theconsensus sequence common to gemini viruses and coconut foliar decay virus.

• One large open reading frame of 858 nucleotides with an associated polyadenylationsignal. This ORF, when translated, contained an NTP binding site usually associated withviral replicases.

• An untranslated region.The PCR primers derived from the sequence of pBT338 were used to amplify this

BBTV circle from a Philippine isolate of BBTV. The product was similar in size to that obtained from the Australian isolate and was subsequently cloned and sequenced.The Philippine sequence had the same features as the Australian sequence and the nucleotide sequence of the large ORF has very high sequence similarity with theAustralian isolate.


DNA from at least three other unique circles from BBTV virions has been cloned andpartially sequenced. Attempts are now being made to clone and sequence the completecircles and determine their function.

Analysis of SCSV has revealed that the genome is composed of at least seven uniquecircles of ssDNA each containing a single large ORF. It would appear at this time thatBBTV will have a very similar genome organization. Coconut foliar decay virus sharesmany properties with BBTV and SCSV (small isometric particles containing ssDNA). Onecircle of this virus has been cloned and sequenced. This sequence contains one large ORF(possible replicase) and five other smaller ORFs. No other different DNA circles havebeen reported from this virus.

Attempts have also been made to determine the N terminal amino acid sequence ofthe coat protein; these attempts have been unsuccessful, suggesting that the N terminusis blocked.

A possible second virusThere is some evidence that a second virus may be associated with banana bunchy topdisease. Initially, Dale et al. (1986) reported the presence of “luteovirus-like” dsRNA ininfected but not healthy bananas; this has since been confirmed by three other groups.Secondly, Iskra et al. (1989) reported the purification of 28 nm VLPs from BBTV infectedbananas. Thus there is a real possibility of a second virus, probably an RNA virus, involvedin the disease. This situation can be resolved only by further experimentation.

There are a number of instances where it has been demonstrated that the associationof two different viruses together cause a particular disease. In fact, there is already oneinstance of a DNA and a RNA virus combining to cause a disease: rice tungro bacilliformand rice tungro spherical viruses.

Diagnosis and Control of BBTVThe most likely early benefits of biotechnology as applied to bananas will be in the area ofdisease diagnostics and control. This is already happening in relation to the diagnosis ofBBTV, and research on control of BBTV using biotechnology is well established.

Diagnosis of BBTVUntil recently, diagnosis of BBTV infection was based on the detection and identificationof symptoms. The symptoms of BBTV are quite distinctive once fully developed and areeasily recognized by experienced personnel. However, there are two main dangers: first, inmany instances, personnel charged with the responsibility of detecting BBTV-infectedbananas have no practical experience of the symptoms. This is often the case ofquarantine officers in countries where BBTV does not occur. Secondly, there are situationswhere the symptoms of infection are indistinct, masked, or absent; this is particularly trueof infected plantlets in tissue culture, the method of choice for disseminating bananagermplasm internationally.


More advanced and accurate techniques of diagnosis have become available sincemethods for the purification of BBTV particles were developed. Both Su and Wu (1989)and Thomas and Dietzgen (1991) have produced monoclonal and polyclonal antisera andutilized these in Elisa-based detection assays.

Harding et al. (1991) have developed DNA probes for the diagnosis of BBTV and theseappear to be of at least equal sensitivity to the Elisa-based systems. More recently, wehave investigated the polymerase chain reaction (PCR) for “ultra-sensitive” and rapiddetection of BBTV in infected tissue. Initial experiments indicate that it is possible todetect the virus in less than 5 µg of tissue.

The development of these advanced diagnostic techniques greatly decreases the riskof inadvertent dissemination of BBTV to “uninfected” regions, and should improve theeffectiveness of other current and future control strategies.

Control options for BBTVThe primary objective of the international research effort on BBTV is to control the virusand therefore the disease. There are essentially two strategies for plant virus control:avoidance and resistance. Avoidance includes minimization and exclusion. Resistancecan be subdivided into selection, conventional breeding, and genetically engineeredresistance.

When the options for controlling BBTV are evaluated, there are a number of importantpoints that must be considered: (a) Musa spp. appear to be the only important naturalhosts of BBTV; (b) bananas are a vegetatively propagated perennial crop; (c) BBTV doesnot occur in all banana-producing countries or continents; and (d) bananas are producedboth as a large-scale plantation crop and as a domestic subsistence crop. Almostcertainly, therefore, different BBTV control strategies must be used for differentsituations.

Avoidance

Virus minimization depends very heavily on the ability to be able to diagnose an infectionrapidly and sensitively. With the availability of sensitive and specific serological andmolecular tests for BBTV, virus avoidance becomes a more attractive option.

Exclusion. This operates at two levels. The first is the regional, international, orintercontinental level where the approach is to exclude the virus from an area where itdoes not occur. This is usually implemented through quarantine. Quarantine may involvethe total exclusion of all plant species known to be hosts of the virus, exclusion of thoseplant species if the particular plants had been transported from or through an “infected”country or region and, finally, quarantine can involve the testing of plants to determinewhether they are infected with a particular virus prior to release. Quarantine isparticularly relevant to the control of BBTV as the virus does not occur in South andCentral America. There is, however, pressure to import new Musa germplasm into theAmericas as the major banana breeding programs are in the Americas whereas the centerof diversity of Musa is in Southeast Asia where BBTV occurs. Further, there are a numberof countries in “infected” continents that have not recorded BBTV and wish to continue to


exclude the virus, usually by means of quarantine. Also, BBTV has been successfullyexcluded from regions of Australia using a regional quarantine policy (Dale 1987).

The second level at which the exclusion strategy can be implemented is within regionsor countries where a particular virus occurs. This normally involves the establishment of avirus-tested scheme where planting material is made available that is known to be virus-free or in which the percentage infection is very low. This has the effect of reducing theamount of virus inoculum within the crop and has been used very successfully with anumber of vegetatively propagated crops including potatoes and citrus. A number ofcountries have used such a policy with BBTV and, at least in Australia, this strategy hascontributed to the high level of control of BBTV in that country.

Eradication. This strategy involves the destruction of plants that are infected with orare possible hosts of the virus of interest. This may involve the removal of infected cropplants and/or the removal of noncrop host plants. These noncrop hosts may be a speciesother than the crop or be the same species but growing wild. Again, eradication played animportant role in the control of BBTV in Australia. Almost certainly, eradication of BBTV-infected bananas in other countries would involve not only the removal of infectedbananas in plantations and gardens but also the removal of wild or noncultivated bananas.

Resistance to BBTV

Selection. For most crop plants, there are numerous cultivars differing in variousagronomic characters. It is often possible to select cultivars resistant to a particular virusby screening. This normally involves assembling a cultivar collection, inoculating eachcultivar and finally examining each cultivar for evidence of infection. This usually involvesboth a visual inspection and screening with a suitably sensitive and specific diagnosticsystem. Such a selection procedure will identify cultivars that are truly resistant to thevirus and also those that are tolerant. Unfortunately, there are no confirmed reports ofany banana or plantain cultivars being resistant to BBTV. It would appear that a greatereffort should be made to identify resistance or tolerance in banana cultivars.

Conventional resistance breeding. Where no cultivars have been identified withresistance or tolerance or where the resistant cultivars are unsuitable, it is possible withmany crops to breed resistance into a crop by conventional breeding, that is, crossing aresistant cultivar or species with a suitable other parent. Again, this strategy is unlikely tobe successful for the control of BBTV, at least in the short term. There are three majorreasons for this: (a) no confirmed BBTV resistance has been recorded in any Musa spp.;(b) there are no current commercial cultivars of bananas that are the product of abanana breeding program; and (c) none of the current banana breeding programsincludes BBTV resistance as a target, nor do they screen for BBTV resistance.

Genetically engineered resistance. Recently, a number of techniques have beendeveloped that can induce artificial resistance in a plant to a particular virus. The mostsuccessful of these techniques is coat protein gene-mediated resistance. Since it was firstreported (Powell et al. 1986), this technique has been shown to be applicable to thedevelopment of resistance against a wide range of RNA viruses in a number of differenthosts and is now accepted as a standard strategy in the “molecular breeding” of virus-


resistant plants. However, it is unknown at this time as to whether such a strategy will beeffective against DNA plant viruses such as BBTV.

Despite this lack of prior knowledge, our research is concentrated towardsinvestigating coat protein gene-mediated resistance against BBTV in bananas. We are,however, investigating a number of alternative approaches that may be applicable todevelopment of resistance to BBTV.

Our research, in 1992, is centered on three major objectives.1. The cloning, sequencing, and analysis of the BBTV genome initially to identify the

coat protein gene.2. The development of a convenient and efficient banana transformation system. Our

efforts have concentrated on the use of microprojectiles using reporter genes. At thistime, this appears to be the most promising technique.

3. The identification of suitable plant promoters that will express appropriate genes inbananas. To date, our efforts have concentrated on constitutive monocotyledonpromoters.

ConclusionsThere appear to be excellent prospects for the application of biotechnology in the controlof BBTV. Advances have already been made in the diagnosis of the disease using bothserological and DNA-based techniques, and these new diagnostic systems are beingadopted rapidly, particularly where international movement of germplasm is concerned.

The greatest benefits are probably still to come. The most important objective will bethe development and distribution of genetically engineered bananas resistant to BBTV.

There may also be benefits derived from the study and utilization of the virus itself.

ReferencesCHU PWG, HELMS K. 1988. Novel virus-like particles containing circular single-stranded DNA associated with

subterranean clover stunt disease. Virology 167:38-49.DALE JL. 1987. Banana bunchy top, an economically important tropical plant virus. Advances in Virus Research

33:301-325.DALE JL, PHILLIPS DA, PARRY JN. 1986. Double-stranded RNA in banana plants with bunchy top disease. Journal

of General Virology 67:371-375.HARDING RM, BURNS TM, DALE JL. 1991. Small single-stranded DNA associated with banana bunchy top disease.

Journal of General Virology 72:225-230.ISKRA ML, GARNIER M, BOVE JM. 1989. Purification of banana bunchy top virus. Fruits 44:63-66.MATTHEWS REF. 1982. Classification and nomenclature of viruses. Intervirology 17:1-200.POWELL AP, NELSON RS, DE B, HOFFMAN N, ROGERS SG, FRALEY RT, BEACHY RN. 1986. Delay of disease development

in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-743.SU HJ, WU RY. 1989. Characterisation and monoclonal antibodies of the virus causing banana bunchy top.

Technical Bulletin no. 115. Tapai: ASPAC Food and Fertiliser Technology Center. 10 pp.THOMAS JE, DIETZGEN RG. 1991. Purification, characterisation and serological detection of virus-like particles

associated with banana bunchy top disease in Australia. Journal of General Virology 72:217-224.WU RY, SU HJ. 1990. Purification and characterisation of banana bunchy top virus. Journal of Phytopathology

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93GA Strobel, AA Stierle, R Upadhyay, J Hershenhorn, G Molina

The Phytotoxins of Mycosphaerellafijiensis, the Causative Agent ofBlack Sigatoka Disease, and TheirPotential Use in Screening forDisease Resistance

GA Strobel1, AA Stierle1, R Upadhyay1,3, J Hershenhorn1, G Molina2

IntroductionBananas and plantains are the primary food source for millions of people in many areasof the world, including Central Africa, Southeast Asia, Central and South America, andthe Caribbean. People in these regions are generally faced with the problems ofenormous population growth and recurring food shortages, conditions that augment theimportance of high-yield, low-cost crops such as bananas and plantains (Stover,Simmonds 1987, pp.397-438). They yield a sweet, nutritious fruit and produce a starchthat can be used to prepare a variety of staple foods. But bananas and plantains providemore than just complex carbohydrates. They also yield a diverse array of usefulsecondary products such as fibers, wrappers, confectioneries, vegetables, catsup, beer,wine, and vinegar (Stover, Simmonds 1987; Valmayor 1986).

Commercial cultivation of bananas in tropical America, the Philippines, andCaribbean and Pacific islands has emerged as a significant economic resource. In theseregions they not only maintain their importance as a food staple, but also contribute tothe GNP, provide employment and fiscal earnings, and generate foreign currency. Of allthe plants grown as food, bananas and plantains produce the highest yield for the lowestcost (Stover, Simmonds 1987, pp.402-422). Unfortunately, this vital resource is currentlybeing threatened by the devastating leaf disease known as black Sigatoka.

Black Sigatoka is the most severe form of the Sigatoka disease complex involvingthree closely related fungal pathogens: Mycosphaerella musicola Leach ex Mulder,causative agent of yellow Sigatoka, first identified in the Sigatoka district of Java in 1902;

1Department of Plant Pathology, Montana State University, Bozeman, Montana 59717-0314, USA; 2FHIA, Apdo 2067,San Pedro Sula, Honduras; 3Present address: Directorate of Plant Protection, Quarantine & Storage, NHIV,Faridabad, Haryana, India

M. fijiensis Morelet, causative agent of black leaf streak, described in Fiji in 1964; andM. fijiensis var difformis Mulder and Stover, causative agent of black Sigatokadiscovered in Honduras in 1972 (Fouré 1986). As the Sigatoka diseases have progressedin time their virulence has increased. Their host range has broadened to includeplantains, which are resistant to yellow Sigatoka disease. Black Sigatoka has supplantedyellow Sigatoka as the major threat to the cultivation and economic importance ofbananas and plantains (Buddenhagen 1986).

The black Sigatoka epidemic in Honduras and several other countries in 1972-73resulted in crop losses approaching 47% (Jaramillo 1986, p.46). Crop yields for Horn-typeplantains have been reduced by 50% (Stover, Simmonds 1987, p.293). Control costs forthis disease can be astronomical, totalling more than US$17 million per year in CostaRica alone; no other disease or pest control program in that country can compare(Jaramillo 1986). The cost of controlling black Sigatoka disease in Central America,Colombia, and Mexico surpassed US$350 million in less than 8 years. Large fruit-producing companies must spray fungicide mixtures up to 30 times annually to controlblack Sigatoka, expending up to 30% of their gross incomes. Annual fungicide costsamount to 10’s of millions of dollars (Stover, Simmonds 1987). Small landowners cannotcontrol the disease in their fields, lacking both the equipment and the funds required forfungicide application. The impact of this plant disease on the poor people of the tropicshas been enormous.

An alternative solution is the isolation and dissemination of banana and plantaincultivars resistant to the Sigatoka disease complex. Such a program could involve eitherbreeding for resistance or the screening of existing cultivars for inherent resistance.Breeding bananas and plantains is a slow, tedious process, due to serious problems withpolyploidy and poor seed yields and pollen infertility (De Langhe 1986). Furthermore,movement of the germplasm of bananas and plantains and different pathogenicbiotypes in the screening process could inadvertently introduce new forms of Sigatokaor other diseases into disease-free areas. The isolation and identification of wild-typeresistant cultivars or the selection of somaclonal mutants may circumvent thesedifficulties.

Unfortunately, finding such resistant cultivars can be an expensive, time-consumingprocess. Using traditional methodology, mature plants are challenged by fungal sporesproduced on naturally infected leaves according to fastidious inoculation schemes toartificially induce the disease. This process may require over 12 months to establishunequivocally the susceptibility or resistance of a particular cultivar to the disease(Stover 1986, pp.114-118). Fortunately, a preliminary report in 1989 indicated thatdisease symptoms could be induced by the crude extract of the pathogen, suggesting thepresence of one or more phytotoxins (Upadhyay et al. 1990). Because of the extremeimportance of this disease we began a comprehensive investigation of the phytotoxins ofM. fijiensis. It was hoped that phytotoxins isolated from the fungus could provide a rapidindication of cultivar susceptibility or resistance using a simple leaf assay. This paperdescribes the isolation, characterization, and biological significance of the phytotoxinsisolated to date.

94 The Phytotoxins of Mycosphaerella fijiensis


Fungal culture The culture of M. fijiensis (IMI 105378) used in this study was originally isolated fromMusa sapientum by R Leach in 1964 in Fiji, and was supplied by the CommonwealthMycological Institute, London. M. musicola (357) was provided by RA Fullerton, DSIR,New Zealand; M. fijiensis cv difformis was isolated by GC Molina, FHIA, Honduras. Allcultures were maintained at 26°C on modified M-1-D medium to which 12 mL L-1 coconutmilk was added (Pinkerton, Strobel 1976).

Mass culture and extraction The fungi were grown in 2-L Erlenmeyer flasks containing 800 mL modified M-1-Dmedium (autoclaved) (Pinkerton, Strobel 1976). The medium was inoculated with twomycelial disks (1 cm2) cut from the edge of a vigorously growing fungal colony. Each flaskwas shaken at 140 rev min-1 for 28 days at 26°C with 12 hours of light (50 EM-2 S-1). Afterincubation, an equal volume of methanol was added to the fungal culture, which was leftfor 24 hours at 4°C. The culture was filtered through eight layers of cheesecloth and thevolume of the filtrate was reduced by 2/3 by rotary evaporation at 40°C. Theconcentrated filtrate was exhaustively extracted (3x) with an equal volume of ethylacetate. The three extracts were combined and reduced to a brown oil (ca 90 mg L-1).For the investigation of fungal metabolites at timed intervals, cultures were grown for 10,15, and 20 days following the protocol outlined above. The crude extract yields were ca35, 75, and 86 mg L-1, respectively.

Bioassay A simple leaf puncture bioassay was used to guide us in the isolation of phytotoxins(Sugawara et al. 1985). Five test varieties of bananas and plantains including GrandeNaine (Musa acuminata Colla, AAB), Horn plantain (M. acuminata x M. balbisiana),Saba (M. balbisiana, ABB), IV-9 (new line of FHIA), and Bocadillo (AA) werepropagated through meristem culture to produce plantlets in test tubes. Plantlets wereready for testing at the 3-5 leaf stage when plants were approximately 10 cm tall. Testfractions were dissolved in 10% methanol and applied to the nicked surface of the fullyopened heart leaves in 5 µL droplets. The punctures were encircled with Vaselinepetroleum jelly to maintain droplet integrity. Leaves were placed in sterile plasticchambers with moistened filter paper to provide humidity. Crude extracts were tested at25, 50, and 100 µg per puncture wound; pure compounds were tested at 0.01, 0.1, 1.0, 5.0,and 10 µg per puncture. Necrotic tissue around the wound was measured at 24 and 48hours after treatment. Activity indexing utilized a 0-5 scale (+), ranging from no necrosis(0) to lesions 12 mm in diameter (5+). All tests were repeated three times. Methanol(10%) and water were used as controls. Organic extracts of sterile (noninoculated)medium did not exhibit phytotoxicity.


Results and Discussion

Isolation of phytotoxins The ethyl acetate soluble extracts of the culture filtrate of M. fijiensis were chromato-graphed on Sephadex LH-20 [CHCl3-MeOH, 1:1 (v/v)]. Fractions 3, 4, and 5 werephytotoxic. Fraction 4, the most active fraction, was further resolved by reverse-phasehigh-performance liquid chromatography on an Altex C-18 column using a gradientelution of acetonitrile-water. This step concentrated the primary phytotoxins in the sixthand eighth of eight fractions. Repeating the HPLC procedure on the sixth fractionyielded 2,4,8-trihydroxytetralone 1. Flash silica gel chromatography of the eighthfraction yielded juglone 2 as the first eluant.

Fraction 3 was resolved into nine fractions by reverse-phase HPLC using a MeOH-water gradient. The third fraction was the known phthalide isoochracinic acid 5.Fraction 5 yielded 2-carboxy-3-hydroxycinnamic acid 3 after chromatography on the LH-20 column, as described above. Fraction 6 was 3,4,6,8-tetrahydroxytetralone 6(4-hydroxyscytalone). Table 1 summarizes the data for the isolation of compounds 1-6.

Table 1. Comparison of molecular formulae and separation procedures ofphytotoxins isolated from Mycosphaerella fijiensis.

Compound Molecular Final Rf-TLCa % Yieldb

formula isolation

1 C10H10O4 RP-HPLCc 0.39 0.63

2 C10H6O3 Flash silica 0.95 0.02

3 C10H8O5 Sephadex LH-20 0.0 0.03

5 C10H8O5 RP-Flashd 0.57 0.03

6 C10H10O4 Sephadex LH-20 – 0.50

a: Silica gel developed in chloroform-methanol, 10-1.b: % yield = (mass of compound isolated/mass of total organic extract) x 100.c: 25%-75% acetonitrile in water over 25 min.d: 40%-70% MeOH in water over 20 min.

Structural elucidation of phytotoxins (each shown as a numbered structure in Fig.1)

The molecular formula of compound 1 was determined to be C10H10O4 by high-resolutionmass spectrometry, indicating a compound with six sites of unsaturation. Phenolic andketone functionalities were indicated by infrared absorptions at 3590 and 1640 cm-1.Both 2D COSY and 1D decoupling experiments indicated a trisubstituted benzene ringwith three adjacent protons and a cis-1,3-diol system. Detailed analysis of the massspectral and 1H NMR data resulted in the proposal of structure 1, 2,4,8-trihydroxytetra-lone. Compound 1 was proposed as a melanin shunt-pathway product by Stipanovic andBell (1977), although it was isolated only when an appropriate precursor was supplied to



Figure 1. The seven phytotoxins of Mycosphaerella fijiensis. Each is numbered according tothe order shown in the text. Phytotoxin 7 was previously described by Upadhyay et al.

the fungus Verticillium dahliae (Fig.1). The compound had also been isolated byFujimoto et al. (1986) from Penicillium diversum var aureum. However, no biologicalactivity has ever been ascribed to the compound.

Compound 2, juglone, has been previously isolated from several plant sourcesincluding walnuts (Juglans nigra) (Binder et al. 1989) and species of Penicillium(Fujimoto 1986) and Verticillium (Greenblatt, Wheeler 1986). It was readily identifiedby spectral data analysis: both spectroscopic and physical characteristics were identicalto the authentic compound purchased from the Aldrich Chemical Company.

Compound 3 has a molecular formula of C10H8O5, as determined by chemicalionization mass-spectrometry, indicating seven sites of unsaturation. 1H NMR analysisindicated three adjacent protons on a tri-substituted benzene ring, with the highest fieldaromatic proton, δ6.95 ppm, ortho to a hydroxyl group. Two mutually coupled (J=16Hz)olefinic absorptions at δ8.32 and δ6.18 ppm suggested a trans-α ,ß-unsaturatedcarboxylic acid derivative. This was a very polar compound which streaked on silica TLCplates unless 0.5% trifluoroacetic acid was added to the solvent system. This behaviorsuggested the presence of carboxylic acid functionality; infrared analysis supported thisidea with broad OH stretch absorption from 3150 to 3550 cm-1 and carbonyl absorption at1720 cm-1. The compound was methylated by dropwise addition of freshly prepareddiazomethane (Aldrich, no date). Examination of the 1H NMR spectrum of the singleproduct 4 indicated the addition of three methyl groups at δ3.94, δ3.83 and δ3.77 ppm,which indicated the presence of either three carboxylic acid moieties or two acids and aphenolic moiety in the original compound. Difference nOe experiments with themethylated product led to the proposed structure of the novel compound 2-carboxy-3-hydroxycinnamic acid.

Compound 5, isoochracinic acid, C10H8O5, is also a bicyclic compound with the samearomatic substitution pattern found in 1 and 2. It contains both a fused-ring aromaticlactone and a carboxylic acid, as indicated by the infrared absorptions at 1723 and 1718cm-1. Isoochracinic acid has been isolated from Hypoxylon coccineum and Alternariakikuchiana, the causative agent of black leaf spot of pear (Kamida, Namiki 1974; Trostet al. 1980). The biogenesis of isoochracinic acid has been of particular interest. It is oneof the few naturally occurring phthalides. During this study we were able to establish 3as a possible precursor of 5. If left in contact with CHCl3-MeOH (1:1) for 24 hours at25°C, cyclization would occur spontaneously, resulting in a racemic mixture of 5.

Compound 6 has a molecular formula of C10H10O4, with six sites of unsaturation. It hasthe same aromatic moiety as 1 and 2 but differs in the aliphatic portion. Examination ofthe spectral data indicates the structure shown, 4-hydroxyscytalone, which is also a knownmetabolite in the melanin shunt pathway (Bell et al. 1976).

2,4,8-trihydroxytetralone 1. MS: m/z(%) 194 (13.4), 176 (5.6); HRMS: 194.0550(observed), 194.0552 (actual); 1H NMR (MeOH-D4): 7.54 (t, J=8.1Hz), 7.21 (d, J=8.1), 6.86(d, J=8.1), 4.89 (dd, J=11.4, 5.1), 4.41 (dd, J=13.3, 4.8), 2.63 (dt, J=4.8, 11.4) and 1.98 (dd,J=13.3, 5.1)

juglone 2. MS: m/z 174; 1H NMR (MeOH-D4): 7.68 (t, J=7.5Hz), 7.59 (d, J=7.5), 7.31 (d,J=7.5), 7.00 (s), 7.01 (s)


2-carboxy-3-hydroxycinnamic acid 3. MS: m/z 208; 1H NMR (MeOH-D4): 8.32 (d,J=16Hz), 7.39 (t, J=7.1 Hz), 7.09 (d, J=7.1), 6.95 (J=7.1)

2-carboxy-3-methoxycinnamic acid dimethyl ester 4. HRMS: 250.0821 (observed),250.0817 (actual); 1H NMR (CDCl3): 7.63 (d, J=16Hz), 7.38 (t, J=8.0), 7.19 (d, J=8.0), 6.94(d, J=8.0), 6.37 (d, J=16)

isoochracinic acid 5. HRMS: 208.0364 (observed), 208.0368 (actual); 1H NMR (MeOH-D4): 7.52 (t, J=7.5Hz), 7.03 (d, J=7.5), 6.86 (d, J=7.5), 5.81 (dd, J=6.0, 5.8), 2.85 (dd,J=15.4, 5.8), 2.71 (dd, J=15.4, 6.0)

4-hydroxyscytalone 6 MS: m/z(%) 210 (22.1), 191.9 (9.2), 137 (39.1); 1H NMR (MeOH-D4): 6.73 (d, J=2.5Hz), 6.21 (d, J=2.5), 4.30 (d, J=3.0), 3.97 (ddd, J=3.0, 4.8, 8.1), 2.96 (dd,J=4.8, 17.5), 2.66 (dd, J=8.1, 17.5).

Biological activity Each of the compounds isolated displayed phytotoxicity at one or more test concentrationson one or more cultivars of banana or plantain. Compound 1 exhibited the greatest hostselectivity, analogous to the fungal pathogen itself, particularly at the 5 µg 5 µL-1 level.That is, the variety IV-9 is resistant to black Sigatoka disease and was insensitive to 1 upto the 10 µg 5 µL-1 application rate in the leaf bioassay test. Saba, a tolerant bananacultivar, was only slightly reactive to 1, unlike the extremely disease-susceptible varietiesBoca and Horn plantain which developed large necrotic lesions after application of toxin1. Compound 1 shows definite potential as a screening tool for toxin sensitivity in tissueculture systems, especially with the recent development of such systems (Novak et al.1989).

The most biologically active phytotoxin was juglone 2, which induced the formationof necrotic lesions on all cultivars of bananas and plantains at 0.1 µg (Table 2).Unfortunately, it was not host-selective at any test concentration, making it unsuitablefor cultivar screening. Juglone was isolated at extremely low concentrations (Table 1)and therefore may not play an important role in symptom induction. The novelphytotoxin 2-carboxy-3-hydroxycinnamic acid 3 demonstrated some host selectivity atthe 5 µg 5 µL-1 level, which again paralleled that of the fungus. Toxins 5 and 6 wereconsiderably less active than the other phytotoxins isolated and displayed no hostselectivity. Fijiensin 7, previously reported as a phytotoxin from the fungus, exhibits alow level of bioactivity and no host selectivity (Upadhyay et al. 1990). In this study wefound no evidence of phytotoxins in the aqueous phase of the fungal culture extracts andno evidence of additional phytotoxins in the ethyl acetate extract. It appears on the basisof yield, level of bioactivity, and host selectivity, that 2,4,8-trihydroxytetralone 1 is themost important phytotoxin produced by M. fijiensis.

It is interesting to note that all of the phytotoxins isolated might possess a muchgreater level of bioactivity than appears in Table 2. The leaf puncture method is areliable but relatively crude technique which has recently been measurably improved bya fluorescence labeling method employing flow-cytometry (Bergland et al. 1988).Compounds with low solubility can have more ready access to intact protoplasts, and



may demonstrate toxicity levels that are orders of magnitude greater than thosedetermined by the leaf puncture method.

Our study also involved analysis of toxin production at different times during thefermentation process. We were particularly interested in determining when 1 wasproduced in culture. It has been noted in field studies that a 3-4 week lag time occursbetween inoculation of a host with the pathogen and onset of disease symptoms (Stover1986). It seems unlikely that a toxin that is capable of inducing disease symptoms in thelaboratory in a matter of hours would require 3 weeks for symptom induction in the field.To this end, we grew the fungus for intervals of 10, 15, 20, and 28 days. The ethyl acetatesoluble extract of each culture was analyzed in precisely the same manner as before. No

Table 2. Comparison of the reactions of various banana/plantain cultivars to thephytotoxins produced by Mycosphaerella fijiensisa.

µg/puncture Grande Naine Horn plantain Saba Boca IV-9

Compound 10.10 µg - - - - -1.00 - + - - -5.00 + ++ - + -

10.00 ++ +++ + +++ -

Compound 20.01 µg - - - - -0.10 + + + + +1.00 +++ +++ ++ +++ +++5.00 +++ ++++ ++ ++++ ++++

Compound 31.00 µg - - - - -5.00 + + - + -

10.00 +++ ++++ + ++++ ++++20.00 ++++ ++++ ++ ++++ ++++

Compound 51.00 µg - - - - -5.00 - + + + +

10.00 + ++ ++ ++ +20.00 ++ +++ +++ +++ ++

Compound 61.00 µg - - - - -5.00 - - - - -

10.00 - + + + +20.00 + + + + +

a Toxins were applied at the rate indicated in 5 mL of solution (2% EtOH in water) as described in Materialsand methods. Readings recorded above were made 48 hours after treatment.


evidence of either 1 or 2 was found until the cultures had reached 28 days (or past 20days), although isoochracinic acid 5 was found in all of the cultures. This interestingdiscovery correlates the observed lag time between inoculation and onset of diseasesymptoms in the field with the production of 1 in culture. Further investigation of thephytotoxins of the 10-, 15-, and 20-day cultures is in progress.

One extremely important aspect of this study concerns the determination of anymorphological or chemical differences among the three fungi of the Sigatoka complex,M. musicola, M. fijiensis, and M. fijiensis var difformis. Preliminary data show someinteresting chemical trends. All three fungi were grown and processed as describedabove. M. musicola causes the least virulent form of the Sigatoka disease complex, and it

Figure 2. Proposed pentaketide pathway of melanin biosynthesis and shunt pathways leadingto the synthesis of 1, 2, and 6 (Greenblatt, Wheeler 1986).

does not appear to produce 1 or 2. M. fijiensis var difformis, causal agent of the mostsevere form of black Sigatoka, produces toxin 1 at 6-10 times the level of its less virulentcounterpart M. fijiensis. Detailed comparisons of phytotoxin production by these threefungi will be presented at a later date.

It is interesting to note the biosynthetic origin of the major toxin, 1, and relatedmetabolites in the fungal extract. Examination of the literature (Stipanovic, Bell 1977;Bell et al. 1976a,b; Wheeler, Stipanovic 1985) indicated that 2,4,8 trihydroxytetralone 1,juglone 2, and 4-hydroxyscytalone 6 are likely melanin shunt-pathway metabolites(Fig.2). Wheeler studied the biosynthesis of melanin in 20 species of ascomycetous andimperfect fungi, and found that, in all species tested except Aspergillus niger, themelanin precursor is 1,8-dihydroxynaphthalene, not indole, as was previously suspected(Wheeler 1983). In a series of elegant experiments using melanin-deficient mutants,these investigators carefully mapped not only the melanin pathway but also the majorshunt-pathway metabolites (Tokonsbalides, Sisler 1979). Many of the compoundsisolated from M. fijiensis may arise via these pathways.

Melanin occurs in the cell walls of many fungi, apparently protecting the cells fromdesiccation and ultraviolet radiation. It may also protect fungal cells from othermicroorganisms (Greenblatt, Wheeler 1986). The production of melanin can also be animportant determinant of pathogenicity: melanization of the fungal appressorium is anecessary prerequisite to invasion of rice by Pyricularia oryzae (Woloshuk et al. 1980,1983), of cucumbers by Colletotrichum lagenarium (Kubo et al. 1985), and of beans by C. lindemuthianum (Wolkow et al. 1983). Application of the fungicide tricyclazole, a systemic inhibitor of melanin biosynthesis, can control these fungal infections by interfering with melanization of the fungal cells (Greenblatt, Wheeler 1986;Tokousbalides, Sisler 1979; Woloshuk et al. 1983). Examination of the biosyntheticmelanin pathway, however, indicates an interesting departure from the norm.Tricyclazole inhibits pathogenicity by blocking melanization, which leads toaccumulation of shunt metabolites in the fungal cells (Tokousbalides, Sisler 1979). In M.fijiensis, it is the shunt-pathway metabolites that are the probable agents causing celldeath. We could predict that application of tricyclazole could actually increase thevirulence of the fungi by increasing the production of these phytotoxins.

We ground and extracted, with EtOAC, 500 g of infected banana leaf tissue in order tofind evidence of any or all of the phytotoxins within the plant. Chromatography on an LH-20 column, as previously described, did not yield any fraction that possessed biologicalactivity. Likewise, no evidence for the presence of any of these compounds could befound by TLC (Table 1), or NMR. This is not surprising, considering the strong possibilitythat in the process of fungal infection, these phytotoxins could conceivably be oxidized,reduced, or derivitized into other compounds not having biological activity. Comparablestudies using plant material in the very early stages of infection may prove more fruitfulin discovering these substances in the tissue. Such information would be helpful in morefirmly establishing their role in the infection process.

As a test for its potential usefulness, a purified preparation of tetralone from M.fijiensis, was applied to a series of meristem plantlets at varying concentrations. The


banana and plantain cultivars showed differential activity in concentrations as low as 5 µg µL-1. This activity paralleled the known resistance or susceptibility of thesecultivars to M. fijiensis. Therefore, we feel that preparations of the toxin have potentialusefulness in plant breeding/selection programs in all parts of the world where bananasand plantains are grown.

Acknowledgments

The authors wish to acknowledge the financial assistance of NSF grant DMB-8607347, anAID grant in cooperation with G Molina and the Montana Agricultural ExperimentStation.

ReferencesALDRICH CHEMICAL COMPANY. No date. Diazomethane Generators. Technical Information Bulletin AL-103.

Milwaukee, WI, USA: the Company.BELL AA, STIPANOVIC RD, PUHALLA JE. 1976a. Tetrahedron 32:1353-1356.BELL AA, PUHALLA JE, TOLMSOFF WJ, STIPANOVIC RD. 1976a. Can. J. Microbiol. 22:787-799.BERGLUND D, STROBEL S, SUGAWARA F, STROBEL GA. 1988. Pl. Sci. 56:183-188.BINDER RG, BENSON ME, FLATH RA. 1989. Phytochemistry 28:2799-2801.BUDDENHAGEN IW. 1986. Pages 95-109 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA,

eds). ACIAR Proceedings no.21. Canberra, Australia: ACIAR.DE LANGHE EA. 1986. Pages 19-23 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA,

eds). ACIAR Proceedings no.21. Canberra, Australia: ACIAR.FOURÉ E. 1986. Pages 110-113 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds).

ACIAR Proceedings no.21. Canberra, Australia: ACIAR. FUJIMOTO Y, YOKOYAMA E, TAKAHASHI T, UZAWA J, MOROOKA N, TSUNODA H, TATSUNO T. 1986. Chem. Pharm. Bull.

34:1497-1500.GREENBLATT GA, WHEELER, MH. 1986. J. LIQ. Chrom. 9:971-981.JARAMILLO RC. 1986. Page 42 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds).

ACIAR Proceedings no.21. Canberra, Australia: ACIAR.KAMEDA K, NAMIKI M. 1974. Chem. Lett. (1974 vol.):1491-1492.KUBO Y, SUZUKI K, FURUSAWA I, YAMAMOTO M. 1985. Pest. Biochem. Physiol. 23:47-55.NOVAK F, AFZA R, VAN DUREN M, PEREA-DALLOS M, CONGER BV, XIALONG T. 1989. Biotech. 7:154-159.PINKERTON F, STROBEL GA. 1976. Proc. Nat. Acad. Sci.(USA) 73:4007-4011. STIPANOVIC RD, BELL AA. 1977. Mycologia 69:165-171.STOVER RH. 1986. Pages 114-118 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds).

ACIAR Proceedings no.21. Canberra, Australia: ACIAR.STOVER RH, SIMMONDS NW. 1987. Bananas. New York, USA: Wiley. SUGAWARA F, STROBEL GA, FISHER LE, VAN DUYNE GD, CLARDY J. 1985. Proc. Natl. Acad. Sci. (USA) 82:8291-8294.TOKOUSBALIDES MC, SISLER HD. 1979. Pest. Biochem. and Physiol. 11:64-73.TROST BM, RIVERS GT, GOLD JM. 1980. J. Org. Chem. 45:1835-1838.UPADHYAY RK, STROBEL GA, COVAL SJ, CLARDY J. 1990. Experientia 46:983-984.WHEELER MH. 1983. Trans. Br. Mycol. Soc. 81:29-63.WHEELER MH, STIPANOVIC RD. 1985. Arch. Microbiol. 142:234-241.WOLKOW M, SISLER HD, VIRGIL EL. 1983. Phys. Plant Path. 23:55-71.WOLOSHUK CP, SISLER HD, TOKOUSBALIDES MC, DUTKY SR. 1980. Pest. Biochem. Physiol. 14:256-264.WOLOSHUK CP, SISLER HD, VIRGIL EL. 1983. Phys. Plant Path. 22:245-259.VALMAYOR RV. 1986. Pages 50-55 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds).

ACIAR Proceedings no.21. Canberra, Australia: ACIAR.


104 Molecular Approaches to Identifying Fusarium Wilt Resistance

Molecular Approaches to IdentifyingFusarium Wilt Resistance

RC Ploetz

IntroductionFusarium wilt has a long and destructive history in the world’s banana-producing regions(Ploetz et al. 1990; Stover 1962). Also known as Panama disease, it is caused by avariable soilborne fungus, Fusarium oxysporum f.sp. cubense. Fusarium wilt almostdestroyed the export trade during the first half of the 20th century. Although continueddamage on Gros Michel (AAA), the cultivar used by exporters at that time, was avoidedby the conversion to cultivars of the Cavendish subgroup (AAA), the later clones are nowsuccumbing to a new variant of the pathogen, race 4, in subtropical production areas inthe eastern hemisphere. Although significant damage has yet to occur on the Cavendishclones in the tropics, there is concern that race 4, or something like it, will develop inthe important export plantations found in these regions.

Less publicized, but potentially more destructive than the epidemics on Gros Michel,are outbreaks that affect nonexported, locally consumed cultivars. Susceptible clonesare used as staple foods or important dietary supplements in eastern Africa, Brazil,Southeast Asia, and other production regions (Alves et al. 1987; Ploetz et al. 1990;Valmayor 1987). Since preferred cultivars are heavily damaged or have been eliminatedby fusarium wilt in many of these areas, there is an urgent need to develop or identifyresistant replacements that would be accepted by local consumers.

Resistance to Fusarium WiltBanana breeding began in 1922 at the Imperial College of Tropical Agriculture inTrinidad and in 1924 in Jamaica (Stover, Buddenhagen 1986). The impetus for theseprograms, and one that subsequently began and still continues in Honduras [FundaciónHondureña de Investigación Agrícola (FHIA)], was fusarium wilt. Others at thisworkshop will address past and current breeding efforts (see Rowe 1993; Vulsteke,Swennen 1993), but it is instructive here to note the record of the early programs andhow resistance to fusarium wilt among products of these programs was assessed.

University of Florida, Tropical Research and Education Center, 18905 SW 280th Street, Homestead, FL 33031, USA

Despite their long history, acceptable commercial replacements for the AAA dessertcultivars have not been developed by the breeding programs (Buddenhagen 1990; Stover,Buddenhagen 1986). Genetic constraints, such as triploidy and sterility of somedesirable parents, impede straightforward progress in this area. In addition, theexceptionally good agronomic attributes of Gros Michel and the Cavendish cultivars havebeen difficult to match in the resistant products that have been produced to date (in thefinal analysis, producing a fusarium-wilt resistant AAA dessert banana with horticulturaltraits that equal those of the current Cavendish ideotype may be an unrealistic goal).Although success in breeding resistant AAB dessert or ABB cooking bananas may bemore easily achieved, breeding for these targets has started only recently (Buddenhagen1990; Rowe 1990; Shepherd et al. 1987; Vulsteke 1993). In general, the resistance of AABand ABB replacements produced in Brazil, Honduras, and Nigeria is either not wellcharacterized or not known.

New genotypes produced by the breeding programs are evaluated in field trials.Unfortunately, field screening has provided erroneous information about the resistanceof products of these programs (for examples of “resistant” clones succumbing tofusarium wilt after deployment to different areas, see Stover, Buddenhagen 1986). Untilrecently, screening sites were limited to the few areas in which active breeding programsexisted. Unfortunately, these sites provided imperfect screening environments. Eithernarrow pathogenic variability in F. oxysporum f.sp. cubense at the sites compromisedtheir effectiveness (e.g., FHIA in Honduras), or edaphic factors at a location restricteddisease development (i.e., disease-suppressive soils at the EMBRAPA site in Brazil).Even now that the value of geographically diverse screening sites is recognized, it is notclear that field trials in different locations will provide the necessary information. SinceF. oxysporum f.sp. cubense is a diverse pathogen, and because the factors thatdetermine whether or not fusarium wilt develops on a given clone in a given area arecomplex and not completely understood, determining whether new clones possess globalor durable resistance to fusarium wilt will be difficult (Ploetz 1990).

Banana breeding is time-consuming and expensive. Thus, methods either to expeditethe selection process or to generate new, resistant genotypes would be very useful.Below, I briefly review and discuss means by which fusarium wilt-resistant bananas couldbe produced in the future.

