IAEA-TECDOC-800
In vitro mutation breeding of bananas and plantains
Final reports FAO/IAEAofan co-ordinated research programme organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture from 19881993to
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IN VITRO MUTATION BREEDING OF BANANAS AND PLANTAINS IAEA, VIENNA, 1995 IAEA-TECDOC-800 ISSN 1011-4289 © IAEA, 1995 Printe IAEe th AustriAn y i d b a June 1995 FOREWORD
Mutation breedin bees gha n successfully applie cerealso dt , legume othed san r seed- propagated crops whicn i , inducee hth d mutants segregat homozygotess ea vegetativeln I . y propagated crops, many important mutant cultivars have been released t onlno ,y ornamental plant t alssbu o major fruit tree crops majoe Th . r objectiv previouf eo s studie bees e sha nth development of cultivars with increased resistance or tolerance to biotic or abiotic stresses, e.g. disease or lodging resistance, quality improvement for higher market values, early or late maturity for better agriculture practices, etc.
Therurgenn a s e applicatioi e th t nee r fo d f mutatioo n n breeding techniquen i s vegetatively propagated crop se bananas sucth e northers ha mosn th I f . o t n developed countries, banana is known primarily as a dessert fruit. However, bananas and plantains (cooking bananas majoe ar ) r source f starcso tropican hi l countries alse ar o d importanan , t expors a t items. Currently, yields ofMusa crop declinine sar plantings ga attackee sar y db various disease organisms, e.g. black sigatoka in Latin America, Fusarium wilt in Southeast Asia, and banana bunchy-top virus (BBTV) disease in Pakistan.
Unfortunately, improvement of bananas and plantains by conventional breeding technique difficuls si problemo t e du t s associated with sterility. Eve promisina f ni g disease resistance gene were foun relatea i h d d species t wouli , vere db y difficul incorporato t t t ei into superior cultivars thesn I . e instances mutation practicaa breedin e b y lgma methor dfo the improvement of these crops. Recent development of in vitro culture techniques makes the application of mutation breeding possible with bananas and plantains. Large numbers of somatic embryos, or somatic tissues, can be irradiated in a small container. Thus, the treatmen large th f e o tpopulation s normally require mutatior dfo n breedin possibles gi .
A co-ordinated research programme (CRP organizes )wa studo dt feasibilite yth f o y developing mutation breeding protocol solvinn Musa,i n si d probleme an gth s likele b o yt encountered in the implementation of such an approach. This TECDOC contains the final report participante th f so CRPe reporth e f so Th . groupt. s Nova froF hi lat . e d meth Dr k, an presentethas wa tSecon e th t da d Research Co-ordination Meeting (RCM Josen ) helSa ,n di Costa Rica is also included. Dr. Novak who passed away on 19 July 1993, was a pioneer an drivinda g force behin banane dth a research program t IAEA'sa s Seibersdorf Laboratory and elsewhere.
This document was jointly prepared by the Scientific Secretary, E. Amano, and by R.L. Jarret, United States Departmen Agriculture'f to s Agricultural Research Service, whose contributio acknowledgeds ni . EDITORIAL NOTE
preparingIn this publication press,for IAEAthe staffof have pages madethe up from the original manuscripts as submitted by the authors. The views expressed do not necessarily reflect those of the governments of the nominating Member States or of the nominating organizations. Throughout textthe names Memberof States retainedare theyas were when textthe was compiled. The use of particular designations of countries or territories does not imply any judgement by publisher,the legalthe IAEA, to status the as of such countries territories,or of their authoritiesand institutions delimitationthe of or of their boundaries. The mention of names of specific companies or products (whether or not indicated as registered) does implyintentionnot any infringeto proprietary rights, should construednor be it an as endorsement or recommendation on the part of the IAEA. The authors are responsible for having obtained the necessary permission for the IAEA to reproduce, translate or use material from sources already protected by copyrights. CONTENTS
INTRODUCTION ...... 7 .
Improvemen bananaf o t s (Musa cvs.) throug vitron hi anther culture ...... 9 . M. Perea-Dallos Genetic improvement of bananas through in vitro mutation breeding in Costa Rica . . 13 A.T. Valerin, R. Salazar, W. Navarro Radiation mutation induction in 'China Tianbao' banana ...... 17 Huang,Wei,F. W. Yang, P. Li, Zheng, J. Guo,C. J. Huang,D. He, R. X. Huang, B. Chen, W. Lin Improvemen bananf o t a (Musa cvs.) throug vitron hi culture technique induced san d mutations ...... 25 S.H. Siddiqui, A. Khatri, LA. Khan, G. Nizamani, R. Khan Effect of toxic filtrate of Fusarium oxysporum f.sp. cubense on the development of banana (Musa cvs.) shoot tips ...... 31 B.M.J. Mendes, B.I.P. Rodrigues, A. Tulmann Neto vitron I mutation breedin developmene th r gfo bananaf o t s with resistanc raco et , e4 fusarium wilt (Fusarium oxysporum f. sp. cubense) ...... 37 U.K. Smith, S.D, Hamill, P.W. Langdon, K.G. Pegg Somatic embryogenesi bananan si plantaind san s (Musa clone speciesd san ) .....5 4 . A.D. Krikorian Improvement of Musa through biotechnology and mutation breeding ...... 57 F.J. Novak, Afza, R. Morpurgo, R. Duren, Van Sacchi, M. M. KhatriA. Study on mutation breeding of banana, 'Kluai Khai' ...... 65 B. Silayoi, K. Wanichkul, S. Keawsompong, P. Saraduldhat, N. Singhaburaudom
CONCLUSIONS AND RECOMMENDATIONS ...... 79
PARTICIPANTS IN THE CO-ORDINATED RESEARCH PROGRAMME ...... 85 INTRODUCTION
Less than a decade ago fewer than a handful of scientists worldwide were involved in biotechnological research with bananas and plantains. This situation has changed dramatically in the last ten years. More than a dozen laboratories are now actively involved in tissue culture, molecular genetic, transformatio basid nan c plant pathology research with Musa. For the most part these research efforts are conducted in support of, or in direct collaboration with, on-going plant breeding programmes. currene Mucth f ho t interes Musan i t improvemen attributee b n ca t threo dt e factors. increasinn a ) 1 Thes: ge ar eawarenes vulnerabilite th f o s f bananao y plantaind an s o t s attac y varioub k s disease organism d insecan s t n acknowledgemenpestsa ) 2 , e th f o t difficult f breedino y g these crops using conventional techniquesrecognitioe th ) 3 d f an ,no the social and economic importance of these crops to many millions of people worldwide. An impressive amoun f progreso t s alreadha s y been e developmenmadth n i e d an t application of biotechnological approaches for Musa improvement. A few examples include : tissue culture protocols for the in vitro propagation of Musa clones via organogenesis, and somatic embryogenesis have been reported and are being further refined. successful cryopreservation of Musa cell suspensions has been achieved. molecular genetic markers have been used to study evolutionary and taxonomic relationship f Musaso r estimatinspeciefo d an s g genetic diversit Musan yi germplasm collections. a genetic linkage map of Musa, using molecular markers, has been constructed. progress has been made in the application of genetic transformation technology using the biolistic process. conditions for mutation induction, utilizing in vitro culture techniques in combination with 60C oray7 r EMSo s , have been established irradiation-induced an , d mutante ar s undew no r field evaluation. an in vitro Musa germplasm repository has been established. Recent advance genn i s e isolation technology microsatellitd an C YA , e gene mapping, and transformation technologies suggest thae nexn yearth tte t s a ver wile yb l exciting period for Musa biotechnology researchers. In much the same way that tissue culture and mutation induction techniques have been melded into an effective system for Musa improvement, so will molecular genetic and transformation technologies merge. Ultimately all of these approaches will be used to support both conventional and mutation breeding programmes as Musa improvement efforts become increasingly multi-disciplinary. The reports presented in this TECDOC represents the progress made to date in the application f mutatioo n breedin n vitroi d an gcultur e technique e productioth r fo s f o n disease resistant banana plantainsd an s . This wor s largeld inspirean kwa , ydby directed by, the late Dr. F. J. Novak, in whose laboratory protocols for the regeneration of banana somatia vi c embryogenesis e irradiatioth d an , f somatino c embryo r mutatiosfo n induction, were first developed. These significant advancements opened the way for the broader application of mutation induction and tissue culture techniques for the generation of disease resistant bananas and plantains.
Next page(s) left blank IMPROVEMENT OF BANANAS (Musa cvs.) THROUGH IN VITRO ANTHER CULTURE
M. PEREA-DALLOS Departament Biologiae od , Faculta Cienciase dd , Universidad Nacional de Colombia, Bogota, Colombia
Abstract
Agricultural products play a great role in the Colombian economy, and the banana is one of the most important. Since 1981, one of the more serious problems effecting production of this crop is the fungus Mycosphaerella fijiensis . difformis,sp that causes black sigatoka disease. e recenMosth f o tt efforto t s control this disease have been directed toward e identificatiosth f cloneno s toleran r resistano t o thit t s disease. One alternative approach is the use of anther culture to obtain resistant haploid plants. Diploid clones (Musa - AA) have been used as a model in this study. The results presented here identify the most appropriate stag f antheeo r developmen r callufo t s inductio proliferationd nan treatmentd an , s that reduce tissue browning.
1. INTRODUCTION mose Bananath f t o plantainimportan d o an s tw e sar t stapl casd ean h crop r severasfo l million people developinth n i e g countries [1]. Sinc e beginninth e f large-scalo g e banana production, Colombimose th f t o importantbee s e aha n on banan plantaid aan n export producers. Most plantain production occurs on farms that are located in the mountains, where coffee is the main crop. Under these conditions plantains are grown to furnish food for the coffee growers (as a "survival crop"). A "survival" crop implies securit e for th f chea mo n i y p e farmere food th t onlth r no f ,fo y o s growing regions t als Colombia'r bu ,ofo s various social groups. The genetic improvement of bananas and plantains through conventional breeding methods has been hampered, due to the lack of understanding about the genetics of the plant, the pathogens attacking the crop, the interaction and co-evolution of host and pathogens, and the various factors effecting sterility that result in the production of non-viable seed [2]. Sterility in edible bananas is resule complea th f o t f factorxo s thadirectle ar t y relate ploide th o dt y (triploidy plane d th an f t o ) the attendant meiotic abnormalities associated with triploidy. Fruit developmen e resulth f s o i tt parthenocarpy. These characteristic geneticalle ar s y controlle havy ma e d arisedan n through gene mutations in fertile diploids [3, 4, 5, 6, 7]. A combinatio traditionaf no l cross-hybridizatio biotechnologf o n e techniqueus e th d ysan could provide a powerful tool for the development of new Musa clones with desirable features such as, for example, resistanc r toleranceo diseaseso et . Djploid clone f boto s h wild (seed-fertiled an ) edible banana e essentiaar s s startina l g materia r bananfo l a breeding s sourcea , f genetio s c resistanc r toleranceo e majoth o ret banana diseases. Source f resistanco s mano et y diseased an s insects attacking Musa (except Banana Bunchy Top Virus), have been identified in the extensive collection diploif so d Musa acuminata . balbisianaM d an [8].
. 2 ANTHER CULTURE
The importance of anther culture to crop improvement has been discussed elsewhere. Significant advance anthen i s r culture techniques have been decadeslaso e madth tw t n ei . Anther culture of monocotyledonous plants has been extensively researched over a number of years, since many important crop plants belon thio gt s group. Problems associated with strong genotypic effects and high proportions of albino regenerants have seriously hindered the progress in this area [9]. A large numbe f plano r t species have been successfully regenerated from cultured antherr o s pollen grains harveste t differenda t state developmentf so . However Musan i , clones, especialle yth widely cultivated clones, most of the genotypes do not produce viable pollen grains or egg cells. This phenomeno f man o s prevente e ha ny us valuabl e th d e clone conventionan i s l hybridization improvement programs. Anther culture of more genotypes needs to be attempted in order to determine the interaction of genotype with media composition and environmental conditions. In e importancvieth f wo f haploideo plann i s t breeding s highli t i , y desirabl o investigatet e various factors concernin e effecth gf environmeno t d median t a composition e inductioth n o f celo n l division and differentiation. Anther culture is not currently used frequently in Musa breeding e stricprogramth o tt dependence du s f plano e t regeneratio genotypen o n . However, there ar e attempt resolvo st e this problem. Very recently, Bakr Horrd yan y [10] regenerated plantlets from cultured banana anthers derived from A A diploids plantlete Th . s were diploids thaoriginated ha t d from unreduced microspores.
3. MATERIALS AND METHODS
3.1. Plant material Experiments were carried out using three selected diploid clones of banana: Bocadillo, Pisang lilind Malascensisan , . These materials were collected from nurser ye Institutth field t a s e Colombiano Agropecuario (ICA) at Carepa-Uraba. In keeping with the objectives of the study to determine the appropriate state of inflorescence development suitable for culture, inflorescences were chosen at different stages of development, for example when the bunch emerged 15 cm, and whe . Thibunce cm nth emerge d s 5 latte3 hha - 0 rd 3 developmenta l stage corresponde time th eo dt when the bunch began to bend downwards. Considering the asynchronous development of anthers in Musa, several hands presenwere th en i selectete studiesus r e mosdfo Th .t appropriate stagf eo the developmen whes inflorescence wa t nth 1-1d eha 2 hands.
Pollen grains were cultured when they uni-nucleate werth n ei e stag whed exine an nth d ean inline layers were observable. The collected inflorescences were stored at 4 °C for 4 to 7 days.
3.2. Culture procedur medid ean a
These materials were disinfecte (v/v% 5 ) n bleaci d h (Clorox) solutio5 minutes4 r fo n , followe severay b d l rinse steriln i s e water isolatioe Th . f antherno carries swa t unde dou r aseptic condition thed an sy were placed onto Murashig Skood ean g basal medium [11], supplemented with various concentration f (3,6-dichloro-o-anisi1 o s g/ 2 , ^M 5 2 r o , c20 aci , d15 (dicamba), 10 , 5 t a medie caseith s adjuste f awa o n 5.8H o sucrosehydrolysatt 1 dp g/ . e 0 Fou6 Th . d r an eanther s were placed in each culture vessel and then incubated in the dark at 28 °C.
To avoid explant browning due to the phenolic compounds, several concentrations of activated charcoal (0.5, 1.0, and 2 %) and polyamines (spermine 5, 50 and 100 /*M; spermidine 250 and 500 yM and 0 /iM, 50 putrescin )d weran e0 testede25 . Afte e appearancth r f calluseo , explants were subcultured weekly.
. 4 RESULT DISCUSSIOND SAN S Callus was first observed 26 days after culture initiation. Callus development was observed in all anther inflorescencee th f so . Activated t effectivcharcoano s n reducini wa el e browninth g f callio t gvariou bu , s concentration f polyamineso s were very usefu reducingn i l this browning. Intensive blackenins gwa a serious problem durin e establishmenth g f antheo t r cultures. However e additioth , f o n polyamine e mediuth o t s m helpe controo dt l this phenomeno enhanced nan e calluth d s induction frequency. As seen from the effect of polyamines on browning, it is important to include these compounds during culture initiation. Also, it is necessary to identify the optimal concentration.
10 Mengol d Bagnan i i [12] reporte conversiow dlo thae th t n rat f grapo e e somatic embryos into plantlete verth yo t hig se hcouldu polyamine db e content relativ e levelth o set foun zygotin i d c embryos.
Dicamba
100- D 10 15 80- 20 25
60-
40-
20-
0 Bocadillo Pisanm hi g Malascensis
Fig.l. Effect ofdicamba on the growth of callus from anthers ofdiploid bananas.
TABLE I. Percent of anthers forming callus when cultutred in the presence of dicamba Concentration Genotype of Dicamba Bocadillo Pisang lilin Malascensis
5 88 75 50 10 75 58 37 15 49 35 25 20 32 28 12 25 15 10 8
11 Acknowledgements authoe Th indebtes i r Joine th o tdt Progra m IAEA/FA r supporOfo f par o tf thi o t s workd an , . BayonaR . alsDr ,o ot Directo f Laboratorio r CENIBANANe od . MayorgaM . Dr d , ODirectoan r of Regional 1C A at Carepa-Uraba who provided the plant materials. The author also acknowledges AUGURA and the Universidad Nacional for their support of this research. Gratitude is expressed typine . manuscript Espitie hels th M th hi n f pi o r gt o afo .
REFERENCES [I] CRONAUER, S. S. and A. D. KRIKORIAN. Multiplication of Musa from excised stem tips. Ann. Bot (1984)3 .5 .
] NOVAK[2 . J Musa . F , (Banana Plantains)d an s Biotechnologn I . f perenniayo l fruit crops. F.A. Hammerschla R.Ed gan . Lite (Eds)(1992). [3] KRIKORIAN, A. D. and S. S. CRONAUER Banana. In Handbook of plant and cell . . SharpEvansA W . . D ,Ed , culture. P.V2 l . AmmiratVo . . YamadaY d oan . MacMillan Company Yorw kNe . (1984). ] ROWE[4 . BreedinP , g banana plantainsd san . Plant Breeding Review (1984s2 135-155: ) . ] SHEPHERD[5 . BananK , a breeding paspresentd an t . Acta Horticultura (19876 e19 ) :37-43. ] SIMMONDS [6 evolutioe Th bananase th . f nW o . N ., Longmans. London (1962). [7] SIMMONDS, N. W. Bananas. 2nd ed. Longmans. London (1966). [8] NOVAK, F. J., R. AFZA, M. VAN DUREN, M. PEREA DALLOS, B. V. CONGER and TANG XIOLANG Somatic embryogenesis and plant regeneration in suspension culture of dessert (AA and AAA) and cocking bananas (ABB) Musa spp. Biotechnology 7 (1989) :154- 159. ] KASHA. ZIAUDDIN[9 A , J. . K , . REINBERGE , haploidf . o FALK E e d Us induceSn an si . d mutation in barley and wheat. In Proceedings of second FAO/IAEA Meeting on Use of Induced Mutation in Connection with Haploids and Heterosis in Cereals, (eds.): M. Maluszynski and Z. Barab.(Katowice. Poland). Published by Cereal Research Institut 6701. Szeged. Hungary (1991).
[10] BAKRY, F and HORRY, J. P. First evidence for androgenesis in banana (Musa acuminata Colla). In: Plant Cell, Tissue and Organ Culture (1992)(in press). [II] MURASHIGE, T. and F. SKOOG A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant (19625 1 . ) :473-497. [12] MENGOLI . BAGNN & . I M , Polyamine somatid san c embryogenesi highen si r plants: In . Newsletter of International Association for Plant Tissue Culture. 68 (1992) :2-8.
12 GENETIC IMPROVEMEN BANANAF TO S THROUG VITROHN I MUTATION BREEDIN COSTGN I A RICA A. VALERINT . . SALAZARR , . NAVARRW , O Plant Genetics Program, Universidad Nacional, Heredia, Costa Rica
Abstract
Fifty corms of the banana cultivar Gran Enano {AAA), were collected from a commercial plantation, surface sterilized with 10 % bleach (0.5 % NaOCI), reduced in size, disinfected again with 2 % bleach, and rinsed three times with sterile distilled water under aseptic conditions Shoo explantp ti t lengthsn i (0.m 5c ) were excised and immersed in antioxidant solution Half of the explants were cultivated on the medium recommended by Novak et at., and half on the medium described by Sandoval. Fifty shoot tips from in vitro plantlet rinsed an sd, werthre°C 0 eo houre3 treate tw timet r a s fo sd(v/vS % witwit EM 5 )h h0. sterile distilled water. The explants were cultivated on MS + 1 mg/l BA and then transferred to MS + 4 mg/l BA Material was transferred to test tubes with MS for root development. Rooted plantlets were hardened in a glasshouse for 12 weeks, placed in a nursery, and finally planted in the field at Los Diamantes Experimental Statio thin I ns field location plant exposee sar naturao dt evaluatee b l inoculun ca d musinan scale gth e proposed by Stover as modified by Gaul. Considerable phenotypic variation was observed among the plants produced in vitro from EMS-treated shoot tips. After 11 months, 13 clones were selected for further evaluatio o theit e r du ntoleranc o blact e k sigatoka. These clones were further propagate n ordei d o t r establis commerciaha l plantin r economigfo c evaluation.
1. INTRODUCTION According to the National Banana Corporation (CORBANA), last year Costa Rica exported 74.1 million boxes of bananas (18.14 kg each), produced on 28 296 hectares. One of the most striking problem banane th plantaid n i s aan n production area contros si f blaco l k sigatoka, caused by Mycosphaerella fijiensis var. difformis. This diseas firss ewa t reporte Costn di a Ric Octoben ai r of 1979 [1, 2], and it has now spread throughout the banana and plantain production areas. Control of black sigatoka has been accomplished using fungicides. This practice, considerably increases the cost of production and environmental concerns. The most effective way to solve problems with black sigatoka is through the use of varieties resistant to this disease. However, use of traditional plant breeding methods to produce resistant hybrid vers si y difficul thin i t s generally seed sterile crop. Mutation inductio alternativn a s ni e tool for improvemen f banano t a cultivarr developmenfo d an s f blaco t k sigatoka resistant genotypes. The use of in vitro plant tissue/cell culture techniques make this approach possible. These are the reasons that the current project involving genetic improvement of banana and plantain through in vitro mutation breedin startes gwa Costn di a Rica.