Resistance MechanismsAlthough resistance to diseases has been identified in other crops when pathogenesiswas not well understood, knowledge of this process is often useful during theseendeavors. For fusarium wilt on banana, the interaction between the pathogen and hostis complex. Work to define ways in which F. oxysporum f.sp. cubense and bananainteract was begun by Wardlaw (1930) who noted the formation of tyloses in banana inresponse to vascular colonization by this fungus. Although much remains to be learnedabout these processes, a good basic understanding of the steps leading from rootcolonization to death of a susceptible cultivar, or defense of a resistant plant, is now

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available (those interested in in-depth treatments of this subject should refer toBeckman 1987, 1989, 1990).

I am not aware of specific studies on infection of banana by F. oxysporum f.sp.cubense, but results from work with other plants and F. oxysporum suggests thatresistance in other hosts to different formae speciales of this fungus is expressed afterinfection occurs; the roots of resistant and susceptible plants are both infected(references in Beckman 1987). Resistance and susceptibility are ultimately determinedby a series of chemical and physical events that occur in the xylem (Beckman 1987; Pegg1985). Resistant hosts effectively wall off the invading parasite by forming gels, gums,tyloses and other xylem-occluding products (e.g., Vander Molen et al. 1977a,b). In turn, asuccessful pathogen breaches host defenses by virtue of, among other things, an array ofhydrolytic enzymes that degrade host cell walls and reaction products (Pegg 1985).

Phenolic compoundsPhenolic compounds that impregnate host reaction products are thought to play animportant role in the resistance process, either making physical barriers stronger orchemically impervious to the previously mentioned hydrolytic enzymes of the pathogen(Beckman 1987; MacHardy, Beckman 1981; Pegg 1985). Enzymes that are instrumentalin the formation of these compounds have been previously studied in many differenthosts, including banana (Mace, Wilson 1964; Mace et al. 1972; Mueller, Beckman 1978).For example, phenol-oxidizing enzymes such as peroxidase, tyrosinase and laccase, areassociated with many different vascular diseases (Pegg 1985). In general, these enzymesare inducible. Their role in resistance may be a timing phenomenon; that is, hosts whichquickly produce sufficient levels of the important products in response to the pathogenresist disease development.

Peroxidases and polyphenoloxidases are stored, preformed, in various, localized sitesin banana (Mace et al. 1972; Mueller, Beckman 1978). Different isoforms of the enzymes(isozymes) and the quantities produced are known to differ among different bananagenotypes (Jarret, Litz 1986a,b) and, since the levels and number of peroxidase isozymesproduced are greatest in roots of banana and other hosts, it has been postulated thatthey may help protect plants against infection by root pathogens (Bonner et al. 1974;Lagrimini, Rothstein 1987). The involvement of these enzymes in the resistance processand the considerable enzymatic variation that exists among different banana genotypesindicate that enzyme assays could help characterize and identify fusarium wiltresistance. If specific forms or relatively high quantities of these enzymes are correlatedwith resistance in the host, it is conceivable that products of breeding programs or pre-existing clones could be quickly and effectively evaluated via enzyme analyses, withoutresort to conventional disease screening trials.

The potential for this approach is borne out by recent, unpublished work byMorpurgo (as cited in Novak 1992). He indicated that constitutive levels of peroxidase inSH-3362, a race-4 resistant, synthetic AA hybrid produced at FHIA, were greater by 10-fold than in Pisang Mas, a susceptible AA cultivar. Additional work in this area iswarranted.


Somaclonal ResistanceSomaclonal variation is a common phenomenon that has drawn the attention of thoseinterested in all aspects of crop improvement (Larkins, Scowcroft 1983). Somaclonalvariants with enhanced resistance to various diseases are known for many plants,including banana (Hwang 1990). Resistant somaclones have been recovered from cellor tissue cultures with and without applying selection pressure prior to field screening.Hwang (1990) generated about 20,000 plantlets of Giant Cavendish via conventionalmeristem culture and screened these, without prior selection, in a field infested withrace 4. Six somaclones with tolerance to race 4 were identified.

Obviously, field selection of disease-resistant somaclones is a time-consuming andlabor-intensive procedure. Although preselection of “resistant” somaclones in thelaboratory prior to field testing could speed this process, such an approach requiresreliable selection criteria. Perhaps the simplest procedure by which cell or tissuecultures have been screened is by direct exposure to pathogens (Heath-Pagliuso et al.1988; McComb et al. 1987). Cell or tissue cultures, however, are often very sensitive tothis sort of treatment and it may not be possible to distinguish resistant fromsusceptible selections in vitro. At least with the different fusarium wilt systems that Iam aware of, both resistant and susceptible materials die after challenge (Heath-Pagliuso et al. 1988; pers. com. Kistler; Ploetz 1990).

Several pathogen-free methods have been used that are generally less destructive,more reproducible, and amenable to manipulation than the direct exposure method.Since they are pathogenic determinants (i.e., responsible for pathogenesis), host-specific toxins are ideal for this purpose. For example, T-toxin, produced by the fungusBipolaris (Helminthosporium) maydis, has been used to select somaclones of maize(Zea mays) with resistance to southern corn leaf blight (Gegenbach et al. 1978).However, from a selection standpoint, host-specific toxins are, unfortunately, notalways involved in pathogenesis.

Nonspecific toxins produced by pathogens may be useful, even when therelationship between tolerance of a toxin and resistance to a disease is not clear.Fusaric acid is a good example of such a host-nonspecific toxin. It is produced in vitroby many formae speciales of F. oxysporum and has been detected in infected hosts,including banana (see Beckman 1987 and references therein). Although its role as awilt toxin is controversial, fusaric acid has been used to select fusarium wilt-resistantvariants of tomato (Lycopersicon esculentum) (Shahin, Spivey 1986), and suggested asa selective agent for fusarium wilt-resistant musk melon (Cucumis melo)(Mégnégneau, Branchard 1988). Results with strawberry (Fragaria X ananassa)(Toyoda et al. 1991) and banana (Epp 1987; Krikorian 1990), however, have been lessencouraging. Fusaric acid is highly toxic. During work on banana, there appeared to bean either/or phenomenon: either meristems and cell cultures died at a givenconcentration of the toxin or, at lower concentrations, no growth reduction or mortalitywas observed (Epp 1987; Krikorian 1990). Since sublethal concentrations of fusaricacid that could be used to select tolerant somaclones were never identified, fusaric

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acid may not be a useful tool for selecting fusarium wilt-resistant somaclones ofbanana.

When culture filtrates of pathogens have been used as screening agents in planttissue cultures, the results have also been mixed. Arcioni et al. (1987) and Gray et al.(1986) reported using culture filtrates to identify, respectively, fusarium wilt-resistantalfalfa (Medicago sativa) (F. oxysporum f.sp. medicaginis) and brown stem rot-resistant soybean (Glycine max) (Phialophora gregata). However, Mégnégneau andBranchard (1991) and Vardi et al. (1986) indicated that culture filtrates were not usefulfor identifying, respectively, fusarium wilt-resistant musk melon (F. oxysporum f.sp.melonis) and different cultivars of Citrus spp. with resistance to Phytophthoracitrophthora.

A wide range of compounds and stimuli induce resistance in plants to disease agents(Kuc 1985), some of which have been used to select disease-resistant somaclones inplant cell or tissue cultures. For example, Buiatti and colleagues (Buiatti et al. 1985,1987a,b) used cell wall components of F. oxysporum f.spp. dianthi and lycopersici aselicitors during work on, respectively, carnation (Dianthus caryophyllus) and tomato.Clones with altered phytoalexin production, compared with the parental lines, wererecovered in both systems, and they suggested that such a screening technique couldprovide a new method for selecting pathogen-resistant genotypes. Krikorian (1990) usedpotassium vanadate as an elicitor in cell cultures of Cardaba, an ABB cooking banana.He indicated that small percentages of cells survived treatment and that organizedpropagules were recovered. At this time, I am not aware of results from pathogenicitytrials with material produced during this work.

Although Hwang’s results (1990) make it clear that somaclonal variation couldprovide new sources of resistance in banana to fusarium wilt, I am not aware of resultsother than his in this area. Certainly, as cell or tissue culture techniques for banana arerefined or new ones are developed, investigation of somaclonal variation for resistancepurposes should be expanded, especially when conventional breeding strategies areunsuccessful.

Markers for ResistanceThe identification of markers for disease resistance has received significant attention asa means to increase the efficiency of screening programs (Behare et al. 1991; Medina,Stevens 1980; Michelmore et al. 1991; Rick, Fobes, 1974; Robinson et al. 1970; Sarfatti etal. 1989). The objective of these studies is to identify phenotypic or genetic traits thatare linked to resistance genes. Marker identification relies on populations of hostprogeny that segregate for resistance and susceptibility and nonambiguous means bywhich the presence or absence of the marker and disease response of the progeny can bescored. For example, Robinson et al. (1970) identified an anthocyaninless gene intomato (ah) that was linked to a gene for resistance to tobacco mosaic virus (Tm2). Byselecting progeny with green stems, breeders were also able to select for resistance tothis virus. Rick and Fobes (1974) observed linkage between resistance in tomato to


Meloidogyne incognita (rootknot nematode) and a particular acid phosphatase allele(Aps-1). Thus, progeny that possessed Aps-1 could be selected as nematode-resistantwithout recourse to nematode challenge.

Recently, two additional types of genetic markers have been used to expediteresistance breeding. In fundamentally different ways, restriction fragment lengthpolymorphisms (RFLPs) and randomly amplified polymorphic DNAs (RAPDs) identifyvariations in host DNA sequences that can be exploited as markers for resistance genes(for detailed discussions of these techniques refer to Beckman and Soller 1986;Michelmore et al. 1991; and Tanksley et al. 1989). For example, RFLPs have been used asmarkers for genes for resistance to Stemphylium spp. (Sm) and race 2 of F. oxysporumf.sp. lycopersici (I2) (Behare et al. 1991; Sarfatti et al. 1989), whereas Michelmore et al.(1991) described screening bulked, segregating host populations for RFLP or RAPDmarkers and provided data on the use of RAPD markers for identifying resistance genesin lettuce (Lactuca sativa) to downy mildew (Bremia lactucae).

Although resistance markers can be powerful tools for plant breeders, theiridentification can be quite time-consuming and expensive. In addition, they may relyupon considerable background information that has been generated for a given hostgenome. Compared with lettuce or tomato, two of the above hosts for which impressivelinkage maps exist, almost nothing is known about banana (see González de León 1993).Also lacking in banana is a progeny population that segregates for resistance to fusariumwilt. Presumably, segregating populations could be generated by breeding programs atFHIA, or elsewhere, which have diverse collections of resistant and susceptible parents.

Information on the inheritance of fusarium wilt resistance among different Musaacuminata subspecies and M. balbisiana was generated by Vakili (1965). Geneticcontrol of resistance was not complex [for example, he determined that resistance torace 1 in Lidi (AA) was controlled by a single dominant gene]. Unfortunately, additionalresearch in this area has apparently not been conducted after the publication of Vakili’s(1965) work. However, if we assume that resistance to different races of fusarium wilt inother banana genotypes is controlled by single genes, it should be possible to identifymarkers for resistance to this disease in the future (see Gonzáles de León 1993 forinformation on genetic mapping of the M. acuminata genomes). His, and other researchon this topic, could begin to provide the foundation on which marker work couldproceed.

Genetic TransformationGenetic transformation of plants to enhance either disease or insect resistance isdiscussed by others at this workshop (Dale et al. 1993; Fauquet, Beachy 1993; De Waele1993). Transformation with coat protein genes of viral pathogens is now a widely useddisease-control strategy (see Dale et al. 1993; Fauquet, Beachy 1993), but transformationto enhance resistance to fungal-incited diseases has received little attention. Althoughexperience in the latter area is scant, production of transgenic banana with resistance tofusarium wilt is conceivable (Murfett, Clarke 1987).

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Several substantial hurdles must be cleared before such an objective can be realized.First, it will be necessary to develop transformation protocols for banana (see Dale et al.1993; Bakry et al. 1993). Many different methods have been developed for transformingplants (for a comprehensive list and discussion of the available methods, see Potrykus1991); however, I know of no published reports on banana transformation. Since a reliabletransformation technique could be used for diverse and important applications, work inthis area is of great interest (see Novak 1992). Unfortunately, the widely used andsuccessful method, involving genetically engineered strains of Agrobacterium tumifaciensas DNA vectors, is not effective with all plant species. In particular, monocotyledons arenot usually amenable to A. tumifaciens-mediated gene transfer, but there are notableexceptions (Potrykus 1991). Some “transitional” monocotyledons, such as asparagus(Asparagus officinalis) (Potrykus 1991) and black pepper (Piper nigrum) (pers. com. RELitz) are easily transformed. Other monocotyledons, such as maize, have been transformedvia agroinfection, but this appears to be more difficult than for the previously mentionedmonocotyledons (Grimsley et al. 1987). Still, it should not be assumed that attempts totransform banana by this route would be futile (Murfett, Clarke 1987).

Other methods are used less frequently and, to date, have been less successful thanthe Agrobacterium-based systems. This may change as new transfer techniques aredeveloped and refined. Microprojectile or biolistic transformation is one of the moreunusual approaches to the problem of introducing foreign DNA into plant cells (seeKlein et al. 1987; Sanford et al. 1987; Dale et al. 1993; Bakry et al. 1993). Transformationwith microprojectiles has several advantages. Two of several listed by Potrykus (1991)are that it may be used on many different types of tissue (i.e., specific tissues are usuallynot needed), and that it is host-range nonspecific. Thus, the biolistic approach may beespecially useful for species, such as bananas, that might not lend themselves toAgrobacterium-mediated gene transfer (Dale et al. 1993).

DNA has been introduced into plant protoplasts by a variety of techniques (seePotrykus 1991 for details). Like the microprojectile strategies, protoplast transformationavoids the host-range constraints of A. tumifaciens-based systems. Physical methods fortransforming protoplasts include electroporation and microinjection, whereas chemicalmethods rely on polyethylene glycol or other osmotica for DNA transfer. The genetictransformation of banana with these techniques would rely on procedures by whichprotoplasts could ultimately be used to regenerate plants. Since totipotency for bananaprotoplasts has only recently been reported (referenced in Novak 1992), it may be sometime before these systems can be used to produce transgenic banana plants. Results forother crops indicate that these protocols can be used rarely for more than a few cultivarsor types of given crops. Although gene transfer via banana protoplasts may not befeasible in the near future, continued attention should be given to this possibility.

Obviously, for any transformation technique to be useful, a gene or genes of interestmust have been previously identified and cloned for use. In addition, marker gene(s)must be incorporated with the chosen gene(s) to facilitate selection of transformedtissues or cells. An effective promoter for banana must also be found and used. I amaware of no work on banana in these areas.


Recently, plant transformation for enhanced chitinase production has beenconsidered as a means to increase resistance to diseases caused by fungi (Jones et al.1986; Nitzsche 1983). Chitin is a cell-wall component of fungi, but not plants (Bartnicki-Garcia 1968). Plants normally produce constitutive, low levels of chitinases, enzymesthat have been shown to inhibit fungal growth in vitro (Schlumbaum et al. 1986). Sinceinfection by fungal pathogens induces an increase in the levels of these enzymes inplants, and because there are no known substrates for chitin in higher plants, chitinasesare thought to play a role in the inducible defense systems of plants.

Chitin-specific strategies are aimed at raising the constitutive levels of chitinaseproduction in hosts. It is hoped that plants with altered chitinase production will, as aconsequence, possess enhanced protection against infection and colonization by fungalpathogens that have chitinous cell walls. In general, these strategies involvetransforming plants with genetically engineered constructs of constitutive promotersand foreign chitinase genes from either bacteria or higher plants (Broglie et al. 1991;Chet et al. 1991; Jones et al. 1986; Linthorst et al. 1990). Recently, Broglie et al. (1991)reported enhanced resistance in tobacco to the soilborne fungus Rhizoctonia solaniafter transformation with a bean chitinase gene under constitutive control of the 35Spromoter from cauliflower mosaic virus. When transgenic seedlings were planted in soilinfested with this pathogen, seedling survival was increased and disease developmentwas delayed relative to nontransformed plants. Since transformation for chitinaseproduction is a very new strategy, it will probably be some time before its usefulnessagainst diseases other than those caused by necrotrophic fungi is known. It is hoped thatvascular wilt diseases, such as that caused by F. oxysporum f.sp. cubense, will also beamenable to these schemes.

Manshardt (1992) discussed two additional classes of compounds that target chitin.Lectins that agglutinate and precipitate chitin are known and one from Utrica dioica,UDA, has been shown to have activity against several plant-pathogenic fungi. Partialknowledge of its amino acid composition and its small size (8500 MW) make the eventualidentification and isolation of a gene that encodes the production of UDA hopeful. Theother class of chitin-specific compounds, nikkomycins, are nucleoside peptide antibioticsproduced by the actinomycete Streptomyces tendae. Nikkomycins are inhibitors of chitinsynthases, the enzymes responsible for chitin production, and they are capable ofinhibiting fungal growth. Although their production is encoded by more than one gene(probably several), work on the isolation of these genes and their use to engineer plantscapable of producing nikkomycins is under way.

Receiving less attention than the chitin-specific compounds are low molecular weightlytic peptides that disrupt cell membrane integrity (discussed in Manshardt 1992). Theyhave been isolated from several different animals, and genes for their production havebeen isolated. Tomato and potato (Solanum tuberosum) have been transformed withmodified genes which produce one class of these compounds, cecropins, and enhancedresistance to Pseudomonas solanacearum, a bacterial pathogen of both hosts, wasobserved. Currently, little is known of the potential for controlling fungal-inciteddiseases with such strategies.

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Utsumi et al. (1988) recently cloned a gene for detoxifying fusaric acid from thefungus Cladosporium werneckii in Escherichia coli. When they treated either tomatocallus or cuttings with the transformed bacterium prior to treatment with fusaric acid,callus viability was dramatically increased and wilting in cuttings was reduced. Aspreviously mentioned, fusaric acid has been used to screen various plants for resistanceto fusarium wilt. Although it has not been used successfully to identify fusarium wilt-resistant banana somaclones (Epp 1987; Krikorian 1990), transformation with this, orother fusaric acid-detoxifying genes, should be attempted if gene-transfer protocols forbanana become available in the future.

Although conceptually intriguing, transformation of banana for fusarium wiltresistance will require much more work before it becomes possible. Certainly, muchcould be learned from successes with other crops, and it is hoped that some of theseapplications will be used in the future to control this important disease.

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PLOETZ RC, HERBERT J, SEBASIGARI K, HERNANDEZ JH, PEGG KG, VENTURA JA, MAYATO LS. 1990. Importance offusarium wilt in different banana-growing regions. Pages 9-26 in Fusarium Wilt of Banana (Ploetz RC,ed.). St Paul, MN, USA: APS Press/Amer. Phytopathol. Soc.

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115RC Ploetz

116 Potential of Gene Transfer for Engineering Resistance against Nematode Attack

Potential of Gene Transfer forEngineering Resistance againstNematode Attack

D De Waele

IntroductionGenetically engineered plants expressing completely new traits are no longer a dream ofthe future. Recent advances in recombinant DNA and tissue-culture technology have madeit possible successfully to transfer genes from any organism, be it a microorganism, plant,or animal, to higher plants. The performances of these “new” so-called transgenic plantsare already being evaluated. The first results, covering a variety of applications, arepromising.

Gene transfer has also a great potential for engineering resistance against nematodeattack in agricultural crops. Nematologists, however, have been surprisingly slow inrecognizing the potential of this new technology. As a result of this lack of interest, thedevelopment of a genetically engineered nematode-resistant plant is far behind theengineering in plants of such traits as virus and insect resistance. Fortunately, thissituation is changing. During the last 2-3 years, several research groups in Europe and theUSA, in the public as well as in the private sector, have initiated albeit modest R&Dprograms aimed at engineering nematode resistance in plants.

The present paper consists of three major parts. First, the present status of engineeringnew traits in plants is summarized. This information should demonstrate to the reader thatthis new technology is working. Secondly, it is shown that plant genetic engineering offersan interesting alternative for managing nematodes on bananas (= bananas and plantainthroughout) in view of the existing limitations of the traditional management methodsused. It is also shown that this technology is complementary to conventional plantbreeding. Thirdly, the major strategies that are currently being developed to engineernematode resistance in plants are discussed. This discussion permits the identification ofthe different steps in the development of a program aimed at the genetic engineering ofnematode resistance in bananas. It should then become clear that a lack of vital, mostlybasic knowledge is constraining the development of such a program.

It is hoped that the presented information will offer the reader a good idea of the typeof research that can and should be initiated in order to obtain this knowledge.

Plant Genetic Systems NV, Jozef Plateaustraat 22, 9000 Gent, Belgium

Status of Engineering New Traits in PlantsSince 1983, when the first successful transformation of a plant cell with a foreign gene andthe regeneration of a fertile plant from this cell was reported, about 30 different plantspecies have been transformed (Fraley 1992). The list of transgenic plants includes suchimportant horticultural and field crops as cucumber, eggplant, lettuce, melon, pea,strawberry, tomato, alfalfa, cotton, maize, oilseed rape, potato, rice, soybean, sugar beet,sunflower, and tobacco. For most of these plants, Agrobacterium tumefaciens, a plantpathogen causing tumerous crown galls upon infection, was used as the vector to introducethe genes in the plant cells. For plants resistant to Agrobacterium-mediatedtransformation, including most monocotyledonous plants, alternative transformationtechniques, such as electroporation, polyethylene-mediated transfer, microinjection, andparticle bombardment, were developed (Uchimiya et al. 1989).

Today, transgenic plants resistant to viruses (Beachy et al. 1990), bacteria (Anzai et al.1989), fungi (Broglie et al. 1991), insects (Brunke, Meeusen 1991), and herbicides(Botterman, Leemans 1988) exist. Also available are transgenic plants that produce higheramounts of nutritious proteins (Altenbach, Simpson 1990) or new biologically activepeptides used in the pharmaceutical industry (Krebbers, Vandekerckhove 1990). Hybridseeds can now be obtained through genetic engineering of nuclear male sterility (Marianiet al. 1990) while the ripening of fruit can be controlled (Oeller et al. 1991) and the color offlowers can be changed (Meyer et al. 1987) through the genetic engineering of antisensegenes. The agronomic performances of many of these transgenic plants and the expressionof some of the new traits are being evaluated in field trials throughout the world. Thecumulative total of field trials with genetically engineered organisms (also including othertransgenic organisms) was estimated at about 500 by the end of 1991.

Commercialization of the first transgenic plants is expected in the second half of the1990s. However, in addition to a number of remaining technical limitations, regulatoryapproval and public perception will also be important factors determining the future use oftransgenic plants.

Genetic Engineering and Conventional PlantBreedingIn contrast with what is sometimes postulated, plant genetic engineering will not replaceconventional plant breeding. On the contrary, it provides the breeder with a novel andpowerful tool to overcome some limitations inherent in conventional plant breeding.

In traditional plant breeding, genes are transferred among sexually compatible indivi-duals from the same or closely related species. The source of resistance is limited; thegenetic basis is rather narrow. Through genetic engineering it is possible to bring genesinto a plant from any organism. The resistance sources are unlimited; the genetic basis iswide. In traditional plant breeding, traits from wild plant varieties are transferred into acommercial plant variety without knowing the genetic or molecular basis responsible forthis trait. In genetic engineering, characterized genes, coding for well characterized gene

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products, are transferred. Since the entire genomes of both parents are combined,conventional plant breeding is time-consuming. Since in genetic engineering the gene istransferred in “one step”, while other characters of the recipient plant are in principle notaffected, the amount of backcrossing needed is considerably reduced.

A good illustration of the unlimited and well characterized transfer of genes inherent ingenetic engineering is the successful development of tobacco plants resistant to the diseasewildfire (Anzai et al. 1989). Wildfire is caused by a toxin produced by Pseudomonassyringae pathovar tabaci upon infection. The resistance was obtained by engineering in thetobacco plants a detoxifying enzyme, derived from the pathogen itself.

Through genetic engineering it will be possible to add new traits to superior plantvarieties obtained from a conventional plant breeding program.

Nematode Pests of Bananas In most regions of the world, nematodes are recognized as important pests of bananas(Gowen, Quénéhervé 1990). Crop losses caused by nematodes to bananas are very high.Average annual yield losses due to damage by nematodes on bananas are estimated at 19.7%worldwide, representing an estimated annual monetary loss of about US$178 million basedon 1984 commodity prices (Sasser, Freckman 1987). In Africa, estimated crop losses varyfrom 20 to 80% (Sarah 1989). In Central America, reductions of 50% in fruit yields and anincrease of 60% in lodging have been observed (Roman 1986).

In general, three types of plant-parasitic nematodes can be distinguished. Theectoparasitic nematodes live completely outside the plant and pierce the outermost plantcell layers with their stylet in order to feed. The migratory endoparasitic nematodes livecompletely inside the plant tissues but are able to move freely between the root and thesoil. They feed on normal plant cells inside the plant. Eggs are laid either outside or insidethe plant. Migratory endoparasitic nematodes have no complex interaction with their host.The sedentary endoparasitic nematodes also live inside the plant but the adult femalebecomes sedentary (immobile). Eggs are laid outside the plant. Sedentary endoparasiteshave a complex interaction with their host. They induce normal plant cells to formspecialized feeding structures. These structures serve as food transfer cells and are vital forthe survival of the nematodes.

The most damaging and widespread nematodes attacking bananas are migratoryendoparasites: the burrowing nematode Radopholus similis (Cobb) Thorne, the root-lesionnematodes Pratylenchus coffeae (Zimmerman) Filipjev & Schuurmans Stekhoven and P.goodeyi Sher & Allen, and the spiral nematode Helicotylenchus multicinctus (Cobb)Golden (Gowen, Quénéhervé 1990).

The migratory behavior of the migratory endoparasites in the cortex of bananas, feedingupon and destroying the parenchymous cells, causes the formation of cavities and,consequently, the disruption of the root system. Since, in bananas, the root system is notonly vital for absorption and transportation of water and solutes but also for providing afirm anchorage in the soil, the presence of these nematodes often results in toppling of thebananas. The symptoms caused by these nematodes are very similar. The roots show dark,


brown or red, lesions. The above-ground symptoms are characterized by dwarfing, fewerand smaller leaves, leaf chlorosis, premature defoliation, small bunches with reducedweight, and lodging of plants.

The sedentary endoparasitic root-knot nematodes Meloidogyne incognita (Kofoid &White) Chitwood, M. javanica (Treub) Chitwood, M. arenaria (Neal) Chitwood, and M.hapla Chitwood also occur in banana corms and roots (Gowen, Quénéhervé 1990).Juveniles (J2) emerge from the egg, invade a root and induce specific feeding structures,the so-called giant cells, which progress within the vascular cylinder, and also induce thecortical cells to multiply and form galls. The J2 swell and molt three times to form thefemales which enlarge rapidly and become immobile and globose. The adult femaleproduces several hundred eggs which are laid via a canal outside the host tissue. Root-knotnematodes cause root injury while restriction of the total water flow through the infectedroots by the giant cells results in stress and a reduction in the uptake of water andnutrients. Infected plants show stunted root growth. The most obvious symptoms are gallingon primary and secondary roots.

In addition to the damage directly caused by nematodes, nematode-induced root lesionscreate a food base for weak, unspecialized fungal parasites of banana (Fusarium spp.,Cylindrocarpon spp., Rhizoctonia spp.), enabling the fungi to invade the stele and toincrease the amount of root necrosis. Aggravation of fusarium wilt or Panama disease, oneof the most devastating diseases affecting bananas, caused by Fusarium oxysporum f.sp.cubensis in the presence of nematodes, has been reported (Newhall 1958; Loos 1959).

Limitations of Traditional Nematode Managementin BananasWhere nematode parasitism of bananas cannot be prevented by using clean plantingmaterial in nematode-free soil and growing the plants under strict quarantine conditions(which is a difficult task since nematodes are present in every agricultural field anddisperse easily), nematode management on bananas is mainly based on crop rotation andchemical control (Gowen, Quénéhervé 1990). However, the successful application of bothmethods is severely restricted. In those areas where bananas are grown continuously, croprotation is useless while chemical control is usually also not applied when traditional mixedcropping systems are being used. Not only is the high price of nematicides often prohibitivefor the small farmer, but most nematicides are also extremely toxic for nontargetorganisms, including the user, and may pollute the environment. In the USA and Europe thedetection of DBCP, EDB, D-D, carbofuran, and aldicarb in groundwater has resulted in thesuspension or restriction of the use of these nematicides (Thomason 1987). This example isbeing followed by more and more governments in developing countries. Recently, even afirm that produces a nematicide (Rhône-Poulenc) has initiated a worldwide suspension onthe sale and use of one of its products (aldicarb) on bananas, following the detection ofhigher-than-expected residue levels in one of five trials being conducted in Central andSouth America (Agrow, 14 June 1991).

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No banana variety specifically resistant to nematodes has been released so far. Althoughsources of resistance, be it only against Radopholus similis, are known, the development ofa nematode-resistant banana variety is extremely difficult because of the geneticcomplexity of this crop, its low fertility, and the long period required for the nematologicalevaluation of crossings (Pinochet 1988).

In view of the limitations of the traditional management methods used, geneticengineering offers an attractive alternative for managing nematodes on bananas.

Strategies for Engineering Nematode Resistancein PlantsSince the existent recombinant DNA technology is not suited for the engineering ofcomplex pathways in plants, strategies for engineering nematode resistance in plants arebeing focused on proteins which can be engineered by transferring a single gene, codingdirectly for the protein.

The strategies for engineering nematode resistance in plants that are currently beingdeveloped can be classified as: (1) strategies aimed at the direct killing of the nematode;(2) strategies aimed at using or reinforcing existing plant defense mechanisms; and (3)strategies aimed at interfering in the compatible nematode-plant interaction. Most of thesestrategies are being developed. No transgenic plants resistant to nematodes have beenobtained so far.

In the first strategy, external or internal nematode structures are targeted with eitherlytic enzymes or oral toxic proteins.

Examples of lytic enzymes are the chitinases and collagenases. Since chitin is animportant component of the nematode egg shell (Bird 1976) and collagen of the cuticle ofjuvenile and adult nematodes (Reddigari et al. 1986), it is hoped that the expression ofthese lytic enzymes in plants will lyse the external nematode structures and kill thenematodes (Havstad et al. 1991). The expression of chitinases in plants will work onlyagainst the eggs of migratory endoparasitic nematodes and not against the eggs ofectoparasitic nematodes, which do not enter the roots, or the eggs of the sedentaryendoparasitic nematodes which are deposited outside the roots. The expression ofcollagenases will of course not work against ectoparasitic nematodes. The development ofthis strategy is seriously hampered by the incomplete knowledge of the precise nature ofboth the egg shell and the cuticle of plant-parasitic nematodes. This lack of basicknowledge contrasts sharply with the data currently available on the same structures inanimal-parasitic nematodes.

A good example of the search for oral toxic proteins is the screening for toxins producedby Bacillus thuringiensis. This gram-positive soil bacterium is better known worldwide forits insecticidal crystal proteins, which have been used successfully as bio-insecticides andto engineer insect resistance in plants (Lambert, Peferoen 1992). Bacillus thuringiensisisolates with (ovicidal and larvicidal) activity against nematodes have been found, but thisactivity was almost exclusively effective against free-living and animal-parasitic nematodes


(Bottjer et al. 1985; Meadows et al. 1990). Only some nematocidal activity againstPratylenchus scribneri Steiner, a migratory endoparasite, has been reported so far(Bradfisch et al. 1991). The molecular basis of the observed ovicidal and larvicidal activityis unknown. Screening of B. thuringiensis isolates for oral toxic proteins active againstplant-parasitic nematodes is seriously hampered by the obligate biotrophic way of feedingof most plant-parasitic nematodes. This implies that plant-parasitic nematodes feed onlyupon living plant cells.

The cloning of genes conferring resistance to nematodes forms the subject of thesecond strategy. Since the recombinant DNA technology currently available permits onlythe transfer of traits encoded by single genes, only single, dominant resistance genes arebeing cloned. Examples of such genes are the Mi gene from Lycopersicon peruvianumwhich is effective against all Meloidogyne species attacking tomato except M. hapla(Williamson et al. 1991). The molecular basis of the resistance is unknown. Several routesto clone these resistance genes can be followed (North 1990). Mapping of the genes relativeto polymorphic markers followed by chromosome walking is favored by most researchers.Cloning of resistance genes is a time-consuming activity. Because only resistance genesconferring resistance to sedentary endoparasitic nematodes have been identified so far, thisstrategy is not applicable to ectoparasitic and migratory endoparasitic nematodes species.

The third strategy aims at interfering in the nematode-plant interaction. Although thisstrategy includes such activities as the search of repellants and of proteins blocking thenematode-plant recognition, most research efforts are aimed at the inhibition ordestruction of the feeding structures induced by the sedentary endoparasitic nematodes.This limits eventual application to these nematodes.

The expression in plants of antibodies, the so-called plantibodies, directed againstproteins secreted by the nematodes, and which are essential for the induction and/ormaintenance of the feeding structures, is one example of a route followed to disrupt thefeeding structures (Schots et al. 1991). This method is based on the assumption thatexpression of these antibodies will inhibit the biological activity of the proteins. Anotherroute followed is based on the idea that the sedentary endoparasitic nematodes specificallyinduce genes that are switched on only in the feeding structures. Induction of genes innematode-infected plant roots has been reported (Gurr et al. 1991). The regulatorysequences of such genes could be used to direct cytotoxic proteins to the feedingstructures. The development of both methods is hampered by a lack of basic knowledge onthe precise function of the secreted proteins on the one hand and the specificity of theinduced genes on the other.

Steps in the Development of a Program aimed atthe Genetic Engineering of Nematode Resistancein BananasThe first step in the development of a program aimed at the genetic engineering ofresistance against nematode attack in any crop, and thus also in bananas, is the identifi-

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cation of the most important nematode species. The nematode life cycle, feeding behavior,and interaction with other pathogens and the host plant will all have a decisive influence onthe choice of the strategy to be followed. Also, the ease with which these nematodes can becultured in order to obtain living specimens for bioassays will be of importance.

There is sufficient information that at least three distinct pathotypes of R. similis occurin Central America and the Caribbean (Pinochet 1979; Tarté et al. 1981). Information onthe existence of other pathotypes outside this region or of pathotypes of the other migratoryendoparasitic nematodes is completely lacking but necessary for the correct identificationof the target nematodes.

Bananas, as well as most other subtropical and tropical crops, are often parasitizedsimultaneously by several nematode species (Gowen, Quénéhervé 1990). This implies thatthe genetic engineering of resistance to only one of these species may result in theincreased occurrence of one or more of the other nematode species against which theresistant genes offer no protection. The strategy should thus be aimed at engineering globalresistance.

The second step in the development of a program aimed at the genetic engineering ofnematode resistance in plants is the identification and purification of the protein on whichthe chosen strategy will be based and the cloning of the gene that encodes this protein.

There are many biological substances that show activity against nematodes, but most ofthese products are secondary metabolites. Secondary metabolites are usually the productsof complex biochemical pathways involving many different proteins. Engineering nematoderesistance in a plant with a secondary metabolite would involve the cloning of a completemetabolic pathway. For this task, the existent recombinant DNA technology is not suited.Therefore, strategies for engineering nematode resistance in plants must focus on proteinsthat can be engineered by transferring a single gene, coding directly for the protein. Veryfew of these proteins have been identified so far.

In their quest for biological control of nematodes, nematologists have identified arelatively large number of bacteria and, especially, fungi showing activity against plant-parasitic nematode species (Kerry 1990). Unfortunately, in most cases, the molecular basisfor this activity has not been investigated.

Once a protein has been identified, the encoding gene has to be cloned. A clone consistsof multiple copies of identical DNA sequences that are produced when they are inserted andreplicated in cloning vehicles using recombinant DNA technology. The gene must then betransferred to cells of bananas, using one of the transformation techniques, and thetransformed cells regenerated. Finally, the transgenic bananas must be thoroughlyevaluated on resistance to the target nematodes and on agronomic performance.

Constraints for the Engineering of NematodeResistance in BananasMajor constraints obstructing the development of virtually every program aimed at theengineering of nematode resistance in bananas are: (1) insufficient knowledge on the


occurrence of pathotypes or races of the migratory, especially R. similis, and sedentarynematodes parasitizing bananas; (2) the obligate biotrophic way of feeding of all banananematodes; (3) the primitive level of nematode-plant interaction of most banananematodes, except the Meloidogyne species; (4) the rather high difficulty of establishingmonoxenic cultures of banana nematodes; and (5) the occurrence of multispeciesinfections on the same banana.

In addition to these constraints, there is a general lack of basic knowledge of manyexternal and internal structures and processes of plant-parasitic nematodes and virtuallyno information on the molecular and genetic basis of most nematode-plant interactions.

ConclusionAll indications are that recombinant DNA technology, as a complementary tool toconventional plant breeding, will have a major and beneficial impact on the agriculture oftomorrow. Although genetic engineering offers certainly no cure-all for obtainingnematode-resistant crops, opportunities to use this technology also for obtaining nematode-resistant bananas, be it against one or more nematode species simultaneously, should notbe neglected.

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125N von Mende, P Burrows, J Bridge

Molecular Aspects of Resistance toNematodes

N von Mende, P Burrows, J Bridge

Plant parasitic nematodes are important pests in tropical and subtropical agriculture.These microscopic plant parasites are frequently divided into three major groupsaccording to their migratory and feeding behavior. The first group, the migratory,ectoparasitic nematodes, remain outside the root and insert their stylet into epidermal ormeristematic cells to feed. In general they cause little damage to plant tissue unlike thesecond group, the migratory, endoparasitic nematodes. Endoparasitic nematodes not onlykill the cells they feed upon but, by burrowing through the root tissues, they causeextensive destruction leading to cavitation and secondary infection (Dropkin 1980). Thethird group includes the sedentary, endoparasitic nematodes which have evolved highlysophisticated relationships with their hosts. These nematodes enter roots as vermiformjuveniles and induce certain root cells to transform into nurse or transfer cells. Thefunction of the nurse cells is to act as a metabolic sink that provides the sedentarynematodes with a continuous supply of nutrients. This feeding strategy allows the femaleto swell and produce a large number of eggs (Jones et al. 1981).

The nematodes causing the most damage to bananas and plantains are migratoryendoparasites, and include Radopholus similis, Pratylenchus goodeyi, P. coffeae, andHelicotylenchus multicinctus. These cause destruction of roots, initially stunting theplant but ultimately causing disruption of the anchorage and toppling of the plant. It isalso common to find such sedentary endoparasites as Meloidogyne spp. andRotylenchulus reniformis parasitizing the root system. In addition to members of thesefive genera, over 146 other nematode species are associated with Musa spp. throughoutthe world (Gowen, Quénéhervé 1990).

A variety of measures is used to control the damage by these parasitic nematodes inbanana plantations. To prevent the introduction of these pests into new plantations,carefully selected pared suckers (free of nematode damage) are heat-treated or dosedwith nematicides before planting. Currently, a more favored approach is to grownematode-free plants from meristematic tissue. Nematicides are used widely by growersproducing fruit for the international export market. Less specialized production servinglocal markets may not justify the high cost of chemical treatment. There is considerableinterest in nonchemical, environmentally friendly, alternative methods of nematodecontrol. Attention has focused on producing nematode-resistant plants through

Research Group on Nematodes in Tropical and Subtropical Agriculture, IACR, Rothamsted Experimental Station,Harpenden, Herts, AL5 2LQ, and CAB International Institute of Parasitology, St Albans, UK

conventional breeding and modern biotechnological means, and on the use of naturallyoccurring biological control agents (De Leij, Kerry 1991).

Resistance to pests and diseases in crops can be obtained either using availableexperimental lines/germplasm accessions in conventional breeding programs or throughmolecular genetic and plant transformation technologies. Molecular techniques canovercome some limiting factors inherent in plant breeding, e.g., selected genes can beinserted into a new host thus bypassing infertility and other breeding barriers. Thisapproach has been used successfully for the production of transgenic plants resistant toviruses (Grumet 1990) and insects (Brunke, Meeusen 1991). Programs have also beeninitiated recently to produce nematode-resistant transgenic plants. The most importantquestion, however, is which gene to select for insertion into the plant to induceresistance. Two strategies have been considered. Firstly, to exploit the plant’s naturaldefense mechanisms, and secondly to insert genes which, directly or indirectly, disruptnematode development. Both of these strategies are discussed below.

Some progress has been made in the selection of nematode-resistant banana clonesbut less headway has been made in identifying genes for resistance. Some clones appearto be resistant to some species of nematode and not others. For example, Pinochet andRowe (1978, 1979) found two clones of the diploid Pisang Jari Buaya cultivar wereresistant to R. similis but not to P. coffeae. In contrast with the lack of informationconcerning resistance to migratory nematodes, several genes resistant to sedentaryendoparasitic nematodes have been identified through conventional plant breedingprograms (Cook 1991). There is growing evidence, through extensive studies of plant-nematode interactions, that in many cases a gene-for-gene system exists in which avirulence gene in the nematode has been identified which is matched by a resistancegene in the plant (e.g., Jones et al. 1981). One resistance mechanism is thehypersensitive response involving rapid cell necrosis which stops the nematode fromfeeding and becoming established on the plant. A few resistance genes have beenidentified, such as the Mi gene which confers resistance to Meloidogyne incognita andthe H1 gene conferring resistance to the potato cyst nematode Globodera rostochiensis.Current efforts are under way to isolate and characterize these genes (Barone et al. 1990;Klein-Lankhorst et al. 1991).

In addition to the hypersensitive response, plants have many other defensemechanisms such as enzyme inhibitors, lytic enzymes, and lignification (Lamb et al. 1989;Bowles 1990), but their role in resistance to nematodes has not been elucidated.

The aim of the second strategy for producing nematode-resistant plants is to disruptnematode development. One approach involves the use of antisense constructs whichinhibit the translation of specific transcripts essential for host-plant recognition andparasitism. The success of this approach is dependent upon a thorough understanding ofhost-parasite interactions at the cell and molecular level. Research at RothamstedExperimental Station is currently under way to identify signal molecules and generegulation involved in host-recognition and induction of nurse cells. For this purposeendoparasitic nematodes and Arabidopsis thaliana are being used as a model system(Sijmons et al. 1991). A. thaliana is ideally suited for molecular studies because of its

126 Molecular Aspects of Resistance to Nematodes

small genome size, short life cycle, and well developed classical genetics. Furthermore, awide range of well characterized ecotypes and mutants are available.