. 2 MATERIAL METHODD SAN S
2.1. First step Fifty corms of the banana clone "Gran Enano" (AAA), were collected from a commercial plantation. In the laboratory they were washed using tap water to eliminate soil residue. Corms were reduced in size by removal of leaves until they were about 8 cm. in length, disinfected in 10 % Clorox (0.52 sodiu5% m hypochlorite) containin dropo g tw f tweeso minute0 n2 20/10r fo sl 0m and then rinsed three times with sterile distilled water under aseptic conditions. Additional leaves were removed unti shooe . longs abouth l cm wa t .2 t They were then disinfecte% d 2 agai a n ni Clorox solution containin dropo g tw f twee o s n 207100ml minutes0 2 r fo , . Explants were rinsed three times with sterile distilled water and further reduced in size to 0.5 cm in length. Shoot tips
13 were then immerse minute0 1 a sterilr fo dn i s e antioxidant solution containing L-cysteine (7.5 mg/1). Shoot tips were rinsed three times with sterile distilled water.
One-half of the total number of explants were cultured following the protocol recommended by Novak Duren ,Va Afznd [3]an ae explant .Th s were cultured individuall flaskn yi s (7x) 6cm basaS M containin lf o mediu l m 0 mg2 (XI) supplemented wit : hlmg/ 1 thiamine indole-3M (i 5 , - acetic acid (IAA) /ti0 M 1 , benzyladenin eagare othe1 (BA)sucrosg/ 1 Th .8 e rg/ th hald 0 ,f 4 ean o f explants were culture mediue th n do m describe Sandovay db l [4]. This medium consisteS M f do basal medium (X2) supplemented with: 1 mg/1 BA, 30 g/1 sucrose and 7 g/1 agar (Table I). Media pH wer medid e adjustean a8 wer5. o dt e sterilize autoclaviny . db °C 1 12 t ga
Tabl . eI SUPPLEMENT MEDIO ST A
Culture medium (quantity per liter)
Component X-l X-2
E(1/2MS)
thiamine 1 mg i mg 1 mg 0.1 mg g m 0 ,.1 0.1 mg IAA 5 ^zM BA 10 pM. 20 fjM 1.0 mg i ,. 0 mg ISA - 1 pM - sucrose 40 g 40 g 40 g 30 g 30 g 30 g agar 8 g 8 g 7 g 7 g 7 g Basal medium Murashige and Skoog I : Initiation, P : Proliferation, Rooting
Flasks containin explante gth s were transferre growta o dt h room wit temperaturha C ° 8 2 f eo and 16 h (light) photoperiod. After 28 days, the explants on XI initiation media were transferred to XI proliferation medium (P) supplemented with 20 fM BA and kept on an orbital shaker at 50 rpm. The explants cultured on X2 initiation medium were transferred to X2 proliferation medium, supplemented wit. mg/h5 BA f 1o
2.2. Second step Fifty shoot tips produced in vitro were 'treated with a 5 % (v/v) solution of ethyl methanesulfonat suggestes a eC ° (EMS houro Nova0 y . d3 [3]b tw al t r sa t .)ke fo Shoot tips were then rinsed three times with sterile distilled water. The treated explants were cultured on MS medium supplemente 0 daysd3 d the witr an ,n fo 1 mg/ h transferreA B S 1mediu M o t dm supplemented with 4 mg/1 BA as shown in Fig. 1. MjV4 plantlets (Fig. 1) were transferred to individual test tubes (150 x 25 mm) containing rooting medium (E) for root development. Mutagenic Treatment 60 Days 30 Days 30 Days M1V3 30 Days MXV4 30 Days Rooting 30 Days Nursery Fig. 1. Diagram of procedures used for production of mutated banana cv. Gran Enano.
14 2.3. Third step Rooted plantlets were transferred to soil and maintained in the greenhouse for 12 weeks. A plant4 23 tota1 s f wero l e plante Diamantee th n di s Experimental Statio Atlantie th n ni c regiona ; location thas higha t h concentration f naturao s l inoculu f blacmo k Sigatoka fungicideo N . s were applied to these plants and the field was placed under minimum agronomic management to observe response th plantf eo s under natural epidemic condition. Three evaluation month5 1 s werd an s eafte1 conducte1 plant4 , r 9 10 plantin t 1 a s n do g using the method of Stover as modified by Gaul [5]. During each evaluation the level of infection, and the specific leaf infected, were recorded. Plant vigor, fruit quality, and leaf shape were also examined. After evaluation some plants were selecte cormd dan f theo s m were take r furthenfo r micropropagation in the tissue culture laboratory.
. 3 RESULT DISCUSSIOD SAN N
Fifty shoot tips from shoots produced in vitro, were treated with EMS and 1 600 plantlets were produced from them. After rooting, 1 234 plants were planted in the field. Of these, 1 104 reached an adult stage of growth (nine months old).
As observe Novay . db [6] al t ,ke man y different mutant forms were observed afte MjVe th r 4 vegetative generation, mainly relate changeo dt lean i s f color, shap distributiond ean . Some dwarf mutants were also observed. After the first evaluation, 128 plants showed different levels of tolerance to black Sigatoka suggesting that the first infection occurred on the fifth or later leaf. From these 128 plants, nine mutants were identified as tolerant because they were infected only after the seventh or later leaf, and because infection at this stage permits the continued development plane oreasonabld th f an t e fruit production. After the second evaluation four additional clones were selected as tolerant, for a total of 13 putative black Sigatoka-tolerant mutants. The third evaluation confirmed the previously identified tolerance in the selected mutants, 16 months after the plants were put into the field. These clones are being micropropagated now in order to establish a large-scale commercial planting for economic evaluation.
Table II. RESULTS OF EVALUATIONS IN BANANA FOR TOLERANCE TO BLACK SIGATOKA
Numbe f planto r s Position of ———————————————————————————— first infected leaf 1st evaluation 2nd evaluation
7, 8 or 9 9 13 6 r o 5 119 160 4 169 366 3 , 12 , 812 365
Total 1104 1104
4. CONCLUSIONS
The method proposed by Novak et al. [3] to induce mutations in banana was used successfully in our experiments. Many clones showed changes in color, shape and orientation of their leaves. Some dwarf mutants were observed. Most importantly, different responses to black Sigatoka infection were observed amon e mutagen-treateth g d materials. Thirteen clones were selectes a d tolerant to this disease and they are now promising sources of germplasm for banana cultivation.
15 REFERENCES
[1] GONZALES ; JARAMILLOM , , 1979R , . Sigatoka Negra. ASBANA (C.R.) 3(10):3. 9 , 7 , [2] ROMERO, R. 1986. Observaciones sobre la incidencia de Sigatoka Negra (Mycosphaerella fijiensis var. difformis) en el cultivo de banano en la zona Atlántica en Costa Rica. ASBANA (CR). 10(25) 22-25. [3] NOVAK, F. J.; AFZA, R. and Van DUREN M. 1987. In vitro mutagenesis for plantain and banana (Musa spp.) genetic improvement.Plant Breeding Unit. Joint FAO/IAEA. Programme. Vienna, Austria, (s.f.. 37 ) [4] SANDOVAL . (1985J , ) Micropropagacio Muscease nd . ASBANA. (CR).9(24) 21-23.
[5] ROMERO MARÍNR , . D (1990), . Estrategias par l combate a a Sigatokl e d e a Negra (Mycosphaerella fijiensis Morelet l impacte y ) o sobr a resistencil e s benzimidazoleslo a . ASBANA. (CR). 14(33): 15-18. [6] NOVAK DUREN N , OMARd AFZA J. . an S VA . Mutatio . . F . , R M M ,, n inductioy nb gamma irradiatio vitrn i f ono cultured shoot-tip f bananso plantaid aan n (Musa cvs.) Tropical Agriculture (Trinidad) (1989) 67,21-28.
16 RADIATION MUTATION INDUCTION IN 'CHINA TIANBAO' BANANA
P. YANG, C. LI, F. WEI Beijing Institut f Nucleaeo r Engineering, Beijing . HUANGW . ZHENGJ , . HUANGD , E H . R , Zhangzhou Fruit Vegetabled san s Development Center, Fujian J. GUO, X. HUANG, B. CHEN, W. LIN Zhangzhou Plant Quarantin Monitorind ean g Center, Fujian China
Abstract
This paper present e resultsth f irradiatioso n experiments using 'China Tianbao' banana (Musa cv., AAA group) usin vitrn gi o culture methods lonm gm intac0 .6. Explanto tt shoo0 3. f ts o tipsshood an , t tips dissected longitudinally, were irradiated with ^Co. Difference radiatioe th n si n dose-survival response th f eo explants were observed amon e differength t culture groups LDse Th o. dosage r intacfo s t shoot-tipd san longitudinally dissected shoot-tips were 42.5 Gy and 38.69 Gy respectively. A regression equation of probabilit f ydeato uni) (Y ht percentag f longitudinallo e y dissected shoot-tip o dost s e logariths wa ) m(X established as Y = 4.1703X - 1.6029. In the irradiated materials which resumed growth after 45 days, statistical analyses suggested that there was no significant correlation between the rate of proliferation of treated materials and radiation dose, or between the proliferation rate and the technique used to prepare the materials (intac . longitudinallvs t y dissected). Experimental procedures were improve decreaso dt e microbial contamination.
1. INTRODUCTION
'China Tianbao' banana is the dominant commercial banana cultivar of the Fujian Province locatesouth-eastere th n do n coas f Chinao t s uniquIt . e flavo s veri r y much appreciated bott ha home and abroad. However, this cultivar is susceptible to hurricane damage and infection by banana bunch p viruto y s (BBTV) disease which greatly diminishe e yieldth s f thio s s banana cultivar. Therefore, improvement of 'China Tianbao1 is important as it relates to the continued production of this cultivar in SE China. Banan plana s i a t tha s clonalli t y propagated s difficuli t I .breeo t t d commercial triploid bananas using classical hybridization methods due to the sterility of most clones. Genetic variation for many trait s limitei s n banani dd selectio an a f improveo n d genotypes, obtained through controlled hybridizations s veri , y difficult n manI . y instance e onlth s y practical improvement method appeare mutatiob o t s n breeding d desirablan , e mutation-induced charactere b n ca s produced, and subsequently maintained through vegetative propagation. Mutation induction can be used to improve one or more of the undesirable characteristics of this banana cultivar, while maintainin s superioit g r characteristics. Mutation breeding using Co gammn a e ab rayn ca s important means of banana improvement. 60 paste Inth , irradiatio suckerf no s resulte onln di y limited mutation induction. Recently, with the development of biotechnological techniques, banana tissue culture methods can be used to produce the large populations of explants required for irradiation experiments [1, 2, 3]. In addition mutation i e us s nit inductioo t n experiments, tissue culture procedure usee b generato dt n ca s o es called somaclonal variants e productioTh . f somaclonao n l variant y resul ma n svaluabli t e genotypes.
17 The purpose of this research was to establish protocols for the improvement of 'China Tianbao' banana mutatioy b , n breeding specifie Th . c objective projece th f o s t wer productioe eth n of shorter plants with resistance or tolerance to banana bunchy top virus disease. Sensitivity of 'China Tianbao' banana' to 60Co gamma rays, and methods of mutation breeding, are also discussed.
2. MATERIALS AND METHODS
The experimental materials were 'China Tianbao' banana (Musa cv., group AAA) which were obtained fro mcommerciaa l orchar Zhangzhon i d u City, Fujian Provincef Chinao . R .. P , Healthy suckers were selected and used to initiate shoot-tip cultures. Shoot-tip cultures were propagated to provide sufficient shoot-tips for experimental use.
Five irradiation experiments were performed beginnin Octoben gi r 1990firse th t n I thre. e experiments, in vitro shoot-tips (2.0 - 5.0 mm in length) were divided into two groups. Group A (intact shoot-tips) were place conican di l flasks. Grou (longitudinallpB y dissected shoot-tips) were placed in sterile petri dishes with a few drops of sterile water. All culture vessels were sealed with
parafilm. After irradiation witCho gamma rays materiale th , s were immediately transferreo t d culture medium. Unfortunately, 60 these materials were contaminated werd an ,e eventually discarded. The remainder of this report focuses on the two successful experiments. The fourth experiment was begun on September 4th, 1992. The experimental materials were again divided into two groups; Group A (intact shoot-tips), and Group B (longitudinally dissected shoot-tips. Eleven levels of radiation were tested; 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 dosa Gy t a eGy/min0 , 1 rat f eo . lengthn i Sixt m ym , shoot-tipswer0 6. e- use0 eacn 3. i ,d h irradiation treatment. In these experiments, the dissection and culture of the experimental materials were carrie t asepticalldou laboratorye th n yi . Five shoot-tip explants were place eacn di h culture bottle (conical e flask)culturTh . e t changemediuno s n ordei dmwa o avoit r d possible contamination.
Based on the results of the fourth experiment, in which we determined the radiation dose response of the experimental materials tCo60 o gamma rays, a fifth experiment was carried out the following October. The experimental materials were all dissected longitudinally and placed in conical flasks with solid medium irradiatioe Th . n doses were delivered t fouan da 5 r4 levels, 35 , 0 : dosa t a eGy/min0 1 y rat G f eo 5 5 . Two-hundred shoot-tip explants were use eacn di h radiation dose treatment. Again, the culture medium was not changed after irradiation in order to minimize contamination.
During these previous two experiments, little contamination of experimental materials was observed and the irradiated shoot tips had a high survival rate. These results were satisfactory. Irradiated materials resumed growt day0 h4 s afte irradiatioe th r n treatment. Experimental materials were subculture rooted an d vitron di . After hardening culturee th , d plantlet t intse pu wiloth e b l field. Over 2 000 plants will be planted in a nursery to detect variants.
. 3 RESUL ANALYSID TAN S
3.1. Radiation treatment and growth of explants Table I illustrates the response (survival) of the treated experimental materials from the fourth experiment, which resumed growth afte days5 4 rbot n I intace . hth t shoot-tip group (Group e longitudinallth d Aan ) y dissected shoot-tip group (Grou apparenn a ) ther s pB ewa t correlation between the radiation dose and the death of the shoot tips. There were also differences in the respons f explanteo radiatioe th o st n dose when comparin controe gth treatele grouth d dpan group. Durin e earlgth y stages, untreated explants became blackened. Treated explantt displano d ydi s these symptoms immediately and they did not proliferate. Browning and blackening of the treated explants began fifteen days after irradiation. Examination of leaf primordia of surviving and dead
18 treated explants revealed them to be spongy. Some explants that eventually died developed one or two leaf primordia initially. This effect on leaf primordia might be explained by assuming that the radiation treatment had its greatest damaging effect on the cells located towards the middle of the apical meristem, with less damage occurring to the cells located away from the meristem. This phenomenon agrees wit resulte . hth Probably 5] Pinn , s Yi g[4 reporte i e deate th Sh th ,f y ho db explants is related to physiological defects caused by the radiation treatment.
Table I. GROWTH OF EXPLANTS AFTER EXPOSURE TO DIFFERENT DOSES OF IRRADIATION (45 DAY AFTER TREATMENT)
Dose (Gy) 0 10 20 30 40 50 60 70 80 90 100 Grou pA (intact ) 9 5 9 5 9 5 4 5 6 4 1 5 9 2 8 0 0 0 No.dead No.survived 60 60 60 52 31 9 14 6 1 1 1 Proliferation 1 1 1 9 4 1 1 1 5 4 7 10 1 15 0 15 2 17 Number Grou pB (longitudinall y dissected) No.dead 2 3 10 11 24 40 54 56 57 58 60 No.survived 58 57 50 49 36 20 6 4 3 2 0 Proliferation 2 0 2 3 6 0 1 6 2 6 7 9 10 0 10 5 13 0 13 Number
3.2. Relationship between different radiation doses and treated materials
These dosage response data (Table I) were subjected to statistical analysis. The results of this analysis are shown in Table II.
Tabl . eII STATISTICAL ANALYSI RADIATIOF SO N DOSE RESPONSF EO GROUE TH (LONGITUDINALLPB Y DISSECTED SHOOT-TIPS) Ratf eo death Percent relative Logarithm Probability unit Dose dead to dose of percent death (Gy) plants* control** dgx) (y) 0 3.33 _ _ - - 10 5.00 1.67 1.00000 2.8799 20 16.67 13.34 1.30103 3.8877 30 18.33 15.00 1.47)l2 3.9636 40 40.00 36.67 1.60206 4.6602 50 66.67 63.36 1.69897 5.3425 60 90.00 86 67 1.77815 6.1123 70 93.33 90.00 1.84510 6.2816 80 95 .00 91.67 1.90309 6.3852 90 96.67 93.34 1.95424 6.4985 100 100 00 96.67 2.00000 6.8384
: * Number dead/0 6 X 100 % ** . (Percent dead) - (Valuf eo control)
19 3.2.1.LD5 0longitudinalle dosth f eo y dissected shoot-tips group As shown in Table II, the correlation coefficient (r) of logarithm dosage and percent explantage deat determines hwa 0.9716e b o dt . Thihiga s si h correlation. The regression equation of probability unit (Y) of percent death to log dose was Y = 4.1703X - 1.6029. The significance of the F value was examined; F= 161.2083 (F001 = 11.26). valueF s were determine e significanb o e dlevelt th t sa t indicate Tabln i d ee regressio IIITh . n equation (Y = 4.1703 X - 1.6029) was tenable (Figure 1).
Table III. ANALYSIS TABLE OF THE REGRESSION EQUATION OF DOSE LOGARITHM AND PROBABILITY UNIT OF DEATH PERCENTAGE Mutation sourc f eF o Meam DegreeSu n F f o 0.0s 1 freedom squares square
Regression 15.8629 15.8629 161.2083** 11.26
Surplus 0.7875 0.0984 Total mutation 16.6504
Y Probabi!ity unit f deato h percentage 5.0 - LD50dose point 4.0 -
3.0 Y=4.1703X-1.6029 2.0
1.0
0 0.2/0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Dose -1.0 logarithm
-2.0
-3.0 dose-responseThe Fig. 1. regression of percentage death of longitudinally dissected shoot-tipsto radiation dose in logarithm.
When the death rate reached 50 %, the probability unit of percentage death was 5. If Y=5, and using the equation Y= 4.1703 X - 1.6029, then X = 1.5876. The inverse log of X was the LD50 dose which equaled 38.6 longitudinalle , e.gLDe th 9Gy th r . 5fo 0 y dissected shoot-tip group was 38.69 Gy. relationshicurve e th Th f eo p between percentage deat radiatiod han n dos shows ei Fign ni . .2 This dose response curve is close to a typical 'S' shaped curve. Thus, when the radiation doses are small, increase percentage th n i s f deaeo d explants increase slowly. Whe radiatioe nth n dosee sar
20 in the mid-range of the curve, small increases in radiation result in more rapid increases in the percentag f deaeo curve th d f eexplantso hige furthed th hen t A r. increase dosagn i s e illicit only slight increase percentage th n i s f explanteo t survives no tha o d t . This respons f bananeo a shoot- tip o irradiatiot s n with Co gamma ray s similai se reporte th o t r d response f otheo s r planto t s irradiation [6]. 60
Death percentage
10
0 10 20 30 40 50 60 70 80 90 100 Dose (Gy) Fig. 2. The dose-response curve of longitudinally dissected shoot-tips.
3.2.2.LD50 dose of intact shoot-tips e resultTh f experimento s s examinin e radiatiogth n dose-respons e showar e Tabln i n. eIV seeFroe b nn m tha ca correlatioe Tabl t i th t V probabilite I th dos g d nlo ean coefficiene th y f o ) (r t
unit of percent death was 0.1793 (r = 0.1793 is < =r 0.6319). Thus a significant correlation 5 0 0 regressiodoea t exisd no s an t n equatiot calculatedno s nwa . Fro curve mth LDe Fign th ei 5 , 03 . dos f intaceo t shoot-tip curve shape similas th Th f e42.s wa e o . thao swa curv5e rt Gy th t Fign ei . 2. Death percentage 100
90
80
70
60
50
40
30
20
10
0 10 20 30 40 50 60 70 80 90 100 Dose (Gy) dose-responseThe Fig. 3. curve of intact shoot-tips.
21 Tabl . eIV STATISTICAL ANALYSI RADIATIOF SO N DOSE RESPONSF EO INTACT SHOOT TIPS
Percent Relative Transformed Dose in Probability Radiation dead rate of death log unit of death dose(Gy) plants* death** (%)*** dgx) =y % )(
0 0 ______10 0 0 1.00 1.00000 2.6737 20 0 0 1.00 1.30103 2.6737 30 13 .33 13.33 14 .33 1.47712 3 .9331 40 48 .33 48.33 49 .33 1.60206 4.9825 50 85 .00 85.00 86 .00 1.69897 6.0803 60 75 .67 75.67 76 .67 1.77815 5 .7290 70 90 .00 90.00 91 .00 1.84510 6.3408 80 98 .33 98.33 99 .33 1.90309 7.4573 90 98 .33 98.33 99 .33 1.95424 7.4573 100 98 .33 98.33 99 .33 2.00000 7.4573
*dead/6. . No 0 X 100 % **. (Percentage dead plants) - (Value of control) * * * .% dead was zero at 10 and . 20Gy Thus , 1.00 was added to all the % dead values for ease of calculation.
3.2.3. Effec radiatiof o t proliferatioe th n no nirradiatee ratth f eo d materials valueF s were calculate showe ar valueF d Tabln ni dan . seVI wer t significanteno . This indicates tharate f proliferatioth teo s independennwa radiatioe th f o t n dose thad an o ,t n ther s ewa significant difference betwee rate nf proliferatioth eo f longitudinallno y dissecte intacd an d t shoot- tips.
Table V. RELATION OF PROLIFERATION MULTIPLE AND THE RADIATION DOSE
Proliferation multiple*
Dose (Gy) 0 10 20 30 40 50 60 70 80 90
Group B** 2.8667 2.5000 2.5167 2.0577 1.4516 1.2222 1 1.5 1 1 Group A*** 2.2414 2.3684 2 2.2245 2.1111 1.3000 1.6667 1.5 1 1
*: (Total number)/(Number surviving) **: Intact shoot tips ***: Longitudinally dissected shoot tips
Table VI ANALYSIS OF RELATIONSHIP AMONG TREATMENT PATTERN, RADIATION DOSE AND PROLIFERATION MULTIPLE
Mutation f o Degreem Su f so Mean of source freedom squares square F FO.OI FO.OB
Between doses 9 6.2430 0.6937 0.1034 5.35 3.18
Between treatments 1 0.0044 0.0044 0.0007 10.56 5.12
Error 9 60 .3956 6.7106
Total mutation 19 66 .6430 7.4087
22 3.2.4. LD50 dos intacf eo t shoot-tips
LDe dosTh r intacefo t shoot s calculatetipswa 42. e . b Thi highes 5o Gy dt swa r thae nth 0
LD dos er longitudinall5 (38.fo ) 6Gy y dissected shoot-tips. This suggests thae longitudinallth t y 0
dissecte5 d shoot-tips were more sensitive tCo o gamma rays, perhaps due to injury during dissectio damagr n o meriste e th o et m tissue. 60
4. DISCUSSION
60 4.1. Sensitivit LDd yan 5 0 dos materialf eo s irradiatey Cdb o gamma rays Experimental results indicate that n obviouthera s i e s differenc sensitivitn i e 60o t yCof o intac d longitudinallan t y dissected explants e longitudinallTh . y dissected shoot-tips were more sensitive than the intact shoot-tips and their LD50 doses were 38.69 Gy and 42.5 Gy, respectively. e longitudinallTh y dissected shoot-tips were considere e morb o et d suitabl r irradiatiofo e n experiments. Figur whice3 h illustrate resulte e irradiatiosth th f so n respons f intaceo t shoot-tips indicates that the values are dispersed. Through more rigorous statistical analysis [7, 8], the response appear neithee b o st r typicalineaa r ' -shapeno r 'S l d curve relationship. Thu LDe sth 50 dos 42.f eo 5 Gy is only an estimate. In contrast, the LD50 dose of longitudinally dissected shoot-tips was
determined. 4.2. Effect Cofo60 gamma rays on proliferation rate of explants e proliferatioTh n rat t f correlateexplanto eno s wa s d with eithe e radiatioth r ne dosth r o e metho f explando t preparation (longitudinal dissectio s intact)nv t shoulI . notee db d thatime th t e require r proliferatiodfo s twicnwa e that required durin usuae gth l proliferation cycle. e Baseth n do reports of Zhou Li Nong [9, 10, 11] and Li Bao Rong [12], the maximum proliferation rate of shoot-tip s generalli s y reached e proliferatioafte th days5 1 d r an , n multipl s highesi e 0 days2 t a t. The proliferation coefficient of shoot-tips that formed only one leaf primordia was much lower than that of young shoots. It remains to be determined whether or not the synchronization of proliferation was the result of a long growth time. It should also be mentioned that the size of explants (5. 0normall) - 10. 0mm y use propagatior dfo larges ni r tha size explantf nth eo s (3.0 6. 0- mm) use thesn di e experiments.
4.3. Experimental metho avoio dt d fungal contamination
Three experiments were carrie t earlieou d r during thin si researche Beijinn i on o d tw an g, Fuzhou. During experiments, there were considerable problems with fungal contamination. The source of the fungal contamination may have been environmental, or the result of the long-distance transportatio f experimentao n l materials t mighI . e alsb t o relatee radiatioth o dt n source th n i e laboratory which was not well-suited for aseptic work. Therefore, in the present experiments, all the experimental operations vitron weri e e th cultur carrien i t eou d laboratory e practicTh . f o e sealing with parafilm was replaced by using rubber plugs, and there was no reculture of explants to fresh medium immediately aftee irradiationth r . These steps resulte n satisfactori d y controf o l fungal contamination.
4.4. Selectio mutantf no s
During the period of growth following irradiation with 60Co gamma rays, our observations were mainly focused on changes in leaf color and leaf shape. It was difficult to detect promising mutant t thia s s stagee selectioTh . f favorablo n e mutant characters should emphasizee th n i d nursery and in the field. Our research is presently focused on the examination of the radiation respons f cultureeo d banana tissues selectione r worth Ou n ko . f gammo s a irradiated materials, fundamental investigations on the effects of irradiation on the resumption of growth, the time
23 period required for micropropagation, and observation of plants in the nursery and in the field, will continue.
REFERENCES
. ] NOVAKMutatioal [I t e . J . nF , inductio gammy nb a irradiatio vitrn i f ono cultured shoot-tips of banana and plantain (MUSA cvs). Trop. Agric. (Trinidad) Vol. 67: January (1990) 21-28. [2] NOVAK, F. J. et al. Somatic embryogenesis and plant regeneration in suspension cultures of dessert (AA and AAA) and cooking (ABB) bananas (MUSA SPP) Biotechnology. Vol. 7 Feb. (1989) 154-159. [3] CHEN, ZHENGUANG Tissue culture of plant. Volume of Modern Plant Science. Shanghai Scienc Technologd ean y Press (1987). [4] SHI, YINPING Mutation breedin f fruigo t trees. Modern Fruit Tree Science. Shanghai Scienc Technologd ean y Press (1986). [5] MA, XUEJUN and ZHOU, LINONG Technology study of quick breeding of banana tissue culture. Guangdong Agricultural Science 1st. issue, (1989) (22-24). [6] Mutation breeding. Institute of genetics of China Academy of Science. Science Press (1972).
[7] YANG, SHUQIN Hygienic statistics. People's Hygiene Press (1989). ] LIN[8 , DEGUANG Experiment desig d statisticnan s analysi f tropicao s l plants. Southern China Institute of Tropical Plants. April 1974. [9] ZHOU, LINONG Research of quick breeding of banana through the way of embryo body. Guangdong Agricultural Science. 3rd. issue, (1991) 22-26. [10] YAO N Cultur,JU f apice-tieo f banans quicpo it d k aan breeding . Guangxi Plants. Guihaia 11(2): (1991) 181-185. [II] M. KAZUMITSU et al. Unfabric Materials as being the Support of Original Embryo Explan Bananf o t a Category, Southern Fujian Agricultural Science (19912 . No ,) 48-50. [12] LI, BAORONG Some questions on production of banana seed. Guangdong Agricultural Science. 3rd. issue, (1989) 19-21.
24 IMPROVEMEN BANANF TO A (Musa cvs.) THROUG VITROHN I CULTURE TECHNIQUES AND INDUCED MUTATIONS
. SIDDIQUIH . S . KHATRIA , . KHANA . I , . NIZAMANIG , . KHAR , N Atomic Energy Agricultural Research Centre, Tando Jam, Sindh, Pakistan
Abstract
Rapidly multiplying shoot tip cultures were established from meristem explants of banana (Musa cvs.). Banana cultivars were successfully propagated on modified Murashige and Skoog medium with 4.5 mg/l BAP. Roots were induce plantletn do s culture 2 strengt1/ n mediuS do hM m containing 0.20 mg/l IBA. Plantlets were acclimatize transferred dan soio dr furthet fo l r evaluation. Proembryogenic call! were initiated from basal leaf sheaths and rhizome tissue cultured on SH medium with 6.63 mg/l dicamba. Protoplasts were isolated from rhizome tissue using an enzyme solution containing 3 % cellulase R-10, 1 % macerozyme R-10 and 1 % pectinase. Efforts are in progress to regenerate plants via somatic embryogenesi d froan sm isolated protoplasts A mutatio. n induction experimen y s carrieb wa t t ou d irradiating vegetative shoot apices at 5, 10, 15 and 20 Gy using a ^Co source. Radiosensitivity was assessed by determining the subsequent rate of shoot differentiation.
1. INTRODUCTION
Pakistan is a land of diverse ecology capable of producing a variety of horticultural crops. Banana is a major fruit crop in Pakistan. The cultivated area of banana in 1947 was about 117 hectares, with 365 tones of fruit production. Since that time the area has increased to about 23 100 hectares, with 205 700 tones of fruit production (Fig.!)[!]. The average yield of 8.88 MT/hectare whew ilo s n compare otheo dt r banana growing countrie thesd san e yields e increaseneeb o dt y db growing genetically improved cultivars with high yield superiod an s r quality characteristicse Th . genetic improvement of banana by conventional breeding techniques is hampered due to pollen, ovule and seed sterility [2, 3]. Banana is a vegetatively propagated crop where new cultivars commonly originate through spontaneous mutations [4] l significanAl . t cultivar e triploiar s steriled an d . Thi s madha se th e applicatio f conventionano l breeding methods more difficult [5]e recen rapiTh d . an t d spreaf do banana bunchy top virus (BBTV) in the province of Sindh has almost eliminated the cultivation of banana in this region as no cultivars resistant to BBTV are known to exist. Thus, breeding for resistance require vitrostrategien i w f sne o culture us se suceth techniquess ha . Tissue culture techniques have made a significant contribution to plant breeding and are especially useful in banana [6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. A combination of conventional breeding and biotechnology provides the necessary tools for the development of new clones resistant or tolerant to diseases [2] and for the elimination of virus [16]. Tissue culture facilitates mass propagation, conservatio d easan ny movemen f germplasmo t . Other techniques sucs a h somatic embryogenesi d protoplasan s t cultur f economicallo e y important cultivars offew ne r opportunities for inducing genetic variability (somaclonal variation). e inductioTh f somatio n c mutations could contribut e broadenineth f genetigo c variation i n banana [17]. These techniques were utilized successfully to develop the mutant clone GN60A [12]. Therefore investigations of biotechnological techniques, micropropagation, cell/tissue culture, protoplast culture, and induced somatic mutation, were initiated at AEARC, Tando Jam, to overcome our local problems with banana production. In thie abovth s f papeeo e describtechniquee w rus e eth develoo t s a micropropagatiop n system for rapid clonal propagation, for obtaining disease-free plants for nursery stock and for the productio isolatiod nan f bananno a somaclone mutantd an s s with improved characteristics sucs ha fruit qualit resistancd yan biologicao et environmentad an l l stresses.
25 Area (ha) and production (M.T.) Yield /ha 200 i———————————————————————————————————I—————0 1 i
I 150
x, 100 I- £ ' . r t I .-f. 50
Area in ha ______
1948 1953 1958 1963 1968 1973 1978 1983 1988 1990 Years Fig. 1 Area, production and yield of banana in Pakistan 1948 -1990
. 2 MATERIAL METHODD SAN S
2.1. Micropropagation Clones of banana (Musa cvs.) used in this study were Basrai (Dwarf Cavendish), Williams hybrid, SH3362, Magrabi, Hindi, Grand Nain, GN60A, Highgate and 30-29. Plant material was provided by Dr. F.J. Novak of Seibersdorf Laboratories, IAEA, Vienna, Austria, and Dr. M.K Smith, Maroochy Horticultural Research Station, Queensland, Australia.
Suckers/corms consistin f shooo g t d tiprhizomatouan s s base tissue were use s explana d t materials. Explants were initially outee washe wateth p ta d r n layerdan i r f leaveso s were removed Shoot apices wit 4 leah2- f primordia were obtaine thesd dan e were disinfecte ethano% 0 7 ln i d (one minute), by immersion in 0.5 % sodium hypochlorite solution for 15 minutes, and then rinsed thoroughly in sterile distilled water. More leaves were removed until the remaining shoot tips were 1 cm at the base. Explants were cut into four equal pieces, and transferred aseptically into 150 x 25 mm test tubes containing Murashige and Skoog [18] medium supplemented with 1 mg/I thiamine-HCl, 0.88 mg/1 indole-3-acetic acid (IAA), 2.25 mg/1 benzylaminopurine (BAP), 40 gm/1 sucrose and solidified with 8 gm/1 bacto agar. Cultures were maintained in a growth' chamber at 28 °C on a 16 h photoperiod. After 21 days of culture the shoots were cut longitudinally and transferred into solid wels a , s liquida l mediuS M , m containin r additiona fo mg/5 gP 4. 1BA l shoot multiplication. Cultures in liquid medium were placed on a horizontal gyratory shaker at 60 rpm. Shoot proliferation cultures were subcultured at 28 day intervals. For rooting, individual shoots were placed in half-strength MS medium containing 4 % sucrose and 0.20 mg/1 indole-3-butyric acid (IBA) Rooted plantlets were transferred to jiffy pots, acclimated under controlled conditions, and subsequently transferred to the soil.
2.2. Callus induction Rhizom d leaan ef sheath tissues were excised fro n vitrmi o shoot cultures d uses an a ,d explants for callus induction. The culture media used was comprised of macro- and micro-elements
26 of Schenk and Hildebrandt [19], Staba vitamins, 100 mg/1 inositol, 40 mg/1 cysteine-HCl, 40 g/1 sucrose and 6.63 mg/1 3,6 dichloro-2 methoxy benzoic acid (dicamba) was used.
2.3. Protoplast culture Leaf, leaf base and corm tissues were obtained from proliferating shoot cultures and were used for protoplast isolation. Tissues were digested with a protoplast isolation enzyme mixture (3 % cellulase R-10, 1 % Macerozyme R-10, and 1 % pectinase). Protoplast were separated from the enzyme mixture by filtration and centrifugation at 100 x g for 10 minutes. Protoplasts were washed in culture mediu saltH mstab+ (S s a vitamin 6.6+ s 3 mg/1 mannitol)dicambM 4 0. + a , separated by centrifugation, and finally resuspended in wash medium. Protoplast viability was determined by fluorescein diacetate (PDA) staining and density was determined sing a counting chamber (hemacytometer). Cultured protoplasts were incubated at 25 °C in the dark. 2.4. In vitro mutagenesis Shoot tipf cvso s . Magrab d GN60an i A were isolated from proliferatin n vitri g o shoot cultures. Shoot tips wer t longitudinallyecu , transferre petro t d i dishes containin dropw fe f o sga
sterile distilled water irradiated an , d with gamma rays d froan mC 5 o1 sourc , 10 t dose, ea 5 f o s . IrradiateGy 0 2 d shoot tips were then transferre micropropagatioo t d 60 n medium. Afte weeks4 r , radiosensitivit evaluates ywa termn vitrodn i i f so growth ratsubsequend ean t shoot proliferation.
. 3 RESULT DISCUSSIOND SAN S
3.1. Micropropagation
The color of explants cultured on medium containing IAA and BAP changed from creamy white to green within 7-10 days. Shoot tips initially produced one or few shoots within 10 to 15 days after culture initiation. After culture establishment e explantth , s were transferreo t d multiplication medium containin 5 mg/4. g1 BAP. Longitudinally splitting shoot tips stimulated shoot multiplication during subsequent subcultures on multiplication medium. Splitting of shoot tips stimulated multiple shoot formation. Recultur f splio e t shoot tip o liquit s d medium greatly increased the rate of growth and the rate of shoot proliferation when compared with explants culture solin do d medium average Th . e numbe liquif shootn i o r 0 r cluste1 ds pe s mediuwa r d man sin solii x d mediums alswa o t observeI . d that explants showed tendenc produco t y e phenolic compounds which oxidized rapidly and resulted in the death of some explants. The use of cysteine-HCl (40 mg/1) inhibited oxidation.
lengthm c Shoot5 )- wer3 ( s e separate transferred dan rootino t d g medium. Roots initiation was observed 7-10 days after transfer. Plants with well developed root systems were transferred to jiffy pots containing perlite and irrigated daily with distilled water and twice a week with liquid MS media. After acclimatization for 1 - 2 weeks plants were exposed to natural greenhouse condition r 8-1sfo 2 weeks. Plants were then transplanted int fielde oth . Plants propagate thiy db s procedur e growinar e g vigorousl e fieldth n .i y Surviva f planto l s during transfer from culture vessel to soil was 100 %. Plants are phenotypically identical to the parental clon havd t showean eno symptom y nan f diseaseo s performance Th . f theso e e plantsn i , the field, is under evaluation.
3.2. Callus induction
Rhizom lead ean f tissue explants proliferated proembryogenic callus withi 4 weekn3- s after culturmediuH S n eo m containing Staba vitamin 6.6d san 3 mg/1 dicamba e calluTh . s producen do this medium frequently appeare morphogeneticalle b o dt y competent i.es comprise.wa f smalldo , more or less isodiametric and cytoplasmically dense cells. Virtually all our initial attempts to induc sustaid ean n vigorous callus proliferation from shoot tip r leaso f base tissue were inhibitey db severe explant browning combinatioA . f 6.6o n 3 mg/1 dicambmg/1 1. 1 d thidiazuroan a n (TDZ)
27 improved callus initiation and helped to reduce explant darkening. The use of cysteine-HCl also reduced oxidation. Explants from diploid clon 336H eS 2 proliferated both callu proembryonad an s l structures. Explants from triploid clone GN60A formed semi-compact calli and occasionally proembryonal structures. These proembryogenic structures were transferred to regeneration medium. Shoot formations from these structure t observedno s wa s . progresEffortn i e ar s regenerato t s e plants through the process of somatic embryogenesis. 3.3. Protoplast isolatio culturd nan e
Attempt isolato st e banana protoplasts use enzymn da e mixtures explante th f O . s tested, corm
tissue gav highese eth t yield f protoplastsso . Protoplasts wer t isolateeno d successfully from leaf
5 tissue. Average yields of protoplasts from corm tissue ranged from 2.5 x 10 5 to 8.7 x 10 protoplasts/gram fresh weight of corm tissue. Freshly isolated protoplasts were generally colorless and the population was heterogenous with respect to size. Undigested clumps of cells were not detected in purified protoplast preparations. Eighty percent of the protoplasts stained with PDA were highly fluorescent unde lighV U r t indicatin a ghig h leve f viabilito l y immediately after isolation. These protoplasts were cultured on callus induction medium and remained viable up for 3 week2- s after isolation. Cell wall formatio s observewa n day2 d s after culture initiation. However o callun , s colony formatio s observedwa n . progresn Efforti e ar so induc t s e callus initiatio regeneratd nan e plantlets from isolated protoplasts.
3.4 vitron I . mutagenesis No adverse effect n explano s t viability, growt r shooo h t formation were observed after irradiation with 5 to 20 Gy (Table I). Shoot formation appeared to be slightly stimulated by low doses of irradiation. Shoot proliferation occurred from small nodular growths. The nodules contained small buds which either remained dormant or eventually developed into green shoots. Regenerated plantlets were readily established under non-aseptic growth condition numbea d san f o r plants have been established under field conditions. Observation vegetativn so e growth, suckering, flowering and fruiting will be recorded to maturity for the mutants having desirable agronomic characteristics.
Tabl . eI SHOOT REGENERATIO SUBCULTURENN I D TISSUES DERIVED FROM UNIRRADIATED AND IRRADIATED EXPLANTS. (DATA EXPRESSE AVERAGS DA E NUMBE BUDF RO S AND/OR SHOOTS PER CLUSTER)
Nof o . Irradiation dose (Gy) subcultures ———————————————————————————————— 10 15 20
Clone Magrabi 1st 5.12' 4.25 3.37 2.12 1.37 2nd 6.75 6.00 4.62 4.00 3.25
Clone CN60A 1st 7.75 5.37 3.75 2.87 1.87 2nd 8.37 6.12 4.75 4.25 3.12
Acknowledgement
The authors are indebted to Dr. K. A. Siddiqui, Director, AEARC. Tandojam, and Dr. A. R. Azmi (Ex-Director suppore th r )fo establiso t tissuha e culture laboratory. Als. F . o IAEDr d Aan
28 . NovaJ f IAEko A Seibersdorf Laboratories, Vienn providinr afo g trainin t Viennaga wore Th s k.i partially supporte FAO/IAEy db A Contrac . PAK-5426/R3/RBNo t .
REFERENCES
[I] Pakistan Statistical Year Book. 145. (1990).
[2] ROWE, P. "Breeding banana and Plantains". Plant Breeding Review (1984) 2 : 135-155.
[3] SHEPHERD, K. Banana breeding: Past and Present. Acta Horti. (1987) 196. 37-43. GUZMANE [4D ] , E.V., DELL ROSARIO, A.G., PAGALIWAGAN, P.C. Productiof o n mutant y irradiatiob s n vitroi f o n cultured tissue f coconuo s d banan an td thei an a r mass propagatio e tissuth y eb n culture technqiues : ProcIn . . Induced Mutatio n Vegetativeli n y Propagated Plants II. IAEA, Vienna, STI/PUB/519.(1982) 113-138.
[5] SIMMONDS, N.W. "Bananas" 2nd ed., Longmans, Green & Co. London. 512. (1966). [6] JARRET, R.L. RODRIGUEZ, W., AND FERNANDEZ, R. Evaluation, tissue culture propagation and dissemination of "Saba" and "Pelipita" plantains in Costa Rica. Scientia Hortic. (1985) 25: 137-147.
[7] GUPTA . P , Eradicatio f mosaio n c diseas d rapian e d clonal multiplicatio f banano n d an a plantains through meriste culturep mti . Plant Cell, Tissu Orgad ean n Culture (1986 : 33-39)6 .
[8] PERSLEY, G.J. AND DE LANGHE, E.A. Banana and Plantain breeding strategies. Proc. No. 21 Canberra. (1987).
[9] DORESWAMY SHIJRAd an . R , . ML Micropropagatio f bananno a from male floral apices culture vitro.n di Scientia Hortic. (1989 : 181-188)40 . [10] DALE, J.L. Banana and Plantain. In: Persley G.J. (ed.) Agriculture Biotechnology: Opportunit Internationar yfo l Development. C.A.B. Intern. Wallingford. (1990) 225-240.
[II] NOVAK, F.J. DURAN,N AFZAVA , PERE, M. R. ,, A DALLOS , CONGERM. . , B.Vd an . TANG, XIAOLONG Somatic embryogenesi pland san t regeneratio suspension ni n culturf eo AAAd an deser Cookind A )an (A t g (ABB) banana (Musa spp.) Biotechnology. (1989 : 154)7 - 159. [12] NOVAK, F.J. AFZA, R., VAN DURAN, M. and OMAR, M.S. Mutation induction by gamma irradiatio vitron i f no cultured shoot tip f banano s plantaid aan n (Musa spp.). Tropical Agric. (Trinidad) (1990) 67: 21-28. [13] NOVAK, F.J. Musa (bananas and plantains) In Hammerschlag F.A. and Litz. RE (eds.) Biotechnolog perenniaf yo l Fruit Crops." C.A.B. Intern. Wallingford (1992 . 449-488)pp .