A further approach is the insertion of functional genes that interfere with the feedingor development of nematodes. So far this strategy has only been developed for insectpests using genes encoding, for example, for the cowpea trypsin inhibitor or toxinsproduced by Bacillus thuringiensis (Brunke, Meeusen 1991). Also envisaged is theinsertion of genes for collagenases or chitinases, which would digest collagen (a majorcomponent of nematode cuticle), or chitin (a component of the egg shell), respectively.Some workers are considering incorporating genes encoding for mammalian antibodieswith affinity for important receptors or essential structural proteins that are involved insuccessful parasitism, e.g., salivary proteins or feeding tubes (Hussey 1989).

In conclusion, several diverse strategies involving molecular techniques are availableto develop nematode-resistant plants. It is highly likely that some of these strategiescould be extended to Musa species. Before this can happen in a rational and informedway leading to durable resistance much more needs to be known about communicationbetween nematodes and their respective hosts at the cell and molecular level.

ReferencesBARONE A, RITTER E, SCHACHTSCHABEL U, DEBENER T, SALAMINI F, GEBHARDT C. 1990. Localization by restriction

fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to thepotato cyst nematode Globodera rostochiensis. Mol. Gen. Genet. 224:177-182.

BOWLES DJ. 1990. Defense-related proteins in higher plants.Annu. Rev. Biochem. 59:873-907.BRUNKE KJ, MEEUSEN RL. 1991. Insect control with genetically engineered crops. TIBTECH 9:197-200.COOK R. 1991. Resistance in plants to cyst and root-knot nematodes. Pages 213-240 in Agricultural Zoology

Reviews vol.4. DROPKIN VH. 1980. Introduction to Plant Nematology. New York, USA: John Wiley.DE LEIJ FAAM, KERRY BR. 1991. The nematophagous fungus Verticillium chlamydosporium as a potential

biological control agent for Meloidogyne arenaria. Revue Nematol. 14:157-164.GOWEN S, QUÉNÉHERVÉ P. 1990. Nematode parasites of bananas, plantains and abaca. Pages 431-460 in Plant

Parasitic Nematodes in Subtropical and Tropical Agriculture (Luc M, Sikora A, Bridge J, eds). Wallingford,Oxon, UK: CAB International.

GRUMET R. 1990. Genetically engineered plant virus resistance. HortScience 25:508-513.HUSSEY RS. 1989. Disease-inducing secretions of plant-parasitic nematodes. Annu. Rev. Phytopathol. 27:123-141.JONES FGW, PARROTT DM, PERRY JN. 1981. The gene-for-gene relationship and its significance for potato cyst

nematodes and their Solanaceous hosts. In Plant Parasitic Nematodes vol.III (Zuckerman BM, Rohde RA,eds). London, UK: Academic Press.

KLEIN-LANKHORST R, RIETVELD P, MACHIELS B, VERKERK R, WEIDE R, GEBHARDT C, KOORNNEEF M, ZABEL P. 1991. RFLPmarkers linked to the root knot nematode resistance gene Mi in tomato. Theor. Appl. Genet. 81:661-667.

LAMB CJ, LAWTON MA, DRON M, DIXON RA. 1989. Signals and transduction mechanisms for activation of plantdefenses against microbial attack. Cell 56:215-224.

PINOCHET J, ROWE P. 1978. Reaction of two banana cultivars to three different nematodes. Plant DiseaseReporter 62:727-729.

PINOCHET J, ROWE P. 1979. Progress in breeding for resistance to Radopholus similis from Central America onValery Bananas. Nematropica 9:40-43.

SIJMONS PC, GRUNDLER FMW, VON MENDE N, BURROWS PR, WYSS U. 1991. Arabidopsis thaliana as a new modelhost for plant-parasitic nematodes. The Plant Journal 1:245-254.

127N von Mende, P Burrows, J Bridge

128 Breeding Cooking Bananas for Areas with Marginal Growing Conditions

Breeding Cooking Bananas forareas with Marginal GrowingConditions by using Cardaba (ABB)in Cross-Pollinations

PR Rowe, FE Rosales

IntroductionThe ABB cooking bananas (Bluggoe, Saba, Cardaba, and Pelipita) have a high level ofresistance to black Sigatoka (Mycosphaerella fijiensis), are resistant to drought, and aretolerant of soil nutritional deficiencies. Of these cultivars, Saba is produced in thegreatest amount for a particular area. It is the most important cooking banana in thePhilippines where its estimated annual production is over 1.5 million t (Valmayor 1987).Saba and Cardaba were earlier thought to have a BBB genomic composition, but resultsfrom an isozyme analysis (Jarret, Litz 1986) indicate that both should be classified asABB. All these robust ABB cultivars can be grown in areas with soil and climaticconditions unfavorable for cultivation of the AAB plantains and AAA dessert bananas.

Plantains predominate as the preferred starchy banana in Latin America and theCaribbean, and most of the nearly 5 million t of these AAB cooking types producedannually in this region are consumed domestically (Jaramillo 1987). Plantains are evenmore important in western and central Africa where they account for more than 25% ofthe carbohydrates in the diets of some 60 million people. Over 60% of the world’splantains are produced and consumed in this area of Africa (INIBAP 1987).

The other major nondessert types are the highland AAA cultivars of eastern Africawhich are divided into cooking bananas and beer bananas. For about 20 million peoplein Burundi, Rwanda, Uganda, northern Tanzania, and eastern Zaire, these cookingbananas (boiled green) are the primary staple food. The annual production of about 9million t of these two types of bananas (all for domestic consumption) in this areaconstitutes close to one-half of the African production of bananas and plantains (INIBAP1986).

Before the advent of black Sigatoka in Latin America and Africa, no serious attentionwas given to breeding new disease-resistant hybrids which could substitute for the AABand AAA cooking cultivars. Stover and Dickson (1976) described the 1973 black Sigatoka

Fundación Hondureña de Investigación Agrícola (FHIA), Apto Postal 2067, San Pedro Sula, Honduras

epidemic in Honduras. This disease is now present in most of the plantain-producingcountries of Latin America (Fullerton, Stover 1990). It is already widespread in westernand central Africa (Wilson, Buddenhagen 1986) and was identified in eastern Africa in1987 (Sebasigari, Stover 1988). All the different plantain clones that have been testedare susceptible to black Sigatoka (Swennen, Vulysteke 1990). The AAA highland cookingand beer bananas of eastern Africa are also susceptible to this leaf spot disease(Sebasigari 1990).

With the outbreak of the black Sigatoka epidemic in Honduras, and the drasticeffects of this new disease for Latin America on plantain yield and quality, investigationsof possible approaches to genetic improvement of plantains were begun in the Honduranbanana breeding program. Now, breeding for black Sigatoka-resistant hybrids whichcould replace the AAA clones of eastern Africa has also become critically important.

The four ABB cooking banana cultivars mentioned earlier are seed-fertile whencrossed with diploids, and all were used in 3n x 2n cross-pollinations made in the initialattempts to breed black Sigatoka-resistant hybrids which could substitute for plantains.It was learned later that the French Plantain and Maqueño AAB clones could be used asfemale parental lines in breeding plantains (Rowe, Rosales 1990), but now it appearsthat crosses made onto one of the ABB clones (Cardaba) have provided someexceptional hybrids in breeding for new cooking bananas. The development andapparent value of three of these hybrids (one triploid and two tetraploids) with Cardabaparentage are discussed in this paper.

Development of a Dwarf Triploid with CardabaAncestryGenetic behavior of the ABB clones during meiosis is variable. Cheesman and Dodds(1942) found that when Bluggoe was crossed with diploids, the resultant hybridsincluded diploids, triploids, and tetraploids as well as apparent aneuploids with littlevigor and abnormal leaf characters. Rowe (1976) reported preliminary attempts to usedifferent ABB clones in breeding. Bred diploids of M. acuminata (AA) and accessions ofM. balbisiana (BB) were crossed onto Chato (synonymous with Bluggoe), Pelipita andCardaba (listed as Saba in the article, but later correctly identified as Cardaba). Theweak and abnormal hybrids were discarded as seedlings, while normal progenies weretransplanted to the field for subsequent verification of ploidy in individual plantsselected for further observation.

The hybrids with the best plant and bunch features from this series of crosses weretetraploids from Cardaba x M. balbisiana. Subsequently, a triploid hybrid derived fromcrossing a M. acuminata diploid onto one of these ABBB selected tetraploids had amuch larger bunch (43 kg) than either Cardaba or its tetraploid parental line (Rowe1983). This triploid was tall and was later discarded because of its slow growth, but itprovided indications that hybrids with Cardaba in their pedigree are potentially useful inbreeding for genetic improvement of cooking bananas.

129PR Rowe, FE Rosales

Since Cardaba is a tallplant (Fig.1), one of theprimary objectives was tointroduce the dwarfing geneof bred dwarf diploids intohybrids with Cardaba parent-age. Rowe (1987) reporteddevelopment of the SH-3386dwarf triploid by crossing adwarf diploid (AA) onto atetraploid derived fromCardaba x M. balbisiana. SH-3386 very nearly approachescommercial acceptability, butthin fruit at maturity hasprecluded its evaluation as aprospective cooking banana.

While SH-3386 has nopollen, it does produce a fewseeds when pollinated. Mosthybrid seedlings obtainedfrom crossing diploids ontoSH-3386 are useless thick-leaved dwarfs characteristicof heptaploids, but a few grownormally. One of the tetra-ploid hybrids that has beenselected from among pro-genies of SH-3386 has manydesirable qualities, and meritsevaluation as a possible new commercial type with characters superior to those of thetraditional cooking bananas in certain areas.

A Tetraploid Cooking Banana Hybrid withCommercial PotentialThis tetraploid with apparent potential as a new cooking banana is SH-3565 which wasderived from SH-3386 x SH-3320, and was briefly described in an earlier report (Rowe1990). SH-3320 is a bred diploid (AA) which has a high level of resistance to blackSigatoka. A code name of FHIA-03 was assigned to SH-3565 when this hybrid was chosenas one of the entries for evaluation in the International Musa Testing Program (IMTP)which is supervised by INIBAP.


Figure 1. Plant with bunch of the Cardaba (ABB) cultivarwhich has a tall plant stature.

One traditional cultivar for which FHIA-03 could possibly be a superior replacementis Bluggoe. It is widely grown and is a veryimportant source of food in parts of easternAfrica (e.g., the southwestern slopes of Kilimanjaro and the eastern side ofZanzibar) and the West Indies (Simmonds1966). This ABB clone is susceptible to bacterial wilt or moko disease (Pseu-domonas solanacearum) and race 2 ofPanama disease (Fusarium oxysporumf.sp. cubense) (Stover 1972).

The reactions of FHIA-03 to moko andrace 2 of Panama disease are not yet known.However, one of the characters of FHIA-03 isthat the male flowers are persistent, whichshould reduce contamination by the insect-transmitted strain of the moko diseasebacteria. Further tests for resistance toPanama disease are pending, but FHIA-03has been observed to remain healthy in anarea where surrounding plants were infectedwith race 1 of this pathogen.

At the time cross-pollinations were begun with Cardaba to evaluate the usefulness ofthis clone in breeding, black Sigatoka was not yet present in eastern Africa. Before thearrival of this disease, the most serious restriction on production of the highlandbananas was soil nutritional and mineral deficiency problems which exist in all countriesof eastern Africa (Sebasigari, Stover 1988). Now it appears that possibly the greatestvalue of FHIA-03 could be as a vigorous black Sigatoka-resistant substitute for thesusceptible AAA cooking and beer varieties of that region.

The susceptibility to black Sigatoka of the Nyamwihogora (AAA) cooking banana ofeastern Africa as compared with the resistance of FHIA-03 in Honduras is shown inFigure 2. Under severe disease pressure FHIA-03 has some spotting, but still has aboutfive functional leaves as compared with none for the eastern African clones when thebunches are ready to harvest.

FHIA-03 is an extremely vigorous plant and has produced well under prolongeddrought (4 months) and marginal soil (clay loam) conditions. This hybrid appears to


Figure 2. Plants showing relative reactions tosevere black Sigatoka pressure of the Nyamwihogora (AAA) cooking banana of eastern Africa (left) and FHIA-03 whenbunches are ready to harvest. All the leaves of the former are necrotic while the latter has five functional leaves.

have equal or better vigor than its Cardaba progenitor, even after the introduction of twoM. acuminata diploids in its pedigree. If FHIA-03 is otherwise adapted to the altitudesof eastern Africa, its apparent tolerance to soil nutritional and mineral deficienciesshould also be a valuable trait that would promote its cultivation as a new cookingbanana.

No precise data have been taken on flowering and ratooning speeds, but observationshave been that FHIA-03 is relatively fast in both respects. By counting the number ofpseudostem stumps for harvested bunches in individual stools from the same plot, FHIA-03 was judged to be equal to the AAA clones of eastern Africa in ratooning speed. Anadditional desirable character of this hybrid is that it has a dwarf plant stature (whichcan be compared with the Grande Naine or Valery Cavendish clones depending uponwhether it is grown under poor or optimum conditions). The pseudostems are verystrong, and no doubled plant has been observed even when not propped and withbunches weighing more than 45 kg.

Another attractive feature of FHIA-03 is its eating qualities. When green fruit is fried asthin slices, the flavor and texture are very similar to those of Bluggoe. When boiled whole,the green fruit has a flavor and texture almost identical to those of the AAA clones ofeastern Africa. While the eastern African cooking bananas are eaten only as boiled greenfruit, Bluggoe is cooked both green and ripe. FHIA-03 has a soft texture and sweet-acidflavor when ripe, as compared with a firmer texture and somewhat starchy flavor for ripe


Figure 3. From left: bunch features of the Nyamwihogora and Bluggoe cultivars of easternAfrica compared with the FHIA-03 hybrid, which merits evaluation as a possible disease-resistant replacement for these two traditional cooking bananas.

Bluggoe. It remains to be determined if FHIA-03 compares favorably with Bluggoe whencooked ripe.

Perhaps any deficiencies in ripe FHIA-03 cooking qualities would be compensated forby its pleasant flavor and texture when eaten raw as a dessert banana. It could providefresh fruit with an apple-like flavor in areas where dessert bananas have traditionally notbeen readily available, since ripe Bluggoe and AAA fruit of eastern Africa do not havepleasing fresh flavors.

Another possible use for FHIA-03 would be as a beer banana. The Pisang Awak ABBcultivar is a popular beer banana in eastern Africa, but is susceptible to Panama disease(Sebasigari, Stover 1988). Since FHIA-03 has an ABB clone in its pedigree, this geneticbackground could contribute to its suitability for this purpose.

FHIA-03 appears to have potential as a new hybrid for domestic consumption, asdiscussed above. Yields of FHIA-03 would be expected to be considerably greater thanthose of the traditional clones for which this hybrid could be an attractive alternative.Bunch features of a typical eastern African cooking banana and Bluggoe, as comparedwith those of FHIA-03, are shown in Figure 3. FHIA-03 should not be considered forexport since, like its Cardaba parental line, it tends to ripen rather quickly after harvest.This relatively short green life would present risks that ethylene released from ripeningFHIA-03 could cause premature ripening of other export bananas if shipped together.

Pollen production is sparse in FHIA-03, and only one seed has been produced frompollination of about 30 bunches of this hybrid with a diploid that produces abundantpollen. Since pollen from diploids is much more efficient in fertilization than pollen fromtetraploids, and FHIA-03 produces scant amounts of pollen, it is expected that thistetraploid would remain seedless if grown commercially.

This effective seedlessness is a valuable trait for commercial production, but itessentially eliminates FHIA-03 as a parental line in further cross-pollinations. However,another tetraploid which has been selected from the segregating progenies of SH-3386produces abundant pollen and is seed-fertile.

A Pollen- and Seed-Fertile Tetraploid Breeding LineThis pollen- and seed-fertile tetraploid is SH-3648 (Fig.4) which was derived from SH-3386 x SH-3362. SH-3362 is a bred diploid (AA) which has shown resistance to race 4 ofPanama disease in Australia (Langdon, Pegg 1988).

Triploids cannot be crossed with triploids. However, seed-fertile tetraploids can becrossed with pollen-fertile tetraploids since the even number of chromosome setspermits normal divisions and recombinations during reproduction. Thus, tetraploidsderived from crossing a diploid onto one particular triploid clone can be crossed withtetraploids produced by crossing a diploid onto a different triploid genotype. This 4n x 4nbreeding scheme has provided possibilities for genetic recombinations that heretoforewere not practical. One factor that has made 4n x 4n crosses an attractive approach tobreeding new types of bananas has been the identification of an array of seed-fertiletriploid clones. Also, the development of diploids with desirable agronomic and disease-


resistance features have nowresulted in superior genetic-ally diverse tetraploids from3n x 2n crosses (Rowe, Rosales1993).

The three main groups of cooking bananas areplantains (AAB), easternAfrican highland bananas(AAA), and the ABB clones.Rowe and Rosales (1990)reported the development oftetraploids from crosses ontoa French Plantain and theMaqueño (AAB) cookingbanana. Now, SH-3648 has theCardaba ABB clone in itspedigree and provides anadditional tetraploid formaking 4n x 4n crosses inattempts to produce hybrids

that have combinations of the best features of different cooking bananas.For development of plantain types with potential for expanding the export market,

the most promising 4n x 4n cross is between tetraploids derived from the FrenchPlantain and tetraploids with Maqueño parentage. However, since this paper is abouthybrids with Cardaba as a progenitor, discussion will be limited to crosses involving SH-3648.

All the tetraploids produced so far from crosses onto French Plantain and Maqueñoare tall. The first seeds obtained from crossing the dwarf SH-3648 onto these AAABtetraploids have been produced and are being germinated by embryo culture. These 4n x4n crosses were made with the anticipation that some of the resultant hybrids will havethe eating qualities of plantains plus the dwarfness and vigor of SH-3648. Such hybridswould be expected to be productive in areas where plantains are preferred but presentlyhave limited productivity due to infertile soils.

Another use of SH-3648 is as a pollen parent in crosses onto the triploid Bluggoe,Pelipita, and Cardaba clones. These 3n x 4n crosses are rationalized by taking into


Figure 4. Plant and bunchfeatures of the dwarf SH-3648tetraploid which has Cardabain its pedigree. SH-3648 isbeing used as both a pollenand seed parent in furthercrosses for breeding newcooking banana hybrids.

consideration that meiosis is variable in these ABB clones, and the expectation thatsome of the progenies will be triploids and tetraploids. Such crosses could result inhybrids with the dwarf plant habit of SH-3648 and the preferred cooking qualities of thetraditional tall ABB clones. The first seedlings produced from this series of crosses havealready germinated in embryo culture.

Crosses between SH-3648 and different advanced bred diploids are expected to givegenetic diverseness in triploid hybrids that would be seedless due to the uneven numberof chromosome sets. The first hybrids obtained from crossing the black Sigatoka-resistant SH-3437 diploid onto SH-3648 have been transplanted to the field. Selectedhybrids from this cross are anticipated to provide additional prospective commercialtypes that could have even higher levels of resistance to black Sigatoka than that ofFHIA-03.

ConclusionsMore than 85% of the total world production of about 60 million t of the different types ofbananas are consumed domestically. A large portion of this production consists ofplantains, cooking bananas, and beer bananas. With the exception of the Saba andCardaba cooking bananas, which are cultivated in the Philippines (Valmayor et al. 1981;Valmayor 1987), all the major clones used for cooking or making beer are susceptible todiseases that either are already causing increased production costs and reduced yieldsor are constant threats to the few remaining relatively disease-free areas.

Attempts to use Saba in breeding have not yet resulted in any useful hybrids.However, as has been discussed in this paper, some hybrids with exceptional plant andbunch features have been developed by using Cardaba in cross-pollinations. One hybridwith Cardaba parentage has qualities that indicate it merits evaluation to determinefurther its potential for commercial cultivation, and another provides a superiortetraploid breeding line in subsequent crosses for development of additional newcooking bananas.

Acknowledgment

Grateful thanks are given to the International Development Research Centre (IDRC) ofCanada which has provided the major financial support that made continued breedingfor genetic improvement of cooking bananas possible.

ReferencesCHEESMAN EE, DODDS KS. 1942. Genetical and cytological studies of Musa IV. Certain triploid clones. Journal of

Genetics 43:337-357.FULLERTON RA, STOVER RH (eds). 1990. Sigatoka Leaf Spot Diseases of Bananas: proceedings of an

international workshop held at San José, Costa Rica, March 28 - April 1, 1989. Montpellier, France:INIBAP. 374 pp.

INIBAP. 1986. Banana Research in Eastern Africa. Proposal for a Regional Research and DevelopmentNetwork. Montpellier, France: INIBAP.


INIBAP. 1987. Plantain in West and Central Africa. Proposal for a Regional Research and DevelopmentNetwork. Montpellier, France: INIBAP.

JARAMILLO CR. 1987. Banana and plantain production in Latin America and the Caribbean. Pages 39-43 inBanana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds). ACIAR Proceedings no.21.Canberra, Australia: ACIAR.

JARRET RL, LITZ RE. 1986. Determining the interspecific origins of clones within the `Saba’ cooking bananacomplex. HortScience 21: 1433-1435.

LANGDON PW, PEGG KG. 1988. Field screening for resistance to Fusarium wilt. In Research Report: ACIARBanana Improvement Project for the Pacific and Asia — Fusarium Wilt (Panama Disease) Studies.Canberra, Australia: ACIAR. 12 pp.

ROWE P. 1976. Possibilités d’amélioration génétique des rendements de plantain. Fruits 31:531-536.ROWE P. 1983. L’hybridation pour l’amélioration des plantains et autres bananas à cuire. Fruits 38:256-260.ROWE PR. 1987. Breeding plantains and cooking bananas. Pages 21-23 in International Cooperation for

Effective Plantain and Banana Research: proceedings of the third meeting of IARPB held in Abidjan,Côte d’Ivoire, 27-31 May 1985. Montpellier, France: INIBAP.

ROWE PR. 1990. New genetic combinations in breeding bananas and plantains resistant to diseases. Pages 114-123 in Identification of Genetic Diversity in the Genus Musa: proceedings of an international workshopheld at Los Baños, Philippines, 5-10 September 1988 (Jarret RL, ed.). Montpellier, France: INIBAP.

ROWE PR, ROSALES FE. 1990. Breeding bananas and plantains with resistance to black Sigatoka. Pages 243-251in Sigatoka Leaf Spot Diseases of Bananas: proceedings of an international workshop held at San José,Costa Rica, March 28 - Abril 1, 1989 (Fullerton RA, Stover RH, eds). Montpellier, France: INIBAP.

ROWE PR, ROSALES FE. 1993. Bananas and plantains. In Advances in Fruit Breeding, 2nd edn (Janick J, MooreJN, eds). Timber Press (in press).

SEBASIGARI K. 1990. Effect of black Sigatoka (Mycosphaerella fijiensis Morelet) on bananas and plantains inthe Imbo plain in Rwanda and Burundi. Pages 61-63 in Sigatoka Leaf Spot Diseases of Bananas:proceedings of an international workshop held at San José, Costa Rica, March 28 - April 1, 1989(Fullerton RA, Stover RH, eds). Montpellier, France: INIBAP.

SEBASIGARI K, STOVER RH. 1988. Banana Diseases and Pests in East Africa: report of a survey made in November1987. Montpellier, France: INIBAP.

SIMMONDS NW. 1966. Bananas, 2nd edn. London, UK: Longmans. 512 pp.STOVER RH. 1972. Banana, Plantain and Abaca Diseases. Kew, Surrey, UK: Commonwealth Mycological

Institute. 316 pp.STOVER RH, DICKSON JD. 1976. Banana leaf spot caused by Mycosphaerella musicola and M. fijiensis var.

difformis: a comparison of the first Central American epidemics. FAO Plant Protection Bulletin 24:36-42.SWENNEN R, VULYSTEKE D. 1990. Aspects of plantain breeding at IITA. Pages 252-266 in Sigatoka Leaf Spot

Diseases of Bananas: proceedings of an international workshop held at San José, Costa Rica, March 28 -April 1, 1989 (Fullerton RA, Stover RH, eds). Montpellier, France: INIBAP.

VALMAYOR RV. 1987. Banana improvement imperatives - the case for Asia. Pages 50-56 in Banana and PlantainBreeding Strategies (Persley GJ, De Langhe EA, eds). ACIAR Proceedings no.21. Canberra, Australia:ACIAR.

VALMAYOR RV, RIVERA FN, LOMULJO FM. 1981. Philippine Banana Cultivar Names and Synonyms. IPB Bulletinno.3. Los Baños: University of the Philippines. 16 pp.

WILSON GF, BUDDENHAGEN I. 1986. The black Sigatoka threat to plantain and banana in West Africa. IITAResearch Briefs 7(3):3.


Part 2

In-Vitro Strategiesfor Musa

139O Arias

Commercial Micropropagation ofBanana

O Arias

During this century, the production of bananas has been increased significantly. Theincrease was interrupted temporarily only by the two wars and the depression of the1930s. World production of export bananas over the period increased 18 times, from lessthan half a million tonnes exported in 1900, to 2.3 million t in 1950 and 9 million t in1990.

Despite the fact that the total value of bananas in the international market is nothigh, the impact of this activity in the economies of banana-exporting countries in LatinAmerica has been considerable. Not only has there been a monetary contribution, butalso work and income have been created for a large sector of the population. The exporttrade additionally contributes to national economies through taxes.

Banana growing has led to an important increase in jobs. It has been estimated thatin Latin America 250 000 are employed and 450 000 more are involved in complementaryactivities. This means that 700 000 families or a total of 35 million people depend on thisactivity. Even though during 1981-90 the standard of living has decreased because ofsetbacks in the economies of banana-exporting countries in Latin America, this has notaffected the people involved in banana production because, in most cases, the peopleinvolved earned more than others who work in different areas of agriculture (UPEB1991).

Opportunities for new markets, especially in Europe, has led to an expansion ofbanana plantations. Between 1985 and 1991 the number of hectares cultivated in LatinAmerica increased tremendously: by approximately 118% in Ecuador, 61% in Costa Rica,65% in Colombia, and 67% in Mexico. In other countries the increase was not verysignificant, as shown in Table 1.

The establishment of new banana-growing areas has increased the demand forplanting materials. Growers have mainly used three types: large suckers with an upperdiameter of 10-15 cm; small suckers with a diameter of 5-10 cm; and in-vitro plants. Thefirst two are inconvenient because the vegetative part of the plant which is usedfrequently carries pathogens and pests, especially nematodes such as Radopholussimilis, the borer Cosmopolitus sordidus, race 4 of Fusarium oxysporum f.sp. cubense,and viruses such as cucumber mosaic and bunchy top. In-vitro plantlets provideexcellent alternative planting material free of these pests and diseases for use in thenew areas of cultivation. This advantage has been intensively exploited over the last

Agribiotecnología de Costa Rica S.A., PO Box 25-4001, Alajuela, Costa Rica

Table 1. Estimates of the areas planted with export bananas in Latin America (ha).

Country 1985 1991 Increase

Costa Rica 20 285 33 000 12 415 Colombia 24 191 40 000 15 809Guatemala 7 688 8 500 812Honduras 17 590 19 500 1 910Nicaragua 2 761 3 000 239Panama 13 541 15 000 1 459Ecuador 48 000 105 000 57 000Mexico 12 000 20 000 8 000

Total 134 356 244 000 97 644

5 years by such Latin American countries as Colombia and Costa Rica. The technologyhas been transferred in part from countries with a longer tradition in the use of bananatissue-culture plantlets, such as Israel, Australia, and Taiwan (Reuveni 1986).

The main advantages of the in-vitro propagation technique for bananas are thefollowing.

1. Plants can be rapidly multiplied from a mother plant of known, desirablecharacters.

2. Selected and screened plants can be maintained free of serious diseases and pests. 3. The use of in-vitro plantlets in areas not infected with nematodes avoids the use of

nematocides, at least for a period of 5 years; it also avoids environmental contaminationand residues, and gives significant savings to the grower of approximately US$400 ha-1 a-1.

4. The use of single-cycle high-density banana plantations could be adopted morewidely, especially for the window market. The expected advantages of this practice arethe following: very high yield in a short time; efficient control of flowering and harvestingtime; saving of hand labor; the possible use of poor land marginal for permanentcultivation; saving expenditure on infrastructure (Israeli, Nameri 1987).

5. 98% survival under field conditions. 6. Plants from in-vitro plantlets grow faster in the early growing stages than those

from suckers. 7. Uniformity of flowering. 8. Short harvesting period. 9. In comparison with the suckers, plants are cheaper and easier to propagate and

transport. 10. There are important advantages regarding germplasm conservation and the

possibility of international transfer. 11. The material produced should be true-to-type and conform to the characters of

the mother plant. (Such conformity is currently causing some concern in the industry.)With the increased use of in-vitro plant multiplication, the widespread presence of

somaclonal variation has been detected, from negligible levels up to 40% of a field. This

140 Commercial Micropropagation of Banana

has been found in all countries that have used in-vitro planting material, e.g., Australia,Israel, South Africa, Taiwan, and Latin America (Arias, Valverde 1987; Reuveni 1986).

Experience gained in Costa Rica in the commercial use of in-vitro plantlets suggeststhe incidence of off-type plants produced in vitro varies between 1 and 10%, when bigpopulations of carefully selected plants are planted. Almost all the off-types havemorphological characters in stage V (nursery), making elimination possible at this stage.

Table 2 shows results from the evaluation of 66 299 plants grown in three differentlocations. The percentage of mutants was between 6.5 and 8% for cv Grande Naine and8.5% for cv Williams.

Table 2. Off-types observed in three fields in Costa Rica.

Plantation Cultivar No. of plants evaluated % of off-types

1 Grande Naine 50179 8.02 Grande Naine 10260 6.53 Williams 5860 8.5

When the plants were analyzed individually, we were able to establish the frequencyof occurrence of some of the mutants. It was then apparent that changes in plant statureand bunch characters were the most common off-types, as shown in Table 3.

Table 3. Approximate % distribution of mutants in different clonal classes (pers. com. A Azofeita, O Arias 1991).

Type %

Dwarf ...................................................................................................................................... 40 Leaves upright ....................................................................................................................... 25Valery-like ............................................................................................................................... 12Plantain-like ............................................................................................................................ 5 Variation in the amount of brown to purple pigmentation ................................................. 7 Abnormal foliage ..................................................................................................................... 5 Leaf variegation ....................................................................................................................... 4“Masada” .................................................................................................................................. 2

Dwarfism, as indicated by the mutation of the Grande Naine height class, accountedfor 40% of the mutants.

A second class of mutants comprises those that have: leaves upright; closerinternodes and abnormal phyllotaxy, giving the impression of a travelers palm (Ravenalamadagascariensis); and fruit of no commercial value. The incidence of these plants was25%. Twelve per cent of the plants were found to revert to Valery height with normalbunches. We also found plants with bunches similar in appearance to plantain. At thenursery stage we distinguished these plants by the large size of the leaf blade anddropping leaves. They accounted for 5% of the mutants. These plants may have a heightsimilar to cv Grande Naine, or even a little smaller, but with abnormal phyllotaxydistribution and persistent bracts on the bunch.

141O Arias

The absence of or abnormal pigmentation was also related to somaclonal variation.The plants concerned had darker and thickened leaves that also showed purplepigmentation on the lower side of the leaf. They represented 7% of the total of mutants.This pigmentation is different from the known yellow to green variegation (thataccounted for 4%). Occasionally plants had abnormal foliage characters: irregular shapeof the lamina, sometimes with a portion missing or a wavy edge of the leaf thataccounted for 5% of the mutants. We found that 2% of the plants looked normal but had greasy spots on the lower side of the leaf, which the Israeli investigators called“Masada”. These plants produced small fruits of no commercial value.

If careful screening is carried out, almost all the somaclonal variations can bedetected at the nursery stage, as already noted. In this case we found in the field anapproximate error of 1.5%, which corresponded to dwarf plants most of the time becausethey were the most difficult to observe at the nursery stage.

In conclusion, the experience obtained in Latin America over the last 5 years inwhich thousands of plants were produced and planted in the field, has produced enoughtechnology for the large-scale use of in-vitro plants, especially for the establishment ofnew plantations and the substitution of traditional varieties in old fields. Usingsomaclonal variants, the Taiwanese researchers have also demonstrated the possibility ofusing these plants for screening for disease resistance (Wang, Ko 1987).

The tissue-culture production of bananas in Latin America is probably the mostimportant example of the application of biotechnology in agriculture. In spite of theprogress achieved, we still have an enormous lack of information on the horticulturalmanagement of the plants at nursery level (stage V).

The development of more practical methods for the early detection of mutants in thelaboratory would assist the commercial laboratories considerably, and thus help in thecoming years in the development of genetically engineered plants with resistance to andtolerance of the most important pests and diseases, within the overall objectives ofbiotechnology research for improved banana cultivation.

ReferencesARIAS O, VALVERDE M. 1987. Producción y variación somaclonal de plantas de banano, variedad grande naine,

producidas por cultivos de tejidos. Asbana 28:6-11.ISRAELI Y, NAMERI N. 1987. A single-cycle high-density banana plantation planted with in vitro propagated

plants. Israel: Jordan Valley Banana Experimental Station. 14 pp.REUVENI O. 1986. Performance and genetic variability in banana plants propagated by in vitro techniques. Bet

Dagan, Israel: The Volcani Center, Department of Subtropical Horticulture, Agricultural ResearchOrganization. 26 pp.

UPEB. 1991. La actividad bananera mundial en 1990. No.91-92:9-20. WANG HSC, KO WH. 1987. Mutants of Cavendish banana resistant to race 4 of Fusarium oxysporum f.sp.

cubense. Pages 42-46 in Taiwan Research Institute Annual Report 1987.

142 Commercial Micropropagation of Banana

143FJ Novak et al.

Improvement of Musa throughBiotechnology and MutationBreeding

FJ Novak, H Brunner, R Afza, R Morpurgo, RK Upadhyay1, M Van Duren, M Sacchi, J Sitti Hawa2, A Khatri3 (IAEA); and G Kahl, D Kaemmer, J Ramser, K Weising (University ofFrankfurt)

IntroductionThe genetic system of Musa is extremely complicated to study because of the seriousproblems of sterility, interspecific hybridity, heterozygosity, and polyploidy that arecommon in most of the clones. The asexual behavior is often an inseparable barrier inusing cross-breeding as a tool for genetic improvement. Consequently, to date, thebreeding of bananas and plantains having desirable characters and acceptability at thefarmers’ level has been without much success. Therefore, the complexity of Musagenetics needs a more sophisticated system to support conventional breeding programs,and prospects of using potentials of biotechnology in this crop are very high (Persley, DeLanghe 1987; Dale 1990; Novak 1992). This report describes the results of developmentof the following systems of Musa biotechnology: (a) in-vitro mutation induction; (b) cell(protoplast) culture and somatic embryogenesis; (c) markers of genetic diversity; (d) in-vitro screening and selection for resistance against Fusarium oxysporum f.sp. cubenseand Mycosphaerella fijiensis.


MaterialsClones of the following Musa species and cultivars have been used as experimentalmaterial: M. acuminata subsp. burmannica (clone IV-9), M. balbisiana, Pisang Mas

Plant Breeding Unit, Joint FAO/IAEA Programme, IAEA Laboratories, A-2444 Sebersdorf, Austria; and PlantMolecular Biology, Department of Biology, University of Frankfurt/Main, D-6000 Frankfurt/Main, Germany.Permanent addresses: 1PPO (Plant Pathology), Directorate of Plant Protection, Quarantine, and Storage,Department of Agriculture and Co-operation, N.H.-IV, Faridabad, Haryana, India; 2Fruit Research Division,Malaysian Agricultural Research and Development Institute (MARDI), PO Box 12301 General Post Office, 50774 Kuala Lumpur, Malaysia; 3Atomic Energy Agricultural Research Centre, Tandojam, Pakistan

(AA), SH-3362 (bred diploid clone AA), Grande Naine (AAA), Saba-Cardaba (ABB),Pisang Rastali (Silk banana, AAB).

Shoot-tip cultureShoot tips (2-5 mm in size) consisting of the meristematic dome with 2-4 leaf primordiawere cultured on MS medium with 10 µM 6-benzylaminopurine (BAP) and 5 µMindoleacetic acid (IAA). The medium was solidified with gelrite (1.75 g L-1). Multipleshoot formation was achieved by subculturing shoot tips on MS medium supplementedwith 20 µM BAP. Shoots were elongated and rooted on MS medium with 5 µM 2ip, 1 µMIBA. Detailed methods of in-vitro production of banana plantlets were described byVuylsteke (1989).

Induced mutagenesisMeristem tips, 1-2 mm in size, were excised from in-vitro growing shoots and irradiatedin a gamma cell with a 60Co source with 15-60 Gy at a dose rate of 8 Gy min-1 (Novak etal. 1990). Uniform longitudinally dissected meristem tips were dipped into an aqueoussolution of 50 µM filter-sterilized ethylmethanesulphonate (EMS) with 2% v/v dimethyl-sulphoxide (DMSO) for 3 h at a constant temperature of 28°C (Omar et al. 1989). Theexplants were then thoroughly washed with ample amounts of sterile water prior toculture. The nutrient media were the same as described for shoot-tip culture.

Embryogenic callus and cell suspension, plant regenerationThe extreme basal parts of the leaf sheaths were cut from young leaves of axenicmeristem-derived plantlets. Corm tissues deprived of the apical meristem were cut intosegments (5 x 5 mm). Both types of explants were plated on petri dishes on Schenk andHildebrandt (1972) medium supplemented with a modified mixture of Staba’s vitamins(Novak et al. 1989), 40 g L-1 sucrose, 40 mg L-1 cystein HCl, 30 µM Dicamba. The cultureswere maintained in the dark at 28°C for 4-6 weeks. Embryogenic calli were subculturedinto liquid, hormone-free medium MS (half strength) and incubated on shakers (120 revmin-1) for 1 week. Cell suspensions were transferred into half-strength SH medium with20 µM Dicamba for long-term culture of cell suspension (more than 1 year). The cellsuspension maintained in the Dicamba medium lost its regeneration capacity within a 2-3 month period. To regenerate plants, the fresh suspensions of cells and proembryogenicstructures were transferred into half-strength, hormone-free MS and then plated ontosolid medium composed of half-strength MS + vitamins + 40 g L-1 cystein + 20 g L-1

sucrose, 1 g charcoal, 5 µM zeatin, 4 g L-1 agar and 1.75 g L-1 gelrite. In this double-layersystem, somatic embryos converted into plants within 2 weeks. The detailed protocol isdescribed elsewhere (Novak et al. 1989).

Encapsulation of embryosCarefully selected, complete bipolar somatic embryos were transferred to a sterile 3%(w/v) solution of sodium alginate (Sigma, medium viscosity), prepared in MS basal

144 Improvement of Musa through Biotechnology and Mutation Breeding

medium (half concentration) without calcium chloride, supplemented with Stabavitamins, 20 g L-1 sucrose, 100 mg L-1 inositol, 40 mg L-1 cystein-HCl and 5 µM zeatin.

Embryos, together with the alginate solution, were then taken up in a sterile pipettewith an opening of 4 mm, and at close distance carefully dropped into a 50 µM solutionof calcium chloride dihydrate on a gyratory shaker (60 rev min-1) at room temperature.

The beads were left in the solution for 30 min to allow proper hardening. Selected,totally encapsulated beads were then transferred to the double-layer medium describedabove.

Protoplast isolation and cultureThe enzymatic mixture of Macerozyme R-10 (1%), cellulase Onozuka R-10 (3%) andpectinase (1%) released a large number of palisade mesophyll cells from leaves ofaseptic plants and parenchyma cells of rhizome tissue during 12 h of cultivation at 28°C.The suspension of intact protoplasts, cell debris, starch grains, and raphides was washedin CPW13M, transferred to conical tubes and after 10 min of centrifugation at 100x g theprotoplasts were collected from the surface of the media. The washing procedures wererepeated twice to obtain pure fractions of protoplasts. The protoplasts were cultured inthe half-strength MS with 20 µM Dicamba in thin layers of liquid media in petri dishes.

Protein extraction and electrophoresisSoluble proteins were extracted from young folded leaves by using SDS buffer. Sampleswere homogenized and centrifuged at 13 000 rev min-1 at 4°C for 15 min. A 12%polyacrylamide gel with a 4% stacking gel was used for electrophoretic separation ofproteins at 1500 V/h. The gel was stained with comassie blue.

DNA fingerprintingExtraction of DNA. High molecular weight DNA was isolated from lyophilized leaves

or other organs according to a modified cetyl-trimethylammonium bromide (CTAB)method (Weising et al. 1991). The lyophilized material (0.5 g) was ground to a finepowder and subsequently transferred to 15 mL of hot (60°C) 2 x CTAB extraction buffer.After gently swirling the resulting cell lysate for 30 min, nucleic acids were isolated byextraction with an equal volume of chloroform/isoamylalcohol (24:1) followed byprecipitation with 0.6% volume of isopropanol. After centrifugation pellets wereresuspended in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8), and DNA was further purified byultracentrifugation in CsCl/ethidium bromide followed by extraction with TE-saturated1-butanol and ethanol precipitation.

Restriction of DNA, agarose gel electrophoresis, and gel-drying. During initialexperiments the purified DNA was digested with Hinf I, Alu I, and Taq I (according tosupplier’s instructions; 6 units of enzyme per mg DNA). Since Hinf I gave the best resultsit was used in all subsequent experiments. After digestion the fragmented DNA waselectrophoresed in 1.0% agarose gels and TAE buffer (40 mM Tris-acetate, 20 mM sodiumacetate, 1 mM EDTA; pH 8.3). The gels were dried on a vacuum gel dryer.

145FJ Novak et al.

Probe labelling, radioactive hybridization, and autoradiography. The gels weredenatured, neutralized and hybridized to 32P endlabeled oligonucleotides essentially asdescribed (Ali et al. 1986). Temperatures of hybridization and stringent washing stepswere 35°C for (GATA)4, 43°C for (CA)8, and 45°C for (GTG)5. One and the same gel wasused for different hybridization probes. Before reprobing, probes were stripped off the gelby washing in 5 mM EDTA at 60°C (2 x 15 min).

DNA amplification fingerprinting. The polymerase chain reaction was performedwith 100 ng of high molecular weight genomic DNA in a volume of 50 µL. The tubescontained 150 µmol L-1 of each dNTP, 20 pmol of the primer(s) and 25 units of DNApolymerase from Thermus aquaticus (Ampli-Taq polymerase, Perkin-Elmer/Cetus). Thereaction mix was overlaid with 30 µL mineral oil, incubated for 5 min at 94°C, andamplified in a waterbath thermocycler for 30 cycles consisting of 1 min at 94°C, 1 min at annealing temperature, and 2 min at 72°C. The last elongation step was extended to 10 min. One and the same DNA preparation served as a source for both oligonucleotideand amplification fingerprinting.

Fusarium culture and bioassays of plant-fungus interactions

Fungal culture and filtrate production. Fusarium oxysporum f.sp. cubense (FOC)was cultured on Czapek-Dox medium and transferred to potato dextrose agar forsporulation. One mL of conidial suspension containing 450 000 conidia mL-1 wastransferred to a 500-mL Erlenmeyer flask containing 250 mL of liquid Czapek-Dox forcrude filtrate production. Cultural conditions were: 28°C, continuous light for sporulationon PDA, 28°C dark condition for production of mycelium and crude filtrate. After removalof mycelium, the liquid phase was filtered through three layers of cheese cloth, refilteredon Watmann paper no. 1 and finally filtered through a 0.22 µm Millipore membrane.Production of crude filtrate activity was assayed with a spectrophotometer following theabsorbance of fusaric acid at 270-272 nm.

Plant-fungus (FOC) coculture. The fungus (FOC) was inoculated on a slant potatodextrose agar (PDA) medium in tubes and allowed to grow for about 3 weeks inincubators at 28°C and continuous light.

The plantlets were rooted in vitro on solidified MS medium with 20 µM BAP and 2 g L-1

charcoal, for about 3-4 weeks under 16 h day-1 light.For the treatment, uniform growing fungus and plantlets were selected. The plantlets

were washed thoroughly with sterile water and the roots trimmed. Meanwhile liquid MSmedium with 5 µM 2ip and 1 µM IBA was added on top of the cultured fungus. Theprepared plantlets were then introduced into the above tubes and placed in incubators at18°C, 25°C, and 32°C with 16 h day-1 light.

For the control, the plantlets were prepared in the same manner but grown in fungus-free PDA slant medium topped with similar liquid MS medium, as described above.

Observations for the wilting symptoms were carried out, such as the first appearanceof the yellowing of leaves and final death of the plants.


Bioassay of crude filtrate. Banana shoot tips cultured as already mentioned weretransferred to a medium containing 3, 6, 9, and 12% of crude filtrate obtained after 21days and placed in the same condition as before for 3 weeks. After that time, the plantmaterial was analyzed for different parameters such as plant height, total fresh weight,shoot production, and root fresh weight.

Plant inoculation. Rooted plantlets were obtained and after removal of gelrite theroots were cut and plants dipped into a conidial suspension (500 000 conidia mL-1) for 10min and then transplanted in Erlenmeyer flasks containing sterile perlite.

Total soluble peroxidase activity. At regular intervals infected plants were removedfrom the flask and after removal of roots and leaves the remaining corm tissues withsome pseudostem tissue were crushed in a mortar using phosphate buffer, pH 6.8, asextraction buffer. The homogenized tissues were collected and placed in Eppendorftubes and centrifuged for 15 min at 14 000 rev min-1. The supernatant was transferred tonew Eppendorf tubes and immediately assayed for total peroxidase activity. Peroxidaseswere assayed with guaiacol as the hydrogen donor. The reaction mixture consisted of0.3% v/v guaiacol and 15 µL of hydrogen peroxide in phosphate buffer, pH 6.8. Five µL ofsupernatant were added to 3 mL of reaction mixture and changes in absorbance werefollowed at 470 nm. The reaction was stopped after 2 min.

Isoelectrofocusing (IEF) of total soluble peroxidases. Isoelectrofocusing ofperoxidases was performed in a horizontal apparatus (Pharmacia Multiphor II) at 4°C.Ampholines (Pharmacia LKB) with a pH range of 3-10 were used as electrolyte carriersand prefocused for 20 min. Fifteen µL of sample per lane was added. Running conditionswere fixed at 8 W, the mA from 33 to 10, and the voltage up to 2500 V. After focusing, thegels were soaked in 100 mL 0.1 M phosphate buffer at pH 6.8 containing 0.3% v/v guaiacoland 15 µL of hydrogen peroxide. After 15 min of incubation the gels were analyzed forthe stained bands.

Isolation of toxins of Mycosphaerella fijiensis and bioassayThe pathogen (MF) was grown in a modified M-1-D plus coconut water liquid medium inan Erlenmeyer flask at 25±1°C and 12 h day-1 light and dark cycles on a rotary shaker at120 rev min-1 for 28 days. After the incubation period was over, an equal volume ofmethanol was added in the culture and placed overnight at 4°C. The culture was thenfiltered and the filtrate evaporated to reduce the volume on a rotary evaporator undervacuum at 36°C. The filtrate so obtained was extracted three times with an equalamount of ethylacetate which was evaporated under reduced pressure and mildtemperature to obtain the crude extract (CE). CE containing bioactive compounds wasfractionated after passing through a column loaded with Sephadex LH-20, usingchloroform and methanol (1:1) as solvent system. The tetralone, isoochracinic acid and2-carboxy-3-hydroxycinnamic acid of 90% purity were isolated using the protocol ofStierle et al. (1991). Juglone was obtained from Aldrich Chemical Company. Fijiensinwas isolated using the method described elsewhere (Upadhyay et al. 1990). Bioassayswere conducted on leaves of plantlets from different cultivars comprising highly

147FJ Novak et al.

susceptible to resistant cultivars using the leaf puncture bioassay test method (Upadhyayet al. 1990).

Results and Discussion

Mutation breedingIn-vitro mutagenesis in shoot-tip culture of banana is schematically represented inFigure 1. This system was performed in the following steps: (1) vegetative shoot apiceswere micropropagated before mutagenic treatment; (2) tiny meristems permitted


Figure 1. Schematic representation of the in-vitro mutation breeding system in Musa (Novaket al. 1990).

149FJ Novak et al.

Figure 2. Schematic representation of biotechnological methods supporting the conventionalbreeding system of Musa (Vakili 1967). The techniques in dotted rectangles are not fullyestablished for banana.

treatments on a large number of propagulas and even the use of mutagenic chemicalswhich otherwise may have difficulty in penetrating target primordial cells; (3) replatedsubculture on a proliferation medium forced formation of adventitious buds whichreduced chimerism in vegetative offsprings; (4) an in-vitro system was used for massscreening of the plantlets’ response to pathogenic fungi or their toxic products; (5)plants were regenerated and evaluated in field conditions.

The variants in M1V4 vegetative progenies of irradiated and/or chemical mutagen(EMS)-treated shoot tips comprised phenotypic changes in morphological (plant stature,leaf shape), physiological (sucker growth and multiplication, flowering time, fruitripening), and agronomic characters (bunch quality). The outstanding plant of thecultivar Grande Naine (putative mutant GN 60GyA) was identified and multiplied forfield-testing. The mutant plant showed differences in the zymograms of soluble proteinsand esterase isozymes (Novak et al. 1990). Induction of somatic mutations maysubstantially contribute to the broadening of the genetic variation among Musa cloneswith obligate vegetative reproduction (e.g., Cavendish). The system of in-vitromutagenesis, however, may also be important for the breeding of fertile diploids beforetheir use for crossing. Figure 2 schematically outlines the biotechnology supporting theMusa conventional breeding system proposed by Vakili (1967). Two steps of mutagenesisare proposed: (1) mutation-breeding of female diploids before crossing, or (2) mutation-induction and selection among F1 recombinant diploids before polyploidy induction.

Somatic embryogenesisRealization of somatic embryogenesis (Fig. 3) in economically important Musa cultivars(AAA and ABB) opened new approaches to somatic cell manipulation and breeding(Novak et al. 1989). Somatic embryos may be encapsulated and used for propagation andin-vitro storage; however, the frequency of “germinating” artificial seeds was very low(less than 0.1%). Embryogenic suspension represents a superior source of geneticvariation (“true somaclonal variation”) as well as a unique unicellular system formutation induction and selection. Plants of Grande Naine regenerated via somaticembryogenesis were sent to FHIA, Honduras, and Maroochy Experimental Station,Queensland, for field observation of phenotypical variation. The material grown in agreenhouse at Seibersdorf will be analysed for DNA fingerprinting patterns to determinethe genetic (in)stability of the somatic embryogenesis process. Our experience insomatic embryogenesis of different Musa clones clearly showed that a modified protocolmust be empirically developed for each particular genotype. Embryogenic suspension isthe preferred source for protoplast isolation and culture (Fig. 4). However, the attemptsto induce cell wall formation and callus-colony proliferation were unsuccessful.

Genetic markersDifferences among four different clones in the soluble protein electrophoresis wererecognized as reliable markers for discrimination among Musa clones of genomic group A.However, consistency in the number of polymorphic bands on electrophoreto-graphs was


151FJ Novak et al.

Figure 3. Somatic embryogenesis in Musa. A: embryonic callus. B: cell suspension. C: somaticembryos. D: encapsulated somatic embryos (“artificial seeds”). E: germination of somaticembryos. F: regenerated plants.


strictly dependent on the precise determination of devel-opmental stages of leaves before protein extraction. Theyoung, fully folded leaves were found as standard materialfor sample preparation (Fig. 5). A band of 30 000 d hasbeen identified only in SH-3362, while a band of 32 000 dcharacterized Grande Naine (Fig. 6). Quantitative differen-ces were identified among clones (Sacchi, Novak, 1992).

DNA oligonucleotide and DNA amplificationfingerprinting were successfully used to detect geneticdiversity between 13 out of a total of 15 representativeMusa species and cultivars. DNA fragments specific forthe A genome as well as the B genome can be found withboth techniques, which also permit discriminationbetween triploids (AAA) differing in their susceptibilityor resistance towards Fusarium (e.g., Gros Michel,Cavendish). Both the oligonucleotide and the ampli-fication fingerprints (RAPiD) are somatically stable, i.e.,do not exhibit any differences between tissues of one andthe same plant. The probes and restriction endonucleasesused for DNA fingerprinting (see Material and Methods)did not distinguish among individuals of the same clone,though a unique fingerprint was found for each clone(Fig. 7). However, certain amplimer combinations

Figure 4. Protoplast suspension of Grande Naine (AAA).

Figure 5. SDS electrophoresisof soluble proteins extractedfrom different leaves ofGrande Naine (AAA).

153FJ Novak et al.

discriminate between an original clone and a mutant in amplification fingerprinting(Fig. 8; Kaemmer et al. 1992). DNA fingerprinting produces a dendrogram closely related to the evolutionary history of the banana clones under study (Fig. 9). Our futurecooperative research aims at detecting individual differences in breeding populationsafter crossing or mutagenesis.

Bioassays of plant-fungus (FOC) interactionsCrude filtrates obtained from the FOC culture expressed toxic activities which dependedon the age of Fusarium culture and final concentration in the banana tissue culturemedium. Shoot tips of bananas were affected in proportion to the concentration of crudefiltrate present in the culture medium. No conclusive differential reaction has been

Figure 6. SDS electrophoresisof soluble proteins extractedfrom folded leaves of fourdifferent cultivars of Musa:DC = Dwarf Cavendish; HG = Highgate; GN = Grande Naine; 3362 = SH-3362. Figure 7. DNA oligonucleotide fingerprinting of different

clones of Musa. a = Pisang Mas; b = SH-3362: a bred diploid;c = Musa acuminata ssp. burmannica; d = M. balbisiana; e =Highgate; f = Dwarf Parfitt; g = Williams; h = Grande Naine; i = GN 60A: an induced mutant of Grande Naine; k = Tafetanverde; l = SH-3436: a bred tetraploid; m = AVP-67; n = Horn; o = Pelipita; p = Cardaba. The group of AA clones isgenetically unrelated (a,b,c). Note the differences betweenGros Michel (e) and Cavendish (f,g,h,i) groups. There are Bgenome aspecific fragments in d,m,n,o,p.


Figure 8. DNA amplification fingerprinting (RAPiD) of various Musa clones (see Fig. 7 for gelposition). At the top is simplex RAPiD and at the bottom is duplex RAPiD. Molecular weightstandards are indicated in kb. RAPiD fingerprinting detects genetic diversity among all 15clones. Both amplimer combinations discriminate clones within the Cavendish group (f,g,h,i).RAPiD fingerprinting indicates (among others) differences between the original clone GrandeNaine (h) and the mutant GN 60A (i).

obtained with respect to the known response of the pathogen on banana cultivars in vivo,suggesting that toxic compounds excreted by the pathogen in culture might not beresponsible for the different levels of resistance expressed by the clones. On the otherhand, the very real fact that crude filtrates showed a toxic activity supported the ideathat toxin(s) play some role in pathogenesis; but this could be due to a postinfectionalprocess. Since the establishment of pathogenesis is a multiple step process it is likelythat the different levels of resistance shown by Pisang Mas and SH-3362 in vivo were dueto some mechanism of early plant-pathogen interaction including wall-to-wallrecognition eliciting defense processes.

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Figure 9. Dendogram of the phylogenetic relationships among various Musa cultivars.

Wilting symptoms on plants cocultured with FOC began to appear at about 2-3 weeksafter treatment for all three temperatures under study, although the degree of infectionvaried, the most severely infected being the treatment under 32°C, whereby the olderleaves turned brown and there was no or a very scarce new root growth. By weeks 5-6, allthe treated plants succumbed to the infection for 32°C, 70% for 25°C, and about 50% for18°C. In contrast, the control for all temperatures showed good shoot and root growththroughout. The results indicated that the major temperature (32°C) seemed to have

enhanced the infection and thus the appearance of the wilting symptoms. With theencouraging results obtained so far, this technique could be used as a potential tool forearly screening for FOC resistance in vitro.

Levels of peroxidase (PRX) activity differ in the two clones. SH-3362 showed a 10-foldhigher activity in uninoculated plants than Pisang Mas. SH-3362 exhibited a sharpincrease of peroxidase activity after inoculation with both race 1 and 4. Differences havebeen recorded on time of induction and activity in the two fungal races. In fact, PRXactivity of plants treated with race 1 increased 5 days after inoculation while theincrease was recorded 8 days after inoculation in plants treated with race 4. When SH-3362 was treated with race 4 the PRX activity continued to increase up to 11 days,followed by a decrease while the plants inoculated with race 1 PRX activity started todecrease immediately after inoculation and rose once again after 13 days. These resultswere confirmed by IEF of infected and uninfected banana plants grown in growthchambers, showing that the increase of activity was mainly due to the basic class ofperoxidases. In Pisang Mas we were not able to find a similar pattern both in totalactivity and in IEF. Our preliminary results on PRX seem to indicate that this enzyme isinvolved in an active defense mechanism against FOC, but that this mechanism is notthe only one that counteracts Fusarium infection in banana, as demonstrated by thelack of response in Pisang Mas plants infected with FOC race 1. This is not surprising; infact, the role of peroxidases has been studied in many plant-pathogen interactions andtheir role has not been unequivocally established in any plant pathogen system. Moreinvestigations are needed in banana to establish the actual role of peroxidases in thedefense response to Fusarium.

Bioassays of Mycosphaerella toxinsEach toxin isolated from MF exhibited phytotoxic activity and induced necrotic lesionsonly within clones of bananas and plantains except for juglone, which damaged everyplant tissue tested. However, juglone has shown a certain degree of differentiation atvery low concentrations (0.01 mg 5 µL-1) with a wide array of plants tested fromdicotyledon and monocotyledon groups. Since juglone is produced by the pathogen intraces (Stierle et al. 1991), its differential activity at lower concentrations has specialimportance and needs further investigations at biochemical and molecular levels. In apreliminary test, juglone was found to stimulate shoot formation in poor shoot-formingbanana clones at lower concentrations (<20 µM-1). This observation indicates itspotential use as a growth regulatory substance.

Fijiensin, isoochracinic acid and cinnamic acid have been found active at 5-10 µg 5 µL-1 levels of concentration, but they did not show a significant differential activitywithin clones of different cultivars of bananas and plantains. Tetralone expressed hostselectivity and a clearcut differential activity among different clones of banana andplantain cultivars even at the higher test concentrations (10 µg 5 µL-1). The varieties IV-9 (M. acuminata ssp. burmannica) and Saba, known as cultivars resistant to andtolerant of the Sigatoka disease complex in the field, exhibited no reaction or a very mildreaction in vitro, whereas susceptible cultivars such as Boca, Horn plantain, and Grande


Naine developed large necrotic areas, thereby showing conformity with the fieldobservations. These findings suggest that tetralone could be an important host-specifictoxin with a fair chance of its use in inducing new clones and screening varieties forresistance in tissue culture against the Sigatoka disease complex. However, suchexperiments must also be supplemented with the pathogen itself to establish a selectionpressure in tissue culture using new techniques.

ConclusionThe current status of Musa biotechnology is very close to making a significantcontribution to the breeding of new cultivars. Tissue-culture techniques are beingdeveloped for the induction of heritable variation with a high potential for Musabreeding. In-vitro mutagenesis may contribute to cross-breeding programs to extend thegenetic pool for recombination. Somatic embryogenesis has been realized ineconomically important cultivars. This technique is especially important in the study of“true” somaclonal variation in Musa and for the use of this unicellular system inmutation induction. Disease resistance is a main target for biotechnological research inMusa. Reliable selection systems of resistant genotypes against FOC may be soonavailable in juvenile plant stages, while in-vitro cell selection seems to be a long-termtarget for basic research before its application in practical breeding programs. Tetraloneand juglone produced by MF offer great potential as a screening tool in the future, withreference to black Sigatoka disease.

Acknowledgments

The Musa clones and strains of FOC were kindly provided by PR Rowe, FHIA, La Lima,Honduras, and M Smith, QDPI, Maroochy Experimental Station, Australia. Thanks arealso due to GA Strobel, Department of Plant Pathology, Montana State University,Bozeman, MT, USA, for extending scientific advice regarding toxins produced byMycosphaerella.

This research was supported by the IAEA Fellowship Programme granted to S Hawaand A Khatri, as well as by a Special Service Agreement granted to RK Upadhyay by theJoint FAO/IAEA Division.

ReferencesALI S, MÜLLER CR, EPPLEN JT. 1986. DNA fingerprinting by oligonucleotide probes specific for simple repeats.

Hum. Genet. 74:239-243.DALE JL. 1990. Banana and plantain. Pages 225-240 in Agriculture Biotechnology: Opportunity for

International Development (Persley GJ, ed.). Wallingford, Oxon, UK: CAB International.KAEMMER D, AFZA R, WEISING K, KAHL G, NOVAK FJ. 1992. DNA oligonucleotide and DNA amplification

fingerprinting of wild species and cultivars of banana (Musa spp.). Bio/Technology 10:1030-1035.NOVAK FJ. 1992. Musa (bananas and plantains). Pages 449-488 in Biotechnology of Perennial Fruit Crops

(Hammerschlag FA, Litz R, eds.). Wallingford, Oxon, UK: CAB International.

157FJ Novak et al.

NOVAK FJ, AFZA R, VAN DUREN M, PEREA-DALLOS M, CONGER BV, XIAOLANG T. 1989. Somatic embryogenesis andplant regeneration in suspension cultures of dessert (AA and AAA) and cooking (ABB) bananas (Musaspp.). Bio/Technology 7:154-159.

NOVAK FJ, AFZA R, VAN DUREN M, OMAR MS. 1990. Mutation induction by gamma irradiation of in vitro culturedshoot-tips of banana and plantain (Musa cvs.). Tropical Agriculture (Trinidad) 67:21-28.

OMAR MS, NOVAK FJ, BRUNNER H. 1989. In vitro action of ethylmethanesulfonate on banana shoot tips. ScientiaHort. 40:283-295.

PERSLEY GJ, DE LANGHE EA (eds). 1987. Banana and Plantain Breeding Strategies. ACIAR Proceedings no. 21.Canberra, Australia: ACIAR.

SACCHI M, NOVAK FJ. 1992. Use of protein electrophoresis for characterization of genetic diversity in Musa.Personal communication.

SCHENK RU, HILDEBRANDT AC. 1972. Medium and techniques for induction and growth of monocotyledonousand dicotyledonous plant cell cultures. Can. J. Bot. 50:199-204.

STIERLE A, UPADHYAY R, HERSHENHORN J, STROBEL GA, MOLINA G. 1991. The phototoxins of Mycosphaerellafijiensis, the causative agent of Black Sigatoka disease of bananas and plantains. Experientia 47:853-859.

UPADHYAY RK, STROBEL GA, COVAL SJ, CLARDY J. 1990. Fijiensin, the first phytotoxin from Mycosphaerellafijiensis, the causative agent of Black Sigatoka disease. Experientia 46:982-984.

UPADHYAY RK, STROBEL GA, COVAL SJ. 1990. Some toxins of Mycosphaerella fijiensis. Pages 231-236 in SigatokaLeaf Spot Diseases of Bananas (Fullerton RA, Stover RH, eds). Montpellier, France: INIBAP.

VAKILI NG. 1967. The experimental formation of polyploidy and its effect in the genus Musa. Amer. J. Bot.54:24-36.

VUYLSTEKE DR. 1989. Shoot-tip culture for the propagation, conservation and exchange of Musa germplasm.Rome, Italy: IBPGR.

WEISING K, BEYERMANN B, RAMSER J, KAHL G. 1991. Plant DNA fingerprinting with radioactive and digoxigenatedoligonucleotide probes complementary to simple repetitive DNA sequences. Electrophoresis 12:159-169.


159M Perea-Dallos

Contribution to the study ofBanana Anther Culture

M Perea-Dallos

IntroductionBanana and plantain are two of the most important staple crops for several millions ofpeople in the world (Cronauer, Krikorian 1984). The food value of Musa cultivars as asource of such minerals as calcium, phosphorus, potassium, magnesium, and iron, is alreadywell known. They also have high contents of vitamins A and C, which have an importantvalue in human nutrition, and additionally high amounts of carbohydrate (Novak 1992).

Since the beginning of banana production, Colombia has been one of the mostimportant countries for the export of bananas and plantains. The main banana-producingarea, Uraba, covers an area of 42 000 ha, with a yield of 246 000 t a-1.

Most plantain farms are located on the mountains, where coffee is the main crop.Under these conditions, plantain is grown to provide needed supplies for the growers as a“survival crop.” It gives “security and cheap food,” not only for peasants in the growingareas, but also for different Colombian social groups.

The genetic improvement of banana and plantain through conventional breedingmethods is hampered because the pollen grains and ovule do not produce fertile seeds(Novak 1992). Sterility in edible bananas is the result of a complex of factors, the mostimportant of which is triploidy and attendant meiotic anomalies, and parthenocarpy andsterility have arisen through gene mutation in fertile diploids (Krikorian, Cronauer 1984;Rowe 1984; Shepherd 1987; Simmonds 1966).

The combination of traditional cross-breeding with biotechnology could provide apowerful tool for the development of new clones with desirable features, such as, forexample, resistance to or tolerance of major diseases. Diploid clones of both wild andedible bananas are essential starting material for banana breeding as such sources of gene-tic resistance or tolerance, except for bunchy top disease which has been identified amongthe extensive collections of diploid accessions of Musa acuminata (Novak et al. 1989).

Anther Culture The importance of crop improvement through anther culture has been widely discussed,and significant advances have been achieved in the last two decades.

Departamento de Biologia, Facultad de Ciencias, Universidad Nacional de Colombia, AA 23227, Bogotá, Colombia

Anther culture of monocotyledons has been extensively researched for many years,because of the importance of the crops that belong to this group. However, the lowfrequency of success, as a result of the very strong genotypic effects and, in some cases, ahigh proportion of albino regenerants, have seriously hindered progress in this area(Kasha et al. 1991).

To date (1993), a great majority of plant species have been successfully regeneratedfrom isolated anthers or grains of pollen at different stages of development. However, inMusa clones, especially the cultivated clones, most of the genotypes do not give any pollengrains, and eggs of diploid clones are haploids. This phenomenom excludes many valuableclones from hybridization.

In view of the importance of haploids in plant breeding, more genotypes need to betried in order to determine the role of genotypes in regard to media composition andenvironmental conditions, with specific reference to cell division and differentiation.

Anther culture is not frequently used in Musa breeeding due to genotypicallydependent differentiation of the genome, but there are efforts to resolve this problem.


Plant materialExperiments were carried out with three selected diploid clones of banana: Bocadillo,Pisang Lilin, and Malascensis. This material was collected from nursery fields of theInstituto Colombiano Agropecuario (ICA) at Carepa-Uraba.

In order to study the appropiate stage of inflorescence, they were chosen at differentstages of development, as for example, when the bunch had emerged (15 cm), and whenthe bunch had grown to about 30-35 cm (i.e., when it began to bend down). To allow forthe asynchronic development of anthers, several hands were selected for research. Themost appropiate stage of development was when inflorescences had 1-12 hands. The pollengrains were thus cultured when they reached the uninucleate stage and when the exineand intine layers were observable.

Method of anther cultureAfter collection, the inflorescences were stored at 4°C for 4-7 days. This material wasdisinfected in 5% sodium hypochlorite (Clorox) for 45 min, followed by several rinses insterile water.

The isolation of anthers was carried out under aseptic conditions and then placed ontoMurashige and Skoog (1962) basal medium, supplemented with various concentrations ofDicamba (3,6-dichloro-o-anisic acid at 5, 10, 15, 20, 25 µM), 2 g L-1 casein hydrolysate, and60 g L-1 sucrose; the pH was adjusted to 5.8.

Four anthers were placed in each vessel, and then incubated under dark conditions at28°C. To avoid browning due to the phenolic compounds, tests were carried out at severalconcentrations of activated charcoal (0.5, 1.0, and 2.0%), and, after the appearance of thecalli, subcultures were done every week.

160 Contribution to the study of Banana Anther Culture

ResultsThe first evidence of callus appeared after 26 days of culture. The uniformity of celldevelopment was observed in all anthers of the inflorescence.

The reduction of browning was noneffective through the activated charcoal treatmentand the calli became dark after several days (1 week). However, cell proliferationcontinued and, after 8 weeks of anther culture, globular-like structures were formed.

Table 1 and Figure 1 show the effect of callus formation and growth from the antherculture of diploid cultivars of banana in different concentrations of an auxin (Dicamba).

Table 1. Base data from which Figure 1 is derived.

Concentration % Callus formation and growthof Dicamba

(µM) Bocadillo Pisang Lilin Malascensis

5 88 75 5010 75 58 3715 49 35 2520 32 28 1225 15 10 8

161M Perea-Dallos

Figure 1. The effect of callus growth from anther cultures on three diploid bananas.

Discussion As callus induction manifests rapid and intensive blackening, it is necessary to makesubcultures every week and incubate them in dark conditions.

Efforts are in progress to develop structure-like embryos.

AcknowledgmentsThe author is indebted to the IAEA/FAO Joint Programme for its support for part of thiswork. Also to R Bayona, Director of the Laboratorio de CENIBANANO and M Mayorga,Director of Regional ICA at Carepa-Uraba for the provision of plant material. Also toAUGURA and the Universidad Nacional for the support of this research. Gratitude isadditionally expressed to M Espitia for his help in typing the manuscript.

ReferencesCRONAUER SS, KRIKORIAN AD. 1984. Multiplication of Musa from excised stem tips. Ann. Bot. 53. KASHA KJ, ZIAUDDIN A, REINBERGS E, FALK E. 1991. Use of haploids in induced mutation in barley and wheat. In

Proceedings of the Second FAO/IAEA Meeting: Use of Induced Mutation in connection with Haploids andHeterosis in Cereals, Katowice, Poland (Maluszynski M, Barabas Z, eds).

KRIKORIAN AD, CRONAUER SS. 1984. Banana. In Handbook of Plant and Cell Culture, vol 2 (Sharp W, Evans DA,Ammirato PV, Yamada Y, eds). New York, USA: Macmillan.

MURASHIGE T, SKOOG F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant. 15:473-497.

NOVAK FJ. 1992. Musa (bananas and plantains). In Biotechnology of Perennial Fruit Crops (Hammerschlag FA,Litz RE, eds). Wallingford, Oxon, UK: CAB International.

NOVAK FJ, AFZA R, VAN DUREN M, PEREA-DALLOS M, CONGER BV, XIOLANG T. 1989. Somatic embryogenesis andplant regeneration in suspension culture of dessert (AA and AAA) and cooking bananas (ABB), Musaspp. Biotechnology 7:154-159.

ROWE P. 1984. Breeding bananas and plantains. Plant Breeding Reviews 2:135-155.SIMMONDS NW. 1966. Bananas, 2nd edn. London, UK: Longman.SHEPHERD K. 1987. Banana breeding, past and present. Acta Horticulturae 196:37-43.

162 Contribution to the study of Banana Anther Culture

163MR Söndahl, C Noriega

Bioreactor MicropropagationTechnology for RapidCommercialization Opportunities in Plantation Crops

MR Söndahl, C Noriega

Importance of MicropropagationVegetative propagation includes budding, cuttings, and grafting, and micropropagationrelates to all in-vitro techniques of cloning. Most current commercial plantmicropropagation methods involve exploring the presence of axillary or apical meristemsof donor genotypes (Kurtz et al. 1991). The cultures are maintained on semisolidmedium, and the process requires three steps: explant inoculation and shootdevelopment, shoot multiplication, and rooting/hardening-off. Each step requires a greatamount of labor and laboratory space for incubation of the semisolid cultures.

An enormous potential for short-term return can be realized through the utilizationof vegetative propagation in perennial crops. This technique seems especially attractivefor many tropical species that possess a high degree of heterozygosity and suffer fromsystemic infections. Examples of economically important crops where vegetativepropagation is being used include: palms (oil palm, date palm, peach palm), banana,agave, bamboo, orchids, coffee, cacao, plantain, colocasia, cassava, potato, pineapple,and many others (Söndahl et al. 1983).

Most of the time micropropagation provides an economic advantage for thepropagation of a particular crop. In other cases, there is a technical advantage in cloninga selected genotype, as, for example, when seed propagation procedures will not sufficebecause there is heterozygosity and segregation in the next generation. A case-by-caseanalysis is necessary to determine the pros and cons in applying micropropagation to acrop. Usually in-vitro propagation can provide very large numbers of cloned materialsrapidly, using small, confined facilities. If these advantages are applied properly to aparticular vegetatively propagated species, micropropagation techniques will becommercially superior to other conventional methods of propagation.

Micropropagation methods have also been applied extensively for diseaseelimination. Most of the systemic parasites (virus, bacteria, fungi) are present in the

DNA Plant Technology Corporation (DNAP), 2611 Branch Pike, Cinnaminson, NF 08077, USA

vascular system of plants. The culture of small meristems or cells can eliminate the greatmajority of such diseases because the vascular system is not yet differentiated or iscompletely absent. Sometimes, heat treatment is associated with meristem culture formore efficient disease elimination. There are cases, however, when cuttings or graftingscannot be applied and micropropagation techniques would be the only commercialsolution for crop propagation (oil palm being one example). Many tropical plants fit intothis category mainly due to their high levels of phenols and tannins which may inhibitrooting and grafting.

Many other reasons for using vegetative propagation by conventional methods wouldalso apply to micropropagation methods; for example, maintenance of heterozygosity,cloning superior individual plants, presence of sexual sterility, or incompatibilityproblems. Production of sufficient numbers of plants of a unique genotype for fieldevaluation followed by further developmental studies may be another objective ofmicropropagation, especially in the case where seed production is not possible. Finally, italso may be useful to apply micropropagation to establish seed orchards for bulk seedproduction or hybrid seed synthesis (cloning parental lines).

Great advantages can be gained by applying micropropagation methods to perennialcrops, due to the long time required for breeding. Here, one can easily evaluate thecommercial advantages of cloning selected individual trees, thus bypassing all the timeinvolved in out-crossing and selecting homozygous seed donors. The advantages would notonly relate to the time to establish high-value commercial plantations, but also to thepreservation of heterozygosity and plasticity in such cloned populations. Fruit trees, oiland coconut palms, ornamental trees, and woody species for pulp and lumber are themain candidates for micropropagation methods. Certainly the most appropriatetechnique (meristem culture, somatic embryo, or shoot production) will be determined,in part, by the kind of tree species, the economic value of each individual tree, and thesize of the operation.

Vegetative propagation by culturing apical and axillary meristems has been exploredquite effectively and proved to be a reliable as well as a commercial method for manyplant species. However, in many perennial trees, the number of available meristems is notsufficient to support a large-scale vegetative propagation, when use of somatic embryoswould be the next best choice (Gupta et al. 1991). High-frequency production of somaticembryos has been described for more than 60 plant species. However, the challenge isnow to produce such embryos from cultured tissues of the most important crop species.Moreover, there is a culture medium/genotype interaction and the production of largenumbers of somatic embryos will require optimization for the elite genotypes. With time,field trials will provide information to ascertain the degree of fidelity of cloned plantsderived from somatic embryos, as well as organogenic shoots. In this particular aspect ofclonal fidelity, the experience gained with one plant species should not be extrapolated intotum to other species. The micropropagation method adopted, the source of initialexplant, the genetic background of the stock plants among others, may determine thevalue and fidelity of a particular cloned species. Quick and reliable methods forevaluating clonal fidelity as early as possible in the cloning process are highly desirable.

164 Bioreactor Micropropagation Technology for Rapid Commercialization Opportunities

Somatic embryogenesis has been established as a means of regenerating plants fromsingle cells. Somatic embryos can be produced via “direct” and “indirect” embryogenesis.Somatic embryos obtained by direct embryogenesis are more limited in numbers thanembryos derived from indirect embryogenesis. In the latter case, the cells undergoextensive proliferation (formation of friable embryogenic tissue) before differentiationinto normal embryos occurs (Söndahl et al. 1985). This self-cloning process may or maynot produce a certain degree of variation among plants derived from either direct orindirect embryogenesis.

New AdvancesWidespread use of micropropagation for major crops in agriculture and forestry is stillrestricted as a result of relatively high production costs. These high costs inmicropropagation are mainly due to high labor costs, limited rates of growth anddevelopment of plantlets in culture vessels, and low percentage survival of plantletswhen transferred to greenhouse conditions (Kozai et al. 1991).

New micropropagation processes are being developed whereby the cloned plants willbe produced inside bioreactor vessels taking advantage of regeneration systems viasomatic embryogenesis, or organogenesis, or even axillary shoot multiplication(Ammirato, Styer 1986; Cazzulino et al. 1991). The introduction of robotics in severalsteps of the process is also under development. The advantage of these newmicropropagation technologies is the possibility of producing a much larger number ofcloned plants in a smaller laboratory space, with reduced labor input, with, probably, apotential reduction in the total process time.

A theoretical example with an embryogenic cell suspension system can illustrate thepotential of the bioreactor micropropagation technique:

A 1-L bioreactor vessel charged with embryogenic suspension cells at a density of 1 x106 cells mL-1 and capable of 0.1% embryo regeneration would yield the followingnumbers of cloned material:- somatic embryos = 1 000 000- germinated embryos = 500 000- plantlets = 375 000- young plants in greenhouse = 280 000Note: These figures assume efficiencies of germination at 50%, plantlet development at 75%, and

hardening at 75%.

The use of a bioreactor will increase several-fold the multiplication capacity of oneparticular cell line. Also, several units can be operated simultaneously for multiple lines.Bioreactors will permit a high degree of culture control and the process is amenable torobotics. Low-cost plant bioreactor units can be devised for commercial micro-propagation use. The risks of this process will be the incidence of contamination and thedanger of some degree of variability among cloned individuals due to excessivemultiplication cycles (long-term cultures). To overcome these problems, maximumaseptic techniques and multiple bioreactor units must be implemented. In addition, it


may be possible to monitor clonal fidelity of the propagated plants by rapid molecularscreening methods, e.g., RAPD (random amplified polymorphic DNA).

The micropropagation process via bioreactor vessels (Fig.1) will achieve itsmaximum efficiency if the regeneration process is based on somatic embryogenesis.Nevertheless, it will also provide advantages to propagation systems based on organo-genesis, or axillary shoots. As the regeneration process changes from embryogenesis to organogenesis to axillary shoots, the multiplication rate will decrease. A generalscheme for exploring the micropropagation process via bioreactor cultures is outlined inFigure 1.


Figure 1. Bioreactor micropagation flow.

EXPLANTS||

CALLUS||

EMBRYOGENIC TISSUE||

CELL SUSPENSIONS ———————— BIOREACTOR||

CELL MULTIPLICATION||

EMBRYO REGENERATION||

EMBRYO MATURATION||

EMBRYO GERMINATION ———————————|||

PLANTLET DEVELOPMENT||

HARDENING-OFF||

NURSERY||

FIELD

Cloning in a liquid phase via bioreactor vessels has the potential to (a) reduce bothcost and total time; and (b) substantially increase the availability of high-quality clonedmaterials. For example, a coffee micropropagation process based on liquid cultures willproduce somatic embryos in large quantities. A recent communication from a tissue-culture laboratory in France demonstrated production rates of 460 000 robusta coffeeembryos per liter after 7 weeks in Erlenmeyer flasks, or 600 000 embryos per 3-Lbioreactor vessel after 8 weeks (Zamarripa et al. 1991). Our preliminary data witharabica coffee tissues (Yellow Catuai) revealed that the production of 1 million coffeeembryos would require an initial inoculum of 80 g f.w. of embryogenic cells cultured infour 5-L bioreactor vessels. Efforts are being made to develop similar embryogenicsystems for oil palm and roses. We are also interested in exploring the bioreactor culturetechnique for micropropagation of bananas and pineapple. These two crops are veryamenable to propagaton via axillary shoot multiplication, and so the development ofsimilar protocols for bioreactor vessels seems to be highly feasible. Several otherresearch groups in the USA, Europe, and Japan are working to develop bioreactormicropropagation systems for celery, alfalfa, pinus, poinsettia, gladiolus, and lilies.

Over several years we have researched the application of bioreactor vessels and/orliquid cultures for large-scale micropropagation. Initial efforts were made in a joint R&Dprogram with Arthur D. Little Inc., which focused on adapting a spin filter bioreactordesign to embryogenic cell systems such as carrot and celery (Ammirato, Styer 1986;Styer 1986). This line of research has continued to further develop this technique forcoffee, oil palm, and roses. If successful with these species, other ornamental plants,industrial plant species, or forest trees should follow.

The bioreactor micropropagation technology will offer advantages to this field, but itwill also bring new technical challenges. This propagation process will produceapproximately 0.5 million units per bioreactor unit every 5-10 weeks, and so newmethods need to be devised to handle such large numbers to go through the next phasesof the micropropagation process, i.e., germination, plantlet development, and hardening.Also, there is a great deal of potential to introduce robotics and automation in thisprocess as well as the use of imaging systems to screen embryos at the propermorphological stage.

Recent developments of robotic micropropagation systems have been reviewed byKozai et al. (1991). Toshiba Corporation (Japan) developed a robot that mimics a seriesof manipulations by an operator at the laminar flow hood, and it is testing the systemwith such plants as potato, carnation, eucalyptus, and redwood. Silsoe Research Institute(England) is working on robotic micropropagation with a vision system that identifies Y-shaped nodes. Commonwealth Industrial Gases (Australia) is also developing roboticmicropropagation with a vision system for daisies (Chrysanthemum cinerariaefolium),which it claims is able to produce more than 10 million plantlets per year. Kirin Brewery(Japan) and Twyford (USA) are jointly developing a robotic multiplication systemwithout vision analysis for Ficus benjamin and ferns. Miwa and Mitsubishi (Japan) aredeveloping a system for explanting bulb scales as a rate of 4800 scales per day. KomatsuLtd (Japan) has a system for plants with upright shoots, such as potato. Mitsui and


Ohtake Techno Union (Japan) jointly developed a robotic multiplication for sectioning(10-20 cm each) and transplanting segments of miniature roses.

Automation of micropropagation systems will be a key development step for laborsaving and better environmental control, thus meeting the increasing demands for low-cost and high-quality transplants in agriculture, forestry, and horticulture. Commercialmass production of transplants in the nursery industry is expected to increase in theorder of 10 times, 100 times, and 1000 times if the production cost is decreased by 50%,75%, and 90%, respectively (Kozai et al. 1991). An increasing number of farmers andgrowers will be inclined to buy transplants rather than raise them by themselves if thetransplants are available at a higher quality and a lower price. Such a drastic reductionof production costs is most likely to be achieved by combining bioreactor micro-propagation with automation. Banana and plantation crops would be natural candidatesto receive the benefits of future micropropagation technology, which should stimulatethe clonal propagation market and facilitate production management.

ReferencesAMMIRATO PV, STYER DJ. J. 1986. Strategies for large-scale manipulation of somatic embryos in suspension

culture. Pages 161-178 in Biotechnology in Plant Science: Relevance to Agriculture in the Eighties(Zaitlin M et al., eds). New York, USA: Academic Press.

CASSULINE D, PEDERSON H, CHIN CK. 1991. Bioreactors and image analysis for scale-up and plant propagation.Pages 147-177 in Scale-up and Automation in Plant Propagation (Vasil IK, ed.). New York, USA: AcademicPress.

GUPTA PK, TIMMIS R, PULLMAN G, YANCEY M, KREITINGER M, CARLSON W, CARPENTER C. 1991. Development of anembryogenic system for automated propagation of forest trees. Pages 75-93 in Scale-up and Automationin Plant Propagation (Vasil IK, ed.). New York, USA: Academic Press.

KOZAI T, TINE KC, AITKEN-CHRISTIE J. 1991. Considerations for automation of micropropagation systems. Pages503-517 in Automated Agriculture for the 21st Century. Chicago, USA: American Society of AgriculturalEngineers.

KURTZ SL, HARTMAN RD, CHU IYE. 1991. Current methods of commercial micropropagation. Pages 7-34 in Scale-up and Automation in Plant Propagation (Vasil IK, ed.). New York, USA: Academic Press.

SÖNDAHL MR, MAKAMURA T, SHARP WR. 1985. Propagation of coffee. Pages 215-232 in Tissue Culture in Forestryand Agriculture (Henke RR et al., eds). New York, USA: Plenum Press.

SÖNDAHL MR, SHARP WR, EVANS DE. 1983. Biotechnology of cultivated crops. Pages 98-114 in Biotechnology: theChallenges Ahead. Singapore: Science Council.