[14] SIDDIQUI, S.H., KHATRI, A., KHAN, LA. and KHANZADA, M.H. "Improvement of banana (Musa spp.) through in vitro culture techniques and induced mutations". IAEA 2nd Co-Ordination Meeting, San Jose, Costa Rica (1991).
[15] KHATRI, A., SIDDIQUI, S.H., KHAN. I.A.; NIAZAMANI, G.S., KHANZADA, M.H. and DAKAR N.A tissuA . e culture techniqu r bananefo a improvement Vistaw Ne n i s. In . Plant Genetics. ProcGeneticaw Ne . l Approache o Crot s p Improvement, Karachi, Pakistan (1992) press)n (I . .
29 [16] DREW, R.A., SMITH ,ANDERSON d M.Kan . , D.W. Field evaluatio f micropropagateno d bananas derived from plants containing banana bunchy top virus. Plant Cell Tissue and Organ Culture (1992) 28: 203-205. [17] MENENDEZ . T Applicatio, f mutationo n method banano t s a breedin Procn I g . Induced Mutations in Vegetatively Propagated Plants IAEA Vienna PI-501/8. (1973) 75- 84.
[18] MURASHIGE, T. and SKOOG, F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant. (1962 : 473-49715 ) .
[19] SCHENK, R.U d HILDEBRANDTan . . A.C. Mediu d techniqueman r inductiofo s d an n growth of monocotyledonous and dicotyledonous plant cell culture. Can. J. Bot. (1972) 50. 199-204.
BIBLIOGRAPHY
VUYLSTEKE, D., and DE LANGHE, E. Feasibility of in vitro propagation of banana and plantains. Trop. Agric. (Trinidad) (1985 : 323-3284 2 )6 . WONG, W.C n vitroI . propagatio f bananao n s (Musa spp.): Initiation, Proliferatiod an n development of shoot tip culture on defined media. Plant Cell, Tissue and Organ Culture (1986) 6: 159-166.
30 EFFEC TOXIF TO C FILTRATE FusariumF SO oxysporum . cubensesp . f DEVELOPMENE ONTH BANANF TO A (Musa cvs.) SHOOT TIPS
B.M.J. MENDES, B.I.P. RODRIGUES, A. TULMANN NETO Centre de Energia Nuclear na Agricultura, Universidade de Sao Paulo, Piracicaba, Brazil
Abstract
The objective of this study was to evaluate the effect of toxic filtrate of Fusarium oxysporum f.sp. cubense vitron i culture th developmen n o e f banano t a (Musa) shoot tips. Shoot tips fro e varietiemth s Nanicao (AAA - resistan o Panamt t a - diseasesusceptibl A (A Pisand s an ) o gPanamma et a disease) were isolated and cultivated in culture medium containing different concentrations of toxic filtrate of Fusarium culturefirse th t n I assay. concentratione th , f toxiso c filtrat 12.d e (v/vtestean 0% d 9 , )an d6 , wer3 , e0 then, based on these results, a second assay was conducted using 0, 1.5, 3, 4.5 and 6 % of Fusarium culture filtrate. Five replicate r treatmenpe s t were used. Each replicate consiste erlenmeyen a f do r flask containing 1 5 ml of culture medium and five explants. The flasks were incubated on a shaker at 50 rpm and a daylength of 16 hours. After four weeks total fresh weight, fresh weight of shoots, and the length of shoots, were measured differenco N . response th n ei f resistaneo susceptibld an t e varietie s detectewa s d under low (0-3 %) or high ( > 6 %) concentrations of Fusarium filtrate. At intermediate concentrations (3 s possiblwa t i o discriminatt e ) % 5 4. - e betwee e resistannth susceptibld an t e varieties e baseth n o d measured parameters, primarily total fresh weigh fresd an th weigh f shootso t . These data suggest thae th t us f tissueo e cultur Fusariumd ean culture usefiltratee b dy togethesma studo t r y hostpathogen interactions.
1. INTRODUCTION Fusarium Wil r Panamo t a Disease, cause Fusariumy db oxysporum f.sp. cubense bananan i s i , mose th f t o destructiv e on e tropical diseases e microorganis[1]Th . s initiallmwa y identifiea s a d pathogen on clones of Gros Michel (AAA) and on dessert bananas such as the Brazilian varieties Maga (AAB) and Prata (AAB). Recently, the disease has also been reported to attack varieties of Cavendise th h group subtropica e occuo (AAAt th d n i ran ) l region f Australiaso , South Africd an a Taiwan [2]. Tissue cultur bees eha n use toos d a stud o t l ] usey[3 disease du bananS d bananaf an so an Su . plantlets grow vitron ni determino t e race pathogenicitd san Fusariumf yo isolatesr selectiofo d an , n for resistance to the disease; Mourichon et al. [4] inoculated banana plantlets with Mycosphaerella fijiensis and demonstrated a correlation between the degree of resistance in the field and the results obtained usinvitron i n ga assay. Lepoivr Acund e an ] use [5 a techniquda f toxieo n diffusion into the culture medium in an attempt to establish a system of in vitro selection for resistance to Black Sigatoka. Toxic filtrate of Fusarium culture has been used in the selection for disease resistance in several crops including potato [6] carnatiod , alfalfan ] [7 a n reporte [8]] bananan I [9 . p n a dEp , inabilit establiso yt hsensitivita y test with plantlet r leaso f discs using toxic filtrate from culturef so Fusarium oxysporum f.sp. cubense or fusaric acid, the main component of the toxic filtrate. In assays with leaf discs, differences were found whe e discnth s were place contacn i d t with either fusaric acid or toxic filtrate, an indication that the filtrate contains toxic components in addition to fusaric acid [10]. Becaus e importancth f o e f understandino e e pathogeth g n e elicitorth d an s response of the host to the pathogen at a cellular level, it was considered essential to more precisely evaluat e effec th ef toxi o t c . filtratoxysporumF f o e f.sp n vitroi . cubensee th n o development of banana shoot tips.
31 2. MATERIALS AND METHODS 2.1. Plant materials The assays were carried out with banana plantlets of the varieties Nanicao (AAA - resistant to Fusarium wilt) and Pisang mas (AA - susceptible to Fusarium wilt), which were obtained from in vitro culture. Culture f theso s e varieties were initiated from shoot tipswerd an ,e subsequently propagated from lateral shoots on culture medium containing MS (Murashige & Skoog) salts [11], to which 30 g/I of sucrose, 10 mg/1 of thiamine, 5 mg/1 of benzylaminopurine (BAP) and 8 g/1 of agar were added.
2.2. Fungal culture filtrate In order to obtain toxic filtrate of F. oxysporum f.sp. cubense, the fungus was cultivated in erlenmeyer flask containing Czapek-Dox (CD) liquid culture medium, for a period of 21 days. After that time perio culture dth s filtereewa d throug hMillipora e filter (0.2 filtrate 2 th /*m d e)an stores freezera wa n di .
2.3. Toxic media
The basic growth media consisted of MS inorganic salts [11] supplemented with B5 vitamins [12], cysteine (40 mg/1), sucrose (40 g/1), N6 2-isopentenyladenine (2-iP) (1 mg/1) and indole-3- butyric acid (IBA) (0.02 firse mg/1)th t n I assay. , filter sterilized toxic filtrate e s addeth wa o dt medium, after autoclaving, to provide final concentrations of 0, 3, 6, 9 and 12 %. In the second assay, toxic filtrate was tested at concentrations of 0, 1.5, 3.0, 4.5 and 6.0 %. The media (15 ml) were poured into 250 ml erlenmeyer flasks. Concentrations of toxic filtrate were calculated based on the concentration of fusaric acid in the filtrate, which was determined using a spectrophotometer.
2.4. Culture procedure Shoot tips were isolated from banana plantlets growing in vitro. Each assay consisted of five replicates per treatment. Five explants were cultured per flask. The flasks were placed in a growth roo d illuminateman maintaineC ° 7 d2 t wita d h cool-white fluorescent light bulb provido t s a e day length of 16 hours. Cultures were evaluated after four weeks of incubation, by measuring total fresh weight of the plantlets, the fresh weight of the shoots, and the length of the shoots.
3. RESULTS AND DISCUSSION f tissuo e eus culturee Th technique studo st y host-pathogen interactions selectioe th n i d n an , for resistance to diseases, has been reported for several host-pathogen systems including; tobacco- Phytophthora parasitica var. nicotianae [13, 14], voteto-Phytophthora infestans , 15][6 , alfalfa- Phytophthora megasperma [16], aKzlfz-Fusarium oxysporum f.sp. medicaginis , 17][7 , potato- Pseudomonas solanacearum [18] and elm-Ceratocystis ulna [19]. If the pathogen produces substances hostthae toxi e selectioth e ar t th o ,ct f planno t material resistan pathogee th o y t t nma generally be achieved by addition of the toxin to the culture medium used to grow cells from the host in vitro. e cas Ith nf banan o e e studth a f e diseaseselectioo yth d r resistancan sfo n e have been associated with tissue culture technique t leasdiseasesa o n tw ti s : Panama Blacd diseasan k] [9 e Sigatoka [5], which are caused by pathogens that produce substances toxic to the host. It is evident, from the results of the first assay in our study, that the addition of toxic filtrate of F. oxysporum f.sp. cubense to the culture medium interferes with the growth of the banana shoot possiblet no tips swa (Tabl,t I however . eI) observo t , e differences betwee resistane nth t (Nanicao) and susceptible (Pisang mas) varieties under the concentration used in this assay. When the concentration of toxic filtrate was 6 % or more, shoot tips of both varieties did not develop, indicating thaconcentratione th t wer ) vitro n t adequati % esno e 2 teste 1 th differentiatio r - efo 0 d ( n
32 of varieties based on their known reaction to Panama disease. The definition of the percentage of toxic filtrate utilized depends of the concentration of the toxic substances found in the filtrate, and this varies accordin pathogee conditione th th o g t d culture. e oxysporumcase nF an th f th f eo so n I . f.sp. cubense, fusaric acid is apparently the main component of the culture filtrates. It is known that concentration f fusario s c acid higher than 10' inhibiM growte th t f bananho a shoot tip] [9 s and that 0.8 mM of fusaric acid in the cultur5 e medium was used for selection of barley callus resistant to Fusarium [20]. In the host-pathogen systems described in the scientific literature, the percentage f toxio s c filtrate e cultureaddeth o dt mediu r carnation-Fmfo rang% 0 e5 fro- . m0 oxysporum f.sp. dianthi [8] and elm-Ceratocystis ulmi [19] and from 0 - 25 % for alfalfa-/7. oxysporum f.sp. medicaginis [1]. thosn I e studies percentage th , f filtrateo e chose r selectionfo n assays were 20, 50 and 7.5 %, respectively. The low amount of toxic filtrate of Fusarium required in culture medium, relative to the other pathogens, is probably due to the high concentration of fusaric acid in these filtrates. In the pototo-Alternaria solani system [21] 0.8 - 3.2 % of culture filtrate were used to stimulate the regeneration of shoots from callus.
Tablet. EFFECT OF TOXIC FILTRATE OF F. oxysporum f. sp. cubense ON THE DEVELOPMEN BANANF TO A SHOOT TIPS. FIRST ASSAY. AVERAG5 F EO REPLICATIONS. Filtrate Plant Shoot Shoot concent. fresh weight(g) fresh weight(g) length(cm) (%) Nanicao P.mas Nanicao P.mas Nanicao P.mas
0 0 .83AB 0 .53A 0 .74A 0 .38A 3 .37A 2 .79A 3 1.22A 0 .39A 0 .96A 0 .34A 4 .68A 2 .96A 6 0 . 26sc 0 .05s 0 .25s 0 .05s 1.48B 0 .92B 9 0 .17c 0 .04s 0 .1?B 0 .04B 1.03B 0 .67B 12 0 .13c 0 .03s 0 .133 0 .03B 1.OOB 0 .73s
Values followed by different letters in the same column are significantly different by Tukey's test (5 %).
Tabl . eEFFECII F TOXIO T C FILTRAT . oxysporumF E . F cubensesp TH O E . f N O DEVELOPMENT OF BANANA SHOOT TIPS-SECOND ASSAY. AVERAGE OF 5 REPLICATIONS.
Plant Shoot Shoot Filtrate fresh weight (g) fresh weight (g) length (cm) concentr (%) Nanicao P.Mas Nanicao P.Mas Nanicao P.Mas
0 1.33A 0.69A 1.02A 0.50A 4.69A 3.89A 1.5 1.09A 0.58AB 0.79AB 0.40AB 4.83A 3.44A 3,0 1.21A 0 .44ABC 0.84AB 0 . 2 SABC 5.30A 2.98A 4.5 0.82A 0.25c 0.51B 0.18BC 4.14A 2.29A 6.0 0.31B 0.20c 0.30c 0.16c 2.12s 1.79A
Values followed by different letters in the same column are significantly different by Tukey's test (5 %).
33 WEIGHT (proportion) TOTAL WEIGHT (proportion) 1.2
1.4 —*— Nanicco 1 1.2 "••. MasP -3 0.6 1>
(A) 0.8 0.6 0.6 0.4 0.4 0.2 0.2 ? A 0 3 6 9 12 5 4. 3 1.5 % FILTRATE X FILTRATE
t , SHOOT WEIGHT (proportion) SHOOT WEIGHT (proportion)
(B)
369 12 1.5 3 4.5 % FILTRATE . FILTRAT7 E
. LENGTH (proportion) LENGTH (proportion) 1.2
1'
0.8
(C) 0.6
O.4 Nantcoo 0.2 P. Mooo
0 1.5 3 4.5 X FILTRATE
Fig. Effect 1. toxicof filtratereductionthe totalon the of fresh weight (A), shoot fresh weight shoot(B)and lengthbananaof (C) shoot tips.
34 In the second assay, intermediate concentrations of toxic filtrate in the culture media (0 - 6 %) were tested. In this assay a difference between the varieties Nanicao and P. mas was observed at toxic filtrate concentration of 3 - 4.5 % (Table II). Shoot length was not statistically different. It can be observed from Fig.l that both varieties responded similarly at low (0 - 1.5 %) and high (6.0 %) concentration f toxio s c filtrate. However t intermediata , e concentration ther) % s e(3.5 4. 0- was a marked effect of the toxic filtrate on the development of shoot tips of the susceptible variety. The total fresh weight of Pisang mas plants cultured in medium containing 3 % toxic filtrate was 63 % of that of the control whereas for the resistant variety Nanicao the total fresh weight was 90 % e controloth f . Whe e concentrationth f toxino c filtrate culturth , totan i e% e lmediu5 4. s mwa fresh weight varietiee th f so s Nanicao (resistant Pisand an )(susceptibles gma % 6 3 d an ) wer% 1 e6 controe oth f l cultures, respectively othee Th .r traits followe similada r pattern.
possibilite Th f discriminatinyo g between banana varietie vitron i n i s tests using toxic filtrate of F. oxysporum f.sp. cubense allows the utilization of tissue culture techniques to determine both the pathogenicit isolatese th f yo hose th , t biochemical response toxio st c substances producef o y db pathogene th selection i d an r ,diseas nfo e resistance. These factors enabl rapie accuratd eth dan e assessmen f resistanco t e usin glarga e numbe f planto r smala n si l laboratory space.
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[5] LEPOIVRE, P. and ACUNA, C. P. Production of toxins by Mycosphaerella fijiensis var. difformis and induction ,of antimicrobial compounds in banana : their relevance in breeding for resistanc blaco et k sigatoka : . StoveFullerto(Eds& In H . .. A R r) . SigatoknR a leaf spot diseases of bananas, (pp 201-207). INIBAP, Montpellier. 1989. ] BEHNKE[6 . M ,Genera l resistanc lato et e bligh f Solanumo t tubl-rosum plants regenerated from callus resistan culturo t t e filtrate f Phytophthorao s infestans. Theor. Appl. Genet6 5 . (198O) : 151-152.
] ARCIONI[7 , PEZZOTTIS. , DAMIANId an . vitron M ,I . F ,selectio alfalff no a plants resistant Fusariumo t medicaginis. Theor. Appl. Genet (19874 7 . 700-705: ) .
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[9] EPP, M. D. Somaclonal variation in bananas: a case study with Fusarium wilt. In: Persley G.J. & De Langhe E.A. (Ed5) Banana and Plantain Breeding Strategies (pp. 140-150) ACIAR Proceedings No. 21. 1987.
35 [10] KRIKORIAN . D Baselin . A , e tissu celd elan culture banann si studiee us a r improvemensfo t schemes. In: Ploetz R. C. (Ed) Fusarium Wilt of Banana (pp 127-133). APS Press, St. Paul, Minnesota. 1990. [11] MURASHIGE SKOOGd an reviseA . T ,. F ,d mediu r rapimfo d growt biossayd han s witti tobacco tissue cultures. Physiol. Plant (19625 1 . 473-497: ) .
[12] GAMBORG, O. L., MILLER, R. A. and OJIMA, K. Nutrient requirements of suspension cultures of soybean rc)ot cells. Exp. Cell Res. 50 (1968) : 151-158. [13] HELGESON, J.P., KEMP , HABERLACHD. . J , , G.TMAXWELLd an . , D.P tissuA . e culture System for studying disease resistance : the black shank disease in tobacco callus cultures. Phytopatholog (19722 y6 : )14391443 . [14] HELGESON , HABERLACHP. . dominanJ ,A UPPERd . an D . . T t C . ,gen G , e conferring disease resistance to tobacco plants is expressed in tissue cultures. Phytopathology 66 (1976) : 91-96. [15] BEHNKE, M. Selection of potato callus for resistance to culture filtrates of Phytophthora infestans regeneratiod an resistanf no t plants. Theor. Appl. Genet (1979)5 5 . : 69-71. [16] MILLER, S. A., DAVIDSE, L. C. and MAXWELL, D. P. Expression of genetic susceptibility, host resistance nonhosd an , t resistanc alfalfn ei a callus tissue inoculated with Phytophthora megasperma. Phytopatholog (19844 y7 345-348: ) .
[17] HARTMAN , d KNOUSMCCOYL. an . . R . SelectioC J ,. . T ,T , f alfalfo n a (Medicago sativa) cell lines and regeneration of plants resistant to the toxins produced by Fusarium oxysporum f.sp. medicaginis. Plant Science Letter (1984)4 s3 : 183-194. [18] HUANG , HELGESONY. , SEQUEIRAd an . P . J , . DifferentiaL , l response potatf so o tissue culture inoculatioo st n with strain Pseudomanasf so solanacearum. AnnuaS AP : l In Meeting , Florida/USA. 19B6 (Abst. No. 642).
[19] , PIJUTDOMIR , M. LINEBERGER . C. P f ,. o S SCHREIBERd , e an Us . D . . R R , . L , culture filtrates of Ceratocystis ulmi as a biossay to screen for disease tolerant Ulmus americaw. Plant Science 70 (1990): 191-196. [20] CHAWLAWENZELd an vitrn I . S o . . selectioH G ,, r fusarinfo c acid resistant barley plants. Plant Breedin (1987)9 g9 : 159-163. [21] LYNCH, D. R., COLEMAN, M. C. and LYON, G. D. Effect of Alternaria solani culture filtrate on adventitious shoot regeneration in potato. Plant Cell Reports 9 (1991): 607-610.
36 IN VITRO MUTATION BREEDIN DEVELOPMENE TH R GFO F TO BANANAS WITH RESISTANC RACO ET , EFUSARIU4 M WELT (Fusarium oxysporum . cubense)sp . f
M. K. SMITH, S. D. HAMILL, P. W. LANGDON Queensland Departmen Primarf o t y Industries, Maroochy Horticultural Research Station, SCMC, Nambour, Queensland
K. G. PEGG Queensland Departmen Primarf o t y Industries, Division of Plant Protection, Meiers Road, Indooroopilly, Queensland Australia
Abstract
In 1985 the Queensland Department of Primary Industries initiated an in vitro mutation breeding program for banana improvement. The primary objective was to develop a banana that was resistant to race , 4 Fusarium wilt which could effectively substitut e Cavendisth r fo e h cultivars currently grown. Micropropagated bananas were exposed to gamma radiation from a ^Co source and the LDgo was established at 40 Gy. However, the optimal dose selected for irradiating explants was 20 Gy. At this dosage visual changes were apparent and plant survival, at 73 %, was sufficiently high to make the technique practica largea n o lr scale 0 .plant 00 Ove 0 s2 r have been evaluate r rac fiele ou e4 th t d a n di screening site and nursery at Wamuran in southeastern Queensland. Of the irradiated plants examined only putative th e mutants from Dwarf Parfitt extra-dwarn a , f variet f Cavendishyo , show potentia raca s e4 a l resistant Cavendish cultivar. Plants are intermediate in height between Dwarf Cavendish and Williams, the industry standard. Preliminary results indicat putative eth e mutant fastee sar r cyclin mord gan e cold tolerant than Williams. More importantly they also appea retaio t r resistance nth raco et showe4 mothee th y nb r plant, Dwarf Parfitt.
1. INTRODUCTION Fusarium wilt of bananas, caused by Fusarium oxysporum f.sp. cubense, is recognized as one mose oth f t destructive plan collapse banane tTh diseaseth . f worle o 2] a th , exporn dsi [1 t trades in the 1940's and 1950's, based on the susceptible cultivar 'Gros Michel', was saved only by replanting with resistant Cavendish cultivars. In 1981 another race of Fusarium Wilt, race 4, was identified which was capable of attacking Cavendish bananas [1]. The inability to control this disease by chemical or cultural practices and the lack of resistance in commercially acceptable cultivars means that developmen f bananao t s with resistanc raco et , Fusariue4 m f higwilo s hi t priorit banann yi a improvement. Cavendish are sterile, parthenocarpic triploids and, as such, have not featured in conventional breeding programs. The synthesis of new hybrids which can substitute for Cavendish also has its problems [3]. The difficulty associated with banana breeding can best be summed up by the fact that despite ove 0 year7 rf continuouo s s breedin gbanan w effortsne o an ,cultiva r thas i t commercially acceptabl Cavendisa s ea h replacemen bees ha t n produced factn I . , bananae on e sar of very few crops in which only clones derived from natural somatic mutations are cultivated. Due to the difficulty and costs associated with conventional banana breeding, alternative strategie r genetifo s c improvement have been actively pursued e sucOn h . strategy involves mutation induction. Mutation induction has become an established tool in plant breeding with hundred f improveso d varieties having been release growero dt s from many different crop species maie [4]Th n. advantag f mutatioeo n inductio vegetativela n ni y propagated crop, suc bananas ha ,
37 character w abilite ife sth a otherwis n chango a r yt o f so e eon e outstanding cultivar without altering the remaining and often unique part of the genotype. This is not yet possible using conventional approache banano t s a improvement summarA vitron i . e resultn th a mutatiof f yo o s n breeding program for the development of a race 4 resistant Cavendish replacement, suitable for the subtropical conditions of south-east Queensland and northern New South Wales, is presented.
2. MATERIALS AND METHODS Strains of Fusarium oxysporum f.sp. cubense were characterized by determination of vegetative compatibility groups analysiy [5]b , f volatilo s e components produce steamea n do d rice RAPD-PCy b substrat d an ] e[6 R analysis. Conditions for the initiation and growth of micropropagated bananas have been described by Hamill et al. [7] and Smith and Drew [8]. Briefly, Williams (AAA), New Guinea Cavendish (AAA) and Dwarf Parfitt (AAA) were initiated on a Murashige and Skoog [9] basal medium supplemented with 10 /*M benzylaminopurine, 2 % sucrose and 0.8 % Difco-Bacto agar. This medium also supported rapid shoot multiplication. Cultures were incubated at 28 °C with a 16 h photoperiod. Cool white fluorescent tubes provided a light intensity at the culture surface of 80 fjE nrV1. Before acclimatisation in the glasshouse, plants were subcultured to a hormone-free medium for root initiation and for the development of vigorous, single shoots. Plants were established in fiele th d when plasti 1 the 5 y2. c reache bagsn i m .c heighd a 0 2 f o t
The irradiation procedure involved the transfer of explants, containing the shoot tip and ensheathing leaf bases obtained from in vitro grown plantlets, to Petri dishes which contained a multiplication medium. Cultures were incubate day7 r sdfo before exposur gammo et a radiation . froDosagGy m 60 a calculates 0 C 5 o ewa t a source 0 dosee 4 d , Th usin.s30 use, g20 d , wer10 , e0 a Fricke dosimeter [10]. Plants were then r subculturethreo o e tw cycle r fo d s before disease resistance screening. Smit. [11al ]t he describe e Fusariudth m wilt rac screenine4 g experiments whic f Fusariumo he involvedus e th cultur) (1 : e filtrate vitro,n i s ) rooinoculationp (2 di t ) (3 d an s direct plantin a Fusarium n i g infested field sit t Wamuraa e n south-easteri (latitud) °S 7 2 e n Queensland. Any survivors from the first two screening trials were also tested in the field. The sit t Wamuraea commerciaa s nwa l bloc f Cavendisko h that first became infested with rac durine4 g pathogee 1976Th . n eventually 198y bloce sprea b becomth d 4d kha dan e commercially unviable. The site was leased by QDPI for development as a race 4 screening site and nursery. Levels of inoculum were maintained and increased by redistributing infected material on site. The Fusarium wilt population was intensively sampled and characterized by VCG and volatile analysis.
Plant harvesto s thatw r to survivese withouon r dfo t developin symptomy gan f Fusariuo s m wilt were divided into sucker bitreplanted d san an infesten di d sites. This usuallswa y repeated for a second and third cycle to overcome the possibility of 'escapes' and to verify that unique clones may have arisen following micropropagation and irradiation.
3. RESULTS AND DISCUSSION 3.1. Characterization of strains of Fusarium wilt designatioe Th Cavendis y f racs givei no an e4 o nt h resistance-breaking strai f Fusariumno oxysporum f.sp. cubense (FOC). However, these strains (biotypes) may be of separate origin and y diffethe n virulencma yi r d evean en hos t n internationaa range n I . l breedin r selectioo g n programm e precisth e e classificatio e pathogeth f o n n use r screeninfo d r resistancgfo e muse b t known in relation to isolates of the pathogen from other areas. Previously pathogenicity tests usin glimite a differentiaf o t dse l cultivars wer majoe eth r means for differentiatin racee th gf Fusariu o s m pathotypeso wiltTw . , wer2 , d racee an distinguishe1 s d by their pathogenicity on Gros Michel and Bluggoe, respectively. Stover [12] further separated rac isolatee1 s into yellowin non-yellowind gan g clone ssymptom e baseth n do s produce Gron do s Michel. Other techniques have now been used to reliably identify genetic diversity within the
38 fungus. One approach has been to use vegetative compatibility which is based on the genetics of e funguth s rather thae host-pathogeth n n interaction. Strain f fungo s i whic e abl ar ho fort e m heterokaryon e vegetativelar s y compatibl comprisd an e e vegetative compatibility groups (VCGs). Eleven VCGs have been characterized in FOC from a world-wide collection [13]. Using a number of isolates from south-eastern Queenslan northerd dan Soutw nNe h Wales, vegetative compatibility studies have confirmed that Australia isolate4 nd racan s1 ebelon separato gt e VCGs [5]n I . Australia, race 4 is composed of three VCGs (VCG 0120; VCG 0129; VCG 01211), while race 1 isolates belonVCGo tw o gst (VCG 0124 0125)G ;VC .
Another criteria which has been used to identify variation among Fusarium wilt isolates was productioe th f volatileno steamen o s d rice. Stover [14] cultured isolate steam-sterilizen o s d rice and classified 'odoratum r 'inodoratumo ' ' isolates base thein o d r productio a stron f o n g volatile odour. Moore et al. [6] found that production of volatiles on steamed rice by Australian isolates correlated well with racVCGd ean l rac Al .isolatee4 s produced distinct volatile odours which gave characteristi s chromatogramga c s wher e numbeth e f peako r s equate o VCGt d . Similar chromatograms were obtained for race 4 isolates from Taiwan and South Africa. All race 1 isolates and rac 2 isolatee s (VCG 0128 t produc) no teste d di de odorous volatile compound gavd an se chromatograms without significant peaks. Chromatographic analysis differentiated qualitatively between volatile odours produced by isolates in the race 4 VCGs 0120 and 0129. Recently RAPD- s beeha n R use PC o analyzt d e genetic variatio d resultn an withi C s havFO n e bee n closi n e agreement with previous classifications base n hoso d t range, vegetative compatibilitd an y chromatogram profiles of volatile compounds (S. Sorensen, pers. comm.).
3.2. Effect of gamma irradiation on in vitro growth of Cavendish cultivars
e LD r Th micropropagatefo 50 d William s approximatelwa s , howeveGy 0 4 yr shoot multiplication and general vigour of plantlets was poor (Fig. 1). The optimal dose range was 20 e othe th uses r r wa dfo Cavendis d Gyan h cultivar thin i s s program t thiA s. dosage visual changes were apparent and plant survival, at 73 %, was sufficiently high to make the technique practical on a larger scaleradiosensitivite Th . Cavendise th f yo h cultivars use thesn di e experiments compares well with that foun Novay . db [15 al r Grant ke ]fo d Nain.
3.3. Determinatio racf no , Fusariue4 m wilt resistanc Musae th n ei genepool
In additio o mutatiot n n breeding trial e havw s e also evaluate a rangd f Musao e species, cultivars and accessions for resistance to race 4 at our screening site at Wamuran. These plants were obtained from loca oversead an l s germplasm collection breedind an s g programs. Includen di this materia s Musawa l germplasm collected from PapuGuinew Ne a a [16]. Currentl8 36 y accessions have been screened and of these, only 6 accessions have shown resistance to Fusarium wilt race 4 (Table 1). They included two wild species, Musa ornata and M. velutina; two superior breeding diploids from the FHIA breeding program, SH-3362 and SH-3142; and Dwarf Parfitt, an extra-dwarf Cavendish variety. Mysor s susceptiblewa e when youn t tolerategbu e th d s raca 4 e stool matured.
Though all of these plants showed good levels of resistance, they were not immune to Fusarium wilt. Under extreme condition f cold droughto s an d s existea , n southeasteri d n Queenslan n 1991di , even some plants from this group became infecte showed an d d symptomf o s diseasee th factn I . , Fusariuimmunita realistia n i t r no fo ms yi cm wilgoaai o tt l breeding program in banana because regardless of the pathotype or cultivar used, the fungus is able to penetrate and established in the vascular system of the root [1]. Resistance can breakdown under periods when plante th stressepathogee e sar th d dan active s ni . Howeve levee th resistancf f o li r e observed from these accession expressee b n suitablsca a n di e commercial cultivar, tha possibilite nth f seriouyo s epidemics of the disease may be considerably reduced. We believe Fusarium wilt can be effectively managed with resistant cultivars.
39 o > '>
CO
40 50
en c:
o o>
10 20 30 40 50
DOSE (Gy)
Vigour of surviving plantlets was rated on a scale of 1 to 5: (1) little to no growth, chlorosis and necrosis of leaves excellen) (5 o t t growt vigorousd han , green shoots. Clon PJTje5 , Clon• fs^1 e 1 Clon8 , j5 9 e9
Fig. 1. Effect of gamma irradiation on in vitro survival and vigour of selected Williams clones.
40 Table I. Musa SPECIES AND CULTIVARS CONSIDERED TO SHOW RESISTANCE TO RACE 4 FUSARIUM WILT (Fusarium oxysporum f.sp. cubense).
Species/Cultivar Genotype Origin Comments
Dwarf Parfitt AAA Local selection Membe f Cavendiso r h from New South subgroup. 1 m at bunch Wales emergence commerciao N . l value
SH-3362, SH-3142 AA Improved diploids Importan males a t , from FHIA diploid parents but breeding program of no immediate commercial use
Mysore AAB Imported into Dessert fruit, Queensland from moderate yielding Jamaicd an a Honduras, originally from Indian subcontinent
Musa ornata, Local selections Ornamental bananas, M. velutina from New South colourful bracts Wales
Based on observations from 368 accessions grown on heavily-infested soils at Wamuran in south-eastern Queensland.
3.4. Reaction of gamma irradiated Cavendish cultivars to race 4, Fusarium wilt strategieo Tw s wer vitroen i adoptee th mutatio r dfo n breeding program firse Th t . approach involved irradiating the race 4 susceptible Cavendish cultivars, Williams and New Guinea Cavendish, to improve resistance but retain their agronomic characteristics. The second approach involved irradiating the agronomically inferior Cavendish cultivar, Dwarf Parfitt, to improve performanc retait ebu n resistance .plant 0 Ove00 0 s r2 hav e been evaluate fiele onld th e dan yth n di putative mutants derived from Dwarf Parfitt show the most promise as a race 4 resistant Cavendish replacement under the subtropical conditions existing in Queensland and New South Wales. e firsTh t strateg f irradiatino y g superior t diseasbu , e susceptible, Cavendish cultivars ha s yielde promisinw fe da g markedla line t sbu y improved leve f resistanco lt beeno ns observedeha . Planting material collected fro survivine mth g plants, includin thaw tfe survivede gth culture filtrate and root dip inoculation experiments, has been replanted for a second, third, and now for a fourth
cycle of evaluation. Of the few remaining plants, all are characterized by a dwarf, robust stature (averag t bunching)a m e 9 heigh1. f . o t Unfortunately these plant alse ar so pron o "chokinget a , 1 phenomenon where the bunch fails to emerge fully from the crown of the plant.
The second strategy was that of irradiating Dwarf Parfitt, an extra-dwarf Cavendish banana that has shown a high level of resistance to race 4. Unfortunately Dwarf Parfitt are very small, wit averagn a ht bunching a e extremelm ar e 0 d heigh1. an , f yo t pron o 'chokinget ' (Fig. 2) . Following irradiation of approximately 500 explants at a dose of 20 Gy, 35 MI¥3 plants were recovered which possessed improved characteristics. Plants were larger, they bunched earlier, yield was considerably increased and choking was eliminated (Fig. 2, Table II). They were also apparently more cold tolerant than standard Cavendish cultivars. More importantly they also appeared to retain the resistance to race 4 shown by the mother plant, Dwarf Parfitt.
41 ' i5 rr ' ^8 n ta^ bamm Plams established then i field- (a) Dwarf Parfitt; (b) putative mutant of Dwarf Parfitt following gamma irradiation. Tabl . ePRELIMINARII Y GROWT YIELD HAN D DAT DWARF AO F PARFITT AND ITS PUTATIVE MUTANT IN SOUTHEASTERN QUEENSLAND.
Dwarf Parfitt 'Parfitt' Williams
Heigh bunchint a t g (cm) 104 182 203 Bunch weight (kg) 8,.2 23 .2 22..1 hands/buncf o . No h 9..0 10 .3 10,.4 Plantin harveso t g t 19..8 16 .3 17,.9 (months)
Values are the averages from approximately 20 plants. The data for Williams was taken from non-infested sites as Williams succumbed to Race 4 Fusarium wilt in the original site.
earln Wa stile t ear ya l evaluatioe stagth n ei f thino s materiawise w determino ht d an l e eth genetic stabilit f thiyo s materia makd an l e further comment nature resistancee th th f n eo o ss i t I . interesting that Fusarium wilt of Cavendish is most prevalent in the subtropical regions of the world [2] where periods of cold and drought may place plants under stress. Leaves of our standard Cavendish varieties are typically more yellow that Dwarf Parfitt following winter (photoinhibition - induced chlorosis) and carry fewer photosynthetically competent leaves. We have speculated that e 'weakenedth ' Cavendish cultivar e thereforar s e most susceptible following winte unabld an r o et prevent invasio fungue th f no s intcore oth m durin sprine gth g whe disease nth reacn eca h almost epidemic proportions developiny B . greatega r understandin physiologicae th f go l mechanismf o s resistance theropportunitn a s ei y tha reliabla t e screening techniqu developee b n e ca applie d dan d either in culture or in the nursery to screen the large numbers of plants produced from mutation or conventional breeding programs. The lack of a suitable small plant screening technique continues majoe th f ro obstaclee on e b effectiveln o si t y breedin Fusariur gfo m wilt resistance.
Acknowledgements
We thank L. Pedwell for his support and the use of his land as a race 4 screening nursery. The funding support of the Queensland Banana Industry Protection Board, the Horticultural Researc d Developmenhan t Corporatio e Australiath d an n n Centr r Internationafo e l Agricultural Researc gratefulls hi y acknowledged.
REFERENCES
[1] PEGG, K.Gd P.Wan . . LANGDON, Fusarium Wilt (Panama Disease) a review: n I . "Banan Plantaid aan n Breeding Strategies: Proceeding internationan a f so l workshop helt da Cairns, Australia, 13-17 October, 1986" (Eds. G.J. Persley and E.A. DeLanghe) ACIAR Proceedings No 21, Canberra, Australia. (1987) pp 119-123. [2] PLOETZ, R.C., 'Fusarium Wilt of Banana', APS Press; St Paul, Minnesota, USA. (1990). [3] ROWE, P., Breeding bananas and plantains. Plant Breeding Review. (1984) 2: 135-155.
[4] MICKE, A., B. DONINI, and M. MALUSZYNSKI, Induced mutations for crop improvement - a review. Trop. Agric. (Trinidad) (1987) 64: 259-278.
] BRAKE[5 , V.M., K.G. PEGG, J.A.G. IRWIN d P.Wan , . LANGDON, Vegetative compatibility groups within Australian populations of Fusarium oxysporum f.sp. cubense, the cause of Fusarium wilt of bananas. Aust. J. Agric. Res. (1990.) 41: 863-870.
43 ] MOORE[6 , N.Y., P.A. HARGREAVES, K.G. PEG J.A.Gd Gan . IRWIN, Characterisatiof no strain f Fusariumo s oxysporum f.sp. cubense productioy b f volatilesno . Aust . BotJ . : 39 . 161-166. [7] HAMILL, S.D., S.L. SHARROCK and M.K. SMITH, A comparison of techniques for the initiatio f bananno a tissue cultures from field collected suckers. Plant Cell, Tissu Orgad ean n Culture (1993)(in press).
[8] SMITH, M.K., and R.A. DREW, Current applications of tissue culture in plant propagation and improvement. Aust . PlanJ . t Physiol. (1990 267-289: )17 . [9] MURASHIGE, T., and F. SKOOG, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. (1962) 15: 473-497.
[10] O'DONNELL, J.H., and D.F. SANGSTER, "Principles of Radiation Chemistry", Edward Arnold, London, U.K.(1970) [11] SMITH, M.K., S.D. HAMILL, P.W. LANGDON K.Gd an , . PEGG n vitroI , mutation breedin developmene th r gfo f bananao t s with resistanc raco et , Fusariue4 m Wilt (Fusarium oxysporum f.sp. cubense). In: 'In vitro Mutation Breeding of Bananas and Plantains I, Report of the first FAO/IAEA research co-ordination meeting held at Vienna, Austria, 29 May-2 June, 1989' IAEA, Vienna, Austria, (1990 66-78p )p .
[12] STOVER, R.H., Studies on Fusarium wilt of bananas. III. Influence of soil fungitoxins on behaviou f Fusariumo r oxyporum f.sp. cubense soin i l extract diffusatesd an s . Can . BotJ . . (1958)36: 439-453. [13] PLOETZ, R.C., and J.C. CORRELL, Vegetative compatibility among races of Fusarium oxysporum f.sp. cubense. Plant Diseases (1988) 72: 325-328.
[14] STOVER, R.H., Studie Fusariun o s m wil f bananaso t . VIII. Differentiatio f cloneo n y sb cultural interaction and volatile substances. Can. J. Bot. (1962) 40: 1467-1471.
[15] NOVAK, F.J., R. AFZA, M. VAN DUREN and M.S. OMAR, Mutation induction by gamma irradiatio vitron i f no cultured shoot-tip f banano s plantaid aan n (Musa cvs). Trop. Agric. (Trinidad) (1990 : 21-28)67 . [16] SHARROCK, S., Collecting mission in Papua New Guinea. In: 'Musa Conservation and Documentation: Proceedings of a workshop held in Leuven, Belgium, 11-14 December 1989'. INIBAP/IBPGR, Montpellier, France, (1990) pp 57-58.
44 SOMATIC EMBRYOGENESIS IN BANANAS AND PLANTAINS (Musa CLONES AND SPECIES)
A. D. KRIKORIAN Departmen Biochemistrf to Celd yan l Biology, State University of New York at Stony Brook, Stony Brook, New York, United States of America
Abstract
Broad principles and specific points of importance in the establishment, maintenance and manipulation of suspension culture f Musaso capabl f generatineo g somatic embryo presentes si d agains backgrouna t f do published literatur Stone th d y ean Broo k experience. Strategie f competenso t culture initiation, embryogenic culture maintenanc d vulnerabilitan e e embryogenie wholth th f f o eo y c culture proces o t oftes n unappreciated stresse emphasizede ar s . Inadequate monitorin e bencth t ha ge embryogeni th leve f o l c culture process readily leads to the kinds of problems that have earned Musa the undeserved reputation of being a recalcitrant species. Available protocols emphasize that there are alternative ways to move forward in a specific case and allow one to choose a specific route to fulfill a particular objective. The process of somatic embryogenesi s viewei s d her s onla e smala y l multi-facetea par n i t d approac o bananht d an a plantain improvement culturae Th . l requirement vitron si f Musao emphasize that ther stils ei l muc learo ht n abou developmentae th t l biolog f thiyo s important grou f tropicalspo .