STYER DJ. 1986. Applications and bioreactor technology to seed production and plant propagation. Pages 112-127 in Proc. Agriculture and Life Sciences in China. Seed Certification and Seed Technology. Beltsville,USA: Inst. Intl. Dev. Edu. Agric. Life Sci.

ZAMARRIPA A, DUCOS JP, TESSEREAU H, BOLLON H, ESKES AB, PETIER V. 1991. Développement d’un procédé demultiplication en masse du caféier par embryogenèse somatique en milieu liquide. Proc. 14th Intl. Conf.Coffee Science, San Francisco, USA. pp.44.


169D Vuylsteke, R Swennen

Genetic Improvement of Plantains:the potential of conventionalapproaches and the interface within-vitro culture and biotechnology

D Vuylsteke, R Swennen1

IntroductionGenetic improvement of banana and plantain has gained renewed impetus over thepast few years (Persley, De Langhe 1987). One of the reasons for this interest may befound in the increased awareness among both the donor and the international andnational research communities of the importance of banana and plantain as a localfood crop and as a source of revenue for the many smallholders that grow the crop,particularly in sub-Saharan Africa. Furthermore, the interest in banana and plantainbreeding also emanates from the increased disease and pest pressure that isthreatening the crop’s productivity and sustainability. Apart from the potential ofcultural and/or biological interventions to control some of the diseases and pestsaffecting Musa, host-plant resistance is often perceived as the most appropriatetechnology to check yield losses due to biotic (and abiotic) stresses.

Yet banana breeding is generally considered to be notoriously difficult due to thespecific biology of the preferred and most widely cultivated varieties. Triploidy and lowlevels of fertility have resulted in the perception that the crop is intractable in terms ofconventional breeding approaches. The fact that 70 years of classical breedingendeavors in the Caribbean and Central America have achieved limited success — noman-bred cultivar has yet been grown successfully (Rowe 1984) — is certainly notstrange to this view. But one must bear in mind that the primary objective of thoseearlier programs, i.e., the production of a disease-resistant banana for the export trade(only 10% of world production), may be of limited relevance to the breeding targets andmethodologies for the bulk of the locally consumed bananas and plantains.

Because of the crop’s recalcitrance to genetic improvement, novel approaches havebeen explored. Biotechnology has been proposed as a possible avenue for providing an

International Institute of Tropical Agriculture, Oyo Road, PMB 5320, Ibadan, Nigeria. 1Present address: Laboratoryof Tropical Crop Husbandry, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, 3001 Heverlee, Belgium

array of techniques to overcome the limitations of conventional techniques (Dale1990). In some cases, biotechnology has even been advocated as the sole option tobanana and plantain improvement, for example in producing materials resistant tobanana bunchy-top virus (BBTV).

In this paper, we would like to demonstrate the potential of conventional, yetmodified, breeding approaches by providing an example of plantain improvement workin which tissue-culture techniques were fully integrated. In-vitro culture techniquesand biotechnology have been and will continue to be valuable tools in the improvementof the crop, but we would like to emphasize, as many others have done, that this shouldbe pursued only when integrated in breeding programs in order to maximize benefitsfrom the limited human and financial resources devoted to genetic improvement.

Plantain Improvement at IITAIn 1987, the International Institute of Tropical Agriculture (IITA) initiated a plantainand banana improvement program in response to the rapid spread of the virulent blackSigatoka disease (Mycosphaerella fijiensis Morelet), which is generally considered the major constraint to plantain and banana production worldwide. The programtargets the incorporation of durable host-plant resistance to Sigatoka leaf spot in theplantains (Musa spp., AAB group) of western and central Africa, the highland cookingand beer bananas (Musa spp., AAA and ABB group) of eastern Africa, and ABB cookingbananas.

Black Sigatoka disease causes yield losses of 30-50% in plantains (Stover 1983; IITA1992) and further compounds the pest complex attacking the crop in sub-SaharanAfrica. This warrants investigations into appropriate control strategies and provides thecontext for IITA’s work in this area.

Because of earlier reports on the extreme difficulties encountered in plantainbreeding (De Langhe 1976; Rowe 1984), the program started by screening the plantaincollection (113 cultivars) for female fertility. Several different plantain cultivars wereidentified as seed-fertile as they produced viable seed upon hand-pollination. Twenty-nine French plantain cultivars set seed at averages ranging from less than 1 to over 20seeds per bunch. Also, 8 False Horn plantains were found to be able to produce seed,yet generally at average rates below 1 seed per bunch. In addition to genotype, seasonwas also found to have a marked influence on seed-set (Swennen, Vuylsteke 1990). Theearly identification of these 37 seed-fertile plantain cvs, and their relatively high seed-set, challenged the concept of female sterility in plantains and set the pace for IITA’sincreased efforts in pursuit of conventional breeding.

The breeding program was strongly supported by complementary plant tissue-culture work. Simple in-vitro culture techniques, such as shoot-tip culture and embryoculture, greatly facilitated the breeding activities by surmounting some of the obstaclesimpeding breeding progress.

The most fertile plantains, among which the French plantain cvs Bobby Tannap,Obino l’Ewai, Mbi Egome 1, and Bungaoisan were rapidly propagated in vitro to a total

170 Genetic Improvement of Plantains

of 6000 plants, to set up a massive pollination scheme. Annual seed production fromcrosses of plantains with different diploid male parents averaged around 10 000. Onlyabout 40% of these were viable seeds, containing a zygotic embryo embedded inendosperm. In-vitro embryo germination varied from 5 to 30%, depending on the crossand the season (Vuylsteke et al. 1990a). Of the many seedlings produced, about halfwere rogued at the nursery stage because of leaf abnormalities typical of aneuploidyand euploidy in excess of tetraploidy.

About 250 plantain progenies — diploids, tetraploids and triploids — were field-established for evaluation. Noteworthy was the occurrence of a larger number of diploid(78%) than tetraploid (22%) progenies from the triploid (AAB plantain) by diploid (AA)crosses. Triploid progenies were very few. The frequency of tetraploids also differedaccording to the plantain cultivar used in crosses with the same male diploid. Forexample, cv Obino l’Ewai gave 42% tetraploid progenies, while cv Bobby Tannapproduced only 12% tetraploids.

Among the field-established progenies, 20 plantain-derived tetraploid hybrids wereselected for their reduced susceptibility to black Sigatoka disease, their high yields andgood fruit parthenocarpy (Table 1). They also had generally shorter plant stature andbetter ratooning, but required about 1 month longer for the bunch to reach maturity.Most tetraploid hybrids demonstrated a capacity for seededness due to increasedpollen fertility.

The wild diploid banana clone Calcutta 4 was the male parent of 17 of the 20selected hybrids. Nevertheless, fruit parthenocarpy and bunch size of the tetraploidhybrids was not inferior to that of their French plantain parents (Fig.1). This indicatesthat the poor bunch characters of this wild, nonparthenocarpic banana were generallynot transmitted to the tetraploid progenies. Conversely, the high level of black Sigatokaresistance of Calcutta 4 was readily inherited in its offspring.

Variation in growth and yield parameters (Table 1), qualitative morphological traits,and black Sigatoka reaction (Table 2) were observed within the same tetraploid familyobtained from crosses of plantain with the male diploid parent Calcutta 4, a true-breeding line. This observation was surprising because one would expect an entirelyuniform progeny from the combination of unreduced female gametes and the malegametes of a true-breeding species. Indeed, Calcutta 4 is a true-breeding line becauseits selfed progeny did not show segregation for black Sigatoka reaction ormorphological traits (Simmonds 1952; IITA, unpublished results). Therefore, we inferthe occurrence of segregation and recombination in the triploid plantain genomeduring the modified megasporogenesis, even when there is restitution of all three setsof maternal chromosomes.

This inference challenges the “dead end” target of the primary triploid by diploidcross (Stover, Buddenhagen 1986; Bakry et al. 1990). The concept of banana/plantainbreeding being essentially breeding-improved diploid pollinator lines (Rowe 1984;Shepherd 1987; Simmonds 1987; Stover, Buddenhagen 1986) may thus need to bereviewed, as variability is also increased by segregation on the female triploid side.


Table 1. Preliminary evaluation of growth and yield parameters and of blackSigatoka resistance in the plant crop of some selected tetraploid plantain hybrids(nonfungicide treated) as compared with their respective triploid female plantainparents (fungicide-treated).

Cultivar and Male Plant YLS2 at HTS3 at Fruit Bunchderived hybrids parent1 height flowering harvest filling weight

(cm) (cm) time (days) (kg)

Obino l’Ewai - 370 7.0 188 92 12.4TMPx 548-9 C4 290 11.0 310 131 16.5TMPx 2637-49 C4 360 11.6 225 127 16.6TMPx 4698-1 C4 345 10.5 275 130 20.0TMPx 5511-2 C4 345 10.0 165 118 17.8TMPx 5706-1 C4 340 7.5 250 133 13.6TMPx 6930-1 C4 330 10.3 240 134 18.5TMPx 1658-4 Pl 320 9.3 185 134 21.5

Bobby Tannap - 340 7.0 171 92 14.0TMPx 582-4 C4 300 11.0 270 135 14.3TMPx 4479-1 C4 295 9.0 290 114 13.2TMPx 2796-5 Pl 335 10.0 205 123 21.31 Male parents: C4 = Calcutta 4; Pl = Pisang Lilin.2 Youngest leaf spotted (Stover, Dickson 1970).3 Height of tallest sucker.

Table 2. Segregation of black Sigatoka reaction and other qualitative traits amongselected tetraploid progenies obtained from crosses of the triploid French plantain(Musa spp., AAB group) cultivars Obino l’Ewai and Bobby Tannap with the true-breeding wild diploid M. acuminata ssp. burmannicoides clone Calcutta 4.

Cultivar and Black Sigatoka Bunch Neutral Male bud Ratooningderived hybrids reaction orientation flowers imbrication

Obino l’Ewai susceptible pendulous persistent yes inhibitedTMPx 548-9 mod. resistant1 pendulous deciduous yes regulated2

TMPx 2637-49 mod. resistant pendulous deciduous no regulatedTMPx 4698-1 mod. resistant pendulous semipersistent3 yes regulatedTMPx 5511-2 mod. resistant pendulous deciduous yes inhibitedTMPx 5706-1 less susceptible pendulous deciduous no regulatedTMPx 6930-1 mod. resistant pendulous deciduous yes regulated

Bobby Tannap susceptible subhorizontal persistent yes inhibitedTMPx 582-4 mod. resistant subhorizontal deciduous yes regulatedTMPx 4479-1 less susceptible pendulous deciduous yes regulated1Moderately resistant.2Regulated: 1 to 3 suckers develop freely while others remain inhibited.3Semipersistent: some neutral flowers persist but the majority are deciduous.



In-Vitro Culture and Biotechnology in support ofPlantain/Banana Breeding

Shoot-tip cultureIncreased impetus for genetic improvement programs has focused attention on thecollection, movement, and conservation of Musa germplasm. However, efforts to handlegermplasm of this vegetatively propagated crop are typically fraught with a series ofobstacles, such as slow multiplication, bulkiness, and quarantine-related problems ofconventional propagules. Hence, a wide array of plant tissue-culture techniques isincreasingly being used as an enabling and enhancing technology for the handling ofMusa germplasm (Vuylsteke 1989).

Aseptic shoot-tip culture, in combination with third-country quarantine, has beenused as a vehicle for the safe exchange of banana/plantain germplasm (Vuylsteke et al.1990b). Shoot-tip cultures confer considerable advantages for the international transferof germplasm because the mass of plant material involved in the movement is greatlyreduced and contained, and they overcome nearly all of the problems associated withnonobscure pests and pathogens. From 1985 to 1990, 320 new accessions wereintroduced by IITA as in-vitro cultures, thereby quadrupling the number of accessionsheld in the collection. These genetic resources, which provide the basis for the breedingprogram, were introduced in a joint effort with the Nigerian Plant Quarantine Service

Figure 1. The first hand of the black Sigatoka-resistant tetraploid TMPx 548-9 (left) and of itsfemale plantain parent Obino l’Ewai (right).

and the INIBAP Transit Center at KU Leuven (Belgium). IITA has also deposited 17hybrids of banana and plantain at the INIBAP Transit Center for virus indexing in view oftheir possible future international dissemination.

Shoot-tip culture is a well-established, adequate, and relatively simple in-vitromethod for the rapid propagation of selected Musa materials and the production ofclean planting material. Multiplication rates are several orders of magnitude higher thanthose obtained with conventional methods, which is of great value for the multiplicationof newly bred genotypes or to speed up the testing of selections by plant breeders.Micropropagation has been pivotal in the rapid deployment of IITA’s breeding programby supplying large numbers of plants of female and male parents for the crossing blocksand of black Sigatoka-resistant hybrids for the evaluation trials.

Embryo culture/rescueHybrid plant production in the most common triploid Musa clones is hampered by lowseed-set and by low seed germination rates. Seeds of plantain crosses germinate in soilat a rate of only 1%. Aseptic embryo culture techniques, already in use in bananabreeding for 3 decades, are routinely applied to increase seed germination rates by afactor of 3 to 10. An average of about 900 plantain hybrid seeds are handled in vitro on amonthly basis, in support of the breeding program. Investigations into rescuingimmature embryos are under way further to enhance germination rates. Preliminaryresults showed that immature embryos of 50-55 days after pollination had 20.7%germination, as compared with the 1.0% germination of mature embryos (80-85 daysafter pollination).

Cell-suspension cultures and somatic embryogenesisThough we have attempted to demonstrate above that conventional breeding of plantainshas great potential, certain improvement targets remain virtually impossible without theinput of molecular genetic techniques. Such procedures, however, largely depend on thesuccessful regeneration of plants from cells or protoplasts. Prospective benefits that mightaccrue from the integration of biotechnologies into banana and plantain breedingprograms therefore require access to reliable cell culture protocols (Krikorian 1987). Yet,reports on cell suspension cultures of Musa are few. Moreover, most procedures for cellsuspensions are based on callus (Novak et al. 1989; Escalant, Teisson 1989), which hasobvious disadvantages in applications that require clonal uniformity, such as the recoveryof transgenic plants. In addition, some procedures use zygotic embryos to producesuspension cultures (Escalant, Teisson 1989), therefore excluding the edible bananas.Recently, however, an elegant protocol for direct embryogenesis in somatic cellsuspensions initiated from meristem explants was developed at KU Leuven, Belgium(Dhed’a et al. 1991). These embryogenically competent cell cultures have already provento be the material of choice for the cryopreservation of germplasm (Panis et al. 1990; Paniset al. 1991), and protoplast culture (IITA unpublished results), and are currently evaluatedfor plant transformation studies.


Somaclonal variationThe increased genetic variation among plants regenerated from in- vitro culture hasbeen termed as somaclonal variation, an ubiquitous phenomenon. The frequent use of in-vitro culture techniques for the handling of Musa germplasm warrants investigationsinto the occurrence of somaclonal variation in this genus.

Somaclonal variation in banana and plantain plants derived from shoot-tip culturecan be a significant problem in micropropagation and has been described by manyauthors (see Israeli et al. 1991; Vuylsteke et al. 1991, and references cited therein).Somaclonal variation had earlier been proposed as a source of new useful variability forbanana and plantain improvement, but it is now generally agreed that it should not beoverestimated as a beneficial adjunct to breeding. On the contrary, somaclonal variationwill be of particular concern when new genotypes are rapidly propagated in vitro, eitherproduced by conventional means or by recombinant DNA technology.

In this framework, the usefulness of mutagenesis should also be questioned. The authors’ experience with plants recovered from gamma-irradiated meristems wasthat it did not create any valuable variability, but rather resulted in many grosslyabnormal plants.

ConclusionResults from plantain breeding at IITA emphasize that promising black Sigatoka-resistant tetraploid hybrids, which combine high yields, adequate plant height, andimproved ratooning, can be produced when using a wild diploid banana as a male sourceof disease resistance. In addition, seed production in a range of highland cooking andbeer bananas (AAA group), and the recent planting of their first progenies, suggests thatthis major eastern African crop is also amenable to conventional genetic improvement.

Our results challenge the commonly accepted concept of the intractability ofplantain for genetic improvement by conventional breeding methods. The utilization ofinterspecific hybridization, embryo culture, rapid in-vitro multiplication, field-testing,and selection, has resulted in the identification of improved Musa (plantain) germplasmat IITA.

Established tissue-culture techniques have thus contributed significantly to banana/plantain improvement. Also, molecular genetic methods are becomingavailable for investigating genetic variability (Gawel, Jarret 1991). RFLP and RAPDmapping will assist breeding programs by enabling early identification of promising newgenotypes.

Notwithstanding the recent success in conventional breeding at IITA, recombinantDNA technology may be of great benefit in the improvement of Musa cultivars that aredifficult to breed (e.g., Cavendish) and to incorporate genes coding for characters thatare not available in the Musa gene pool, such as BBTV resistance. We, however, advocatethat biotechnology projects be fully integrated into conventional breeding programs inorder to take full advantage of biotechnological developments and products.


ReferencesBAKRY F, HORRY JP, TEISSON C, TEZENAS DU MONTCEL H, GANRY J. 1990. L’amélioration génétique des bananiers à

l’IRFA/CIRAD. Fruits numéro spéciale: 25-40.DALE JL. 1990. Banana and plantain. Pages 225-240 in Agricultural Biotechnology: opportunities for interna-

tional development (Persley GJ, ed.). Wallingford, Oxon, UK: CAB International.DE LANGHE E. 1976. Pourquoi l’amélioration génétique du plantain n’est-elle pas actuellement réalisable?

Fruits 31:537-539.DHED’A D, DUMORTIER F, PANIS B, VUYLSTEKE D, DE LANGHE E. 1991. Plant regeneration in cell suspension

cultures of the cooking banana cv. ‘Bluggoe’ (Musa spp., ABB group). Fruits 46:125-135.ESCALANT J, TEISSON C. 1989. Somatic embryogenesis and plants from immature zygotic embryos of species

Musa acuminata and Musa balbisiana. Plant Cell Reports 7:665-668.GAWEL N, JARRET RL. 1991. Cytoplasmic genetic diversity in bananas and plantains. Euphytica 52:19-23.IITA. 1992. Crop Improvement Division, Plantain and Banana Improvement Program: 1991 Annual Report and

1992 Workplan. Ibadan, Nigeria: IITA.ISRAELI Y, REUVENI O, LAHAV E. 1991. Qualitative aspects of somaclonal variation in banana propagated by in

vitro techniques. Scientia Hortic. 48:71-88.KRIKORIAN AD. 1987. Callus and cell culture, somatic embryogenesis, androgenesis and related techniques for

Musa improvement. Pages 128-135 in Banana and Plantain Breeding Strategies (Persley GJ, De LangheEA, eds). ACIAR Proceedings no.21. Canberra, Australia: ACIAR.

NOVAK FJ, AFZA R, VAN DUREN M, PEREA-DALLOS M, CONGER BV, XIAOLANG T. 1989. Somatic embryogenesis andplant regeneration in suspension cultures of dessert (AA and AAA) and cooking (ABB) bananas (Musaspp.). Bio/Technology 7:154-159.

PANIS B, WITHERS L, DE LANGHE E. 1990. Cryopreservation of Musa suspension cultures and subsequentregeneration of plants. Cryo-letters 11:337-350.

PANIS B, DHED’A D, SWENNEN R. 1991. Freeze-preservation of embryonic Musa suspension cultures. Pages 183-195 in Conservation of Plant Genes: DNA banking and in vitro biotechnology (Adams RP, Adams JE, eds).New York, USA: Academic Press.

PERSLEY GJ, DE LANGHE EA (EDS). 1987. Banana and Plantain Breeding Strategies. Proceedings of an interna-tional workshop held at Cairns, Australia, 13-17 October 1986. ACIAR Proceedings no.21. Canberra,Australia: ACIAR.

ROWE P. 1984. Breeding bananas and plantains. Plant Breeding Reviews. 2:135-155.SHEPHERD K. 1987. Banana breeding - past and present. Acta Horticulturae 196:37-43.SIMMONDS N W. 1952. Segregations in some diploid bananas. J. Genet. 51:458-469.SIMMONDS NW. 1987. Classification and breeding of bananas. Pages 69-73 in Banana and Plantain Breeding

Strategies (Persley GJ, De Langhe EA, eds). ACIAR Proceedings no.21. Canberra, Australia: ACIAR.STOVER RH. 1983. Effet du Cercospora noir sur les plantains en Amérique Centrale. Fruits 38:326-329.STOVER RH, DICKSON JD. 1970. Leaf spot of bananas caused by Mycosphaerella musicola: methods of

measuring spotting prevalence and severity. Trop. Agr. (Trinidad) 47:289-302.STOVER RH, BUDDENHAGEN IW. 1986. Banana breeding: polyploidy, disease resistance and productivity. Fruits

41:175-191.SWENNEN R, VUYLSTEKE D. 1990. Aspects of plantain breeding at IITA. Pages 252-266 in Sigatoka Leaf Spot

Diseases of Bananas. Proceedings of an international workshop held at San José, Costa Rica, March 28 -April 1 1989 (Fullerton RA, Stover RH, eds). Montpellier, France: INIBAP.

VUYLSTEKE D. 1989. Shoot-tip culture for the propagation, conservation and exchange of Musa germplasm.Practical manual for handling crop germplasm in vitro no.2. Rome, Italy: IBPGR. 56 pp.

VUYLSTEKE D, SWENNEN R, DE LANGHE E. 1990a. Tissue culture technology for the improvement of Africanplantains. Pages 316-337 in Sigatoka Leaf Spot Diseases of Bananas. Proceedings of an internationalworkshop held at San José, Costa Rica, March 28 - April 1 1989 (Fullerton RA, Stover RJ, eds).Montpellier, France: INIBAP.

VUYLSTEKE D, SCHOOFS J, SWENNEN R, ADEJARE G, AYODELE M, DE LANGHE E. 1990b. Shoot-tip culture and third-country quarantine to facilitate the introduction of new Musa germplasm into West Africa. FAO/IBPGRPlant Genetic Resources Newsletter 81/82: 5-11.

VUYLSTEKE D, SWENNEN R, DE LANGHE E. 1991. Somaclonal variation in plantains (Musa spp., AAB group)derived from shoot-tip culture. Fruits 46:429-439.


177JV Escalant, C Teisson

Somatic Embryogenesis and CellSuspensions in Musa

JV Escalant1, C Teisson2

IntroductionCultivated bananas and plantains generally originate from interspecific hybridizationbetween two fertile diploid species, Musa acuminata (AA) and Musa balbisiana (BB)(Stover, Simmonds 1987). The most widespread edible genomic groups are: AAA (e.g.,Cavendish), AAB (e.g., plantains), and ABB (e.g., Bluggoe). Over the past few years,diseases such as black Sigatoka (Mycosphaerella fijiensis), fusarial wilt (Fusariumoxysporum), and the banana bunchy top virus (BBTV) have resulted in increasingefforts to improve Musa disease resistance genetically. Because of the high sterilitylevels and polyploidy of banana and plantain, conventional breeding using traditionalhybridization techniques remains difficult (Rowe 1984). Tissue-culture and molecularbiology techniques have great potential for overcoming the problems yet unsolved bytraditional improvement methods (Murfett, Clarke 1987). Such techniques rely onsuccessful regeneration of plants from cell suspensions or protoplasts.

The first report on cell suspensions in Musa described the obtention of slow-growingcell suspensions from immature fruit-derived callus (Moham Ram, Steward 1964). Novaket al. (1989) reported recovery of plants from somatic embryos obtained in cellsuspensions from rhizome tissue in diploid and triploid banana cultivars. More recently,cell suspensions and subsequent plant regeneration have been obtained applyingsomatic embryogenesis to meristematic ‘scalps’ of cooking banana cv Bluggoe(Sannasgala 1989; Dhed’a et al. 1991).

In this paper, we report on the progress achieved on wild diploid species and triploidcultivars of Musa in the collaborative work between CATIE and CIRAD biotechnologylaboratories.

MethodsDepending on diploid or triploid material, different methods can be used to obtain cellsuspensions and subsequent plant-regeneration techniques.

1CATIE/CIRAD-IRFA, PO Box 104, 7170 Turrialba, Costa Rica; 2CIRAD-BIOTROP, Laboratoire de Culture in vitro, BP5035, 34032 Montpellier Cedex 1, France

Cell suspension in diploid wild speciesEmbryogenic calli were obtained using the method described by Escalant and Teisson(1989). When callus embryogenic characteristics were evident, showing first somaticembryos in its surface, calli were transferred into 25 mL of liquid medium. Cultures werekept in 125-mL Erlenmeyer flasks on a rotary shaker and refreshed every 2 weeks using thesame medium. Once a high cell density was obtained (visual estimate), calli were sieved(500 µm) and cell suspensions (filtrated) maintained in the same medium, being renewedevery week by decanting the old medium. Cell suspensions were initiated with somaticembryogenic callus at different culture periods.

Inoculated calli released lots of cells into the liquid medium. After 3-5 weeks of culture,when cell density was adequate (visual estimate), callus was removed by sieving it (500 µm). At this stage, microscopic control of cell suspensions was done displaying manysingle embryogenic cells mixed with very small clusters (2-5 cells) and a few elongatedcells. After 5 weeks a rapid cell growth was observed, reaching a weekly average increase of10 µL mL-1. During the growing period, many cell divisions took place and, approximately 8-12 weeks later, somatic embryos appeared in large amounts. This growth was recordedmeasuring the total cell mass as packed cell volume using the PCV method (Reinert,Yeoman 1982, pp.12-14). After this, the most developed embryos were isolated (100 µm)and transferred to a germination medium, resulting in plant recovery frequencies from 20 to 36%. Separation of these embryos was carried out every month. After each filtration, itwas possible to isolate more than 300 somatic embryos. This procedure started in mid-1991and, since then, it has been maintained in the embryo-producing suspensions.

With the second procedure, aliquots (0.5 mL) of filtered (100 µm sieved) cellsuspensions, formed numerous somatic embryos after 3-4 weeks of inoculation in ahormone-free semisolid MI medium. Germination started after transferring embryos to theMG medium, with a 30% germination rate. Using this method, it was possible to have up to70 somatic embryos from 1.5 mL of cell suspension. An advantage of this technique is that itfacilitates the selection of the best-developed embryos, allowing immature ones to remainin the culture medium until reaching maturity. Also the fact that embryos are formed in ahormone-free medium will favor formation of more normal embryos, thus increasinggermination percentage and obtention of complete plants.

Histological sections of differentiated somatic embryos from both methods, showed abipolar structure with the shoot and root apices connected to a storage parenchyma withvascularization, and entirely surrounded by an epidermis.

After 1 month of in-vitro growth, plantlets from somatic embryos germination weretransferred to a greenhouse for acclimatization.

Somatic embryogenesis and cell suspension in triploid Musacultivars

Somatic embryogenesisExplants used consisted of very young male flowers of different cultivars: Musa AAA cvGrande Naine and 901; Musa AAB cv Currare French type; Musa ABB cv Pelipita.

178 Somatic Embryogenesis and Cell Suspensions in Musa

After 3-4 months on an MS-derived semisolid medium, full calli and somatic embryoswere observed: some of them yellow and compact and others more friable and white.

Histological analysis showed that calli are originated from reactivation ofperivascular cell divisions and that they are composed by meristematic cells associatedwith starch and proteic reserves. Transversal sections of somatic embryos revealed shootand root apices connected to a storage parenchyma with trifid vascularization.

Germination of somatic embryos occurred by development of the plumule andformation of a whole plant. After 1 month of in-vitro growth and 2 months in thegreenhouse, plants were transferred to field conditions for further evaluation.

Cell suspensions with triploid cultivars

There are two ways of establishing cell suspensions from triploid cultures: (a) using maleflower callus; and (b) using somatic embryos from male flowers.

a) This technique is based on Professor Ma’s findings, and it consists in transferingcallus with explant into a liquid medium. Even though inoculated calli releasednumerous cells in the liquid medium, during the initial steps of culture, most of themwere meristematic and only a few showed some embryogenic characteristics. During thegrowth period, more embryogenic cells appeared and some somatic embryos wereformed. However, these results are only preliminary, and continuing research is needed.

b) In this technique, somatic embryos from male flowers are used as immaturezygotic embryos for the diploid cell suspensions method.

Grande Naine and Currare somatic embryos responded after approximately 3 weeks,forming small embryogenic callus. When this material became friable, it was transferredto a liquid medium releasing many embryogenic cells and small clusters.

The first results from this research are considered very promising because of theirsimilarity to those obtained from wild diploid species.

ConclusionEmbryogenic diploid cell suspensions were obtained by culturing embryogenic callus.Calli from zygotic immature embryos and triploid somatic embryos were an excellentmaterial for this purpose. The simplicity of this technique permits its application inplant transformation studies and its unquestionable future integration into banana andplantain improvement programs. Moreover, this method could be extended to othercultivars (2n, 3n, 4n) using embryos as raw material to obtain embryogenic callus andplant regenerations from cell suspensions.

ReferencesDHED’A D, DUMORTIER F, PANIS B. 1991. Plant regeneration in cell suspension cultures of the cooking banana cv.

“Bluggoe” (Musa spp., ABB group). Fruits 46:135.ESCALANT JV, TEISSON C. 1989. Somatic embryogenesis and plants from immature zygotic embryos of species

Musa acuminata and Musa balbisiana. Plant Cell. Rep. 7:665-668.

179JV Escalant, C Teisson

MOHAM RAM HY, STEWARD FC. 1948. The induction of growth in explanted tissue of banana fruit. Can. J. Bot.42:1559-1579.

MURFETT J, CLARKE A. 1987. Producing disease-resistant Musa cultivars by genetic engineering. Pages 87-94 inBanana and Plantain Breeding Strategies (Persley GJ, De Langhe E, eds). ACIAR Proceedings no.21.Canberra, Australia: ACIAR.

NOVAK FJ, AFZA R, VAN DUREN M. 1989. Somatic embryogenesis and plant regeneration in suspension culturesof dessert (AA and AAA) and cooking (ABB) bananas (Musa spp.). BioTechnol. 7:147-158.

REINERT J, YEOMAN MM. 1982. A laboratory manual: plant cell and tissue culture. Berlin, Germany: Springer-Verlag.

ROWE P. 1984. Breeding bananas and plantains. Pages 135-155 in Plant Breeding Review (Janick J, ed.).Westport, CT, USA: AVI Publishing.

SANNASGALA K. 1989. In vitro somatic embryogenesis in Musa. Dissertationes de Agricultura no.180. Ph.D.thesis. Heverlee, Belgium: KU Leuven. 172 pp.

STOVER RH, SIMMONDS NW. 1987. Bananas. London, UK: Longman.

180 Somatic Embryogenesis and Cell Suspensions in Musa

181H Leblanc, JV Escalant

Induced Parthenogenesis to ObtainHaploid Plants in Musa

H Leblanc, JV Escalant

IntroductionThis research began in CATIE in 1991, with the following main objectives:* To determine if pollen of Musa wild diploid species was able to germinate after being

treated with different doses of gamma radiation.* To determine if, after such treatment, pollen was still able to stimulate embryo

development without fertilization of the oosphere.* To determine if this technique helped to induce the formation of Musa spp. haploid

plants.

Materials and MethodsPollen from M. acuminata ssp. burmannicoides and M. balbisiana type Tani wasirradiated with different gamma radiation doses (5-100 krad) (60cobalt source), and thenused to pollinate female flowers of M. acuminata ssp. burmannicoides.

Pollen germination and evolution after pollinization were monitored using a fluo-rescent microscope with Z stainer and BV-2A filters. Observation of histological sectionspermitted the embryo evolution to be observed.

Embryo rescue was carried out using tissue-culture techniques to obtain whole plants.The ploidy level of plants was determined using two methods: (a) evaluation of

chromosome number with karyotypic analysis, and (b)evaluation of the ADN nucleusvolume using flow-cytometry analysis.

Results and DiscussionPollen was able to germinate after irradiation with any of the doses used. Observationwith a fluorescent microscope revealed that the germinating tube grows to the ovule andmakes contact with it whatever radiation dose is used.

Fruit and seed development were obtained with all treatments. However, only treat-ments between 0 and 10 krad permitted embryo development. Moreover, endosperm-

CATIE/CIRAD-IRFA, PO Box 104, 7170 Turrialba, Costa Rica

vacant seeds were found in all the doses used, but at a higher percentage at doses inexcess of 8 krad. The lack of endosperm is an important parameter because it can berelated to fertilization absence, confirming the presence of haploid embryos.

Embryo rescue by in-vitro transfer permitted the culture of whole plants from seedsirradiated with treatments of 3, 5, 7, 8, and 10 krad.

Karyotype analysis revealed that many of the plants evaluated were diploid, althoughtwo haploid plants were found among them (one with a 5-krad treatment and the otherwith 7-krad). It is also possible that there were more haploid plants not detectedbecause they have the ability to double their chromosome number spontaneously.

For this reason, it is important to follow up this research in order to find a simple andaccurate technique for detecting homocigous plants.

182 Induced Parthenogenesis to Obtain Haploid Plants in Musa

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Cell-Culture Techniques

W Parrott

IntroductionThe discovery that plant cells and tissues could be grown in vitro created a new disciplinein the plant sciences. Cell-culture techniques quickly progressed beyond academiccuriosities as their potential for plant propagation and plant improvement becameevident. Cell-culture techniques have been extended to ever greater numbers of plantspecies, the techniques have become increasingly more refined, and the biology of thesystems better understood. Presently, cell- and tissue-culture technology for some speciesis much further advanced than for other species. Nevertheless, several universal patternsand similarities in procedures are now clearly evident across the most efficient cellculture systems. This information will be invaluable to optimize cell-culture techniquesfor those species still difficult to handle in vitro.

Definition of Terms In its broadest sense, cell and tissue culture encompasses virtually any techniquewhereby aseptic plant organs, tissues, or cells are grown on a nutrient medium insidesealed containers. Thus, this broad sense has come to include techniques as diverse asmeristem micropropagation, embryo, anther, tissue, and protoplast culture. In thestrictest sense, and in the sense used for this review, cell culture refers exclusively to theculture of nonmeristematic cells, a distinctly different process from micropropagation, inwhich the original meristematic state of the explant tissue is maintained in vitro andused to proliferate numerous plantlets. Micropropagation remains an efficient method formass propagation of, or virus elimination from, desired genotypes. Micropropagation canbe a source of novel variation, but thus far has not been readily amenable to gene transfertechnology.

In contrast to micropropagation, cell-culture techniques are not limited to explantsfrom meristematic tissue. Virtually any part of a plant can serve as the explant. Duringthe culture process, cells within the explant tissue divide to form a dedifferentiated tissue called a callus. From the perspective of plant improvement, cell-culture techniquesare most powerful when the callus can be induced to redifferentiate into shoots(organogenesis) or embryos (somatic embryogenesis) from which whole plants can then

Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602-7272, USA

be recovered in a process called regeneration. Because a callus phase intervenes prior toregeneration, such systems are said to be indirect. Various selection, mutagenesis, orgenetic transformation schemes may be superimposed on the system prior to indirectregeneration. However, at times it is possible for cells within the explant tissue toredifferentiate directly into shoots or embryos. Such direct regeneration systems aremore challenging to use for selection or transformation, but are less subject tosomaclonal variation than indirect systems.

Banana Cell CultureThe overwhelming amount of in-vitro research on banana has centered on micro-propagation techniques and will not be covered here. While organogenesis fromnonmeristematic explants of bananas or plantains has not been reported, de novoregeneration of Musa species has been achieved via somatic embryogenesis (Escalant,Teisson 1988; Cronauer-Mitra, Krikorian 1988; Novak et al. 1989). Somatic embryogenesis(reviewed in Merkle et al. 1990) offers advantages over regeneration via organogenesis.First among these advantages is that complete propagules are formed. As somaticembryos have both shoot and root meristems, separate shoot-induction and root-induction steps are not required. Another advantage is that somatic embryogenesis tendsto be less subject to somaclonal variation than organogenesis, presumably becausesomatic embryos are not very tolerant of somaclonal variation which can disrupt theirontogeny (Hanna et al. 1984; Ozias-Akins, Vasil 1988). This can be particularlyadvantageous during genetic transformation of a given genotype, in which case changesother than the engineered trait are undesirable. As technology continues to develop,somatic embryos will be amenable to large-scale production and artificial seedtechnology. Given the amenability of Musa species to somatic embryogenesis, optimi-zation of efficient embryogenic protocols and their use in genetic transformation is alogical goal.

Factors Affecting Somatic EmbryogenesisSomatic embryogenesis has several distinct stages, beginning with induction of theembryogenic state, followed by differentiation into an embryo, and subsequent matu-ration, germination, and conversion into plants. Induction of the embryogenic stateusually requires the presence of an auxin. The best type and concentration of auxin forinduction of somatic embryogenesis is determined empirically for each system. For Musaornata, 2,4-D [2,4-dichlorophenoxyacetic acid] (Cronauer-Mitra, Krikorian 1988) hasbeen effective, and Dicamba [3,6-dichloro-2- methoxybenzoic acid] has been effective for both bananas and plantains (Novak et al. 1989). Induction on 2,4,5-T [2,4,5-trichloro-phenoxyacetic acid] (Cronauer, Krikorian 1983) or induction on picloram [4-amino-3,5,6-trichloropicolinic acid] or Dicamba, followed by transfer to NAA [Ó-naphthaleneacetic

184 Cell-Culture Techniques

acid] (Escalant, Teisson 1988), has permitted the recovery of somatic embryos ofplantain. These results suggest that there is flexibility in the type of auxin used forinduction of somatic embryogenesis in Musa species. A series of experiments critically tocompare all the various growth regulators under controlled conditions should be usefulfor the optimization of the current regeneration protocols.

Several plant tissues can serve as explants, with the caveat that stage of developmentor maturity of the tissue can be very important. The one tissue that is most frequentlyembryogenic is the immature zygotic embryo and, in several species, somaticembryogenesis occurs exclusively from immature zygotic embryos. However, zygoticembryo explants are of limited use in vegetatively propagated cultivars, as they are adifferent genotype from the parent cultivar. For seedless species such as cultivatedbananas and plantains, the use of zygotic embryo explants is impossible. Meristematictissue in which cells are actively undergoing mitosis, sometimes can be embryogenic.Such tissues include immature leaves of dicotyledons, leaf bases and internodes ofmonocotyledons, and apices of shoots, corms, or rhizomes. For those species in which an extensive callus phase precedes the formation of somatic embryos, virtually any tissue can serve as an explant. While immature zygotic embryos from seeded bananas(Cronauer-Mitra, Krikorian 1988) or from artificial hybrids (Escalant, Teisson 1988) canbe induced to undergo somatic embryogenesis, the meristematic regions from shoot tips(Cronauer, Krikorian 1983) and rhizomes (Novak et al. 1989) are also embryogenic.

The induction of somatic embryogenesis theoretically involves the induction of thesame genetic pathways that lead to zygotic embryogenesis, and as such should be auniversal phenomenon in all angiosperms. Nevertheless, individual genotypes within aspecies can differ widely in their ability to undergo somatic embryogenesis (Parrott et al.1991). Hence such genotypic differences in embryogenic capacity probably reflectdifferences in the ability to activate the embryogenic pathway. When the molecularmechanisms of embryogenesis become understood, it should become possible to obtainsomatic embryos from virtually any genotype. Fortunately, the cultivated bananas andplantains both appear to be genotypes with embryogenic capacity under the currentprotocols.

The method by which auxins induce tissues to become embryogenic remainsunknown, but it probably includes a series of steps. First, the presence of an auxin hasbeen associated with DNA methylation, which putatively results in a decrease ortermination of gene expression programs existing within the cell. In addition, auxins canisolate cells or clumps of cells from the remaining tissue, either by severingplasmodesmata and increasing friability of tissues, or through necrosis of surroundingtissues. Isolation may be reinforced by the formation of callose (Dubois et al. 1990;Dubois et al. 1991). The role of isolation has been reviewed by Williams and Maheswaran(1986) and by Smith and Krikorian (1989). The third step appears to be the imposition ofpolarity, possibly as a consequence of polar auxin transport (e.g., Brawley et al. 1984;Chée, Cantliffe 1989). Accordingly, the imposition of polarity by the application of anexternal electrical field has increased the formation of somatic embryos in alfalfa (Dijaket al. 1986).

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Once an embryogenic state has been induced, the continued presence of an auxin at asufficiently high level in the medium can arrest the development of somatic embryos.Depending on the species, this arrest can occur at any time during ontogeny, ranging fromthe proembryo stage all the way to an early cotyledonary stage. A few species can evenform mature somatic embryos in the presence of an auxin, but these have absent orpoorly developed meristems (Halperin, Wetherell 1964; Parrott et al. 1988), which latermakes it very difficult to convert such embryos into plants. Hence, it is always preferableto remove the auxin from the culture medium as early as possible and, in some cases, addactivated charcoal to the medium to assist in the auxin removal process (Zaghmout,Torello 1988; Ebert, Taylor 1990).

When enough auxin is present in the medium to arrest embryogenesis at a proembryoto globular stage of development, the induced embryogenic state can be maintained forvery long periods of time (Terzi, LoSchiavo 1990). This can give rise to cycles of repetitiveembryogenesis, in which the original somatic embryos, instead of completing theirontogeny, give rise to other somatic embryos in a cyclical process. Such a system ofrepetitive or recurrent embryogenesis can effectively replace a callus phase during in-vitro selection or genetic transformation by Agrobacterium (McGranahan et al. 1988;McGranahan et al. 1990) or by microprojectile bombardment (Finer, McMullen 1991).The cycle of repetitive embryogenesis is broken by the removal of auxin from the medium,which allows somatic embryos to complete their development.

Once somatic embryos have reached a cotyledonary stage of development, theyrequire a maturation stage. The length of the maturation period is approximately as longas that required by zygotic embryos maturing in planta. The maturation process takesplace in the absence of exogenous growth regulators, a phenomenon that may reflect theevents that occur in planta, whereby cytokinin levels (Carman 1989) and auxin levels(Carnes, Wright 1988) in the ovule decrease once embryos are formed. As discussedpreviously, auxins inhibit the normal development of meristems, and cytokinins causeswollen hypocotyls in the resulting somatic embryos.

Nevertheless, there is an apparent requirement for abscisic acid (ABA) for normalmaturation of embryos. The continued presence of ABA beyond a few hours is probablynot necessary, and a short pulse will initiate the synthesis of proteins associated with thelate stages of embryogenesis and desiccation tolerance (Galau et el. 1990). While somaticembryos from most species probably have sufficient endogenous ABA that they do notrequire exogenous applications, some species exhibit improved conversion followingexposure to ABA (Senaratna et al. 1989), and others, notably gymnosperms, have anabsolute requirement for exogenous ABA to achieve normal maturation (Roberts et al.1990).