1. INTRODUCTION
1.1. Distribution, importance and morphology genue Th s Musa (banana plantainsd an s , fam. Musaceae) comprises abou 5 species2 t e Th . main cente f diversito r f thesyo e large herbaceous monocotyledon southeass si t Asi t ediblabu e fruit and ornamental varieties have long been grow tropice th subtropicd n ni san botf so h hemispheres. The morphology and growth habit of these giant herbs is highly distinctive. While in the vegetative condition e shooth , t apical growing poin s mori t r les o et soi a s l level. Leavee ar s produce flankmeristee e th th f n o sdo m with very little vertical displacemen growine th f o t g tipd an , leaves wrap around themselves forming a false trunk or pseudostem [1]. The lateral (leaf-opposed) bud Musan i se adventitiou ar s , (cf3])2 , ..[1 Whe e vegetativth n e growing point undergoes transition to the flowering mode, the true stem tip moves upwards vertically through the hollow of the pseudoste. 5] , m[4 The group as a whole has been inadequately studied taxonomically. To avoid confusion, the best method available for dealing with the cultivated clones involves the use of a short-hand technique that takes into account the amount of the contribution of 'A' genome (from M. acuminata) ' genom'B d an e (fro . balbisiand)mM somd characteristican 5 e 1 s [6] generalizatioA . n ' genomei'A s thae mor e sweetee th th tf th ,e o fruie hencd usablrth s i an t tabl a t ei s ea e fruite th ; more 'B more 1,th e starch , hencis t yi t needei s cooking e betteTh .r know commercialld nan y important clone generalle ar s y parthenocarpi ) althougc triploid33 = hn diploids(3 tetraploidd san s exis wels havd a t an le local importance. An important taxonomic descriptor involves the persistence/ deciduous nature of the male bracts raceme , terminath e bud , axif 10])d th 9 o t e , an ssa d 8 (cf ., en lThi .[7 s phenotypic change represents a significant developmental change and has major implications for fruit production [10]. 1.2. Significanc somatif eo c embryogenesi tissud san e culture-related strategies Bananas and plantains are excellent candidates for virtually all tissue culture strategies [11, 12, 13. 14]. The corms or suckers are large and inconvenient for germplasm movement, frequently
45 harbor pathogens and are often in short supply especially when extensive or large scale new plantings are contemplated. Germplasm maintenance in the field is expensive and space consuming, and on a commercial scale germ plasm production is usually relegated to "seed" nurseries. Breeding of the essentially seed-sterile clones of interest to plantation agriculture is fraught with many difficulties [15, 16]. A numbe f diseaseo r s seriously affect banan plantaid an a n plantings throughou e worldth t . The amount of time and money that must be invested in control measures using chemicals are beyond subsistence farmers, usuall t withino y n e workinreacth f o h g clasd posan s e financial problems eve r largfo n e commercial operations. Breeding strategie o generatt s e tolerancr o e resistanc importano et t diseases suc Fusariums ha wilt (Panama Disease) cause . oxysporumF y db . f sp. cubense (various races), Sigatoka leaf spot caused by Mycosphaerella musicola, Black Sigatoka caused by Mycosphaerella fijiensis var. difformis and to the burrowing nematode Radopholus core similisth md weevian l Cosmopolites sordidus have thu r bee maie sfa nth n breeding objectives but thes eo lea t hav o commerciallt t d ye e y successful clones e improvemenTh . t programe ar s rendere e morth l e al ddifficul t becaus e potentiath e breeo t l d plantain bananad an s s seriousli s y handicapped by the high sterility of the clones of primary interest (cf. [7, 17, 18] and refs. there cited). e reliablTh e applicatio f tissueno , cell, somatic embryogenesi protoplasd an s t procedureo t s plantain bananascase d th f mansan e o n i ys a , other plants necessara componeny s i , ke d y yan an n i t comprehensive biotechnology plan aimed at improvement via mutation breeding and genetic transformation; it is also important for germplasm management strategies such as cryopreservation or storag artificiad ean l see r propaguldo e delivery system , 20]19 .s , [1218 ,
1.3. Shoot tip procedures as a preliminary to suspension/ embryogenesis procedures The means to micropropagate Musa are established and have been widely adopted or adapted in various parts of the world. Technical guidelines for safe movement of germplasm have been delineate r somfo d e time (cfe problemawarw th . no f d mos [21]o ee an f assuminar )o ts g that meristems or in vitro cultures are necessarily clean [22]. There are many possibilities of introducing virus diseases along with germ plas mf adequati e precaution thae t takese no t o ne t ar s plant virue sar s indexe r quarantinedo d [18]. Various publications summarize the procedures to make primary explants and to initiate multiplying shoo , 26])25 t . , culture24 Eve , , ther23 so ns s , considerabli e(cf 21 . , [1116 , e opportunity for changes to be implemented in these methods, and refinements and attempts to understand the implications of explant selection and tissue culture management strategies for performanc f propaguleo e ongoinn a e fiele th ar dn gi s activity [10]. Figur Krikorian i e2 l a t ne [27] provide diagrammatia s c representatio varioue f somno th f eo s aseptic culture procedurer sfo Musa theid an r relationships matura ) (1 : e plant wit hratooa r suckerno . Excisio apee th xf no fro m a vegetative cor througm2 ( r alternativel o , h3) steya fro p mmale ti m th a throug e (2 flora d hbu l 2c), yields a system where leaf-opposed and adventitious buds are generated (4 through 8). Subdivision of these multiple shoots and transfer to cytokinin-containing culture medium (semi- solid or shallow liquid) keeps the system open-ended (9). Further treatment of aseptic shoot tips in vitro with auxins can foster the development of callus, calloid, and embryogenic masses (10-13). These various-sized and variously-differentiated units may be further broken down into still smaller dimensions. regeneratee Thesb n eca d into somatic embryos (14ar organogenicallo ) y responsive masses (15). Eac thesf rearee ho b turn e n i full-size o dnt ca d propagule establishee sb than ca t n di soil (14b, 16-17). Protoplast wor n folloca k f wtheso froy e an mpoint t competenbu s t cell suspensions are preferable for a number of reasons [28, 29, 30]. e meanTh s whereb a yshoo p multiplicatioti t n syste s mdevelopei d maintainean d s ha d fundamental implications for the successful establishment of morphogenetically competent suspension cultures (cf. [12, 27, 31]). M and S liquid medium [32] with 5 mg/1 BAP, 100 mg/1 sucros% inositol 4 d e an provide n adequata s e starting poin r initiationfo t f multiplyino g shoot systems from excised stem tip or floral bud explants. Response in liquid is faster than on semi- solid medium. Rotation on a gyratory shaker is helpful but not critical. Pulsing the explant from liqui semi-solio dt bacd dan liquio k t spee y growtdp ma d u h [23].
46 The methods used to carry out shoot tip culture are highly flexible but tests for bacteria and fungi mus carriee b t t routineldou eacd yan h ste (cfpy alon.wa [13e gth , 33]). Test r cucumbesfo r mosaic virus shoul e dondb s welea l (cf. e.g. [18, 34]). Other precautions will depene th n o d location of the operation. For instance bunchy top virus is not eliminated via meristem culture (cf. [18]should an ) testeappropriats e da b r dfo e [35]. Many adventitious buds are produced in vitro from cultured and subdivided shoot and floral e potentiath d r multiplicatioan fo l ] 40 ste , m39 s ver tipi n, s38 y [36, hig37 ,h (cf. [13, 23]). Carryin proceduree th t gou s entirel liquin yi d mediu withoud man t agitation excep initiatioe th t f no the primary explant can speed up the process even more (Krikorian and Scott, unpublished).
1.4. Callus induction Callus culture and cell suspension culture studies using preclimacteric fruit pulp were carried e 1950 th d 1960 oun an si t s (cf. [41]). Those cultures were relatively slow growino n d an g morphogenesis was encountered (neither was it attempted). Since then, callus induction procedures have improved but growth of somatic tissue still is not necessarily fast or routine from many Musa clones. Multiplying shoot tip cultures provide a good starting point for callus induction since they can demonstrable b y clean. Auxins like dicamb more piclorad th usee aan t b e dbu conventionan mca l ones like 2,4,5- 2,4-d Tan active Dar s wellea . Semi-solid medium provide somewhaa s t better substrate in the initial stages of callus induction for research purposes since one can follow the explant more easily. The report that Gelrite is nominally pivotal to successful callus culture of banana [42] may be explained on the basis of failure to use adequately washed agar (cf. e.g. [43, 44]). Gelrite does has value, however, and can be very helpful, for instance, in detecting subtle contaminants. There can be considerable darkening in explants due to polyphenol oxidation [45] and Gelrite can help minimize this. The most important area for obtaining explants for the production of morphogenetically competent, vigorou sregion e calluvegetative th th e f sar so r floraeo leae l th f ape d basese xan Th . presence of multiple foci of meristematic growth at the base of the multiplying shoots is crucial to getting a sustainable culture in a timely fashion; if these are not present or induced to form, competent callus initiatio maintenancd nan mucs ei h more difficult.
2. SOMATIC EMBRYOGENESIS e presencTh a larg f o ee numbe f activo r e meristemati e basa cd th e are cellan th f l o an i s primary thickening meristem of multiplying shoots in vitro make it an attractive zone from which to induce and secure vigorous and morphogenetically competent cells. This strategy is especially helpful under research laboratory conditions when one is remote from the banana growing regions an regulada r suppl flowerinf yo g materia t availableno s i l . Excised zygotic embryo responsive sar e as well d yielan ,d callus culture suspensiond an s s which turnn i , , yield somatic embryos which develop normally although thee arguabl e choicar yth t eno y materials since e ediblmanth f eo y t clonenormallno o sd y produce seed hencd modey san ean l base zygotin do c embryo flawes si d (cf. [46] for details on the production of somatic embryos from zygotic embryos of an ornamental diploid banana; also [47, 48, 49, 50] for work on excised zygotic embryos of a diploid M. acuminata etc). Obviously preferabls i t i , initiato et e cultures directly from field-grown materials with prove r knowno n qualities withou e intermediatth t e ste f micropropagatiopo otheo n r r fo f ni reason tha savo nt e time [18, 31]30 , . Cronauer and Krikorian [51] first reported production of somatic embryos of Musa from suspension cultures of triploid clones of cooking bananas. Although their shoot apices were not well developed (cf. [52] for histological details), very small cell clusters could develop. What I call neomorphs, i.e. structures that are developmentally aberrant, and embryonal structures with varying level f developmentao s d structuraan l l fidelity derived from suspensions have, since then, been routinely generated by us and others from various clones [12, 27, 31, 48, 53, 54].
47 Fig. 1. Photographs of some stages in the process of generating suspensions and embryogenic . cultures. See text for details. A, nodular growths formed at the base of a micropropagated shoot system of an 'ABB' clone, 'Cardaba' 0.5X; section of the same, 'AAB'C, X; clone10 'Lady Finger' showing globular masses (lighter color interspersed between tissues which have darkened severely) produced from nodular growths similarto those in A and B, 2 X; D, view of globular masses separated from units like those shown in C, 3 X; E, partial view of a nipple flask (containing 220 nil of liquid nutrient medium) showing suspended cells, 0.33 X; F, low pH-maintained culture of embryogenic masses a semi-solid on medium. Noteuniformitythe generaland finenessunitsthe of comprising culture,the magnificationX; 4 embryogenicthe of cells shown afterF in dispersion in liquid medium, 71 X; H, somatic embryos generated on semi-solid medium, 5X.
48 have W e initiated morphogenetically competent globules directl liquin yi d from base f smalso l shoot clusters grown on semi-solid medium. 2,4,5-T was used in our initial studies and has remaine auxin da usee nb thadn witca t h success have W e. also confirmed report Novay sb l a t ke [31] that dicamba and picloram work as well (but picloram perhaps a bit better than dicamba to the extent that picloram cultures show less tendency to oxidize, e.g. show purple or black specks). Somatic embryos have been generated from triploid clones using both in vitro plantlets and full size corms as explant sources [31]. We have routinely regenerated plantlets from cell suspensions of various edible clones since 1985. The procedure starts with shoot tip explants and is indirect and labor intensive. Yields have been quite acceptable (cf. Fig. 1 for representative cultured materials) and some clones give good crops of somatic embryos with good morphological fidelity. The Musa regenerants are, however, e higth generall hf o qualit t no y y that somatic embryos obtained from other monocotyledonous systems routinely worke thin i sn do laborator y suc daylilys ha r eveo , n elegane equath o t l t somatic embryos produced from zygotic embryos of diploid Musa. Similarly derived somatic embryo plantletd an s s have been regenerate othey db r laboratories as well from nodular, calloid growths occurring at the base of proliferating shoots. These nodular growth e somewhaar s t reminiscen f orchio t d protocorm e capabl b e sai ar o d t f yieldin d o e an s g cultures whic generatn hca e somatic embryos [54, 55] t musI .cautionee b t d that Cronaued an r Krikorian [39] showed that adventive growths that grossly resemble somatic embryos can easily be mistake trur nfo e somatic embryos problee On . m thas i stee th tm line tissue that yield celle sth s that can organize has been difficult to define. The precursors of the somatic embryos obtained by Sannasgala [55] and Dhed'a et al [54] are very much like the globular structures described by Cronauer [52] but these are perhaps better viewed as aberrant albeit morphogenetically competent, organogenic globules t proembryosno , . Nova . [31al t ]ke reporte d somatic embryogenesis from suspensions generated from callus fro basae mth l area f shooso t tiplead san f bases. Specifico t s a s developmentae th l e somatiorigith f no c embryos still requires elucidation. Gape th s l exisal n i t published papers particularly in those important steps between initiation of a suspension and generation of a plantlet/propagule. thene e mattem th r , f routinFo o r e productio f somatino c embryo e ediblth n i es (i.e. non- seeded), triploid clones remains in need of more work for better understanding the developmental process and needs resolution for more clones insofar as sustainability of high response once an embryogenic culture has been established.
2.1. Procedures
Most (but by no means all of the many that we have tested) clones yield elegant, vigorous shoot tip cultures, and with 2,4,5-T one can start a morphogenetically competent suspension culture. These suspensions are maintained more effectively with 2,4-D (cf. Fig. IE), and are then puso t culture nexe A hth th t NA o et leve f o f advancementfollowe o le us y b d t obtaininbu , e gth right kind of growth can be and frequently is, slow especially in the beginning, slow. Selection mus carriee b t d outinitiae Th . l culturmaintainee b n eca d using very finely sieved suspensions with less or greater difficulty over the long term but cultures are subject to deterioration over consecutive month f seriao s l subculture and, unit size f celso l clusters required prio organizeo t r d growth are often not small enough to give a highly discrete response (cf. e.g. [53, 56]). Auxins such as picloram and dicamba can also be used to initiate competent cultures. In our hands the picloram respons relativels ei y quicker (cf. [31]). r severaFo l year have w s e undertake alternativn a n e strateg develoo yt p cultures whice har virtually completely comprise f somatido c proembryos (cf. Fig. 1F,G). These cultures retain their ability to produce very early stage somatic embryos which themselves multiply by budding. They are best maintained at the preglobular stage by use of low pH (cf. [44]). Since the "secondary" embryos, can in turn, release additional embryos, as long as the more recently generated and developed somatic embryos can be separated and subcultured, one can have a sustainable and subculturable sourc f somatio e c embryos e leve th r stag o t l f developmenBu o e. t whica t h such
49 cultures are maintainable can vary considerably. In some instances, one can have small proembryos or globular stage embryos; in other cases, the somatic embryos can be well advanced and clearly visible to the naked eye and at a stage wherein the next level would be maturation, "germination" and further growt sproutingr ho wells therd a possibilit e , An eth ,is . f achievinyo sustainind gan ga culture witl levelal hf o developmens n i between te ultimatTh . e a culturstat f o e e condition/maintenance strateg r playo n depends largel t thiya s poin r knowledge timn ou i t f eo n o , what one can get. Response seems to have a clonal origin relationship.
2.2. Strategy to obtain somatic embryos of Musa
e strategTh e follow y w involves initiating, maintainin d manipulatinan g g cell suspensions ) fairlwhic(1 ye ar hminimall t clearlbu y y embryogenically differentiated, but f necessityo , e ar , capabl f undergoino e g still more advanced organized development eithe y adventitioub r s routes from small callus masses, or (2) via somatic embryogenesis involving pre-proembryonic (i.e. embryogenic cell cluster) and/or proembryonic (i.e. globular stage somatic embryo) suspensions from which continued somatic embryo growt developmend han deriven ca t . Figures 1A and IB show a representative nodular meristematic mass (which I deduce are referred to as "scalps" by some workers). When subjected to 2,4,5-T in liquid medium (Fig. 1C) these yield compact globular masses compace Th . t globular masses separatgron ca units w a d ean s of varying size, ranging from discrete unit clustero t s s (Fig. ID). These unit organizn ca s d ean yield a nodular growth mode from which units and hence, plantlets can be proliferated. Competent cell derivee b s n (Figca d ) fro .IE e compac mth t globular masses since they release cells morr eo less readily eithed an , r adventitiou r embryogeniso c pathway achievede b formee y th sma n I r. case, shoot generatee sar d adventitiously which mus rootee b t yielo dt d plantlets case th f somati eo n i ; c embryogenesis procesa , s analogou germinatioo t s n encountere zygotin i d c embryos occurse Th . means, in varying degrees of detail to carry this out, may be found in Krikorian [12], Cronauer and ] referenceKrikoria52 , 46 , ns 40 ther[24 , 38 e, cited emphasizI . e here, however, thastratege th t y is what is crucial and there are sufficient differences among clones for us to be confident that the particulars will vary more than a little. From published reports, it is clear that more than one route of regeneratio beins ni g activate realizer do d- bot h embryogeni organogenicd can . The work of Bakry and Rossignol [57] on "callogenesis" and production of "neoformations" (probably equivalen e structureth o t t s define thin i d s laborator s calloidsya tha , organize?— is t d proliferations that initially "look" callus-like but have an epidermis in place) of various types substantiates the view that a lot of different developmental events can go on in a limited space. Adventitious shoot formation from the cultured floral axis of various so-called "determinate" plantain clones without major callus production has been routinely obtained by us (cf. [10, 37]). This indicate s thau to t sthera hig s i eh potentia r differentiatiofo l f meristemo n e novod s ("meristemoids") from otherwise meristem-free areas.
At Stony Brook we have been able to generate competent cell masses of varying size from cells routinely sieved through numbe 0 sieve8 r s (17 porm 7/x e diameter eved an )n numbe0 20 r porm sieve// e 4 diameter(7 s theid an ) r abilit generato yt e plantlet s beyoni s l doubdal t (cf. [12]). e cloneTh s most workee 'Cardaba cookina ar ( n B o d gAB ' banana d 'Ladan ) y Finger' (AAB)a , dessert banana. Eve , ther nso mane ear y developmental questions suc understandins ha s i t i y gwh that in some cases, small groups of cells seem to need to grow into somewhat larger masses before they organize s therI . a erequiremen r criticafo t l e greatesizeTh ?e numbe th r f cella o r n i s morphogenetically competent mass before competenc s expressedi e e greateth , e chancth r r fo e introductio f chimerano l e developinstatuth o t s g propagule e implicationTh . f thir o clonas fo s l stability and integrity is obvious (cf. [58] and references there cited). For me, it also follows that somatic embryos are not necessarily derived from a single cell and the suggestion by some that variation wil minimae b l l from somatic embryo t foundeno s si fact n di . Culture dynamic origid san n of somatic embryos indicates thae samth t e kind f riso s k obtain with suspension cultured an s somatic embryo s wita s h stem tips [59] n factI . , becaus e numberth f o e f somatio s c embryos producible wit suspensioha suspensio w becausd facte ho nan th f o sf eo n culture e initiatear s d dan maintained, the risk is even greater (cf. [60]). The value of cryopreserving such cultures is not readily apparent (cf. [61]).
50 Fig. 2. Schematic diagram of organized development in Musafrom morphogenetically competent cell cultures. See text for details.
In our somatic embryo producing cultures there seems to be a correlation with ability to "germinate" and further grow with the level of development of the haustorium (cf. [46] and unpublished) s therI .a criticae l nutritional r information-processino - g role playe y thib d s cotyledon-like structure? identifn e Unlesrequirementca th e l al yon s r controllesfo d germination and/or retention of dormancy or "quiescence" prior to desired use, the system is only partial and on s therebi e y limite o restrictet d d earlan dy developmental phenomena. Fig 2 schematicall. y emphasizes (albeit in a grossly simplified fashion) that understanding the range of responses encountered as one progresses from a micropropagated plantlet of Musa (be it diploid, triploid or tetraploid) to a vigorously dividing and morphogenetically competent culture is not a trivial task. From such dividing cells, through variou t "unitssge avenuesn "ca (show e diagrae on th , n ni s ma globules) whic givn hca e ris variouo et s embryonal forms (including neomorphs, shown hera s ea "tadpole" form), organogenic masses (some discrete, others not so discrete) from callus or calloid, and also, of course, bona fide somatic embryos. In some cases all four kinds of structures are obtainable from a single culture! One can hardly call such problems as "biotechnological" ones for differential gene action is certainly at work! More effort needs to be expended in understanding all aspects of Musa somatic embryogenesis, and the greatest benefits from a practical perspective laboratore fielde th th n i n .remainsi , t yme no r fo ,
51 Acknowledgements This paper includes work carriet Stona t you d Brook initiall. SandrDr y . Cronauerb yS a - Mitra and Dr. David L. Smith. More recently, Mrs. Mary E. Scott has played a major role in the work aime t resolvinda g important remaining issue helpind san elevato gt e laboratory methoda o st practical level for applied use by those less experienced with in vitro culture methods. Support from the US Agency for International Development is acknowledged as is support for our somatic embryogenesis wor non-Musan o k speciee Stonth n yi s Brook laborator NationaS y U fro e mth l Aeronautics and Space Administration (NASA).
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[39] CRONAUER KRIKORIANd an . S . . S D Adventitiou, . A , s shoot production from calloid cultures of banana. Plant Cell Reports 6: (1988b) 443-445. [40] CRONAUER KRIKORIANd an . S . . S ,Temporal D . A , , spatia morphologicad an l l aspectf so multiplication in aseptically cultured Musa clones. In: F. Valentine (ed) Progress and Prospects in Crop and Forest Biotechnology. State University of New York College of Environmental Science and Forestry, Colloquium. April 18-20, 1985. Syracuse. Springer, New York. (1988d 45-57p )p .
[41] MOHA STEWARDd e inductioan Th N. Y RAM . f . growtC no H . , F ,explante n hi d tissue of the banana fruit. Canad J Bot 42 (1964): 1559-1579. [42] HUANG, L-C. and CHI D-L. Pivotal roles of picloram and gelrite in banana callus culture. Environ Exptl Bot 28: (1988) 249-258. [43] KRIKORIAN SMITHd . an L Somati. . D D . , A , c embryogenesi carron i s t (Daucus carota). In: Plant Tissue Culture Manual: Fundamentals and Application (K. Lindsey, ed.), Kluwer Academic Publishers, Dordrecht, Netherlands (1992 PTCM-Ap )p 9 1-32.
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[48] ESCALANT, J. V. and TEISSON, C. Embryogenese somatique chez Musa sp. CR Acad Sci Paris ser III: 306: (1988) 277-281. [49] ESCALANT, J. V. and TEISSON, C. Somatic embryogenesis and lantsfrom immature zygotic embryos of the species Musa acuminata and Musa balbisiana. Plant Cell Reports 7: (1989) 665-668.