There are a few reports that maltose can be superior to sucrose (Button 1978;Strickland et al. 1987), but thus far, very little attention has been placed on thecarbohydrate source used during maturation. However, recent reports (Finer, McMullen1991), as well as current results from our laboratory, indicate that the use of 6-12%maltose promotes better embryo development than the traditional sucrose at anyconcentration. The use of maltose in a wider range of species merits further investigation.


Somatic embryos of some species germinate readily and convert into plants, whilethose of other species germinate slowly and only sporadically convert into plants.Subjecting such mature somatic embryos to a desiccation step has been associated withenhanced germinability and conversion into plants in a range of monocotyledonous anddicotyledonous species (reviewed in Parrott et al. 1991). Embryos developing in plantaundergo desiccation at the end of their maturation period, and desiccation has beenassociated with the synthesis of germination-specific proteins (Rosenberg, Rinne 1986,1988). Protocols that have used high sucrose levels (e.g., Janick 1986) or paper bridges tosuspend embryos above the medium (e.g., Cronauer-Mitra, Krikorian 1988) to achieveconversion may, in fact, have been achieving a degree of desiccation through osmoticstress. In addition, the use of ABA during the maturation process can enhancedesiccation tolerance and plant conversion (Kumar et al. 1988; Senaratna et al. 1991) insome species.

Germination of somatic embryos follows the return of embryos to a medium devoid ofall growth regulators. Once seedlings have well developed roots and shoots, they aretransferred to soil and hardened off. If a particular species is sensitive to daylength,attention should be provided to the length of the photoperiod to prevent the prematureinduction of flowering.

Cell Culture, In-Vitro Selection, and SomaclonalVariationOne of the characteristics frequently associated with plants regenerated from cell cultureis that regenerated plants can differ by one or more traits from the plant that served asan explant donor. Such changes tend to occur in higher frequencies and represent moretypes of mutations than those resulting from conventional mutagenesis programs (Larkinet al. 1989). Depending on the research objectives, such variation may either be desirableor undesirable. Consequently, cell-culture protocols may be manipulated either toincrease or decrease the amount of somaclonal variation that is recovered.

In a case where genotypic fidelity is desired, such as during the genetic transforma-tion of an elite cultivar, somaclonal variation is most probably undesirable. As mentionedearlier, the use of somatic embryogenesis instead of organogenesis will limit the amountof somaclonal variation recovered. In addition, as the incidence of somaclonal variationincreases with time in culture, somaclonal variation can be decreased by avoiding the useof long-term cultures and reducing the amount of time in culture to a minimum. Finally,callus tissue is notoriously susceptible to somaclonal variation. Hence it becomesespecially important to minimize the length of any callus phase that may be present. Ifpossible, repetitive embryogenesis should be used as a substitute for a callus phase.

Alternatively, when maximizing somaclonal variation is the objective, such variationcan be increased by increasing the amount of time in culture, especially the callus phase.The micropropagation of adventitious buds can also exhibit large amounts of variability(De Klerk 1990). Of particular interest to banana and plantain improvement, somaclonal

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variation for disease resistance has been reported previously, in a range of species from poplars resistant to Septoria musiva (Ostrey, Skilling 1988) to tomato resistant to Fusarium (Evans 1989) and Pelargonium resistant to Xanthomonas (Dunbar,Stephens 1989).

Perhaps the largest drawback to somaclonal variation is that it remains a randomprocess (Evans 1989). When a specific type of somaclonal variant is desired, such asresistance to a pathotoxin, the process can be made less random through the use of in-vitro selection, a process which can select for dominant mutations when the mechanismof resistance at the cell level is the same as at the whole-plant level. The incorporation of pathotoxins into the culture medium has permitted the recovery of tobacco resistant to Pseudomonas and Alternaria (Thanutong et al. 1983), oilseed rape to Alternaria(MacDonald, Ingram 1986), and celery to Fusarium (Heath-Pagliuso et al. 1988).However, in at least the case of Leptosphaeria resistance in oilseed rape (Newsholme etal. 1989) and Verticillium resistance in alfalfa (MacDonald, Ingram 1986), the resis-tances proved to be either transient or nonheritable, underscoring a potential limitationto in-vitro selection.

Given that apparently some callus formation precedes the formation of somaticembryos, Musa species should be amenable to in-vitro selection by incorporating culturefiltrates of Sigatoka or Panama disease in the culture medium. In addition, the ability togrow banana cells in suspension (Novak et al. 1989) prior to the onset of embryogenesiscan facilitate the efficiency of in-vitro selection.

Protoplast Culture The ability to regenerate whole plants from isolated, single cells of plants represents oneof the greatest achievements of academic importance for plant cell culture. However, theagricultural importance of protoplasts is much less clear at the present time. Techniquesstill in their infancy, such as asymmetrical hybridization or cytoplasm transfer, willcontinue to require the use of protoplasts. Other uses for protoplasts, such asinterspecific hybridization and genetic transformation, have received considerableattention.

Regeneration of plants from protoplast culture is extremely mutagenic and limited tovery few species. It requires tremendous inputs of expertise and resources, and simpleralternatives are available. With the advent of microprojectile bombardment andrepetitive embryogenesis technology, it is now clear that protoplasts are not necessary forplant transformation. While various reports of interspecific hybridization throughprotoplast fusion are available, simpler approaches were not tried prior to protoplastfusion. Approaches such as embryo rescue, in-vitro pollination, ovule culture, etc., shouldbe exhausted before the use of protoplasts can be readily justified. These techniques lackthe glamor of protoplast culture, but are much simpler and more economical. In the caseof Musa species, the greatest return on investment can probably be best achieved by firstdevoting available resources to improving the efficiency of embryogenic and cell selectiontechniques.


Conclusions The potential of cell-culture techniques to help solve a variety of agronomic problems haslong been recognized, but results have usually fallen short of expectations. The use ofcell-culture techniques offers the greatest potential in a genus such as Musa, which ischaracterized by the large-scale planting of elite, sterile genotypes. To the degree towhich it is applicable, conventional breeding of bananas and plantains results in the lossof critical agronomic or horticultural characters, making it of limited value.

The optimization of efficient regeneration techniques for this genus is necessarybefore cell-culture technology can be exploited. Fortunately, enough information isavailable to determine that efficient regeneration protocols are within reach for bothbananas and plantains. Once such systems are in place, genetic transformation and in-vitro selection systems can be incorporated into the cell-culture protocols.

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cotyledon differentiation. Proc. Natl Acad. Sci. (USA) 81:6064-6067. BUTTON J. 1978. The effects of some carbohydrates on the growth and organization of Citrus ovular callus. Z.

Pflanzenphysiol. 88:61-68. CARMAN JG. 1989. The in ovulo environment and its relevance to cloning wheat via somatic embryogenesis. In

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Dekalb XL-12. Plant Sci. 57:195-203. CHÉE RP, CANTLIFFE DJ. 1989. Embryo development from discrete cell aggregates in Ipomoea batatas (L.)

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Plant Cell Rep. 2:289-291. CRONAUER-MITRA SS, KRIKORIAN AD. 1988. Plant regeneration via somatic embryogenesis in the seeded diploid

banana Musa ornata Roxb. Plant Cell Rep. 7:23-25. DE KLERK GJ. 1990. How to measure somaclonal variation. Acta Bot. Neerl. 32:129-144. DIJAK M, SMITH DL, WILSON TJ, BROWN DCW. 1986. Stimulation of direct embryogenesis from mesophyll

protoplasts of Medicago sativa. Plant Cell Rep. 5:468-470. DUBOIS T, GUEDIRA M, DUBOIS J, VASSEUR J. 1990. Direct somatic embryogenesis in roots of Cichorium: Is callose

an early marker? Ann. Bot. 65:539-545. DUBOIS T, GUEDIRA M, VASSEUR J. 1991. Direct somatic embryogenesis in leaves of Cichorium. Protoplasma

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plant tissue culture media in the presence of activated charcoal. Plant Cell Tissue Organ Cult. 20:165-172. ESCALANT JV, TEISSON C. 1988. Embryogenèse somatique chez Musa sp. C. R. Acad. Sci. Paris 306:227-281. EVANS DA. 1989. Somaclonal variation — genetic basis and breeding applications. TIG 5:46-50. FINER JJ, MCMULLEN MD. 1991. Transformation of soybean via particle bombardment of embryogenic

suspension culture tissue. In Vitro Cell. Dev. Biol. 27P:175-182.GALAU GA, JAKOBSEN KS, HUGHES DW. 1990. The controls of later dicot embryogenesis. Physiol. Plant. 81:280-

288. HALPERIN W, WETHERELL DF. 1964. Adventive embryony in tissue cultures of the wild carrot, Daucus carota.

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HANNA WW, LU C, VASIL IK. 1984. Uniformity of plants regenerated from somatic embryos of Panicum maxi-mum Jacq. (Guinea grass). Theor. Appl. Genet. 67:155-159.

HEATH-PAGLIUSO S, PULLMAN J, RAPPAPORT L. 1988. Somaclonal variation in celery: screening for resistance toFusarium oxysporum f.sp. apii. Theor. Appl. Genet. 75:446-451.

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KUMAR AS, GAMBORG OL, NABORS MW. 1988. Plant regeneration from cell suspension cultures of Vignaaconitifolia. Plant Cell Rep. 7:138-141.

LARKIN PJ, BANKS PM, BHATI R, BRETTELL IS, DAVIES PA, RYAN SA, et al. 1989. From somatic variation to variantplants: mechanisms and applications. Genome 31:705-711.

MACDONALD MV, INGRAM DS. 1986. Towards the selection in vitro for resistance to Alternaria brassicicola(Schw.) Wilts., in Brassica napus ssp. oleifera (Metzg.) Sinsk., winter oilseed rape. New Phytol. 104:621-629.

MCGRANAHAN GH, LESLIE CA, URATSU S, MARTIN LA, DANDEKAR AM. 1988. Agrobacterium-mediatedtransformation of walnut somatic embryos and regeneration of transgenic plants. Bio/Tech. 6:800-804.

MCGRANAHAN GH, LESLIE CA, URATSU SL, DANDEKAR AM. 1990. Improved efficiency of the walnut somatic embryogene transfer system. Plant Cell Rep. 8:512-516.

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NEWSHOLME DM, MACDONALD MV, INGRAM DS. 1989. Studies of selection in vitro for novel resistance tophytotoxic products of Leptosphaeria maculans (Desm.) Ces. & De Not. in secondary embryogenic linesof Brassica napus ssp. oleifera (Metzg.) Sinsk., winter oilseed rape. New Phytol. 113:117-126.

NOVAK FJ, AFZA R, VAN DUREN M, PEREA-DALLOS M, CONGER BV, XIAOLANG T. 1989. Somatic embryogenesis andplant regeneration in suspension cultures of dessert (AA and AAA) and cooking (ABB) bananas (Musaspp.). Bio/Tech. 7:154-159.

OSTREY ME, SKILLING DD. 1988. Somatic variation in resistance of Populus to Septoria musiva. Plant Dis.72:724-727.

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192 Somaclonal Variation in Musa sp.: theorical risks, risk management, future research prospects

Somaclonal Variation in Musa sp.:theoretical risks, risk management, future researchprospects

FX Côte1, X Perrier2, C Teisson1

IntroductionOver recent years in-vitro micropropagated Musaceae have been more and more widelyused both for the international exchange of germplasm and the industrial productionof healthy planting material. As a result of the increase in the latter use, the banana isprobably the most intensely micropropagated crop.

The appearance of somaclonal variations has introduced certain limits (Reuveni etal. 1986; Arias, Valverde 1987; Hwang, Ko 1987; Stover 1987; Daniells 1988; Vuylsteke etal. 1988; Krikorian 1989; Smith, Drew 1990; Israeli et al. 1991; Sandoval et al. 1991).However, considerable experience has been acquired, from which the following mainconclusions may be drawn.1. The main somaclonal variations that affect Musa are stable in the field after

several crop cycles (Vuylsteke et al. 1988; Israeli et al. 1991). 2. The percentage of variants varies considerably, including in apparently identical

micropropagation conditions.3. The main characters concerned by the variations are those most frequently subject

to natural mutation (Stover 1988; Vuylsteke et al. 1988).The two first above characteristics led us to approach the problem from the

theoretical point of view: we looked for a mathematical model able to describe thevariations in the percentage of variants over several successive cycles of in-vitromultiplication. The conclusions we drew from this study allowed us to define a numberof pragmatic production strategies to limit the problem by combining risk managementwith early identification of variants. The outlook for future research is described.

1CIRAD-BIOTROP, Laboratoire de Culture in vitro, BP 5035, 34032 Montpellier Cedex 1, France; 2CIRAD-IRFA,Laboratoire de Biométrie, BP 5035, 34032 Montpellier Cedex 1, France

193FX Côte, X Perrier, C Teisson

Theoretical Variations in the Percentage ofVariants over Successive Multiplication Cycles

Hypothesis and method of calculationThe following hypotheses were put forward:• Only stable in-vitro variations are considered. Once a variation has occurred, all

plants derived from this off-type plant are also variants.• The multiplication rate of variants is identical to that of true-to-type plants.• The probability of variation is constant during successive multiplication cycles and is

independent of the rate of proliferation.• Variation can only affect a meristem during the initial stages of differentiation; as

soon as this has taken place, risk of variation is zero in the plantlet concerned. For simplification, we are not concerned with proliferation by direct multiplication of

terminal meristems after splitting or fasciation.In these conditions, if p is the probability that a meristemic mass undergoes

variation, t, the rate of multiplication and n the number of cultivation cycles, applicationof the recurrence relation shows that the percentage of variants will be equal to :

1 - [ 1 - ( p {t -1 } / t)] n x 100 called: function 1(cf Appendix 1 for demonstration).Change in the percentage of variants is thus an exponential function which tends

towards 100 when n tends to infinity. This function depends on the rate of proliferation.Variation in the percentage of variants as a function of the number of cultivation

cycles is shown in Figure 1 for the values p = 0.01, n = 1 to 20, and t = 2. The value of pwas selected with reference to studies by Reuveni and Israeli (1990), Vuylsteke et al.

12

10

8

6

4

2

00 2 4 6 8 10 12 14 16 18 20

% variants

Subcultures

Figure 1. Theoretical variation in the percentage of off-type plants over successivemultiplication cycles.Hypothesis of calculation: y = 1 - [ 1 - ( p {t -1 } / t)]n (cf Appendix 1). n = the number of multiplicationcycles, p = 0.01 is the probability that a meristemic mass undergoes variation, t = 2 is the proliferationrate.


(1991), and Sandoval et al. (1991), who, in bananas of the Cavendish group and inplantains, observed rates of variants of a few percent after a small number of multi-plication cycles. Taking into account the parameters selected, the curve of the function(1) may be considered as a linear one over the first 20 multiplication cycles. The wholeprogeny of variants is off-type, and this is responsible for a large proportion of the totalpercentage of variants: after several cycles this percentage is far superior to theprobability of appearance of variations.

Calculation of the variance of function 1-[1-(p{t-1}/t)]n

Function (1) gives the expectation of the proportion of variants after n multiplicationcycles. This value corresponds to the mean of the rates obtained with an infinite numberof lines derived from suckers established in vitro. However, very different percentages ofoff-types may be obtained, depending on how early variations occur. Let us consider thetheoretical case where t = 2: if only one variation occurs during the first multiplicationcycle, 50% of the population will be off-type. With the same probability, if only onevariation occurs during the second multiplication cycle, 25% of the descendants will beoff-type, during the third multiplication cycle, 12.5%, and so on.

The variance of function (1) cannot be analytically expressed; numerical examples ofpercentage of variants were determined using simulations. The simulations were carriedout, for a given probability, p, by introducing variations in a random manner using acomputer program. With t = 2, and p = 0.01, the population would include an average of7.2% of variants after 15 multiplication cycles. Variance of this average determined after500 simulations is shown in Figure 2. The distribution obtained is not normal; values of 3to 55% of variants per line are observed with a mode of 5%. The peaks around 29 and 53%

50

40

30

20

10

01 7 13 19 25 31 37 43 49 55

% lines

% variants

Figure 2. Estimation of the variance of function: 1 - [ 1 - (p {t - 1} / t)]n. Hypothesis of calculation: n = 15, p = 0.01, t = 2 (see caption of Fig.1). The estimation is determined after 500 simulations of function (1). Results are expressed in percentageof lines exhibiting a given percentage of variants. The appearance of variation is introduced in a randommanner. The line is a population of plantlets derived from one sucker. Expectation of the meanpercentage of variants = 7.2%.

correspond to the rare cases when variations appeared in the first and secondmultiplication cycle respectively.

The main information obtained from the above calculations is:- there is an increase in the percentage of variants during successive cultivation

cycles; and- quite different and unpredictable rates of variants can be generated irrespective

of the source of the material or the probability of the appearance of a variation.

Limiting the Risks of Variation linked to the useof PlantletsProduction managementThe mathematical approach suggested two basic ways to reduce the risk of somaclonalvariations:• Reduce the number of proliferation cycles to limit the multiplication of variant

plantlets.• Use batches of plants from the greatest possible number of lines to reduce the

possibility of rare ‘accidents’ linked to the early appearance of variants.These two strategies are already used, though doubtless not consistently enough, by

industrial production units. They are more difficult to apply in the case of a germplasmbank.

Removal of variants before plantingThe management plan described above should be completed by the earliest possibleremoval of the largest possible number of variant individuals before planting.

In the case of a stable variation, in-vitro reintroduction and acclimatization of theseoff-types means a model will be available to facilitate early visual identification ofmorphological characters of variants. This method has been used to characterize dwarfvariants in cv Grande Naine. During hardening, material obtained from off-types may bedistinguished by morphological characters resembling those of dwarfism in adult plants,mainly reduced pseudostem height, a short petiole and leaf length (Côte et al. 1991).

In the case of a large batch of plants hardened in natural conditions, identification isdifficult. In these circumstances, identifying plants with morphological characters thatfall between those of dwarf and true-to-type plants will be problematic. In order tofacilitate identification in this particular case, we tested the use of gibberellin, as didReuveni (1988) in vitro.

Application of GA3 resulted in lengthening of the petiole in dwarf and true-to-typeplants. Nevertheless their reaction to the growth regulator was different. In variantplants, only growth of the first leaf to emerge after treatment was stimulated, whereas,in true-to-type plants, stimulation was already observed in leaves developing at the timeof treatment (Côte et al. 1991). Consequently there was a temporary accentuation in themorphological difference between the two types of banana.


Conclusion and Outlook for Further Research In order to achieve better control of variation during micropropagation, more studies areneeded on risk management and on phenonema that are responsible for the variations.

Risk managementThe theoretical model proposed is based on a number of hypotheses which certainly donot accurately represent the complexity of the phenomena. The model will undergomodification as our knowledge of somaclonal variation increases. However, in themeantime we hope it will suggest new fields of investigation. Our lack of knowledgeabout the biological significance of risk p is one of its main limitations, but neverthless itis a model that may have broader applications.

Compiling percentages of variation observed in large-scale trials or in industrialproduction would enable us to determine the value p more precisely, and there is a needfor information from the scientists involved in these topics. Compilation should be carriedout using batches of plants produced with identical micropropagation techniques. Thevalue of p calculated in this way would enable us to determine the maximum number ofplantlets that should be produced from one sucker for a given risk of variation.

Already the conclusion to be drawn from the range of percentages of variants usingthe same micropropagation technique demonstrated with this model is that experimentsto identify the origin of variations must be carried out on very large batches of plants.

Budding and its effect on variationThe exact way of budding and proliferation of Musaceae may be of primary importancefor the occurrence of somaclonal variation, as is the case in other species (Swartz 1991).In vitro, the great complexity rapidly attained in the proliferating shoots does notfacilitate precise description. The problem nevertheless deserves attention, particularlythe possible difference between neoformed meristem and simple multiplication of theterminal meristem, either after physical mutilation or due to a phenomenon resemblingfasciation.

Use of variation markersThe use of variation markers has several advantages: it can facilitate productionmanagement, the experimental approach by limiting the need for field trials, and furtherknowledge of the origin of the variations.

The dwarf variation in bananas of the Cavendish group could be used as a model inthe search for early markers. This variation is stable and has similarities with naturaldifferentiation in cultivars in this group. It is also the most common variation in the mostfrequently used bananas for in-vitro multiplication. The study of variation markers inother cultivars, and particularly those used in germplasm banks, is probably much moredifficult, given the diversity of genotype, the range of possible variations, and the limitednumber of individuals of a given genotype.


Production management. An extremely limited number of culture cycles may be thesimplest way to reduce the number of variants. This necessitates the frequentreintroduction of plants in vitro, which in turn increases the cost of plantlet production.The use of early markers may provide a way round this problem. Regular screening wouldmean that only plantlets identified as true-to-type are used for a limited number ofmultiplication cycles. The number of variants could be reduced to a very low value byfrequent screening. This method presupposes a preliminary experimentation to ensurethat risks of variation are identical for a population obtained from a true-to-type plantletor from a sucker taken from the field.

Types of early markers

Morphological markers. Identification of variants on a morphological basis at thetest-tube level would be very useful. However, Israeli et al. (1991) have observed that thismethod is possible only for a few types of variants.

Biochemical markers. Modifications in the endogenous hormone balance arefrequently involved in somaclonal variation. The study of gibberellin metabolism isparticularly interesting in the case of dwarfism.

The use of protein markers can also be envisaged, and has already been tested byReuveni et al. (1986).

Molecular markers. This should be one of the preferred methods for studying theorigin of the variations. There are several research programs under way using molecularbiology tools to classify closely related cultivars. Considering the similarity betweennatural differentiation and the main somaclonal variations, it is tempting to envisagecharacterizing variants using the same methods. A project of this type is already underconsideration involving international collaboration between different research teams.

Better knowledge of the origin of somaclonal variations in the banana has implicationsthat go far beyond the already vast question of micropropagation by budding for massmultiplication and the conservation of germplasm. Efforts are currently under way todevelop somatic embryogenesis as a back-up for genetic improvement programs. It is likelythat similar problems of variation will occur in bananas produced with these techniques.

Appendix 1

Theoretical variations in the percentage of variants oversuccessive multiplication cycles: methods of calculation (see hypothesis of calculation in the text)

If p is the probability that a meristem undergoes variation during the early differenti-ation stage, t, the rate of multiplication, mn, the number of true-to-type plantlets in apopulation after n cycles of multiplication:


• After n + 1 cycles of multiplication, t x mn plantlets are issued from the mn plantletsamong which (t - 1)mn are neoformed plantlets and mn were present at the previouscycle.

• The probability for a neoformed plantlet to be a true-to-type one is (1 - p); thus thetotal number of true-to-type plantlets after n + 1 cycle is

mn+1 = mn + (1 - p) (t - 1)mnmn+1 = mn [1 + (1-p) (t - 1)]

ormn+1 = mn [(t - p(t - 1)].

Thus, as mo = 1; m1 = [t - p(t - 1)]; m2 = [t - p(t - 1)]2 ...and mn = [t - p(t - 1)]n.

• After n cycles of cultivation, tn plantlets are produced and the number of off-typeplantlets is t n - [t - p(t - 1)]n.The proportion of variants is

t n - [t - p(t - 1)]n

tn

and the percentage of variants is 1 - [1 - p(t-1)]n x 100.

t

ReferencesARIAS O, VALVERDE M. 1987. Producción y variación de plantas de banano variedad Grande Naine producida por

cultivo de tejidos. ASBANA 28:6-11.CÔTE FX, FOLLIOT M, KERBELLEC F, TEISSON C. 1991. Morphological characterization of banana vitroplantlets (cv

Grande Naine) propagated from dwarf off-type and true-to-type plants during hardening. Effects ofgibberellin treatment. ACORBAT Symposium 1991, Tabasco, Mexico (in press).

DANIELLS JW. 1988. Comparison of growth and yield of bananas derived from tissue culture and conventionalplanting material. Banana Newsletter 11.

HWANG SC, KO WH. 1987. Somaclonal variation in vitro of banana and its application for screening forresistance to fusarium wilt Pages 151-156 in Banana and Plantain Breeding Strategies (Persley GJ, deLanghe EA, eds). ACIAR Proceedings no.21. Canberra, Australia: ACIAR.

ISRAELI Y, REUVENI O, LAHAV E. 1991. Qualitative aspects of somaclonal variations in banana propagated by invitro techniques. Scientia Hortic. 48:71-78.

KRIKORIAN AD. 1989. In vitro culture of bananas and plantains: background, update and call for information.Tropical Agriculture (Trinidad) 66:194-200.

REUVENI O. 1988. Methods of detecting somaclonal variants in “Williams” bananas. Pages 108-113 in TheIdentification of Genetic Diversity in the genus Musa, (Jarret RL, ed.). Montpellier, France: INIBAP.

REUVENI O, ISRAELI Y. 1990. Measures to reduce somaclonal variation in in vitro propagated bananas. ActaHorticulturae 175:307-313.

REUVENI O, ISRAELI Y, ESHDAT Y, DEGANI H. 1986. Genetic variability of banana plants multiplied via in vitrotechniques. Final report submitted to IBPGR PR3/11. Israel: The Volcani Center.

SANDOVAL JA, TAPIA SACM, VILLALOBOS AO. 1991. Observaciones sobre la variedad encontrada en plantasmicropropagadas de Musa cv “Falso corno” AAB. Fruits 46:533-539.

SMITH MK, DREW RA. 1990. Growth and yield characteristics of dwarf off-types recovered from tissue-culturedbananas Aust. J. Exp. Botany 30:575-578.


STOVER RH. 1987. Somaclonal variation in Grande Naine and Saba bananas in the nursery and field. InBananas and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds). ACIAR Proceedings no.21.Canberra, Australia: ACIAR.

STOVER RH. 1988. Variation and cultivar nomenclature in Musa, AAA, group, Cavendish subgroup. Fruits43:353-357.

SWARTZ HH. 1990. Post culture behavior: genetic and epigenetic effects and related problems. Pages 95-121 inMicropropagation: Technology and Application (Debergh PC, Zimmerman RH, eds). Dordrecht, theNetherlands: Kluwer Academic Publishers.

VUYLSTEKE D, SWENNEN R, WILSON GF, DE LANGHE E. 1988. Phenotypic variation among in vitro propagatedplantain (Musa sp. cultivar “AAB”). Scientia Hortic. 36:79-88.

VUYLSTEKE D, SWENNEN R, DE LANGHE E. 1991. Somaclonal variation in plantains (Musa spp., AAB group)derived from shoot-tip culture. Fruits 46:429-439.


200 Early Detection of Somaclonal Variation

Early Detection of SomaclonalVariation

LA Withers

In-Vitro Technology in AgricultureIn-vitro (tissue culture) techniques have revolutionized many aspects of agriculture overrecent years. Mass propagation in vitro is routine for large numbers of agricultural,horticultural, and forest species. The impact of in-vitro propagation is probably greatestfor clonally propagated species but it is not limited to them (Beversdorf 1990).

Propagation can be linked, with great benefit, to disease eradication and diseaseindexing, sometimes carried out in vitro, with the effect of increasing the scope andsafety of germplasm exchange (IBPGR 1988). Moreover, these processes can be carriedout in locations distant and climatically different from the natural growing region of thespecies in question, thereby remote from sources of infection with pathogens and risks ofweather damage. Mass propagation can make elite material available to growers at amuch earlier stage, thus enabling the efforts of the plant breeder to reach the farmermore rapidly.

Biotechnological methods based on in-vitro culture have already made an impact oncrop improvement and will become more important in the future as transformationtechniques become refined and more widely applicable (Tomes 1990). In the context ofresearch, mass propagation can make uniform genotypes available to scientists, therebyeliminating one variable in experimental design.

Genetic resources conservation is a crucial requirement for crop improvement andproduction. On the one hand, biotechnology and in-vitro culture amplify some of theproblems of genetic conservation, e.g., by increasing the risks of narrowing of the genepool through facilitating monoculture. However, on the other hand, they present new andimproved ways of studying, understanding, and utilizing genetic diversity, and ofconserving genetic resources.

In-vitro conservation was a matter of speculation and curiosity little more than 15years ago. Yet it now occupies a firm place in the portfolio of techniques used tosafeguard genetic resources (Henshaw 1975; Withers 1980, 1989). Slow-growth storagefor short- to medium-term conservation is now routine for many crops including bananaand plantain (Dodds, 1991; Schoofs, 1990; Withers, 1987; 1990). Cryopreservation

International Board for Plant Genetic Resources (IBPGR), Via delle Sette Chiese 142, 00145 Rome, Italy

(storage in liquid nitrogen) is virtually routine for cell-suspension cultures such as areused in physiological experiments, secondary-product synthesis, and some mass-propagation systems (Withers 1991a). Techniques are less well developed for organizedcultures, although recent years have seen dramatic progress and the range ofsuccessfully cryopreserved species is now extensive. Particularly promising are the newtechniques of encapsulation-dehydration and of vitrification, which appear to be able topreserve structural integrity in complex explants such as shoot-tips and embryos(Dereuddre et al. 1990; Towill 1990; Withers 1992).

However, in propagation, in breeding and in conservation, there is one particularspectre that haunts the stage, namely somaclonal variation. The following sectionsexplore the nature and potential impact of this phenomenon in general and in the case ofMusa in particular.

Somaclonal Variation and MusaIn-vitro culture practitioners have been aware for many years of the fact that what comesout of a culture procedure may well not be the same as that which goes into it. Theculture process can lead to both genotypic and phenotypic changes (Larkin, Scowcroft1981; Lee, Phillips 1988; Phillips et al. 1990; Scowcroft 1984). Cultures can exhibitchanges in ploidy, chromosome number and structure, individual DNA base pairs,morphogenic potential, secondary product yields, disease resistance, and isozymecomplement. Change is not an “unnatural” characteristic (Hohn, Dennis 1985; Walbot,Cullis 1985); indeed, somatic mutations have played an important part in theimprovement of crops including Musa (Stover, Simmonds 1987). However, in vitro, itappears that the rate of change can be significantly accelerated. The concept of theabsolute clone in vitro appears to be wrong and, if used incautiously as the basis forprocedures with impact upon the genotype and phenotype of the in-vitro progeny, carriesserious consequences. It is safer to consider the “clone” to be a tight population, themake-up of which can vary with time in terms of the range of genotypes present and thefrequency of the predominant genotype.

In the history of in-vitro linked variation, scientists moved from a position ofignorance of its existence, to awareness and fear of its consequences, but with little opendiscussion of the phenomenon. This was until the potential benefits in crop improvementbecame apparent, coincident with the coining of the appealing and judgmentally neutralname “somaclonal variation” (Larkin, Scowcroft 1981; Scowcroft 1984). A period ofexcitement (and much investment) was followed by one of disappointment at therelatively few useful agronomic variants generated in vitro, and on to a period of realism,the situation today. Somaclonal variation exists and is one of the factors to key into theequation when planning and executing in-vitro procedures.

Why is there special concern over somaclonal variation in the case of Musa? Theanswer to this question is related to the valuable niche that in-vitro technology occupies

201LA Withers

in banana and plantain production, improvement, and conservation. A generalappreciation of the importance of in-vitro technology in these aspects of agriculture isgiven above. Some specifics for Musa can be added to reinforce the argument.

There is a huge demand for planting material in regions of the world where bananaand plantain are important staple and export crops (Israeli et al. 1991; Vuylsteke,Swennen 1990). In-vitro propagation is the obvious and proven method to meet needs.Spread of disease in planting material is a serious problem that can be tackled by theexchange of cultures rather than in-vivo plant parts (Frison, Putter 1989; Schoofs 1990).Breeding for badly needed disease resistance in Musa could be accomplished bybiotechnological approaches (Hwang 1990; Krikorian, Cronauer 1984; Novak et al. 1987;Stover, Buddenhagen 1986). The conservation of Musa genetic resources is problematic.Seeds are produced only by wild species. Sterile genotypes are, conventionally, conservedin field gene banks where costs, risks of disease, weather damage, and vandalism all poseserious threats to security (Withers, Engels 1990).

In-vitro conservation, mainly by slow growth but with encouraging progress incryopreservation, is providing a welcome alternative to the field gene bank. The mostwidely used propagation system of meristem proliferation, also used for slow-growthstorage, is adventitious/semiadventitious (Banerjee et al. 1986). Such a system isconsidered risky from the point of view of somaclonal variation, although, in Musa,variants have been found even in directly regenerating, nonadventitious shoots(Vuylsteke, Swennen 1990). By its very nature, slow growth imposes stresses that couldbe selective.

Successful cryopreservation work to date has involved the use of the apparently evenmore risky culture system of the embryogenic cell suspension (Panis et al. 1990).Promising as this technique is, cell and cell aggregate survival are well below 100% and,in principle, subject to risks of selection. Recovery appears to favor unorganized cellaggregates rather than organized, early-stage embryos. In-vitro improvement strategiesinvolve similar systems or even potentially more unstable protoplasts. Whilst no data areyet available to suggest the existence of stability problems attributable to storage orgenetic manipulation per se, these procedures themselves depend upon the developmentof satisfactory propagation systems.

Evidence to date of somaclonal variation in Musa, gathered mainly, but notexclusively, from the standard mass-propagation system of in-vitro meristem proliferation,bears out the anticipated risks. Many studies have documented cases in Cavendishbananas (Musa spp., AAA group) with a smaller number describing variation in plantains(Musa spp., AAB group) (Arias, Valverde 1987; Hwang 1986; Israeli et al. 1991; Pool,Irizarry 1987; Ramacharan et al. 1987; Reuveni 1990; Reuveni et al. 1986; Smith 1988;Stover 1987; Vuylsteke, Swennen 1990; Vuylsteke et al. 1988).

Qualitative and quantitative details were given in other presentations at thisworkshop. For the purposes of the present topic, some observations on the nature ofsomaclonal variation in Musa, taken largely from the work of Vuylsteke and colleagues(Vuylsteke, Swennen 1990; Vuylsteke et al. 1988) can help us characterize thephenomenon in Musa and put it into perspective.


The variants generated in vitro are limited in number and most are not unique to in-vitro propagated material; they have been observed in the field but at lower frequencies.The type and frequency of variation is genotype-dependent. A spectrum of variants canarise from one explant and, whilst the phenomenon is clearly related to culture andprobably to stress factors present in culture, for most variants it does not generallyappear to be linked in magnitude to length of time in culture. (It must, however, be notedthat the apparent linkage or lack of linkage between time in culture and variation coulddepend upon the multiplication regime and the contribution that one subculturing cyclemakes to the subsequent one.) There is evidence suggesting the variation may be geneticin nature. However, reversion is observed in culture, thus showing that the unstablenature of some genotypes is conserved in vitro. This provides the chance of recovering theoriginal genotype, but the problem of control remains.

A unifying characteristic of the different variants is that they are generally manifestedas changes in the plant’s habit, foliage, or inflorescence structure. Therefore, they areusually detectable only at the nursery, field, or even the fruit-production stage. Earliervisual detection in the laboratory or at the hardening stage before transfer to the nurseryis possible only in a very limited number of cases such as extreme dwarfism (Israeli et al.1991). Thus, for most variants, detection is on an extended time-scale, being months toover a year removed from the culture processes apparently generating the variation.

The consequences of a significant time-lag in detecting somaclonal variation areseveral (Krikorian 1989). Material that reaches growers may not perform as expected. Inextreme cases this could involve severe economic losses through poor yields, compoundedby wasted investment of time, field space and other resources in cultivation of thepropagated plants. In a collection of germplasm maintained in vitro, somaclonal variationcould result in the loss of important genotypes. The damage to the reputation of in-vitrotechniques, already felt in the case of some other crops where stability problems havebeen encountered, could be serious, resulting in a reaction against their development andimplementation out of proportion to the real problem, and delaying or preventing asolution to the problem.

From the conservation point of view in particular, the genotypic component insusceptibility to variation is particularly consequential. Genetic conservation should beapplied to and driven by the needs of the gene pool, not driven by the availabletechnologies. We should not just be conserving the “easy” genotypes. Instability in vitro ispart of the biology of a genotype that must be handled in the best ways currently at ourdisposal. In any case, net risks resulting from several factors are experienced in the genebank. Even if the genotype and associated risk factor is fixed, the way in which it ishandled is not. That is under our control, to improve as our knowledge increases. Thus, iftoday that means risking a certain level of instability in vitro to counterbalancepotentially greater risks of loss of entire accessions in the field, then there is no questionthat the in-vitro approach should be part of the conservation strategy. This is the essenceof the integrated approach to genetic conservation, based on complementarity ofdifferent techniques that is now favored by IBPGR and others (Engels 1991; van Sloten1990; Withers 1991b).

203LA Withers

The transition of scientists through the spectrum of reactions to somaclonalvariation described earlier, from ignorance and fear through to realism, needs to bematched by a strategy for confronting somaclonal variation as one of the realities of in-vitro culture of Musa. This could take the form of acknowledging the existence ofsomaclonal variation, learning how to recognize it, quantifying it, and then learning howto control it. Control is the key to handling any potentially beneficial but alsopotentially detrimental event.

In the case of Musa, we are clearly at the second stage of this process in the sense ofrecognizing variants in the nursery and the field, with some small inroads being madeinto the third and fourth stages of quantification and control. What is needed forsignificant progress, however, is a way of overcoming the time-lag between generation anddetection of variation to permit a real understanding of the linkages between variationand inducing factors.

Early-Stage MarkersTo bring the time scales of generation and detection more closely into line, there is aneed for a system of early-stage markers that could be applied in vitro to identifyvariation as soon as it occurs. This is perhaps ambitious but should be viewed as arealistic expectation, particularly in view of the small number of important variants inMusa set against progress in the areasof biology that could provide thefoundation for such markers.

One could speculate about thetypes of marker that could be devel-oped (see box). This speculation is notbased on extensive pilot work, nor is itwithout foundation. There areindications that at least some of thevariants have a genetic basis, thusindicating the relevance of tests basedon gross observations of chromosomesand more detailed analysis of DNA bymolecular techniques. There arespecific indications that in Musa, as inother plants, some variants are theresult of gross ploidy changes oraneuploidy (Hwang, Ko 1987; Israeli et al. 1991; Lee, Phillips 1988; Phillipset al. 1990). Some variants behave in a way that suggests the action oftransposable elements. Research hasalready been carried out on the


Potential early-stage marker systems

chromosome counting

chromosome structure (karyotyping)

chromosome banding

DNA measurements

RFLP

RAPD

isozyme analysis

protein analysis

chlorophyll content

secondary-product analysis

endogenous levels of growth regulators

response to exogenous growth regulators

in-vitro growth rate

respiration rate

in-vitro morphology

application of isozyme analysis and RFLP (restriction fragment length polymorphism) tothe identification of Musa varieties and somaclonal variants (Espino, Pimentel 1990;Jarret 1990). Very recently, work has started on the application of RAPD (randomamplified polymorphic DNA) to varietal identification and the detection of variants.Preliminary results are very promising (pers. com. Ford-Lloyd and Newbury, BirminghamUniversity, 1991, 1992).

Slower field growth in some variants such as those with a drooping leaf habit might bereflected in the growth rate in vitro, expressed either as rate of accumulation of dryweight or rate of leaf primordium formation. Growth-rate abnormalities may be linked tochanges in levels of endogenous growth regulators or a changed response to exogenouslyapplied growth regulators such as gibberellins, as demonstrated by Reuveni (1990). Somemorphological variants might simply have a different morphology in vitro. For example asalready observed, increase in ploidy is marked by an increase in leaf thickness, the size ofepidermal cells, and stomatal density (see Reuveni 1990).

Protein analysis has been attempted in combination with isozyme analysis, but withlittle success (Reuveni 1990). However, there is scope for more extensive testing of thisapproach. Flavonoid analysis has been used in taxonomic studies (Horry, Jay 1990).Assays for these and other secondary products have potential as the basis for screening,particularly in view of the occurrence of variants with enhanced pigmentation (Israeli etal. 1991). Similarly, variegation involving loss of chlorophyll is a phenomenon with aquantifiable symptom.

The ideas included in the box are not a definitive list; they are designed to stimulatethe minds of scientists working with Musa in the laboratory and the field who are familiarwith somaclonal variants, and of scientists whose work in anatomy, cytology, physiology,biochemistry, or molecular biology could be lent to the development of early markers. It is hoped that, as a result of this workshop, further ideas will be generated and potentialtests discussed.

What is important in designing such tests is that they should provide strongcorrelations, be rapid, be relatively inexpensive, and be reliable. Where it provesnecessary to use more than one test, combinations should be limited in complexity so asto remain practical. An invaluable application of the early-stage markers would be inhelping us design better experiments to try to identify and understand the linkagesbetween somaclonal variation and factors in the culture procedure that could bemanipulated to minimize variation. If the development and application of early-stagemarkers throws light onto the nature of somaclonal variants, that would be a bonus.However, it is neither an objective nor a requirement.

Developing a StrategyThe development of the early-stage markers themselves is only part of the story. Theyneed to be fully tested for their fidelity and a strategy developed for incorporating theminto propagation and conservation protocols, to provide the right tests at the right time toavoid the worst consequences of somaclonal variation. Components of the strategy will be

205LA Withers

the nature of the tests themselves, their number, the procedure for sampling cultures, thetiming and frequency of tests, streamlining and whenever possible mechanizing tests, andthe incorporation of test results into procedural modifications. The scale of any testingoperation will be crucial to its effective use. Testing too many cultures too frequently willdeter progress because of costs and workloads. Testing too infrequently and on too fewcultures will fail to pick up certain variants.

The development of early-stage markers will require concerted efforts by many playersand a considerable investment of effort and resources. However, the benefits would add adimension of security to in-vitro propagation and conservation of Musa, the lack of whichat present is probably one of the major impediments to the biotechnological exploitationof the genus.

ReferencesARIAS O, VALVERDE M. 1987. Produccion y variacion somaclonal de plantas de banano variedad Grande Naine

producidad por cultivo de tejidos. Rev. Asoc. Bana. Nac. (ASBANA) Costa Rica 28:6-11.BANERJEE N, VUYLSTEKE D, DE LANGHE E. 1986. Meristem tip culture of Musa: histomorphological studies of shoot

bud proliferation. Pages 139-147 in Plant Tissue Culture and its Agricultural Applications (Withers LA,Alderson PG, eds). London, UK: Butterworth.

BEVERSDORF WD. 1990. Micropropagation in crop species. Pages 3-12 in Progress in Plant Cellular andMolecular Biology (Nijkamp HJJ, van der Plas LHW, van Aartrijk J, eds). Dordrecht, the Netherlands:Kluwer.

DEREUDDRE J, SCOTTEZ C, ARNAUD Y, DURON M. 1990. Résistance d’apex caulinaires de vitroplants de poirier(Pyrus communis L.ev. Beurre Hardy), enrobés dans l’alginate, à une deshydration puis à une congelationdans l’azote liquide: effect d’un endurcissement préalable au froid. Comptes Rendus de l’Academie desSciences 310 Série III:371-323.

DODDS JH. 1991. In Vitro Methods for Conservation of Plant Genetic Resources. London, UK: Chapman and Hall.ENGELS JMM. 1991. A holistic approach to germplasm conservation. IBPGR Newsletter for Asia and the Pacific

7:1-2.ESPINO RRC, PIMENTEL RB. 1990. Molecular methods for detecting genetic diversity in Musa. Pages 36-40 in The

Identification of Genetic Diversity in the Genus Musa (Jarret RL, ed.). Montpellier, France: INIBAP. FRISON EA, PUTTER CAJ. 1989. FAO/IBPGR Technical Guidelines for the Safe Movement of Musa Germplasm.

Rome, Italy: FAO/IBPGR/INIBAP.HENSHAW GG. 1975. Technical aspects of tissue culture storage for genetic conservation. Pages 349-358 in Crop

Genetic Resources for Today and Tomorrow (Frankel OH, Hawkes JG, eds). Cambridge, UK: CambridgeUniversity Press.

HOHN B, DENNIS ES. 1985. Genetic Flux in Plants. Vienna, Austria: Springer-Verlag. HORRY JP, JAY M. 1990. Molecular methods for detecting genetic diversity in Musa. Pages 41-55 in The

Identification of Genetic Diversity in the Genus Musa (Jarret RL, ed.). Montpellier, France: INIBAP.HWANG SC. 1986. Variation in banana plants propagated through tissue culture. J. Chin. Soc. Hortic. Sci. 32:117-

125 (in Chinese, with English summary).HWANG SC. 1990. Somaclonal resistance in Cavendish banana to fusarium wilt. Pages 121-125 in Fusarium Wilt

of Banana (Ploetz RC, ed.). St Paul, MN, USA: APS Press/American Phytopathological Society.HWANG SC, KO WH. 1987. Somaclonal variation of bananas and screening for resistance to fusarium wilt. Pages

151-156 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds). ACIAR Proceedingsno.21. Canberra, Australia: ACIAR.

IBPGR. 1988. Conservation and Movement of Vegetatively Propagated Germplasm: in vitro culture and diseaseaspects. Rome, Italy: IBPGR.

ISRAELI Y, REUVENI O, LAHAV E. 1991. Qualitative aspects of somaclonal variations in banana propagated by invitro techniques. B.V. Sci. Hort. 49:71-88.


JARRET RL. 1990. Molecular methods for detecting genetic diversity in Musa. Pages 56-66 in The Identificationof Genetic Diversity in the Genus Musa (Jarret RL, ed.). Montpellier, France: INIBAP.

KRIKORIAN AD. 1989. In vitro culture of bananas and plantains: background, update and call for information,Trop. Agric. (Trinidad) 66(3).

KRIKORIAN AD, CRONAUER SS. 1984. Aseptic culture techniques for banana and plantain improvement. Econ. Bot.38:322-331.

LARKIN PJ, SCOWCROFT WR. 1981. Somaclonal variation — a novel source of variability from cell cultures forplant improvement. Theoretical and Applied Genetics 60:197-214.

LEE M, PHILLIPS RL. 1988. The chromosomal basis of somaclonal variation. Annual Review of Plant Physiologyand Plant Molecular Biology 39:413-437.

NOVAK FJ, DONINI B, HERMELIN T, MICKE A. 1987. Potential for banana and plantain improvement through in vitromutation breeding. Pages 67-70 in Proceedings of the 7th ACORBAT Meeting, San José, Costa Rica, 23-27Sep 1985 (Galindo JJ, Jaramillo R, eds). Technical Bulletin no.121. Turrialba, Costa Rica: CATIE.

PANIS BJ, WITHERS LA, DE LANGHE EA. 1990. Cryopreservation of Musa suspension cultures and subsequentregeneration of plants. Cryo-Letters 11:357-363.

PHILLIPS RL, KAEPPLER SM, PESCHKE VM. 1990. Do we understand somaclonal variation? Pages 131-141 inProgress in Plant Cellular and Molecular Biology (Nijkamp HJJ, van der Plas LHW, van Aartrijk J, eds).Dordrecht, the Netherlands: Kluwer.

POOL DJ, IRIZARRY H. 1987. Off-type banana plants observed in a commercial planting of ‘Grand Nain’propagated using the in vitro culture technique. Pages 99-102 in Proceedings of the 7th ACORBATMeeting, San José, Costa Rica, 23-27 Sep 1985 (Galindo JJ, Jaramillo R, eds). Technical Bulletin no.121.Turrialba, Costa Rica: CATIE.

RAMACHARAN C, GONZALEZ A, KNAUSENBERGER WI. 1987. Performance of plantains produced from tissue cultureplantlets in St. Croix, U.S. Virgin Islands. Pages 36-39 in Proc. 3rd Meet. Int. Assoc. Research on Plantainand Banana, 27-31 May 1985, Abidjan, Côte d’Ivoire.

REUVENI O. 1990. Methods for detecting somaclonal variants in ‘Williams’ bananas. Pages 108-113 in TheIdentification of Genetic Diversity in the Genus Musa (Jarret RL, ed.). Montpellier, France: INIBAP.

REUVENI O, ISRAELI Y, ESHDAT Y, DEGANI H. 1986. Genetic variability of banana plants multiplied via in vitrotechniques. Final report submitted to IBPGR (no.PR 3/11). Bet Dagan, Israel: Agricultural ResearchOrganization, The Volcani Center.

SCHOOFS J. 1990. The INIBAP Musa germplasm transit center. Pages 25-30 in Musa: Conservation andDocumentation. Proceedings of INIBAP/IBPGR Workshop, Leuven, Belgium, 11-14 Dec 1989. Montpellier,France: INIBAP.

SCOWCROFT WR. 1984. Genetic Variability in Tissue Culture: impact on germplasm conservation and utilization.Rome, Italy: IBPGR.

SMITH MK. 1988. A review of factors influencing the genetic stability of micropropagated bananas. Fruits43:219-223.

STOVER RH. 1987. Somaclonal variation in Grand Naine and Saba bananas in the nursery and field. Pages 136-139 in Banana and Plantain Breeding Strategies (Persley GJ, De Langhe EA, eds). ACIAR Proceedingsno.21. Canberra, Australia: ACIAR.


STOVER RH, SIMMONDS NW. 1987. Bananas. London, UK: Longman. 468 pp.TOMES D. 1990. Current research in biotechnology with application in plant breeding. Pages 23-32 in Progress

in Plant Cellular and Molecular Biology (Nijkamp HJJ, van der Plas LHW, van Aartrijk J, eds). Dordrecht,the Netherlands: Kluwer.

TOWILL LE. 1990. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Reports 9:178-180.VAN SLOTEN DH. 1990. IBPGR and the challenges of the 1990s: a personal point of view. Diversity 6(2).VUYLSTEKE D, SWENNEN R. 1990. Somaclonal variation in African plantains. IITA Research 1:4-10.VUYLSTEKE D, SWENNEN R, WILSON GF, DE LANGHE E. 1988. Phenotypic variation among in vitro propagated

plantain (Musa spp. cultivars AAB). Scientia Horticulturae 36:79-88. WALBOT V, CULLIS CA. 1985. Rapid genomic change in higher plants. Annual Review of Plant Physiology 36:367-

396.WITHERS LA. 1980. Tissue Culture Storage for Genetic Conservation. Rome, Italy: IBPGR.

207LA Withers

WITHERS LA. 1987. Long-term preservation of plant cells, tissues and organs. Oxford Surveys of Plant Molecularand Cell Biology 4:221-272.

WITHERS LA. 1989. In vitro conservation and germplasm utilization. Pages 309-334 in The Use of Plant GeneticResources (Brown AHD, Marshall DR, Frankel OH, Williams JT, eds). Cambridge, UK: CambridgeUniversity Press.

WITHERS LA. 1990. Prospects and problems of in vitro genebanks. Pages 21-24 in Musa: Conservation andDocumentation. Proceedings of INIBAP/IBPGR Workshop, Leuven, Belgium, 11-14 Dec 1989. Montpellier,France: INIBAP.

WITHERS LA. 1991a. Maintenance of plant tissue cultures. Pages 243-267 in Maintenance of Microorganisms(Kirsop BE, Doyle A, eds). London, UK: Academic Press.

WITHERS LA. 1991b. In vitro conservation. Biological Journal of the Linnean Society 43:31-42.WITHERS LA. 1992. In vitro conservation. In Biotechnology of Perennial Fruit Crops (Hammerschlag F, Litz RE,

eds). Wallingford, Oxon, UK: CAB International (in press).WITHERS LA, ENGELS JMM. 1990. The test tube genebank — a safe alternative to field conservation. IBPGR

Newsletter for Asia and the Pacific 3:1-2.


Part 3

Technology Transfer

211JA Chambers, JI Cohen

Donor Assistance in PlantBiotechnology for the DevelopingWorld

JA Chambers1, JI Cohen2

IntroductionTechnological innovation in agriculture is poised on the threshold of significant achieve-ment in the 20th century. Rapid advancements in the application of biotechnology to crop improvement programs promise to extend and expand the technological basewhich, during this century, marked the critical transition in agriculture to a systemreliant on science and technology to increase agricultural yields. This trend towardsknowledge-intensive agriculture, while well-developed in industrialized countries, leavesdeveloping countries at a distinct disadvantage. Developing countries generally lack thewell-established public and private technological infrastructure present in industrializedcountries which enables the latter fully to realize and apply the benefits of technologicalachievement in agriculture (Parton 1990). This disparity in technological benefit isbecoming especially apparent with the increasing application and integration ofbiotechnology in agricultural improvement programs in industrialized countries.

In recent years, development efforts have attempted to address this technologicaldisparity in agriculture through multilateral and bilateral assistance efforts. Multilateralsupport to the international agricultural research centers (IARCs), such as those in theConsultative Group on International Agricultural Research (CGIAR) system, which hasrecently included INIBAP among its members, has been rewarded as evidenced by thesignificant increases in food production achieved during the green revolution of the1960s. Increases in yield have resulted in increased income-generation for many farmersin developing countries.

Yet despite these successes, food self-reliance continues to be an elusive goal formany developing nations, and worldwide agricultural production continues to suffersubstantial losses of 20-40% from continuing pest, weed, and disease pressure (Walgate

1Biotechnology Specialist, USDA/RSSA, Office of Agriculture, Bureau for Research and Development, Agency forInternational Development, Washington, DC 20523-1809, USA; 2Biotechnology and Genetic Resource Specialist,Office of Agriculture, Bureau for Research and Development, Agency for International Development, Washington,DC 20523-1809, USA. (Note: Opinions presented in this paper are those of the authors and do not necessarily reflect those of the Agencyfor International Development or the United States Department of Agriculture.)

1990). Moreover, worldwide concern for a fragile natural resource base continues tomount in the face of increasing population pressures, decreased availability of primelands for cultivation, and obvious environmental demise. Thus, concurrent with needs forgreater production efficiency are new challenges, requiring continued scientificinnovation to enhance environmental acceptability of agriculture.

In an attempt to meet these challenges, biotechnology is becoming an integral part ofthe scientific foundation which comprises modern-day agriculture. The US Agency forInternational Development (AID) is committed to the application of biotechnology toaddress agricultural production constraints in developing countries. AID’s investment inthis technology is apparent through many forms of support, including its continueddirect support to the IARCs as they continue to integrate the tools of biotechnology withtheir conventional research programs. In addition, AID supports the IARCs through aprogram entitled “Collaborative Research on Special Constraints at the InternationalAgricultural Research Centers”, which is designed to overcome specific obstacles totechnological breakthroughs at the IARCs by funding collaborative research with USscientists. But perhaps some of AID’s most important recent achievements in enhancingagricultural biotechnology capacity within developing countries have been at the level ofthe national agricultural research programs. One example of such bilateral support wasthe first National Conference on Plant and Animal Biotechnology in Kenya, held from 25Feb to 3 Mar 1990, which was jointly sponsored by AID, the Kenya Agricultural ResearchInstitute, and a number of other donors. AID’s bilateral assistance efforts in agriculturalbiotechnology have encompassed a variety of crops, including the traditionally supportedcereals, and have recently included more commercially important crops such as theestate and plantation crops. Such efforts have specific relevance for this workshop onbiotechnology for banana improvement.

Recently, AID’s Office of Agriculture, based in Washington, DC, has furtherstrengthened its programmatic support for agricultural biotechnology with thedevelopment and award of a new project in plant biotechnology for developing countries.The project, which is entitled “Agricultural Biotechnology for Sustainable Productivity”(ABSP) will be discussed in specific detail later in this document. However, it isimportant to recognize that the design of this project derived from a number of inputsand value decisions which formed the basis of the project’s philosophical intent andprogrammatic content (Cohen 1991).

National Research Council Panel ReportIn part, the design of the ABSP project was structured after recommendations advancedby a National Research Council (NRC) panel of the US National Academy of Sciencesentitled “Plant Biotechnology Research for Developing Countries”. This panel, whichincluded members from private industry, universities, Collaborative Research SupportPrograms (CRSPs), IARCs, developing-country programs, and the US Department ofAgriculture, was convened at the request of AID’s Office of Agriculture to considerpriorities in plant biotechnology research that could provide near-term (3-5 years)

212 Donor Assistance in Plant Biotechnology for the Developing World

benefit to agriculture in developing countries. Further contributions to the design of theABSP project originated from correspondence between the Office of Agriculture andindividual AID missions, which were asked to respond to an extensive series of questionsregarding the utility of this type of project for their host countries.

The NRC panel’s report, entitled “Plant Biotechnology Research for DevelopingCountries”, was issued in September 1990 (USNRC 1990). It elucidated a distinct needfor a technical assistance program that would encourage and assist developing-countryscientists in the development and application of biotechnology focused on critical agri-cultural constraints of local importance. A number of general institutional and technicalpriority areas were identified which could be used as a basis for the development of morespecific recommendations after detailed discussion and consultation with local expertsmost familiar with local problems, needs, and opportunities.

Institutional priorities identified included three areas: (a) biosafety: AID shouldassist developing countries to implement and monitor appropriate biosafety regulations;(b) intellectual property: AID should participate in the development of policies topromote international cooperation in intellectual property rights (IPRs); and (c) humanresource development and networking: AID should enhance biotechnology capabilitiesthrough doctoral and postdoctoral fellowships, and nondegree training for plantbiotechnologists. Thus, the report emphasized that the progress and success of any newfunding initiative was predicated on support for the institutional, as well as technicaldimensions of biotechnology.

The technical recommendations were divided into three major categories. Thesecategories outlined areas of technical opportunity which AID should consider in thedesign of a new project in plant biotechnology. They are presented in order of degree ofgeneral panel support, increasing scale of complexity, and the types of research capacitythat are prerequisite to achieving results with more complex techniques. These researchcategories are thus those that appeared to present the greatest chance of ensuring thatapplications of biotechnology contribute to agricultural research in the near term.

The first category includes: (a) tissue culture, micropropagation, and transformation:support the building of developing-country capacity in plant tissue culture technologieswhich can augment conventional plant improvement programs, including micropro-pagation, cell selection, embryo rescue and haploid techniques, protoplast fusion andregeneration; (b) micropropagation: develop capacity to use micropropagation to producevirus-free planting material of forest, plantation, fruit, vegetable, and tuber crops; and (c) crop transformation: support the development of transformation and regenerationtechniques for crops of importance to developing countries, such as cassava and millet.

The second category involves plant disease and pest control and includes the following:(a) Bacillus thuringiensis (Bt) strain and gene identification: assist developing countriesto identify strains and clone genes of Bt, which are effective against insect pests importantto tropical areas. While there are many companies working on this worldwide, insufficienteffort has been focused on tropical pests; (b) antiviral strategies: support development ofantiviral strategies to combat plant viruses that attack beans, cassava, sweet potatoes,groundnut, and tropical fruits and vegetables; and (c) pathogen diagnostics and probes:


support research to develop DNA probes, as well as antisera and monoclonal antibodyprobes for plant bacteria, fungi, and viruses that attack crops of importance in thedeveloping world.

The final category, genetic mapping of tropical crops, summarized interest indeveloping specific genetic maps for many of the major crop plants. It included thefollowing guidance: (a) assist the CGIAR and developing-country crop breeders toacquire capacity to use RFLP maps in plant breeding of rice, maize, sorghum, cowpea,and other crops where these maps are becoming available; and (b) to undertakeresearch on primitive germplasm, especially woody perennial plants.

Value DecisionsSeveral value decisions were also factored into the project’s design process. These valuedecisions, which were developed and agreed upon by technical staff within the Office ofAgriculture and then reviewed by staff within AID missions and national researchprograms, include (1) support for field-proven, applied research programs which areintegrated with conventional agricultural research and sustainable agricultureapproaches; (2) mutuality of benefit to US and developing-country participants; (3)support for and programmatic inclusion of both private nonprofit (university, land-grantinstitutions) and private-for-profit organizations; (4) pursuit of equitable intellectualproperty rights through patents and licensing arrangements which recognize thecontributions of germplasm from the USA and developing countries and technical inputfrom all participating countries; and (5) support for testing and evaluation ofrecombinant products in accordance with USDA/APHIS regulations and host-countryapproval mechanisms. The incorporation of these value decisions into the project’sdesign was based on an understanding of the technology’s potential and limitations fordeveloping-country agriculture, a recognition of changing institutional trends and theirinfluence on AID’s traditional involvement in development-based, agricultural research,and a growing level of experience among staff which derived from the Agency’s initialprojects in biotechnology. A few of these value decisions, and their role in shaping theproject, are detailed below.

Integrated research in agricultural biotechnologyAgricultural biotechnology may be defined as the science which focuses on cellular andmolecular biology and the new techniques deriving from these disciplines targetedtowards the improvement of the genetic make-up and management of crops and livestock.Continuing investment in this technology derives from the perception that, whenintegrated with conventional, science-based techniques, the alternative tools ofbiotechnology provide additional opportunities to address contemporary challenges forsustainable increases in agricultural production. The tools of biotechnology, whenspecifically provided to plant breeders, present opportunities for increased efficiency,targeted modifications, and reliability in crop production, while simultaneously ensuringincreased profitability and environmental compatibility (Schneiderman 1990). However, a


key component to the ultimate success of this technology is that these alternative tools,when applied at the cellular and molecular level, must be integrated with conventionalresearch to assure product delivery to producers and consumers. Integration of thistechnology with conventional research programs is also essential if objectives are to beachieved in a timely manner.

Ultimately, relationships between biotechnologists and plant breeders may takeseveral forms. The least desirable scenario would be that where biotechnology programslack any formal collaboration with breeders and develop and release materialsaccordingly. More acceptable situations may involve truly collaborative interactions,where plant breeders are involved at the project’s inception and throughout, thusestablishing an interdisciplinary team approach, or may involve opportunisticinteractions in which an initial collaboration between breeders and biotechnologists maybe absent but is later formulated based on positive results from early trials. Breeders maythen be more integrally involved in the evaluation and further development of geneticallyaltered materials. The most favored approach, and that which was judged as one criterionfor selection during the ABSP project review process, was based on establishing suchintegration as a function of initial program development (Cohen et al. 1988a). In addition,the project’s emphasis was targeted towards ongoing integrated research programs whichhad a field-proven history of success. This implied that the project would not providefunds to establish new biotechnology centers or to move scientific capacity out of ongoingagricultural research programs. Detailed developing-country studies and appraisals of USinstitutional capabilities confirmed this approach and increased interest in theforthcoming ABSP project (Hall, Klausmeier 1988).

Further integration of biotechnology with sustainable agricultural concerns wasconsidered essential. This would permit a more detailed examination of contributionsfrom biotechnology to sustainable agricultural practices. Such potential contributionsderive from seed-based technologies which could provide more effective and lesschemically-dependent control of weeds, insects, and disease (Hauptili 1990).

Mutuality of benefitBy design, the ABSP project was initially intended to derive mutual benefits fromcollaborative research between the US public- and private-sector institutions andeffective partners in developing countries. In so doing, the enhancement of self-sustaining partnerships was envisioned, in which each country’s participants wouldderive economic benefit from returns on the original investment in research and capital.Such a focus on economic return is unique to a limited number of programs supportedelsewhere in AID’s portfolio. However, the ABSP project was developed as the Agency’sfirst agricultural project in which research would be geared, whenever possible, towardsa realization of economic return and mutual commercial benefit.

These issues were first explored by AID in conjunction with the US private sector,IARCs, and national programs in April 1988 at a symposium entitled “StrengtheningCollaboration in Biotechnology: International Agricultural Research and the PrivateSector” (Cohen 1989). This symposium was sponsored by AID to encourage dialogue on


these issues and was followed by workshops directed at specific aspects of internationalcollaboration in agricultural biotechnology. At that time it became apparent that therewere many opportunities for AID to develop commercially-directed biotechnologyresearch of mutual benefit to both industrialized and developing-country partners(Sawyer 1989). Recognizing the value of mutual benefit also corresponds with guidancedeveloped for implementing the CRSP projects under the authority of the Title XIIAmendment (BIFAD 1985).

Private-sector collaborationThe design of the ABSP project established a significant role and programmatic supportfor the private-for-profit biotechnology sector as well as AID’s traditional researchpartners, the US university community. The decision to include the private sector in adevelopment-oriented plant biotechnology project derived from a series of recent,recognizable trends that have been changing the face of agricultural research in thedeveloped world, and in the USA in particular.

To date (Jan 1992), most biotechnology research in agriculture has been conductedin and targeted towards the markets of industrialized countries, with practicalapplications limited to those problems and scenarios unique to commercial agriculture.Increasingly, the problem for donors, such as AID, has become one of technology accessdue to the increasing privatization and ownership of valuable biotechnological methodsand materials by the private sector in developed countries. This is, in part, due to thecapacity of the new technological tools to shorten the historical lag time between basicbiological discoveries and product development (USNRC 1987). This trend has beenfurther stimulated by the growth of the venture-capital industry in the USA and itssupport for entrepreneurial, academic-like companies (Bull et al. 1982). In addition,decreased Federal funding for agricultural research in the public sector (especially theUS university community) has been a catalyst for greater collaboration between publicinstitutions and private biotechnology companies. This collaboration has beenspecifically aided by two legislative changes in the USA: (1) a 1985 US Patent Officedecision which held a plant to be patentable, thus permitting the protection of a singletrait in a particular plant (Bent 1989); and (2) the Federal Technology Transfer Actwhich authorized government-supported agencies and institutions to collaboratecontractually with private companies, thus bridging the gap between basic research inthe public sector and more applied, product-oriented research in the private sector(Tallent 1989). As a result, universities are now frequently patenting promising researchproducts for their eventual license or sale to private companies. In return, they havebeen financially rewarded through royalty agreements with private industry which areutilized to sustain their continued research efforts. The net effect of the above trendsand changing institutional relationships has been AID’s decreased access to itstraditional partners (the universities) and an expanded role for its nontraditionalpartners (the private companies) in agricultural research. Thus, the ABSP projectincludes mechanisms to engage significantly the technical and managerial resources ofboth the US public and private sectors in one integrated program.


Prior to the completion of the ABSP project design, AID’s Office of Agriculture hadbenefited from the award of a precedent-setting grant to the Plant Sciences Division ofthe Monsanto Agricultural Company. Following extensive negotiations, a grantagreement was signed between AID and Monsanto which would support theimprovement of agronomically important root and tuber crops for Africa through theutilization of Monsanto’s proprietary technologies in plant biotechnology. Specific focusof the project is on the development of virus resistance through moleculartransformation of cassava, sweet potato, and yams using virus coat protein technologiesdeveloped in collaboration with R Beachy. The project supports the placement of twosenior African scientists for a 3-year period at Monsanto’s laboratory in St Louis. Theproject’s ultimate goal is to export improved root and tuber germplasm to Africa, aftercompliance with USDA/APHIS requirements, for further testing, prebreeding andcultivar development. The establishment of this novel agreement between AID and amajor US agricultural company provided some historical perspective for the developmentof the ABSP project in that it illustrated the need for a comprehensive and integratedendeavor in the area of development-assisted transfer of biotechnology. Issues related tothe field-testing of recombinant plants (biosafety) were raised early in the developmentof this agreement. The participating African scientists will gain experience in both state-of-the-art technologies as well as acquiring valuable expertise in US procedures for field-testing of genetically transformed germplasm. In addition, due to the highly proprietarynature of the technology in question, issues related to protection and ownership ofintellectual property were at the forefront of the negotiation process. Under the terms ofthe agreement, Monsanto will grant “a royalty-free license to use in Africa anyproprietary technology (including disarmed Agrobacterium strains, intermediatevectors, selectable markers, promoters, other expression and transformation technologyand coat protein-based virus tolerance technology) developed by African scientistsembodied within any cassava, yam, or sweet potato germplasm developed in thisprogram” (USAID 1990). The specific advantages for all parties concerned with thisgrant are listed in Table 1. In addition, AID’s experience with the Monsanto agreementprovided an invaluable resource for the design of future collaborative efforts with privatecompanies which are a significant component of the ABSP project.

Intellectual property protectionThe 1980 landmark Supreme Court decision in Diamond vs Charkrabarty, which held amicroorganism to be patentable and, in so doing, ruled in favor of the protection ofanimate nature whose existence resulted from human intervention, has directly favoredthe growth of the biotechnology industry in the United States (Beier, Strauss 1985).Since then, numerous biotechnology patents have been filed in the USA by bothuniversities and industry alike, thus catalyzing the inclusion of initiatives related tointellectual property rights (IPRs) in some of AID’s development-assistance efforts. Thedesign of the ABSP project, which provides substantial support for IPRs, reflects therecognized awareness for effective coordination of IPR initiatives in technology transferprograms to developing countries. This awareness stems from a desire equitably to


protect the sources of and contributions to the resulting inventions, whether they arerooted in proprietary technology contributed by advanced laboratories or indigenousgermplasm contributions and “know-how” provided by developing-country collaborators.Thus, a key component of agreements filed under the ABSP project will be an initialunderstanding and delineation of the IPRs which will accrue to participants.

It may be difficult to enforce violations of proprietary agreements which result fromunauthorized use of germplasm or technologies and litigation may not always be possibleor warranted, particularly in developing countries that lack significant legalinfrastructure. Nonetheless, advanced laboratories, both public and private, willdifferentiate between those institutions which present lesser or greater risk. This is animportant point, for once a negative reputation has been formulated, opportunities forfurther collaboration will decrease as other universities or commercial firms becomeaware of the problem. Those who violate legal agreements may gain in the short term,but may eventually suffer as new material is developed and no longer released to aparticular center or national program.

BiosafetyIntegrating biotechnology, and specifically genetic engineering, into development-assisted agricultural research requires an additional consideration of biosafetyprocedures surrounding the transfer of technology to developing countries, which oftenlack appropriate regulatory infrastructure. Complications surrounding the establishmentof such infrastructure in the developing world are due to a variety of influences.Foremost among these is the diverse array of players involved in agriculturalbiotechnology research and testing (Cohen et al. 1988b). These include the IARCs; donoragencies which provide support to IARCs and national programs; governments andresearch institutions of developing countries; governments, research institutions, and private industry of industrialized nations; and various environmental and public


Table 1. Advantages derived from the AID and Monsanto Corporation Agreement(Cohen, Chambers 1992).

a) Secures specific rights for specific proprietary technologies used in accordance withconditions of the grant.

b) Places senior African scientists within a commercial laboratory setting to becomefamiliar with commercial orientation, concerns, and decisions.

c) Familiarizes African scientists with testing protocols and regulatory compliance.d) Transformation provides all parties with gene expression data essential to expand

research efforts.e) Establishes precedence for donor grants to commercial entities and gain acceptance

for opportunities within commercial legal establishment.f) Establishes relationship between national program, private sector, donor, and IARC

for biotechnology research and product development.

advocacy concerns. Each of these groups has a specific agenda and an intrinsicdefinition of what constitutes safe and appropriate use of biotechnology products in theenvironment.

Furthermore, the continuing “product vs process debate” has figured dramatically inthe role of regulatory oversight with respect to biotechnology products. The perceivedpower or specificity associated with particular genetic modification technologies isdirectly related to an increase in regulatory concern or oversight and continues to fuelthis debate.

Nonetheless, despite the confusion and complications in the regulatory arena,enhancement of host-country capabilities in biosafety, including national abilities toevaluate, approve, and monitor specific requests for testing, is essential if thesecountries are fully to participate in and benefit from the exchange of technology.

Recent AID initiatives in biosafety reflect the Agency’s awareness of a need toincrease indigenous capacity in coordination with its sponsorship of technicalprograms. AID has adopted a “case-by-case” approach to projects warranting closeregulatory involvement. In addition to domestic concerns and regulatory responsi-bilities, AID must now consider how grant recipients will effect contained testingconducted in its client countries. Coordination between the principal investigator ofeach grant, as well as with AID, has been rigorously pursued to ensure that applicableregulations and oversight procedures are undertaken (Cohen, Chambers 1990). As anexample, since 1986 the Office of Agriculture includes, as a standard provision of grantssupporting recombinant DNA research, specific language regarding regulatorycompliance and approval from US regulatory agencies (Jones 1987). Thus, there is aformal written requirement by the Office that any risks posed by the research be fullyaddressed in the context of US government regulations prior to obtaining host-countryapproval. In addition, this approval must be obtained in writing, as must that of the AIDin-country mission.

Further support of the Agency’s commitment to this issue is seen in its 1987establishment of the Standing Committee on Biotechnology which functions to provide:(1) an agency forum for advice on issues surrounding biotechnology and on thedevelopment of mechanisms to address those issues that are technical, regulatory, andprogrammatic in nature; and (2) an entity within AID which will provide liaison with USgovernment scientific and regulatory agencies principally concerned with supportingand regulating biotechnology (USAID 1987). The Standing Committee serves a centralpurpose in the review and dissemination of information and the coordination ofdomestic and host-country regulations affecting biotechnology research and offersspecific procedural guidance for AID projects. By establishing this committee, AIDrecognized the role which biotechnology research occupied in its developmentprograms, and also addressed the Agency’s need safely and responsibly to monitor andimplement the products of its supported research. This initiative has been continuedwith the establishment of the ABSP project, which provides significant financialresources in support of regulatory activities which are necessary to safely move anygenetically engineered products developed by the ABSP research effort into the field.


A New AID Initiative: the ABSP Project

The US$6 million, 6-year ABSP project was authorized in 1991 by AID’s Bureau forResearch and Development to “mutually enhance U.S. and LDC institutional capacity forthe use and management of biotechnology to develop environmentally compatible,improved germplasm”. The project provides direct support for research and developmentactivities in the form of grants to US public and private sectors and developing-countryinstitutions for research to address agricultural production constraints in AID’s clientcountries. It provides indirect support for private-sector involvement (both in the USAand developing countries) in agricultural research through activities designed toincrease host-country capability in the competitive management of biotechnology. Thisindirect support is evidenced through the project’s planned activities in intellectualproperty, biosafety, networking, and the establishment of downstream commerciallinkages for product development. As such, the ABSP project is multidisciplinary: itsupports (1) human resource development via long-term, postdoctoral training inadvanced US institutions, both public and private; (2) constraint-oriented, as opposed tocommodity-based, biotechnology research and development within private and publicinstitutions; (3) activities, either through internships or expert consultation, in IPR andbiosafety, to increase host-country capacity; (4) increased host-country capacity in themanagement of biotechnology through commercially-oriented seminars in private USbiotechnology firms; (5) networking through regional and international conferences,trade association memberships within the Association for Biotechnology Companies,newsletters, directories, developing-country laboratory support, and database linkages;(6) product development via an incentive-based system for funding field-testing andcommercialization activities for promising products of laboratory research.

A peer-review process for submitted proposals was utilized in determining the awardfor the project. A Request for Application (RFA) to US public and private institutions wasissued in May 1991. The external review, conducted by the National Research Council,established individual rankings for the public- and private-sector proposals. Based onthis ranking and on a financial analysis conducted by AID’s procurement office, thecooperative agreement for the project was awarded to Michigan State University (MSU)as the lead institution and management entity in a consortium of universities which alsoincludes Texas A&M and Cornell. J Dodds, formerly of the International Potato Center, isthe project’s managing director. The university research component is under thecoordination of M Sticklen, of MSU. The private-sector subgrant was awarded to DNAPlant Technology Corporation (DNAP), based in New Jersey.

Proprietary agreements will be established for all aspects of joint research programsconducted between the various universities, developing-country collaborators, andprivate companies. J Barton, a professor at Stanford Law School, has been retained aschief counsel to advise the project on intellectual property issues.

The technical focus of the university research component is based on the use ofrecombinant DNA technology and crop transformation techniques to geneticallyengineer resistance to a variety of crop pests and pathogens in agronomically important


crops. The expected outputs for this research will be to increase developing-country cropproduction through the availability of genetically resistant germplasm, and ultimately todecrease dependence on chemical inputs. Specific research activities include: (1)genetic engineering of potato for tuber moth resistance; (2) genetic engineering of sweetpotato for weevil resistance; (3) genetic engineering of maize for stem borer resistance;and (4) genetic engineering of cucurbits for potyvirus resistance. Host-countrycollaborators initially identified include Kenya, Ecuador, and Indonesia, althoughadditional collaborators will also be pursued in an attempt to broaden the project’sglobal relevance.

The technical focus of the private-sector subgrant to DNAP is under the direction ofM Söndahl. As opposed to the MSU/Texas A&M/Cornell technical program, which isstrongly oriented towards the use of rDNA technologies, the DNAP program concentrateson the use of cell culture-based techniques of micropropagation to increase yields andimprove quality of several tropical crops varieties. Under the terms of the agreement,DNAP will collaborate with a private company in Costa Rica, Agribiotecnología de CostaRica, to utilize DNAP’s proprietary technology for (1) the development of high-frequencyembryogenic cell cultures for targeted crops as a prelude to developing large-scalemicropropagation systems; (2) the genetic improvement of elite, but currently limited,planting stock; and (3) the utilization of the technology and planting material as thebase for commercial production of targeted crops in Costa Rica. The four crops targetedby this agreement include banana, pineapple, coffee, and ornamental palms.

Ancillary activities on the project’s agenda include support for short-term internshipsfor host-country participants in intellectual property and biosafety at US legal andregulatory institutions, respectively; inclusions of IARCs in the project’s network, whichwill sponsor three regional and one international conference; support for ABC tradeassociation memberships for all project participants; and, funding for field-testing,product development initiatives, and economic impact studies.

To date (Jan 1992), response to the project has been very positive among AID in-country missions, international agricultural research centers, and national programs indeveloping countries. Expansion of the project’s activities, through supplemental fundingby individual AID missions, is currently under development and may result in additionalresearch agreements between institutions not presently supported under the coreagreement.

ReferencesBEIER FK, STRAUSS J. 1985. Patents in a time of rapid scientific and technological change: inventions in

biotechnology. Pages 15-35 in Biotechnology and Patent Protection (Beier FK, Crespi RS, Strauss J, eds).Paris, France: Organization for Economic Co-operation and Development.

BENT SA. 1989. Patenting genes that encode agriculturally important traits. Pages 109-122 in IntellectualProperty Rights Associated with Plants. Madison, WI, USA: American Society of Agronomy.

BIFAD (Board for International Food and Agricultural Development). 1985. Guidelines for the collaborativeresearch support programs under Title XII of the International Development and Food Assistance Act of1975. Washington DC, USA: Agency for International Development.


BULL AT, HOLT G, LILLY MD. 1982. Inportant issues affecting developments in biotechnology. Pages 57-63 inBiotechnology: International Trends and Perspectives. Paris, France: Organization for Economic Co-operation and Development.

COHEN JI (ed.). 1989. Strengthening Collaboration in Biotechnology: international agricultural research and theprivate sector. Washington DC, USA: Agency for International Development.

COHEN JI. 1991. Value decisions responsive to international initiatives in plant biotechnology. (Paper presented atthe CCSA Plenary Session: Ethical and Policy Issues in Global Crop Science, convened by G Heichel.) CropScience (in press).

COHEN JI, CHAMBERS JA. 1990. Biotechnology and biosafety: perspective of an international donor agency. Pages378-394 in Risk Assessment in Genetic Engineering: Environmental Release of Organisms (Strauss HS, LevinM, eds). New York, USA: McGraw-Hill.

COHEN JI, CHAMBERS JA. 1992. Industry and public sector cooperation in biotechnology for crop improvement.Pages 17-30 in Biotechnology and Crop Improvement in Asia (Moss JP, ed.). Patancheru, India:International Crops Research Institute for the Semi-Arid Tropics.

COHEN JI, JONES KA, PLUCKNETT DL, SMITH NJ. 1988a. Regulatory concerns affecting developing nations.Bio/Technology 6:744.

COHEN JI, PLUCKNETT DL, SMITH NJH, JONES KA. 1988b. Models for integrating biotechology into crop improvementprograms. Bio/Technology 6:387-392.

HALL P, KLAUSMEIER WH. 1988. Opportunities to commercialize life science applications in less developedcountries: a strategic plan. Arlington, VA, USA: Resources Development Foundation.

HAUPTILI H, KATZ D, THOMAS BR, GOODMAN R. 1990. Biotechology and crop breeding for sustainable agriculture.Pages 141-156 in Sustainable Agricultural Systems (Edwards CA, Lal R, Madden P, Miller RH, House G, eds).Akeny, Iowa, USA: Soil and Water Conservation Society.

JONES KA. 1987. A.I.D.’s activities in biotechnology: regulatory considerations. Contract DAN-1406-0-7008-00.Washington DC, USA: Agency for International Development.

PARTON TR. 1990. Issues affecting Technical Change and its impact on the International AgribusinessEnvironment. Board on Agriculture, National Research Council. Washington DC, USA: National AcademyPress.

SAWYER RL. 1989. Building bridges of research collaboration through the CGIAR system of centers. Pages 13-19 inStrengthening Collaboration in Biotechnology: International Agricultural Research and the Private Sector(Cohen J, ed.). Washington DC, USA: Agency for International Development.

SCHNEIDERMAN HA. 1990. Innovation in agriculture. Pages 97-106 in Technology and Agricultural Policy. Board onAgriculture, National Research Council. Washington DC, USA: National Academy Press.

TALLENT W. 1989. A new era of government-industry cooperation in research and development. Pages 423-428 inStrengthening Collaboration in Biotechnology: International Agricultural Research and the Private Sector(Cohen JI, ed.). Washington DC, USA: Agency for International Development.

USAID (US Agency for International Development). 1987. Charter for agency Standing Committee on Biotechno-logy. Washington DC, USA: the Agency.

USAID (US Agency for International Development). 1990. Grant Agreement DAN-4109-G-00-0085-00. Improvingcassava for African farmers through biotechnology. Washington DC, USA: USAID Contracts Office.

USNRC (US National Research Council). 1987. Committee on a National Strategy for Biotechnology inAgriculture, Board on Agriculture. Pages 51-144 in Agricultural Biotechnology: Strategies for NationalCompetitiveness. Washington DC, USA: National Acedemy Press.

USNRC (US National Research Council). 1990. Plant Biotechnology Research for Developing Countries.Washington DC, USA: National Academy Press. 44 pp.

WALGATE R. 1990. Finding genes for better crops. Pages 6-22 in Miracle or Menace? Biotechnology and the ThirdWorld. London, UK: Panos Institute.


Part 4

Recommendations

225Working Group 1

Nonconventional Strategies forProducing Banana and PlantainResistant to Pathogens and Pests

Working Group 1

IntroductionThe potential for new technologies was readily apparent to workshop participants. Mapsof the Musa acuminata and M. balbisiana genomes may well be produced within thenext 2 years, and cultivars transformed for useful traits could be available in 3 years. Itwas stressed, however, that these new strategies be viewed as complements and notreplacements for conventional breeding. It was also stressed that reasonable optimismbe used when anticipating results with the new techniques. Since many of thesestrategies (Table 1) are complicated and expensive, significant or rapid progress may beimpeded by technical difficulties and funding constraints.

Construction and Use of a Genetic Linkage Mapof the Musa Genome for the Improvement ofBananas and PlantainsAlthough considerable progress has been made in breeding bananas resistant to some oftheir most important pathogens, very little is known about the genetic inheritance ofthese or other characters. With the advent of new molecular genetic markertechnologies such as RFLPs and RAPDs, it should be possible, as has been the case formany other crops, to construct a genetic linkage map of the banana genome. A publiclyavailable map will serve to mark important agronomic traits and will promote more rapidprogress in existing and future breeding programs. It will also provide a unique tool forstudying and manipulating characters such as sterility arising from structuralheterozygosity (very common in the diploid bananas) or genic factors, andparthenocarpy, a character that has probably played a major role in the domestication ofdiploid bananas.

The construction of a genetic map requires that certain conditions be fulfilled:distinguishable alleles should exist at as many loci as possible; crosses for the generationof segregating populations of adequate size should be feasible; methods for themeasurement and analysis of characters of interest (such as resistance to pathogens)should be available.