[50] MARROQUIN , PADUSCHECKG. . C , , ESCALANTC. , TEISSONd an . V . . J ,Somati C , c embryogenesi pland an s t regeneration through cell suspension Musan i s acuminata n VitroI . Cell Dev Biol 29P (1993): 43-46.
[51] CRONAUER, S. S. and KRIKORIAN, A. D. Somatic embryos from cultured tissues of triploid plantains (Musa 'ABB')- Plant Cell Reports 2: (1983) 289-291. [52] CRONAUER n vitrI o . growtS . S ,h response f Musao s . Ph.D. Dissertation, State University of New York at Stony Brook, Stony Brook, New York, 206 pp. Dissert Abstr Internatl 47:1816-B (1986). Univ. Micro film 8616686sA OrdeD . No r .
[53] BANERJEE, N., SCHOOFS, J., HOLLEVOET, S., DUMORTIER, F. and DE LANGHE, . E Aspect prospecd san somatif o t c embryogenesi Musa,n si . Bluggoe cv ABB, . Acta Hortic 212: (1987b) 727-730. [54] DHED'A, D., DUMORTIER, F., PANIS, B., VUYLSTEKE, D. and DE LANGHE, E. Plant regeneration in cell suspension cultures of the cooking banana cv. 'Bluggoe' (Musa spp. ABB group). Fruits (Paris) 46: (1991) 125-135.
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[59] REUVENI, O. Methods for detecting somaclonal variants in 'Williams' bananas. In: R.L. Jarret (ed) Identification of Genetic Diversity in the Genus Musa. Proceedings of an International Workshop held at Los Banos, Philippines 5-10 September 1988. International Network for the Improvement of Bananas and Plantain (INIBAP), Montpellier, France. (199 ) pp. 108-113.
[60] KRIKORIAN , O'CONNORD. . FITTERA d , , KANNan . S A. . Developmen P . . S . ,M ,R , t karyotypd an , of e stabilit , Hemerocallisyin plants reare tissuea dvi , suspensio protoplasd nan t culture. V International Congress of Plant Tissue and Cell Cultures, 11-16 July 1982, Tokyo, Japan. Proceedings. (1983 429-430p )p .
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Next page(s) left blank " IMPROVEMENT OF Musa THROUGH BIOTECHNOLOGY AND MUTATION BREEDING F. J. NOVAK, R. AFZA, R. MORPURGO, M. VAN DUREN, M. SACCHI Plant Breeding Unit, Joint FAO/IAEA Programme, IAEA Laboratories, Seibersdorf, Austria
. KHATRA I Atomic Energy Agricultural Research Centre, Tandojam, Pakistan
Abstract
Edible Musa, i.e. bananas, plantain d cookinan s g bananas mostle ar , y sterile polyploids witha reproductive system that is extremely difficult to manipulate. The presently used breeding methods based on crossing of near-sterile triploids with semi-wild diploids is time consuming and laborious. The cultivation of bananas in many regions is seriously threatened by several diseases caused by pathogenic fungi (Mycosphaerella, Fusarium}, bacteria (Pseudomonas), viruses (esp. bunch p virus to d nematodesy an ) . Breedin r resistancfo g e require strategiew e implementatione sth d an s f biotechnologyno e currenTh . t application f biotechnologso Musan yi improvement include ) embry(1 ; o cultur r hybridizationefo ) shoot(2 , - culturp ti micropropagationn ei vitron i ) (3 , mutation induction ) somati(4 , c embryogenesi somaclonad san l variation, (5) in vitro screening and selection, (6) artificial seed production and (7) DMA fingerprinting applied genomio t c analysis. Researc bees hha n undertake l thesal n i e area f Musaso biotechnology resulte Th . s and ongoing projects of the Joint FAO/IAEA Programme are: (1) development of in vitro mutation systems in shoot-tip cultures ) developmen(2 , mutana f o t t clone with superior performanc originae th o et l Cavendish type "Grand Nain" ) somati(3 , c embryogenesi celn si l suspension f Musa,so ) protocol (4 e isolatio th r sfo n shord an t term cultur f protoplastseo e studth f } somaclonayo {5 , l variatio genetie th r nfo c improvemenf o t banana ) encapsulatio(6 , f somatino c embryo r artificiafo s l seed production ) selectio(7 , n techniqued an s nursery screening techniques for disease resistance breeding of Musa and, (8) protein electrophoresis and DNA fingerprintin r genomigfo c characterizatio f differenno t Musa clones. Finally, future opportunitier sfo the utilization of tissue culture and molecular biological techniques in Musa improvement are discussed.
. 1 INTRODUCTION The genetic system of Musa is extremely complicated. Sterility caused by different factors, interspecific hybridity, heterozygosit polyploidd yan commoe yar cultivatee mosn nth i f o t d clones. The asexual nature of Musa clones is often an insurmountable barrier to cross-hybridization. The complexit f Musayo genetics necessitate f additionao e us e lsth technolog supporo yt t conventional breeding programs potentiae Th . biotechnologf o l thin yi s cro ver. s pi Thi 3] y , s2 hig , reporh[1 t describes recent resultdevelopmene th n si followine th f o t vitron gi system r Musa:vitron i sfo ) (i mutation induction; (ii) cell (protoplast) cultur somatid ean c embryogenesis; (iii) marker genetif so c diversity; (iv) in vitro screening and selection for Fusarium oxysporum f. sp. cubensejesistea.ee.
2. MATERIALS AND METHODS 2.1. Materials Clones of the following Musa species and cultivars have been used as experimental material: M. acuminata subsp. burmannica (clone IV-9) . balbisiana,M , 'Pisang Mas' (AA), SH-3362 (bred diploid clone AA), Grand Nain (AAA), Cardaba (ABB).
57 2.2. Shoot Tip Culture
Shoot tip ssizen i (2-)m consistinm 5 meristematie th f go c dome wit4 leah2- f primordia were cultured on MS medium with 10 jtM 6-benzylaminopurine (BAP) and 5 //M indole-3-acetic acid (IAA) e mediuTh s solidifie. mwa d with gelrite (1.75 g/1). Multiple shoot formatios wa n achieve subculturiny db g shoo mediuS t M tip n so m supplemented wit fj0 Mh2 BAP. Shoots were elongate mediuS rooted M dan n dmo 6-dimethylallyl-aminopurin M wit/* h5 e (2ip)indole-3M p 1 , - butyric acid (IBA). Detailed method vitron i f o s productio f bananno a plantlets were describey db Vuylsteke [4],
2.3. Induced mutagenesis
Meristem tips, 1 - 2 mm in size, were excised from in vitro growing shoots and irradiated in a gamma cell with a 60Co source with 15 to 60 Gy at a dose rate of 8 Gy/min [5]. Uniform, longitudinally dissected meristem tips were dipped into an aqueous solution of 50 pM filter- sterilized ethyl methanesulphonate (EMS) with 2 % v/v dimethylsulphoxide (DMSO) for 3 h at the constant temperatur explante [6]C ° Th . 8 2 s f wereo e then thoroughly washed with sterile water prior to culture. The nutrient media were the same as described for shoot tip culture.
2.4. Embryogenic callu celd lsan suspension, plant regeneration
The extreme basal parts of the leaf sheaths were cut from young leaves of in vitro meristem- derived plantlets. Corm tissue pieces withou apican a t l meristem wer t intecu o segments (5x5 mm). Both types of explants were plated on petri dishes on Schenk and Hildebrandt [7] medium supplemented wit a modifieh d mixtur f Staba'o e s vitamins 1 sucrose[8]g/ 0 0 ,mg/4 4 , 1 cysteine-HCI sucrose1 g/ dicamb e culture0 M 4 Th /x . d 0 3 aan ,s were maintaine 8 dare 2 th t ka n di weeks6 - 4 .r °Cfo Embryogeni c calli were subcultured into liquid, hormone-free mediuS mM (half-strength) and incubated on shakers (120 rpm) for one week. Cell suspensions were transferred into half strength SH medium with 20 (jM dicamba for long-term culture of cell suspensions (more thae year)e celon nTh l .suspensio n maintaine e dicambth n i d a medium loss regenerativit t e capacity within a 2 - 3 month period. To regenerate plants, the fresh suspensions of cells and proembryogenic structures were transferred into half-strength, hormone-free MS and then plated onto solid medium compose half-strengtf do sucrose1 cystein 1 g/ vitamin+ g/ 0 S 2 0 1 ,h4 M e+ + s g charcoal, 5 fjM zeatin, 4 g/1 agar and 1.75 g/1 gelrite. In this double layer system, somatic embryos were converted into plants within 2 weeks. The detailed protocol is described elsewhere [8]- 2.5. Encapsulatio embryof no s
Intact bipolar somatic embryos were transferre a steril (w/v% o t d e3 ) solutio f sodiuo n m alginate (Sigma, medium viscosity), prepare basaS M ln i dmediu m (half concentration) without calcium chloride, supplemented with Staba vitamins sucrose1 g/ mg/0 0 2 10 , , 1 inositol 0 mg/4 , 1 cysteine-HCI and 5 jtiM zeatin. Embryos, together wit e alginathth e solution, were then steril a drawn i ep n u pipett e witn ha opening of 4 mm, and carefully dripped into a 50 piM solution of calcium chloride dehydrate on a gyratory shaker (60 rpm) at room temperature. The beads were left in the solution for 30 minutes to allow proper hardening. Selected, totally encapsulated embryos were then transferre double th o dt e layer medium described before.
2.6. Protoplast isolatio culturd nan e
e enzymatiTh c mixtur f Macerozymo e , ecellulas R-1%) 1 0( e Onozukd an a ) R-1% 3 0( pectinas release) % 1 e( largda e numbe f palisado r e mesophyll cells from leave f aseptio s c plants and parenchyma cell rhizomf so e tissue durin suspensio e hour2 g1 Th f incubatio . so °C f no 8 2 t na intact protoplasts, cell debris, starch grain raphided an s s washewa s CPW13Mn di , transferreo dt conical tube afted minute0 an s1 r protoplaste f centrifugatioth o s g x 0 s 10 wer t a n e collected from e surface media th e th washin f Th o e. g procedures were repeated twic purifo t e e protoplasth y t
58 fraction. The protoplasts were cultured in the half-strength MS with 20 pM dicamba in thin layers of liquid media in petri dishes.
2.7. Protein extraction and electrophoresis Soluble proteins were extracted from young folded leave usin y bufferS sb gSD . Samples were homogenized and centrifuged at 13 000 rpm at 4 °C for 15 minutes. A 12 % polyacrylamide gel with a 4 % stacking gel was used for electrophoretic separation of proteins at 1500 V/h. The gel staines wa d with comassie blue.
2.8. DNA fingerprinting
Detailed procedure purificationA DN r fo s , restriction enzyme digestio hybridizatiod nan e nar given elsewhere e purifie[9] sampleA Th . DN d s extracted from freeze-dried leaf tissue were digested with restriction enzymes, Hinfi, Acul r Taql.restrictee o Th fragment A dDN s were passed throug electri n denatures fragmenta agarosn A ha wa n i l DN l cdried d ge e efield ge dan e s Th .Th . were hybridized with single stranded DNA oligonucleotides such as CA, GTC, GATA, and hybridization was detected by autoradiography.
2.9. Fusarium (FOC) culture and bioassays of plant-fungus interactions
2.9.1. Fungal culture and filtrate production Fusarium oxysporum . cubensesp . f s culture wa Czapek-Don do x mediu transferred man o t d potato dextrose aga r sporulationconidiaf o rfo l m e l suspensioOn . n containin conidia/m0 00 0 g45 l l Erlenmeye m s transferre 0 wa 50 a ro t dflas k f containinliquio l dm Czapek-Do0 g25 r crudxfo e filtrate production. Culture conditions were; 28 °C, continuous light for sporulation on PDA, 28 °C dark condition for production of mycelium and crude filtrate. After removal of mycelium, the liquid phas filteres ewa d through three layer f cheeseclothso , refiltered through Watmann pape. No r finalld an 1 y filtered throug h0.2a Milliporn 2/u e membrane. Productio f crudno e filtrate activity was assayed wit hspectrophotometea r followin absorbance gth . nm 2 f fusarieo 27 - c0 aci27 t da 2.9.2. Bioassa crudf yo e filtrate
Banana shoot tips culture alreads da y mentioned were transferre mediua o dt m containin, 6 , g3 crudf o % e 2 filtrat1 d ean obtaine9 d afte day 1 placed 2 same r th san en di conditio befors na 3 r efo weeks. Plant material was analyzed for various parameters including plant height, total fresh weight, shoot productio d roonan t fresh weight.
2.9.3. Plant inoculation
Rooted plantlets were rinse removo dt e gelrite roote th , s were trimmed plante th , s were dipped into a conidial suspension (5 x 10s conidia/ml) for 10 minutes and then transplanted into Erlenmeyer flasks containing sterile perlite. 2.9.4. Total soluble peroxidase activity
At regular intervals infected plants were removed from the flasks and, after removal of roots and leaves, the remaining corm tissue pieces with some pseudostem tissue attached were crushed in a mortar wit phosphatha e extraction buffer 6.8homogenizee H p ,Th . d tissues were collected dan placed in Eppendorf tubes and centrifuged for 15 min at 14 000 rpm. The supernatant was transferred to new Eppendorf tubes and immediately assayed for total peroxidase activity. Peroxidases were assayed with guaiaco hydrogee th s a l n donor reactioe Th . n mixtures consistef do f hydroge(v/vo % d j 3 ) 0. guaiaco5 n1 peroxidd an l phosphatn ei e buffe 6.8)H (p r f .o Fiv! /* e supernatan reactiof o l m s adde n3 wa t mixturo dt changed ean absorbancn si e were followe0 47 t da reactioe nmTh .stoppes nwa d afte min2 r .
59 2.9.5. Isoelectrofocusing (IEF totaf o ) l soluble peroxidases
Isoelectrofocusin f peroxidaseo g s performewa s a horizonta n i d l apparatuge l s (Pharmacia Multiphor II) at 4 °C. Ampholines (Pharmacia LKB) with a pH range of 3 - 10 were used as electrolyte carrier prefocused an s minutes0 2 r fo d . Fifteef samplo l s loade/i n wa er lane pe d . Running conditions were 8 W fixed (mA from 33 to 10). After focusing, the gels were soaked in phosphatM 1 0. f o e l buffem 6.8H 0 (p r)10 containin (v/v % f hydroge o 3 l )g 0. /x guaiaco 5 1 n d an l peroxide. After 15 minutes of incubation the gels were analyzed for stained bands.
3. RESULTS AND DISCUSSION
The variants in MjV4 vegetative progenies of irradiated and/or chemical mutagen (EMS)- treated shoot tips included phenotypic changes in morphological (plant stature, leaf shape), physiological (sucker growt d multiplicationan h , flowering time, fruit ripening d agronomian ) c (bunch quality) characters. An outstanding variant plant from the cultivar 'Grand Nain', (putative mutant 'GN6OGyA') was identified and multiplied for field testing. The mutant plant showed difference zymograme th n i s f solublo s e protein esterasd an s e isozyme ] whe[5 s n comparee th o dt original cv. Grand Nain. Induction of somatic mutations may substantially contribute to the broadening of the genetic variation within and among vegetatively propagated Musa clones. The system of in vitro mutagenesis, however, may also be important for the breeding of fertile diploids before their use for crossing. Fig. 1 schematically illustrates the biotechnology supporting the Musa conventional breeding system proposed by Vakili [10]. Two steps of mutagenesis are proposed; (i) mutation breeding of female diploids before crossing, or (ii) mutation induction and selection among Fj recombinant diploids before polyploidy induction.
The ability to induce somatic embryogenesis in economically important Musa cultivars (AAA and ABB s opene) ha approachew ne d r somatifo s c cell manipulatio breedind an n g [8]. Somatic embryos may be encapsulated and used for propagation and in vitro storage. Embryogenic suspensions represen a potentiallt y superior sourc f genetio e c variation ("true somaclonal variation" a s uniquwela )s a l e unicellular syste r mutatiofo m n inductio d selectionan n . Embryogenic suspensions are the preferred source of protoplasts. However, attempts to induce cell wall formation and callus colony proliferation from protoplasts were unsuccessful.
Differences amon 4 gdifferen t e clonesolublth n i se protein electrophoresis patterns were recognized as reliable markers for discrimination among Musa clones of genomic group A. However, consistency in the number of polymorphic bands on electrophoretographs was dependent precise onth e developmental leavee stagth f eo s from whic proteie hth extracteds nwa . Young, fully folded leaves were used as standard material for sample preparation. A band of 30 000 d was present only in 'SH-3362', while a band of 32 000 d was characteristic of 'Grand Nain . Many qualitative differences were identified among clones [11]. 1 Specifi fingerprintA cDN s were sample detecteA DN r sd fo extracte d from different genotypes. The probes and restriction enzymes used (see Material and Methods) failed to distinguish among individual e samth f eo s clone, however a uniqu , fingerprinA DN e s foun r wa eactfo d h clone. Further research using other probe-enzyme combination continuins si effortn gi deteco t s t individual differences in breeding populations after crossing or mutagenesis.
Crude filtrates obtaine C culturdFO froe mth displaye d toxic activity. Toxicits wa y Fusanume th f o e dependen ag finae culture th th ld n concentratioean o t f extrac no e banan th n i t a culture medium. In vitro grown shoot tips of banana were affected by the crude filtrate and its effect was proportional to its concentration in the culture medium. No conclusive differential reaction has been obtained with respect to the known response of in vivo susceptibility or resistance, suggesting that toxic compounds excreted by the pathogen in culture are not responsible differene foth r t level f resistanco s e expresse clonese th y db . Alternativel face yth t thacrude th t e filtrates sho wa toxi c activity support e ideth sa that toxin(s) have some rol n pathogenesisi e , possibly durin post-infectionae gth l stage. Sinc establishmene eth pathogenesif o t multipla s si e step process it is likely that the different levels of resistance shown by 'Pisang Mas' and 'ISH-3362' in o somt e edu mechanisvivoe ar f earlmo y plant-pathogen interaction including wall-to-wall recognition eliciting defense processes.
60 In vitro mutation Antfier/microspore induction and selection Gene (s) transformation; • far disease resistance viral coat protein gene (s): cultures for production and improved fertility horaozygoteN 2 f o s before crossing chltinase gene (s) •
FEMALE MALE PARENT x PARENT 2 N 2 N
Intraspecific protoplast In vitro fusion - somatic fertilization recombination
DIPLOIDS recombination) Embryo culture
In vitro mutation induction and n vitroI polyploidy selectior fo n inductio o product n e improved diploids tetraploids
TETRAPLQIDS (Heterozygotic)
Self pollination, backcrossingr o . crossing with other tetraploids
Embryo culture Micropropagatiof o n superior tetraploids
TETRAPLOIDS (Selected) X DIPLOIDS
Micropropagation of TRIPLOIDS DNA typing of superior triploids (Final selection) selected clones
Fig.l. Schematic representation of biotechnological methods supporting the conventional breeding system Musaof [10]. techniquesThe dottedin rectangles fullynot are established in banana.
61 Levels of peroxidase (PRX) activity differ in the two clones. 'SH-3362' shows a ten-fold higher activity in uninoculated plants than 'Pisang Mas'. 'SH-3362' showed a sharp increase of peroxidase activity after inoculation with either Race 1 or 4. Differences have been recorded in the time of induction and in the degree of activity in the two fungal races. In fact, PRX activity in plants treated with Rac increase1 e days5 s after inoculation whil plantn ei s treated with Race th e4 increas recordes ewa d afte days8 r 'ISH-3362n I . ' treate dactivit X witPR h e yRac th continue , e4 s increaso t reaco et hmaximua m afte day1 thed 1 r an sn begin decreaseo t s n plantI . s inoculated with Race 1 PRX starts to decrease immediately and increases once more 13 days after inoculation. These results were f infecteo confirme F uninfected IE an d y b d d banana plants grow growtn ni h chambers and showed that the increases in activity are due mainly to changes in the basic class of peroxidases were Pisann t w ablI .s eno fino et gsimilaa Ma d r pattern bot totan hi l activitn i d yan IEF. Our preliminary analysis of PRX seems to indicate that this enzyme is involved in an active defence mechanism against FOC t tha ,n bananbu i t a this e mechanisonle thath on ytt no s mi counteracts Fusarium infection, as demonstrated by the lack of response in 'Pisang Mas' plants infected RacwitC t . surprisinghFO 1 eThi no s si facroln e i , f peroxidasesth teo bees ha n studied in many plant-pathogen interaction theid t beean s rno nrols unequivocallha e y establishey an n i d plant pathogen system. More investigation is needed in order to establish a role for peroxidases in the defence respons Fusarium.o et
. 4 CONCLUSION Current application Musaf so biotechnolog vere yar y clos makino et gsignificana t contribution breedine icultivarsnth w ne f go . Tissue culture technique beine sar g develope inductioe th r dfo f no heritable variation useful in Musa breeding. In vitro mutagenesis may contribute to conventional breeding programme y extendinb s e genetith g c base availabl r recombinationfo e . Somatic embryogenesi s beesha n achieve economicalln di y important cultivars. This techniqu especialls ei y important for the study of "true" somaclonal variation in Musa using a unicellular system for mutation induction e inductioTh . f diseaso n e resistanc f e maio th e s ni e us objectiv e th r fo e biotechnology in Musa. Reliable selection systems for genotypes resistant to FOC may soon be available vitron I . cell selection longer-ter a seem e b o st m objectiv basir efo c research.