Table 1. Priorities for the biotechnological improvement of banana and plantain

Target Application—————————————— ———————————————————

Importance1 Diagnosis2 Control, other3———————— ———————— ——————————————

Local Export Approach Need Approach Potential

Diseases caused by:FungiSigatoka H H RAPD Y markers H complex toxin(s) H

various genes MFusarium wilt H M RAPD? Y markers H

various genes MVirusesBanana bunchy top H D MonoAb, N Coat protein gene? M

cDNABanana bract mosaic M D Potyvirus Y Coat protein gene HBanana streak L D PolyAb, N Coat protein gene? M

cDNACucumber mosaic L D MonoAb, N Coat protein gene H

PolyAb,cDNA

Abaca mosaic L D Potyvirus Y Coat protein gene HAbaca bunchy top L D cDNA Y Coat protein gene? MOther agentsBacteria L L ? Y Vector interactions MBanana weevil M L - - Markers H

BT gene(s)? MOther gene(s) M

Nematodes M L Develop’g Y Markers HVarious genes M

Other targets:Ripening, H M - - AntiPG Mpostharvest AntiACC M1Importance of target for locally consumed and exported bananas are high (H), medium (M), or low (L). Other targets werediscussed (D) but determined to be relatively unimportant. 2Approaches for diagnosis include the use of: randomly amplified polymorphic DNAs (RAPDs); monoclonal antibodies (MonoAb);polyclonal antibodies (PolyAb); and complementary DNA (cDNA), to detect disease agents. Broad-spectrum kits, which recognize95% of all known potyviruses, may be useful for detecting the banana bract mosaic and abaca mosaic viral agents. Whether the needis yes (Y) or no (N), work on an approach is based on the status of development for the approach and the specific agent.3Biotechnological approaches to control various diseases or otherwise to improve banana and plantain include: the identification ofresistance markers that would assist breeding efforts; the use of host-specific toxin(s) produced by Mycosphaerella fijiensis to selectresistant variants; and transformation of the host with various genes. Potentially useful genes for transformation include those whichproduce: chitinases, β1,3-glucanases, chitin-specific lectins, and nikkomycins for diseases caused by fungi; viral coat proteins fordiseases caused by viruses; toxins produced by Bacillus thurinigensis, chitinases, and other oral and lytic enzymes for banana weeviland/or nematodes; and antisense polygalacturonases (antiPG) and inhibitors of ethylene production (antiACC) that extend thepostharvest life of fruit. Since none of these strategies has been tested for banana and plantain, the potential for success with thevarious target:approach combinations was evaluated based on current success with other plants.

226 Nonconventional Strategies for Producing Banana and Plantain Resistant to Pathogens & Pests

To date sufficient data are available to indicate that the DNA of many diploid bananaaccessions is highly polymorphic and that the phenomenon may well be a general one.Crosses between some of the accessions (see González de León’s paper in this volume)generate populations from which a saturated linkage map could be constructed.

Marker-assisted selection in banana breeding is therefore a realistic goal which willrequire the creation and/or strengthening of collaborative projects between breedingprograms and laboratories applying the new technologies.

Furthermore, the relatively small size of the banana genome should facilitateadvances in the construction of a physical map which should permit the isolation andcharacterization of specific banana genes for use through nonconventional breedingmethodologies.

Recommendations

Work on the construction and use of a saturated linkage map of the Musa acuminataand M. balbisiana genomes should be continued and enhanced through increasedsupport of collaborative projects.

Immediate action for achieving these goals should include the following.• The development of a wide variety of segregating populations according to specific

breeding objectives.• The construction of a publicly available high-density core map at a level of saturation

that will suit marker-assisted selection strategies.Long-term objective:• The construction of a physical map using long-range mapping strategies (Cosmid and

YAC libraries) which should facilitate the identification and eventual isolation ofgenes of interest.

TransformationAn efficient system for transforming bananas will be needed before this important cropcan be genetically engineered. The essential components of such a system are: (1) anefficient DNA delivery technique (for instance, biolistics); (2) an effective selectionsystem (for instance, antibiotic or herbicide resistance); (3) promoters to drive highexpression in Musa; and (4) an efficient plant-regeneration system. Preliminary resultspresented at this workshop indicate that progress has been made in each of the areas.However, it must be stressed that considerable research and development is requiredbefore an efficient Musa transformation system is available.

Several useful genes exist that could be incorporated into banana and plantain,particularly pest and disease resistance genes (Table 1), and new genes will becomeavailable within a year.

Recommendations

• The development of an efficient Musa transformation system should be given veryhigh priority.

227Working Group 1

• The incorporation of virus resistance, particularly coat protein-mediated resistance,should be the first priority for the incorporation of useful traits into Musa. In thisinstance, incorporation of genes for cucumber mosaic virus coat protein could serveas a useful model system. In addition, development of strategies for conferringresistance to banana bunchy top virus should be pursued.

• Resistance to banana borer weevil, mediated by genes from Bacillus thurinigensisfor toxin production should be investigated.

• Other potentially useful genes, including those for fungal resistance (chitinase,nikkomycin, etc.) and extended postharvest life of fruit, should be investigated.

Identifying and Generating ResistanceNew and more efficient techniques are required for identifying and generatingresistance to plant diseases and pests. These may include apparent host-specific toxins,such as that produced by Mycosphaerella fijiensis, elicitors for host-resistanceresponses in cases of diseases caused by nematodes and other fungi, and irradiation toinduce new sources of resistance.

Virus IndexingOne of INIBAP’s primary activities is to facilitate the exchange or distribution ofgermplasm. Within this activity is the inherent risk of distribution of viruses in tissue-culture material. Early in 1992 there was no information regarding the transmission ofviruses through Musa seeds.

To reduce the possibility of distributing virus-infected material, INIBAP hasimplemented a virus indexing program by which accessions moving through the transitcenters are tested for virus infection in two indexing centers. However, no one center hasa collection of all the known Musa viruses or the accessibility to implement the range ofappropriate diagnostic technologies.

Virus detection techniques continue to be developed and improved as “new” virusesof Musa are being described. It is therefore essential that those developments areincorporated in the Musa virus testing program to increase the efficiency and security ofthe germplasm distribution program.

Recommendations• It is recommended that INIBAP establish at least one central facility for Musa virus

testing and that:• The facility have the capacity to test for all known Musa viruses.• The facility develop expertise in the application of contemporary virus diagnostic

technology including Elisa (using monoclonal and polyclonal antisera), molecularhybridization, and the polymerase chain reaction.

• The facility obtain and maintain in-vivo cultures of all known Musa viruses, to beused as positive controls.


• The facility monitor new developments in diagnostic technology and incorporatethese technologies into the testing program as appropriate.

• The facility monitor and investigate reports of “new” viruses in Musa andincorporate tests for these viruses when available.

• Groups developing diagnostic tests for Musa viruses make these available to thecentral facility.

• INIBAP encourage research on the characterization and diagnosis of Musa viruses.

Understanding Pathogen DiversityThe importance of understanding pathogen diversity is well understood. Currentresearch on variation in Mycosphaerella fijiensis, Fusarium oxysporum f.sp. cubense,Radopholus similis, and BBTV should continue.

Up to early 1992 work on the fusarium wilt pathogen has progressed furthest.Pathogenic and genetic diversity in F. oxysporum f.sp. cubense is extensive and datahave accumulated indicating that the performance of even some tolerant banana clonesvaries from one location to another. Thorough research has just begun on the BLSburrowing nematode and banana bunchy top pathogens. There is evidence that thereexists diversity in each that affects the performance of important cultivars.

Recommendations

• INIBAP-assisted efforts to collect F. oxysporum f.sp. cubense in Southeast Asia, acenter of origin for Musa acuminata and its hybrids, with M. balbisiana, shouldcontinue. Minor support should be made available for maintenance of the Homesteadcollection of this pathogen and for the distribution of accessions it contains.Techniques for directly and indirectly accessing pathogenicity in all the agents wouldbe useful. The development of such techniques should receive high priority.

Screening MethodsTissue culture, somaclonal variation, and irradiation techniques are being used togenerate variants and mutations that may have resistance to serious pathogens of Musa.The detection of useful germplasm generated by these methods is handicapped by thelack of rapid, reliable screening techniques for determining disease reactions. Field-testing is costly and time-consuming. Laboratory and greenhouse screening methodswould be extremely useful and more appropriate.

Recommendation

• Research to develop rapid, reliable techniques for screening Musa germplasm forreaction to Sigatoka and fusarium wilt diseases to detect useful resistance, should beencouraged. This would involve a continuation of investigations in the role of toxinsas possible screening agents for detecting resistance to Sigatoka disease. Work to

229Working Group 1

develop a rapid, reliable test for detecting resistance to fusarium wilt is also seen asvery important.

Cooperation between Biotechnologists andPlant BreedersIt is recognized that new biotechnological or nonconventional approaches to plantbreeding offer many useful techniques and opportunities for success. However, theycannot be successful in a vacuum. Close continued cooperation must exist betweenbiotechnologists and conventional plant breeders if society is to be the beneficiary ofthis technology.


231Working Group 2

In-Vitro Strategies for Musa

Working Group 2

IntroductionThe Working Group on this topic acknowledged the significant impact and progress thathas been made in Musa propagation and improvement in recent years using in-vitrotechniques. The Group also wished to emphasize that these techniques arecomplementary to more established techniques for propagation and improvement ofbananas and plantains.

In-Vitro Techniques for Germplasm Handling

MicropropagationMicropropagation is a useful technique for the mass propagation of disease- and pest-free planting material. Techniques are currently well established and applicable to awide range of genotypes, but there is a continual need to upgrade and develop moreefficient and cost-effective strategies.

Recommendation

• It is recommended that studies involved with the development of more efficientmicropropagation methods that address the issues of somaclonal variation, and ofmore rapid multiplication, should be supported. Also, INIBAP and otherorganizations should endeavor to strengthen in-country capability for in-vitro culturein developing countries, including the training of personnel.

ConservationIn-vitro culture has become an important means of conserving Musa germplasm. Withina complementary conservation strategy, alongside field gene banks, seed and pollenstorage, in-vitro techniques can help achieve secure, efficient, accessible andsustainable conservation of the Musa gene pool. Techniques for medium-termconservation through slow growth are relatively well developed. Progress has been madewith long-term in-vitro storage of germplasm through cryopreservation, but there is stillroom for improvement.

Recommendations• There is a need to establish a rational basis for management of in-vitro germplasm

collections.• Research is needed better to understand the behavior of cultures over extended

periods of slow-growth storage, particularly with respect to variability.• Continued research is required in the area of cryopreservation to improve the

technique to include a range of culture systems, and to define conditions that areapplicable to a diverse range of germplasm.

Germplasm exchangeConsiderable advantages are conferred by the safe movement of pathogen-testedgermplasm by in-vitro methods. Detailed guidelines have been formulated by FAO,IBPGR, and INIBAP. However dissemination of germplasm could be accelerated.

Recommendations• Research and information is needed to develop simple, sensitive, and reliable

diagnostic methods for the early detection of banana and plantain diseases,particularly for BBTV and other obscure pathogens. These techniques and adequatequarantine facilities are needed in developing countries.

• The FAO/IBPGR/INIBAP guidelines should be updated as new knowledge indiagnostics is acquired.

• INIBAP should endeavor to enhance the efficiency of its germplasm exchangeprogram by introducing recently developed diagnostics to the indexing centers.

CollectingThe establishment of an in-vitro germplasm collections is a big undertaking thatfrequently involves collecting and transporting suckers to laboratories many kilometersaway or in different countries. Apart from logistical problems, soil-bearing propagulesincrease the risk of introducing serious pests and diseases.

Recommendation• There is a need to build upon the promising preliminary research that has been

carried out on in-vitro collecting for Musa in order to refine techniques and makethem more widely available.

In-Vitro Techniques for Genetic ImprovementIn-vitro techniques have enormous potential for the genetic improvement of bananasand plantains by overcoming barriers to the full utilization of the gene pool. Manytechniques are available and have been used successfully in Musa. Some of thetechniques available include: embryo rescue; meristem culture; cell and protoplastculture; haploidy; mutagenesis; and chromosome doubling. Those requiring particularattention were identified by the Working Group.

232 In-Vitro Strategies for Musa

Haploid methodsEffective strategies have been developed to produce haploid material for the productionof homozygous clones to facilitate and accelerate practical breeding programs and todevelop a greater understanding of the genetics of Musa. Progress has been made on anumber of fronts.

Recommendation

• Research into induction of haploidy should continue and, at this early stage inapplication of the techniques, it is proposed to convene a meeting with groupsinterested or currently working in this area.

Cell cultureSimple and adaptable cell-culture techniques are being developed that have applicationfor: (a) rapid clonal propagation; (b) transformation; and (c) in-vitro selection.Advances in this area have been very rapid and there is room further to streamlinetechniques and extend the use of these techniques for a wider range of cultivars.

Recommendations

• There is a need further to develop somatic embryogenesis techniques for the masspropagation of desirable clones. The scale-up and automation of techniquesnecessary to reduce the costs of production further should be investigated. Inaddition, field-testing of plants regenerated from cell culture should be investigated.

• It is imperative that research should be supported to develop cell-culture techniquesto enhance the selection and regeneration of transformed plants. Considerableopportunities for utilizing novel gene products in improvement are now at hand. Thestable integration of these genes into whole plants is the goal, and research in thisarea is needed to develop efficient and applicable techniques. The Group alsorecommends that a meeting be convened to facilitate interaction with groupsinvolved in Musa transformation strategies, and it strongly supports research in thisarea.

• In-vitro screening and regeneration of plants from selected cell lines has beenidentified as a means of obtaining plants with tolerance of/resistance to pathogensand pests, with or without the use of mutagenic procedures to increase variation.Further research is needed to refine these approaches to plant improvement.

Protoplast cultureThere are bottlenecks in conventional breeding, and recent positive results withprotoplast culture have demonstrated their use as a means of bringing together plants ofvarious ploidies and with different genomic and cytoplasmic backgrounds to producenovel combinations. Although the use of protoplast technology is usually moretechnically difficult, its uses should not be ignored.

233Working Group 2

Recommendation• Research is still needed on the development of protoplast fusion techniques to

produce somatic hybrids and on electroporation of single-cell systems for use intransformation studies. These techniques need to be extended to cultivars ofagronomic value.

Somaclonal Variation: detection andcharacterizationThe Group acknowledges the occurrence of somaclonal variation as a seriousimpediment in the in-vitro handling and improvement of Musa. Somaclonal variants areusually inferior; they can result in serious economic losses to the farmer and can impactnegatively on the work of scientists.

Somaclonal variation is linked to both inherent and culture-related factors. Incertain cases and applications, particularly micropropagation, inherent factors mightlargely be controlled by the choice of more genetically stable cultivars and explants.Culture-related factors may be manipulated and culture management strategiesdeveloped to reduce the risk of somaclonal variation. However, the knowledge base ispresently inadequate and more research is urgently needed. The Group identified thefollowing research priorities for concurrent attention covering methods for the earlydetection of somaclonal variation and the development of a better understanding of thefactors affecting somaclonal variation and its causes and origins.

Recommendations• The Group recommends the undertaking of a survey of somaclonal variants in Musa.

This will include the identification and characterization of the more significantsomaclonal variants in order to understand the magnitude of the problem and focuson the development of detection methods.

• To facilitate the characterization of somaclonal variation, the Group recommends thecompilation and distribution of a set of standardized morphological descriptors ofsomaclonal variants of bananas and plantains. The Group recommends that thesedescriptors could be prepared and disseminated through INIBAP’s InformationServices.

• Methods for the early detection and characterization of somaclonal variants areneeded and may be based on a combination of morphological, biochemical, andmolecular markers. It is recommended that a concerted research effort be made todevelop appropriate and efficient strategies for early detection.

• The Group recommends the undertaking of research into the factors influencingsomaclonal variation. This should be integrated with research on early markers inorder to provide opportunities to accelerate research and give timely feedback toimprove culture procedures.

• In the course of all of the above research, the fundamental issues of the causes andorigins of somaclonal variation should be addressed.

234 In-Vitro Strategies for Musa

Coordination and Dissemination of Informationon In-Vitro Techniques and ApplicationsIn order that Musa in-vitro technologies may have greater impact, information must becollected and disseminated in a timely fashion to groups engaged in this work. This isparticularly true of the need for information in developing countries. The establishmentof regional networks with an extension focus for growers is also important in this regard.The Group acknowledges the potential of using INIBAP’s information facilities tocontribute to a better flow of information on biotechnology.

235Working Group 2

236 Technology Transfer

Technology Transfer

Working Group 3

IntroductionThe task assigned to this Group of 35 workshop participants was to explore ways offacilitating biotechnology transfer to scientists working in banana- and plantain-producing countries. This topic was developed in a preworkshop paper entitled “AStrategy for Action”, which explored the workshop objectives, organization, and expectedresults.

The Group used the nominal group technique (NGT) of Delbecq et al. (1975), whichcomprises a highly structured brainstorming session, to develop responses to the singlequestion: What activities are needed to facilitate access to newer biotechnologies forbanana and plantain genetic improvement for producing countries?

Following a brief training session in NGT for the participants, the Group identified178 items related to the target question. Following a period of clarification, a few itemswere combined to eliminate clear duplication. The items were then prioritized and,through this balloting process, 12 items were identified as collectively important:1. Financial support.2. Training.3. Develop laboratory infrastructure.4. Bi-directional scientific exchange.5. Link biotechnology laboratories to breeding programs.6. Provision of documents.7. Identify targets with defined value.8. Funding for collaborative research.9. Appoint INIBAP biotechnology coordinator.10. Networking of international programs and IARCs.11. Link research laboratories in producing countries to biotechnology laboratories.12. Transfer technology.

Following prioritization, a few items were eliminated from the list of 178 as beingeither not serious or inappropriate for technical or policy reasons. These were removedby consensus and totaled only four items.

The remainder of this report organizes the responses according to the categoriesidentified through prioritization, conceptually similar items being merged (items ranked5, 10, and 11; items ranked 1 and 8). An additional topic dealing with policy, researchadministration, and public information was created to deal with the remainder of theitems not associated with the foregoing groupings.

Financial support (items 1, 8)

Access to financial support for biotechnology is needed to fund collaborative researchthat will apply biotechnology to the improvement of banana and plantain. Additionally,there is a need to provide access to consumable materials; that might include lowerprices through research subsidies or some other mechanism.

There is also a need to provide logistical support for biotechnology research thatwould include services such as germplasm exchange and access to genetic materials(e.g., cDNA, YAC libraries, etc.).

It was proposed that foreign aid in developed countries should increase to 0.6% oftheir gross national product and that these countries should give consideration tofunding research on banana and plantain biotechnology in other developed-countrylaboratories to support the application of biotechnology in producing countries.

Another suggestion was to place a tax on pesticides to raise funding for biotechnologyresearch. It was also noted that increased funding is needed for research projects onbanana virus, control, and elimination of viruses while exchanging Musa germplasm.

TrainingThere appeared to be general recognition of the need to intensify training efforts as theyrelate to the application of biotechnology to the improvement of banana and plantain.This might be done through the creation of an international training center that wouldundertake responsibilities for preparing teaching programs and materials, training thetrainers, and offering regional and inter-regional training courses. There will be a needto target the trainees. That might include organized training of experts in locallaboratories.

The establishment of biotechnology degree programs in producing-countryuniversities was also suggested. This might have the effect of stimulating Ph.D. researchon banana biotechnology in national programs.

Other responsibilities of a training effort might include refresher training programsand follow-ups to training that would trouble-shoot related issues. Consideration shouldbe given to creating a banana/plantain collection for training purposes and theorganization of a training package for instrument maintenance.

Develop laboratory infrastructureNumerous suggestions were made to develop laboratory infrastructure in a broad sense.Such infrastructure development could include the establishment of referencelaboratories and collections that would support the application of biotechnology tobanana and plantain improvement. Infrastructure development might also include theelaboration of simple methods, nonhazardous techniques, and low-cost technology thatcould be used in producing-country research laboratories.

Additionally, providing laboratory safety procedures and advice on relevantbiotechnology instrumentation could be useful. Moreover, standardized techniqueswould be useful for interlaboratory comparisons of results.

237Working Group 3

It was proposed that an improved nursery infrastructure would facilitate researchactivities, as would improved quarantine facilities.

It was proposed that producing-country quarantine regulations should be evaluatedfor their impact on the importation of biotechnology research supplies.

Access to biological materials could be increased through facilitated exchanges ofmaterials such as DNA, plasmids, cDNA, YAC libraries, and through the establishment ofcollections of Musa pests and pathogens.

Bi-directional scientific exchangeThe participants recommended that attention be given to facilitating bi-directionalscientist exchange in the application of biotechnology to banana and plantainimprovement. This could help create mutual projects with joint responsibilities, and thusstrengthen research activities.

The dimensions of such exchanges should include visits among producing-countryscientists, as well as producing-country scientist visits to advanced laboratories. Thiscould be enhanced if fellowships were made available for producing-country scientists tosupport these exchanges.

Additionally, there is a need for an increase in visits by developed-country scientiststo producing-country research laboratories. This could be assisted by adopting improvedcommunications between overseas experts and producing-country scientists. Scientificexchanges could also be encouraged by holding periodic biotechnology exhibitions orscience fairs in producing countries.

Finally, to support partnerships, producing-country scientists could be invited toserve as editors of biotechnology journals.

Research networking (items 5, 10, 11)

Several dimensions were identified by the participants for networking researchprograms. These included the linking of: national programs to IARCs; producing-countrylaboratories to biotechnology laboratories in developed countries; and biotechnologylaboratories to breeding programs. On this last point, it was noted that there seemed tobe a need to develop the acceptance of biotechnology materials and products bybreeders and agronomists to facilitate adoption of this technology. To realize thepotential of the biotechnologies for the improvement of productivity and stability ofbanana and plantain, the new technologies should be integrated into plant breedingprograms, and satisfy the goals and needs of breeders.

Biotechnology research in institutes of regional relevance needs to be strengthened.This might be done by holding small international meetings in producing countries andby developing interdisciplinary groups to address specific topics.

There is an apparent need to encourage collaboration between university researchscientists and agricultural ministries in producing countries.

The establishment of a tropical biotechnology network in developed-country researchlaboratories could be of assistance in facilitating the application of biotechnology to


banana and plantain improvement. Encouraging research laboratories to work withproducing-country research laboratories on tropical crop problems would also be ofbenefit. In this respect, there may be a need to support these linkages through modelnegotiations (e.g., memoranda of understanding) for producing-country laboratories toengage in work with developed-country research laboratories.

In addition, some consideration should be given to developing common banana andplantain biotechnology research programs between the European Community and theUSA. Other combinations of developed-country laboratories might also be considered.

Efforts should given to promoting the role of women in the application ofbiotechnology to banana and plantain improvement. Efforts should be given todiscourage brain drains (i.e., the loss of talented scientists from producing countries).

Provision of documentsThe application of biotechnology to banana and plantain improvement could befacilitated through research support activities that would distribute certain kinds ofinformation as documents. For instance, there is a need to catalogue and disseminateinformation on existing biotechnology and the impact of those applications vis-à-visbanana and plantain improvement.

Information on biotechnology and the identification of needs of producing countriesshould be developed and disseminated widely. The publication of biotechnology manualsin the languages of user countries was seen as a desirable activity.

The results of biotechnology research pertaining to banana and plantainimprovement should be published in producing-country journals. In addition,publications that simplify the application of biotechnology in producing countries, andinformation on the progress of large-scale production of germplasm evolved bybiotechnology, should be produced. These might take the form of newsletters or someother type of broad information distribution.

Efforts should be made to document the genetic diversity of the Musa gene poolavailable in the producing countries, to evaluate their important characteristics, and todisseminate existing information on collections of Musa so that this information couldbe made available to others. The creation of databases to support biotechnology researchshould be explored.

Identify targets with defined valueThere is a need to set research priorities correctly. This would assist in targetingresearch that would address clearly stated objectives as to what is to be transferred astechnology and who would benefit. There is a need to evaluate objectively theexpectations of biotechnological research and its relevance to national goals.

It was proposed that reviews of biotechnology programs in producer countries beconducted and that encouragement be given to conducting problem-oriented research.Encouragement should also be given to linking biotechnology to the development of

239Working Group 3

sustainable agricultural production. This was seen as linking “ecologically sound”practises through biotechnology research.

Encouragement should be given to applying biotechnology to local nonexport bananaand plantain varieties to improve their productivity and quality, for local and exportmarkets.

Some consideration should be given to ensuring that technologies to be transferredare realistic to the needs of producer countries, and they should be verified throughindependent, expert appraisal.

It was proposed that technology transfer projects be peer-reviewed, especially toensure relevance for eventual adoption. Research on impact assessment (perhapsthrough cost-benefit analysis) of current banana/plantain research could be useful.Additionally, fact-finding missions focusing on biotechnology-relevant problems shouldbe encouraged. These might include a survey of problems in producer countries, and theconvening of regional/national workshops for identifying research needs.

There was a recognized need for sensitivity to, and understanding of, the limitationsof biotechnology transfer for producing countries. This should include an understandingof the socioeconomic constraints of the end-user, and perhaps an agreement of donorssupporting banana and plantain research to be more receptive to producing-countrydecisions on what is wanted.

Documentation of the costs arising from a failure to increase Musa productivityshould help to establish the need for the application of biotechnology to banana andplantain improvement. Moreover, clear assessment of the losses that occur from fieldinfestations should provide further evidence.

It was proposed that the successful completion of a few biotechnology projects couldbe helpful in establishing expectations and acceptance. Successful experiences could beadvertised to promote broader acceptance of this new technology.

Contract research should be undertaken on specific objectives related to the transferof biotechnology for banana and plantain improvement. Some examples of such targetedresearch might be:

- Development of transformation methods.- Postharvest preservation and storage.- Improved pest control.- Mechanisms of resistance.- Diagnostic kits.- Bioprocessing technology.- Biomass conversion.- Alternative uses for plant products.- Research-enabling technologies.- Value-added technologies.It was also proposed that an inventory be made of unsuccessful technology transfer

projects to learn from these experiences. Donors should be advised that technologiesother than biotechnology may sometimes be more appropriate. A policy statement mightbe issued that would caution against accepting “biotechnology at any price”.


241Working Group 3

Finally, it was proposed that biotechnology products must not be imposed onproducing countries and that some organization should assume responsibility formonitoring activities to make sure that this does not happen.

Appoint INIBAP biotechnology coordinatorIt was proposed that INIBAP appoint a biotechnology coordinator. Responsibilities ofthis proposed position might include the coordination of activities to improve researchefficiency and to stimulate greater support for biotechnology research from fundingagencies. These responsibilities might be undertaken through the creation of aninternational advisory center to support the network.

Transfer of technologyIn this section, consideration was given to activities that would facilitate the transfer oftechnology to the user level (as opposed to transferring technology between researchlaboratories).

Efforts should be made to encourage private enterprise in banana- and plantain-producing countries to engage in Musa biotechnology. This might be done through thestimulation of venture capital and the fostering of long-term support for biotechnologyfrom local industries. By involving local commercial entities in the transfer process,adoption could be encouraged as the technology emerges.

Markets for the products of biotechnology should also be developed and these couldinclude the industrial sector. Additionally, by strengthening relationships between thepublic and private sectors, technology transfer could be enhanced. This might entailsubsidies for biotechnology products, especially during early market development.Government support for research and development of biotechnology products may alsoneed to be considered.

Certain support activities could also help the transfer of technology. By providinglegal expertise and access to information, issues of intellectual property rights could beresolved and thus foster technology transfer. Encouragement of open markets, free fromprotectionism, could also foster the transfer of technology. To encourage the applicationof biotechnology to banana and plantain improvement, some consideration should begiven to the equitable distribution of royalties that represent shared contributions andinvestments. This might include recognition of proprietary know-how in producingcountries and sensitivity to the fact that bananas and plantains are staple food crops insome areas.

Another area of support that could facilitate the transfer of technologies tocommercial products would be programs to ensure environmental safety ofbiotechnology products. Biosafety regulations should be based on rational science andsome thought should be given to providing assistance to national programs in need ofdeveloping biosafety regulations and/or guidelines.

In a related way, regulatory delays in developed countries (e.g., USA) could send thewrong signals to producing countries and thus cause delays in deploying biotechnologythere.

A need was recognized to develop better distribution systems to make the products ofbiotechnology available to the end-user. Such a system should have provisions for the rapiddissemination of biotechnology products, and mechanisms for supporting these productsthat might be aided through funding from donor countries to help develop such newmarkets.

It was proposed that appropriate training be given to agricultural extension workers onthe usefulness of successful biotechnology products, for use by interested growers, andthat the products of biotechnology be demonstrated in the field to show success andencourage adoption. Local markets should be prepared in advance for the products ofbiotechnology to enhance acceptance. This might be supported visually by the preparationof a logo for biotechnology products, and the use of slogans such as “Buy Biotech”.

Additionally, farmers from producing countries should be encouraged to becomefamiliar with the expected products of biotechnology research. This should includeeducation about not only the advantages, but any attendant risks, of this new technology,and might be done through local grower groups and the exchange of scientists and farmersbetween producing countries and nonproducing countries. It could also include televisioninterviews with farmers and politicians on subjects pertaining to biotechnology researchapplications for banana and plantain improvement.

Nonproducing countries should be encouraged to eat more and/or different types ofbananas to help expand markets and thus create opportunities for the application ofbiotechnology to banana and plantain improvement.

Activities relating to policy, research administration, and public informationThere is an apparent need for an educational program for decisionmakers, researchadministrators, and donors. The educational program should include the benefits, as wellas the problems, of biotechnology. This activity might be supported through the use of adonor consultant group on biotechnology.

There is also a need to educate the developed world population on the food value ofbananas in many producing regions. This would help to educate the electorate in donorcountries, thus providing motivation and increased political will on the donor side. Toaddress this, it was proposed that donors be encouraged to hire biotechnology experts andthus improve access to information. Such action might be supplemented by lobbyingdonors for research program needs, and perhaps assisting in the placement of pro-biotechnology people in “high places”.

As a corollary activity, it was proposed that effort be given to encouraging politicalmotivation for biotechnology research for banana and plantain improvement in producingcountries. Some specific recommendations included the suggestion that international aidagencies purge politics from their decisionmaking process and work to avoidadministrative bureaucracy.

It was proposed that the applications of biotechnology to benefit banana- and plantain-producing countries be placed on the UNCED agenda and that draft legislation beprepared that would be helpful to producing countries establishing a biotechnology policy.


It was proposed that nongovernmental organizations (NGOs) be identified to explainthat biotechnology is a “good thing”. Also, consumers could be targeted throughadvertising to explain the environmental advantages of biotechnology. This would help toeducate the biotechnology users. A suggestion was made to develop a biotechnologyvocabulary less frightening to the public and to begin the process of consumeracceptance by educating consumers on the products and benefits likely to be derivedfrom biotechnology research applied to banana and plantain. This could be supportedwith the previously proposed slogan “Buy Biotech”.

Films and videos could be developed to help provide public information on thissubject. Such an effort could address the “fear” of biotechnology and might also considerutilizing a well-known personality to act as a global spokesperson for this technology.

ConclusionsA rich assortment of suggestions were developed to provide guidance on how to facilitatethe transfer of biotechnology for the genetic improvement of banana and plantain. Theorganization of the material in this report was intended to report faithfully the intentionof the Group participants, and any misrepresentation should be consideredunintentional and solely the responsibility of the facilitators.

Given the enthusiastic participation of Working Group 3, it should be reasonable toconclude that there is both a recognized need and strong support for deliberateactivities targeted at many levels, from the farmer to the scientist, the politician to thegeneral public, to facilitate the transfer of biotechnology, not only to researchlaboratories in producing countries, but to foster global exchange for its eventualapplication to improve production of banana and plantain.

ReferenceDELBECQ AC, VONDEN VEN AH, GUSTAFSON DH. 1975. Group Techniques for Program Planning: a guide to nominal

group and delphi processes. Glenview, IL, USA: Scott, Foreman and Co.

243Working Group 3

Annexes

247Acronyms and Abbreviations

Annex 1: Acronyms and Abbreviations

AAcc cultivated Musa acuminata diploidAAw wild Musa acuminata diploidABSP Agricultural Biotechnology for Sustainable Development (USAID project)ACIAR Australian Centre for International Agricultural ResearchAID Agency for International Development (USA)AP-PCR arbitrarily primed PCRASPNET Asian and South Pacific Network (INIBAP)BAP benzylaminopurineBBTV banana bunchy top virusBM basal mediumCATIE Centro Agronómico Tropical de Investigación y Enseñanza (Costa Rica)cDNA complementary DNACE crude extractCEC Commission of the European CommunitiesCGIAR Consultative Group on International Agricultural ResearchCGIS Central Germplasm Information System (INIBAP)CIRAD Centre de coopération internationale en recherche agronomique

pour le développement (France)CNPMF Centro Nacional de Pesquisa de Mandioca e Fruticultura Tropical (Brazil) CP coat proteinCPMR coat protein-mediated resistanceCRSP Collaborative Research Support Program (USA)CTA Technical Centre for Agricultural and Rural Cooperation (the Netherlands)CTAB cetyl-trimethylammonium bromidecv, cvs cultivar, cultivarsDMSO dimethylsulphoxideDNA deoxyribonucleic aciddsDNA double-stranded DNAdsRNA double-stranded RNAECS embryogenic cell suspensionEEC European Economic CommunityEIA enzyme immunoassayEM electron microscopyEMBRAPA Empresa Brasiliera de Pesquisa AgropecuáriaEMS ethylmethane sulphonateFHIA Fundación Hondureña de Investigación AgrícolaFOC Fusarium oxysporum f.sp. cubenseFWI French West IndiesGNP gross national product

IAEA International Atomic Energy Authority (Austria)IARC international agricultural research centerIARPCB International Association of Research on Plantains and Cooking BananasIBPGR International Board for Plant Genetic Resources (Italy)ICTA International College of Tropical Agriculture (Trinidad) IEF isoelectrofocusingIEM immunoelectron microscopy IITA International Institute of Tropical Agriculture (Nigeria) IMTP International Musa Testing Program (INIBAP) INIBAP International Network for the Improvement of Banana and Plantain (France)IPM integrated pest managementIPR intellectual property rightsIRFA Institut de recherches sur les fruits et agrumes, CIRAD (France) kb kilobaseKU Leuven Katholieke Universiteit Leuven (Belgium)MF Mycosphaerella fijiensisMGES Musa Germplasm Exchange System (INIBAP)NEP naked-eye polymorphismNRC National Research Council (USA)ORF open reading framePBIP Plantain and Banana Improvement Program (IITA)PCR polymerase chain reactionPDA potato dextrose agarPRX peroxidase QDPI Queensland Department of Primary Industries (Australia) QTL quantitative trait lociQUT Queensland University of Technology, AustraliaR&D research and developmentRAPD random amplified polymorphic DNARAPiD DNA amplification fingerprinting rDNA ribosomal DNA RFLP restriction fragment length polymorphismRMP reference mapping populationRNA ribonucleic acidrRNA ribosomal RNA Sat satellite (e.g., Sat RNA)ssDNA single-stranded DNAssRNA single-stranded RNATAC Technical Advisory Committee (of the CGIAR)USAID United States Agency for International DevelopmentUSDA United States Department of AgricultureUV ultravioletVLP virus-like particleYAC yeast artificial chromosome

248 Acronyms and Abbreviations

249Participants

Annex 2:

ParticipantsA Abdelnour Esquivel, CATIE, PO Box

17, 7170 Turrialba, Costa RicaP Acuna Chinchilla, Corporación

Bananera Nacional, PO Box 6504-1000, San José, Costa Rica

O Arias, Agribiotecnología de Costa RicaS.A., P.O. Box 25-4001, Alajuela,Costa Rica

CJ Arntzen, Department ofBiochemistry, Texas A&M University,College Station, Texas 77843-2128,USA

F Bakry, CIRAD-IRFA, Station deNeufchâteau, 97130 Sainte Marie,Guadeloupe

J Barba, Laboplant, Ave 12 de octubre1035/of. 701, Quito, Ecuador

L Bernier, Bureau Régional de Coopération Scientifique et Technique, French Embassy inCosta Rica, Apartado 10177-1000,Costa Rica

V Broomes, National AgriculturalResearch Institute, Mon Repos, EastCoast Demerara, Guyana

IW Buddenhagen, AgronomyDepartment, University of California,Davis, CA 95616, USA

JA Chambers, US Agency forInternational Development, Bureaufor Research and Development,Office of Agriculture, Room 406-A SA-18, Washington DC20523-1809, USA

JL Dale, Queensland University ofTechnology, Centre for Molecular

Biotechnology (QUT), 2434 Brisbane,4001 Queensland, Australia

E De Langhe, INIBAP, Bât.7, ParcScientifique Agropolis, 34397Montpellier Cedex 5, France

D de Waele, Plant Genetic Systems NV,Jozef Plateaustraat 22, 9000 Gent,Belgium

M Dufour, CATIE/CIRAD-IRCC, ApartadoPostal 11, 7170 Turrialba, Costa Rica

JV Escalant, CATIE/CIRAD-IRFA, POBox 104, 7170 Turrialba, Costa Rica

A Fareck, Vitronov Inc., 35 BallantyneTerrace, Dorval, Quebec H9S 3E4,Canada

C Fauquet, ORSTOM Research Scientist,Co-Director ILTAB, Division of PlantBiology, MRC7, 10666 North TorreyPines Rd, La Jolla, CA 92037, USA

P Ganashan, Plant Genetic ResourcesCenter, Department of Agriculture,PO Box 59, Gannotuwa Peradeniya,Sri Lanka

D González de León, CIRAD-BIOTROP,PO Box 5035, 34032 MontpellierCedex 1, France

R Haïcour, Université Paris XI Orsay,Bât. 360, Laboratoire deMorphogenèse végétaleexpérimentale, 91405 Orsay Cedex,France

RB Horsch, Monsanto Company, MailZone GG4J, 700 Chesterfield VillageParkway, St Louis, MO 63146, USA

RD Huggan, INIBAP, Bât.7, ParcScientifique Agropolis, 34397Montpellier Cedex 5, France

250 Participants

R Jaramillo, INIBAP-LAC CATIE, PO Box 60, Turrialba, Costa Rica, c/o PO Box 4824-1000, San José,Costa Rica

DR Jones, INIBAP, Bât.7, Parc Scientifique Agropolis, 34397 Montpellier Cedex 5, France

G Kahl, Johann Wolfgang GoetheUniversity, Siesmayerstr.70, 6000 Frankfurt/Main, Germany

K Kamate, Université Nationale, Faculté des Sciences et Techniques(FAST), 22 BP 582, Abidjan 22, Côte d’Ivoire

DK Koumba, Projet CIAM, BP 2183,Libreville, Gabon

SL Kurtz, Twyford Plant Labs. Inc., 15245 Telegraph Rd, Santa Paula, CA 93060, USA

RDR MacKenzie, US Dept of Agriculture,Cooperative State Research Service,Suite 330 Aerospace Building, 901 D St S.W., Washington DC 20250-2200, USA

AM Mailu, Kenya Agricultural ResearchInstitute, PO Box 57811, Nairobi,Kenya

K Matasumoto, CENARGEN/EMBRAPA,S.A.I.N. Parque Rural 70770, PO Box02372, Brasilia-DF, Brazil

X Mourichon, Lab. Pathologie Végétale,CIRAD-IRFA, BP 5035, 34032 Montpellier, France

L Muller, PO Box 336-1007, CentroColon, San José, Costa Rica

LC Navarro Mastache, Centro deInvestigación Científica de YucatánA.C., Apartado 87 Cordemex, 97310 Merida, Yucatán, Mexico

G Ndamage, Institut des SciencesAgronomiques du Rwanda, PO Box 138, Butare, Rwanda

B Neuenschwander,IRCC/CATIE/University of Zurich,Apartado 143, CATIE, Turrialba,Costa Rica

CL Niblett, University of Florida, Plant Pathology Department, PO Box 110680, Florida, USA

FJ Novak, IAEA Laboratories, PlantBreeding Unit, PO Box 100, A-2444 Seibersdorf, Austria

BD Oakes, Vitronov Inc., 35 BallantyneTerrace, Dorval, Quebec H9S 3E4,Canada

RO Quezada, CATIE Representative,Fray Cipriano de Utrera, Centro deLos Heroes, PO Box 374-2, SantoDomingo, Dominican Republic

W Parrott, University of Georgia,Department of Crop and SoilSciences, Athens, GA 30602-7272,USA

M Perea-Dallos, National University,Biology Department, Science Faculty,PO Box 23227, Bogotá, Colombia

RC Ploetz, University of Florida, TropicalResearch and Education Center,18905 SW 280th St, Homestead, FL 33031, USA

P Ramirez Fonseca, CIBCM, Universidadde Costa Rica, Apartado 7353-1000,Costa Rica

O Reuveni, Agricultural ResearchOrganization, The Volcani Center,Bet Dagan, PO Box 6, Israel

JG Rivas Ducca, PO Box 3597, 1000 San José, Costa Rica

H Rodriguez, CATIE, PO Box 127,Turrialba, Costa Rica

F Rosales, FHIA, PO Box 2067, San Pedro Sula, Honduras

P Rowe, FHIA, PO Box 2067, San PedroSula, Honduras

251Participants

P Rubaihayo, Makerere University,Department of Crop Science, PO Box 7062, Kampala, Uganda

D Shaw-Wisdom, Scientific ResearchCouncil, PO Box 350, Kingston 6,Jamaica

M Smith, Queensland Department ofPrimary Industries (QDPI),Maroochy Horticultural ResearchStation, PO Box 5083, SCMCNambour, Queensland 4560,Australia

MR Söndahl, DNA Plant TechnologyCorporation (DNAP), 2611 BranchPike, Cinnaminson, NF 08077, USA

G Strobel, Montana State UniversityCollege of Agriculture, Dept of PlantPathology, Bozeman, MT 59717-0314,USA

C Suarez Capello, FUNDAGRO, PO Box4076, Guayaquil, Moreno Bellido 127y Ave. Amazonas, Quito, Ecuador

R Swennen, Katholieke UniversiteitLeuven, Laboratory of Tropical CropHusbandry, Kardinaal Mercierlaan92, B-3001 Heverlee, Belgium

FA Taha, Agriculture College, CairoUniversity, 23 El-Etehad Square,Maadi, Cairo, Egypt

C Teisson, CIRAD, BP 5035, 34032 Montpellier Cedex 1, France

H Tezenas du Montcel, INIBAP, Bât.7,Parc Scientifique Agropolis, 34397 Montpellier Cedex 5, France

N Von Mende, Rothamsted ExperimentalStation, Harpenden, Herts, AL5 2JQ, UK

D Vuylsteke, International Institute ofTropical Agriculture, Oyo Road, PMB5320, Ibadan, Nigeria

LA Withers, IBPGR, Via delle SetteChiese 142, 00145 Rome, Italy

K Wright-Platais, CGIAR Secretariat,The World Bank, 1818 H Street N.W.,Washington, DC 20433, USA

AB Zamora, University of thePhilippines, Los Baños, PO Box 3720,College, Laguna, Philippines

S Zok, Institut de la RechercheAgronomique, Tissue CultureLaboratory, IRA Ekona, PO Box 25,Buea, Cameroon

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