REFERENCES
[1] PERSLEY, G.J. and DE LANGHE, E.A. (eds.) Banana and Plantain Breeding strategies. Proceeding Internationan a f o s l Workshop hel t Cairnesda , Australia, October 1986, ACIAR Proceedings No. 21 (1987). [2] DALE, J.L. Banana and plantain. In: Persley, G.J- (ed.), Agriculture Biotechnology: Opportunity for International Development. C.A.B. Intern., Walingford, (1990): pp. 225- 240. [3] NOVAK, F.J. Musa (banana plantains)d an s : HammerschlaIn . g F.A Litzd . .(eds.)an R , , Biotechnology of Perennial Fruit Crops. C.A.B. Intern., Walingford - in press. [4] VUYLSTEKE, D.R. Shoot-tip culture for the propagation, conservation and exchange of Musa germplasm. IBPGR, Rome (1989). [5] NOVAK, F.J., AFZA, R., VAN DUREN, M. and OMAR, M.S. Mutation induction by gamma irradiation of in vitro cultured shoot-tips of banana and plantain (Musa cvs.). Tropical Agriculture (Trinidad) 67 (1990): 21-28. [6] OMAR, M.S., NOVAK, F.J. and BRUNNER, H. In vitro action of ethyl methanesulfonate on banana shoot tips. Scientia Hort (1989)0 4 . : 283-295.
62 [7] SCHENK, R.U d HILDEBRANDTan . , A.C. Mediu d techniqueman r inductiofo s d an n growt f monocotyledonouho dicotyledonoud san s plant cell cultures. Can . .BotJ (1972)0 5 . : 199-204. ] NOVAK[8 , F.J. DURENN , AFZA VA , PEREA-DALLOS , M. ,R. , , CONGERM. , , B.Vd an . TAN . GSomatiX c embryogenesi pland an s t regeneratio suspension ni n culture f dessero s t (AA and AAA) and cooking (ABB) bananas (Musa spp.). Bio/Technology 7 (1989): 154-159. [9] AFZA, R. et al. Use of DNA fingerprinting for characterization of genetic diversity in Musa preparationn i - .
[10] VAKILI, N.G e experimentaTh . l formatio f polyploidno genue s effecth it n d i st yan Musa. Amer. J. Bot. 54 (1967): 24-36.
[11] SACCHI, M. and NOVAK, F.J. Use of protein electrophoresis for characterization of genetic diversit Musan yi preparationn i - .
Next page(s) left blank 63 STUDY ON MUTATION BREEDING OF BANANA, 'KLUAI KHAT . SILAYOIB . WANICHKULK , . KEAWSOMPONGS , . SARADULDHAP , T Department of Horticulture N. SINGHABURAUDOM Department of Pathology Faculty of Agriculture, Kasetsart University, Bangkok, Thailand
Abstract
This repor composes i t f threo d e parts ) mutatio(1 ; n induction through tissue culture ) stud(2 ,n o y rooting media experimen) (3 , colchicinn o t e treatment.
Shoot tips isolated from suckers of a local banana cultivar, Kluai Khai were divided into four parts and propagated on MS medium supplemented with 15 % coconut water and 5 mg/l of BA. Subcultures were made every month until 200 plantlets were obtained. Shoots were irradiated with 0, 10, 20 or 30 Gy of gamma rays and cultured on either pH 5.6 or pH 6.1 medium. Subcultures were made until the MiVa. No proliferation was observed on pH 6.1 medium for all doses of gamma rays. On pH 5.6 medium, only the Gy-treate0 contro1 d an l d explants proliferated. These were transferre fielde th .o d t However, only non- treated control plants survived.
'Kluai Khai' plantlets were irradiated with gamma rays at doses of 0, 2.5, 5.0, 7.5 or 10.0 Gy and cut longitudinally intpieceso otw l werAl . e culture mediS M n ado supplemente dcoconu % wit 5 h1 t water2 ,
mg/l BA and 1 mg/l NAA. Subcultures were made every 4 weeks until the 8th generation (MiV8). Three media were teste r rootin dmediufo S M g: m without growth regulators mediuS M , md witan mg/h1 A NA l MS mediu l mediAl mg/m 1 . ad wit BA werlan mg/h1 A e ladjusteNA 5.8H p .o d t Observation s were made afte weeks4 r bese Th t . rootin achieves mediuS gwa M n dmo without growth regulators.
To examine the effect of induced polyploidization, in vitro plantlets of 'Kluai Khai' were treated with 0, 500 ,hours2 frequenc7 e 100 r Th o .f 0r colchicin150o 8 o 4 f explanmediu m o yS , 0 M pp 24 n r ei tm fo survival rate and adventitious bud initiation decreased with increasing concentrations of colchicine. The height, pseudostem circumference and the number of leaves produced were significantly different between treatments differenco n t thers bu , ewa fruin ei t developmen treatmenty an n i t . Ploid determines wa y y db measurmen f stomatao t l cellchromosomd san e counts. Plyploidt inducedno s ywa .
. I INDUCED MUTATIO 'KLUAF NO I KHAI' THROUGH TISSUE CULTURE
1.1. INTRODUCTION
'Kluai Khai' r 'Pisano , importane th gf o Mas' e ton commercias ,i l clone bananf so a grown ni Thailand. The fruit of 'Kluai Khai' are smaller than those of Cavendish banana, but the taste is excellent, fragran sweetd cultivates i an tt I exoti.n a s da c frui r Europeafo t Japanesd nan e markets. e demandTh f thoso s e market e increasingar s . 'Kluai Khai' e accountth f o r almos% fo s 0 9 t bananas exported from Thailand. 'Kluai Khai' is susceptible to leaf spot disease. This disease causes a decrease in effective leaf numbers, small bunches, undersized fingers, and low yield. As 'Kluai Khai' is grown over large areas in Thailand, the disease susceptibility of this clone is causing serious production problems.
65 Conventional breedin f banango difficuls ai problemo t e du t s associated with mal femald ean e sterility. Therefore, mutation breeding using tissue culture techniques is expected to accelerate progress in the development of leaf spot disease resistant 'Kluai Khai'.
1.2. MATERIAL METHODD SAN S Suckers of cv. Kluai Khai were used in all experiments. Shoot tips were excised, divided into four parts propagated an , Murashign do Skood ean g media supplemente dcoconu % wit 5 h1 t water an dmg/5 f benzyladenin1o e (BA). Subcultures were made every four weeks untiplantlet0 20 l s were obtained. Plantlets were irradiated with 0, 10, 20 and 30 Gy of Gamma rays. One half of the plantlets wer 1 mediaremaindee 6. 6 medieth 5. cultured H p .H aan p n Subcultureo rn o d s were mad obtaio et n M.^2 M{Vjd an generations. Dat observationd aan s were recorded monthly.
1.3. RESULT DISCUSSIOD SAN N Multiple shoots were produced after cultur weeks8 e f - explant eo mediS 6 Th M .r n afo o s number of plantlets increased at the rate of 3.3 times per month. The effects of explant culture on medi1 6. presentee H aar p r . o II Table n pd6 i d H 5. an sI
Tabl . eI SURVIVA IRRADIATEF LO D PLANTLETS AFTE RMONTH4 N SO pH 5.6 MEDIA
Numbe f survivino r g plantlets
treatment treated plantlets 3 months 4 months
Control 25 25 30 Trl (10 Gy) 25 40 72 ) Gy 0 (2 2 Tr 25 40 70 Tr3 (30 Gy) 25 32 32
Table II. SURVIVAL OF IRRADIATED PLANTLETS AFTER 4 MONTHS ON MEDIp1 H6. A
Number of surviving plantlets Gamma ray Number of — treatment treated plantlets 3 months month4 s
Control 25 15 0 Trl (10 Gy) 25 11 0 ) Gy 0 (2 2 Tr 25 25 0 Tr3 (30 Gy) 25 19 0
No proliferatio 1 medium6. H np occure.e th Chlorotin o d necrotid an c c symptoms were medium6 5. observe plante H th p e d ,s th plantletdan died n O . s treate gammf o dy witG a 0 rayh3 s did not proliferate and died eventually. Lower gamma ray doses of 10 and 20 Gy were not lethal anexplante dth sshoot 0 produce3 d san respectively2 d3 numbee Th . f survivino r g plant higs swa h 5.6H (p ) mediaH p w .lo Aftee onth months5 r survivine th l al , g plants were transferre rootino dt g media died3 Tr ..d an OnlPlantlet 2 yTr plantletf l o scontrogroupe Tr th d n i ss an l rooted dan were maintaine greenhouse th n di e before transplanting field e theth mo .l t Tr Eventuall e th f o l yal plantlets also died.
66 The results of this experiment indicated that the proper pH of the medium should be around 5 6 and that the gamma ray dose should not exceed 10 Gy.
II. EFFECT OF MEDIA ON ROOTING OF IRRADIATED 'KLUAI KHAF PLANTLETS
ILL INTRODUCTION
Accordin resulte th o gt s describe section di e appropriat th , nI e dos f gammeo . a 'K ray r fo s Khai' was about 10 Gy, and the pH of the culture medium should be around 5.6. A factorial experiment was conducted to examine the effects of gamma ray dose and medium type.
II.2. MATERIAL METHODD SAN S
Five doses of gamma rays were used to treat in vitro cultured plantlets of 'K Khai'. The doses were 0, 2 5, 5 0, 7.5, and 10 Gy. The survival rate in the M^ was recorded. In order to obtain chimera free material afte e mutageth r n treatment, plantlets were subculture MjVge th o dt . MjVg plantlets were then cultured on three types of media;
1 MS without growth regulators (MS) + 1NAA S )(M A + lmg/NA f S o 1M 2 + 1NA S 1BA+ A (M )A B mg/ 1 f o 1+ A lmg/+ NA f S o 1M 3 Thirty five replications were mad r eacefo h treatment. Shoot lengt numberd an h f rooto s s were examined aftemonthe on r .
IL3. RESULTS AND DISCUSSION
In all the treatment classes, survival was high The MjVj plantlets were subcultured to the MjV8 generation The number of shoots produced in each generation is indicated in Figure 1
- 75 - 10
2 18 • 16 - -8_
£ oai | 06 i Z 04 02 0 -t————————i—————————i—————————i- MIV2 MIV3 M.IV4 M1V5 M1VM1V76 M1V8
Fig 1 Average number of shoots/generation
The number of roots produced was also scored and these data are shown in Table III. Neither gamme e mediuth th dos y r ra am eno type, affecte e numbeth d f rooto r s produce treatee th y db plantlets
67 Table III. AVERAGE NUMBER OF ROOTS PER PLANTLET ONE MONTH AFTER SUBCULTUR) 35 = N E(
Gamma Rays (Gy) Media type 0 2 .5 5 .0 7 .5 10.0 Average
MS 4 .33 4. 17 3. 67 4. 33 4 .33 4.17nB A NA M+ S 6 .00 5. 33 4. 67 4. 83 5 .17 5.20ns MS + 1NAA + 1BA 4 .83 3. 50 3. 33 4. 67 4 .33 413. ns
Average 5 .05ns 4. 33ns 3. 89ns 4. 61ns 4 .61ns
C.V. (%} 28.5 ns : Not significant
All MiV6 plantlets were cultured on MS, MS +1NAA, or MS + 1NAA +1BA at pH 5.6. The length of roots was measured and these data are shown in Table IV. The gamma ray dose applied did not affect root length. MS medium without growth regulators resulted in the greatest root growth.
Tabl . AVERAGeIV E LENGT ROOTF H O MONT E SON H AFTER SUBCULTURE
Gamma Rays (Gy) Media type 0 2 .5 5.,0 7 .5 10 .0 Average
MS 9 .03 8 .02 8. 41 8 .40 9 .01 8.50a* A NA M + S 6 .07 5 .17 4. 58 3 .70 5 .61 5.03b MS + 1NAA + 1BA 3 .55 2 .22 3. 24 2 .87 2 .96 2.97C
Average 6 .22ns 5 .14ns 5. 41ns 4 . 99ns 5 .82ns
C.V. (%) 46.05 ns : Not significant : Number * s followe same th ey d b alphabetica l superscrip t significantlno e ar t y different.
Root strength was scored by using a grading method from 1 = weak to 4 = strong. These results are presented in Table V.
Tabl . ROOeV T STRENGT MONTE HON H AFTER SUBCULTURE
Gamma Rays (Gy)
Media 0 2 .5 5 .0 7 .5 10. 0 Average
MS 3 .00 2 .67 2 .17 2 .83 3. 33 2.80a* MS + NAA 3 .33 2 .50 2 .67 2 .00 3. 00 2.70a MS + 1NAA + 1BA 1.50 1.00 1.00 1.17 1. 17 1.17b
Average 2 .61ns 2 .06ns 1.95ns 2 .00ns 2. 50ns
C.V. <%} 35.24. significant nNo s: t : Number * s followe same th ey d b alphabetica l superscrip t significantlno e ar t y different.
68 roote + f plantlet1NATh so S AM d wers an culture eS strongeM n do r than root plantletf so s on MS + 1NAA 4- 1BA. The gamma ray dosage did not affect root strength. Roots of plantlets mediuS growM n no m without growth regulators were stronge longed an r r than thos f plantleteo s culture othen do r media. e dose Th f gammo s a rays use thin i d s experiment wer t affecno e e d the th d tlow an di y, surviva r growte treateo l th f o hd plantlets. Morphological variants suc s albina h o plantlets, plantlets with short pseudostems, curly leaves etc., were found. However, these variants did not survive when they were transferre survivine th f o l soilo dt g Al plantlet. s were transferre soio dt l for further observation.
III. EFFECT OF COLCHICINE ON Musa (AA GROUP) 'K. KHAI' IN TISSUE CULTURE
III.1. INTRODUCTION banane Th a cultiva . Khai' 'K rdiploia s i , d clone wit hsomatia c cell chromosome numbef o r 2n = 2x = 22. This clone is designated as a member of the AA Group. It is a parthenocarpic cultivar, as are other dessert bananas. The fruit is small and similar to those of Tisang Lilin'. popula a width n i s i preferre d t m I c .an r 3 Frui- d 2 lon tm d cultiva c siz gan 0 abous e1 i y - b r 8 t the Thai people because of its excellent taste and aroma. It is also commercially accepted in the international market, e.g. Japanes Europead an e n markets larga d e an ,s exporte amouni t i f o td from Thailand, Malaysi Philippinese th d aan expor. e Khai'K Th .f 'o t from these three countries has increased in recent years. plantee Th . dKhai 'K are Thailann f i 'ao s largei d r than tha f Cavendiso t h bananase th t bu , expory b qualitw tlo standardss i y . Nevertheless e tast th ,s excellent i e e smalTh .l fruitw lo , productivity and susceptibility to leaf spot disease are the major production problems of this cultivar. Some diploi th f thes o eo t d e e . Khai'naturmigh'K du f e o b e.t Generally, triploid banana favouree sar d over diploids becaus f theieo r higher productivity, greater vigou larged an r r greatefruite Th . r vigou f polyploio r d clone improvy sma e their resistanc tolerancr eo leao et f spot disease under field conditions. Thu n experimena s s carrie wa t tapplyin ou d g colchicino t e meristematic tissue of 'K. Khai1.
III.2. MATERIAL METHOD SAN D Different dose colchicinf so e were mediuS addeM o dt m without growth regulators. Explants of 'K. Khai' were cultured on each MS + colchicine medium treatment for various periods of time. The following treatments were applied:
1. Control (0- 0) 2. MS + 500 ppm colchicine for 24 hr. (500-24) 3. MS + 500 ppm colchicine for 48 hr. (500-48) colchicin . m hr pp 2 (500-727 0 r 50 efo + ) S M . 4 5. MS + 1000 ppm colchicine for 24 hr. (1000-24) colchicin . m hr 100+ pp (1000-488 S 04 r M e fo . 6 ) colchicin . m hr + 100 pp (1000-722 S 07 M r e fo . 7 ) colchicin . m hr 150+ pp (1500-244 0S 2 r M 8.efo ) 9. MS + 1500 ppm colchicine for 48 hr. (1500-48) colchicin. m hr 150+ pp (1500-722 0S 7 10 r M .efo )
69 A completely randomized design (CRD) was used for data analysis. The following data were collected for analysis: 1. Survival rate after treatment with colchicine 2. Number of shoots produced by plantlet . 3 Survival rat plantlef eo t after transfe soio t r l . 4 Survival rat plantf fiele eo t th Kamphaengsae da n si n Campus 5. Growth rate - Height, pseudostem circumference, number of leaves . 6 Tim shootinf eo g Day shootingo st , total numbe shootinf o y f leaveo rda n gso 7. Fruit Bunch weight, fruits/bunch, hands/bunch - Fruit size (circumference, length, peel thickness) - Soluble solids, flesh firmness . 8 Numbe chromosomef o r s . 9 Leaf spot disease symptoms
III.3. RESULT DISCUSSIOD SAN N
III .3.1. Effect f colchicino s shoon eo t proliferatio survivad nan l rate Data were collecte monte don h afte colchicine th r e treatment. Result showe sar Tabln ni . eVI
Table VI. NUMBER OF AXILLARY SHOOTS/COLCHICINE TREATMENT AFTER SUBCULTURE TO MS + BA MEDIUM
Colchicine Colchicine treatment duration (h) concentratio- n (ppm) 24 48 72 Average
0 3 .25 3.25 3.25 3.25a* 500 3 .25 3 .10 l .50 2.62b 1000 2.25 2.00 1.45 1.90C 1500 2.30 2 .10 1.10 1.83°
Average of Colchicine 2.50a 2.40a 1.35b treatment
C.V. (%} 72 Number: * s followe same th ey d b alphabetica l superscrip t significantlno e ar t y different.
e resultTh s indicated that high concentration f colchicino s longed an e r period f treatmeno s t resulte lowen di r shoot production. Incubatio mediun no f colchicinmo containinm pp r 0 efo 50 g1 72 hours appeared to be the high end of the treatment range.
70 III.3.2. Surviva . Khail rat'K f eo ' after transfe soio t r l The treated plantlets were subcultured on the rooting medium in vitro before being transferred soilo t . Afte acclimatioe th r n procedur transfed ean soilo t r , they were grow plantina n ni g material which consiste mixturf do soilf eo : san dcompos: t manure (1:1:1 monthe on r ).fo Survival data were recorded aftemonte on r h (Table VII). Table VII.SURVIVAL RATE OF 'K. KHAI' SHOOTS
Colchicine Colchicine treatment duratio) (h n
(ppm) 24 48 72 Average
0 80.0 80.0 80.0 80. Oa* 500 80.0 80.0 60.0 73.3ab 1000 70.0 60.0 50.0 60.0C 1500 70.0 70.0 50.0 63.3bc
Average of Colchicine 73. 3a 70. Oa 53. 3b treatment
34.) C.V(% 9. * : Numbers followed by the same alphabetical superscript are not ssignificantly different. Survival rates were higher in the control treatment and lower at the higher concentrations of colchicin witd ean h longer period treatmentf so .
III.3.3. Survival rate in the field at Kamphaengsaen Campus
Three month old plantlets were transplanted to the field at the Kamphaengsaen Campus of Kasetsart University, Nakhon Fathom Province hundreo Tw fift.d dan y gram f 15-0-so 0 fertilizer per plant were supplied every month. On the fourth and fifth months, the formula of fertilizer used was change 20-20-0o dt quantite th t bu ,y remaine samee dth . Survival rats examineewa e don month after transplanting and these data are shown in Table VIII.
Table VIII. SURVIVAL RATE (%) OF 'K. KHAI1 IN THE FIELD
Colchicine Colchicine treatment duration (h) concentratio- n (ppm) 24 48 72 Average
0 90.0 90.0 90.0 90 .Oa * 500 90.0 90.0 90.0 90 .Oa 1000 90.0 90.0 90.0 90 .Oa 1500 80.0 70.0 60.0 • 70 .Ob
Average of Colchicine 86.yns 83.3ns 80.0ns treatment
C.V. (%) 22.8 : Number* s followe same th ey d b alphabetica l superscrip t significantlno e ar t y different.
The more severe treatments, i.e. higher concentrations of colchicine and longer periods of treatment, resulted in lower rates of survival.
71 III.3.4. Growth heighe pseudostee th Th f o t measures mwa d monthly fro groune morigie lase th th th t f o dnt o leaf. The results are shown in Table IX. The data indicate normal growth within each treatment class, and no significant differences could be observed among the treatment classes at this stage.
Table IX. THE EFFECT OF COLCHICINE TREATMENT ON HEIGHT (cm) OF 'K. KHAI'
Colchicine treatment March April May June July August
0 - 0 31.7 47.5 64.0 97.7 149 .7 194 .0 500 - 24 33.8 38.8 46.3 67.3 116 .2 152 .0 500 - 48 31.3 48.0 62.2 92.0 142 .0 182 .3 500 - 72 29.3 42.0 56.3 85.2 137 .8 180 .3 1000 - 24 34.3 47.3 54.0 79.0 123 .0 161 .3 1000 - 48 30.2 39.2 46.0 63.3 110 .2 146 .0 1000 - 72 30.5 42.2 50.8 76.0 122 .5 158 .0 1500 - 24 31.5 40.5 50.8 79.2 136 .0 163 .0 1500 - 48 32.0 43.7 55.2 87.8 140 .0 175 .7 1500 - 72 29.7 41.7 49.8 78.0 131 .7 166 .5
Tes- F t ns ns ns ns ns ns C.V. (%) 15.9 22.0 26.1 27.8 23 .3 20 .3
Pseudostem growth was measured as the circumference in cm at a height of 15 cm from the ground. The results are shown in Table X.
Table X. THE EFFECT OF COLCHICINE TREATMENT ON PSEUDSTEM CIRCUMFERENCE (cm) OF 'K. KHAI'
Colchicine treatment March April May June July August
0 - 0 10.0 13.3 17.7 27.3 37.5 48.3 500 - 24 9.1 12.0 14.7 20.3 30.5 44.5 500 - 48 9.9 13.7 18.6 27.3 38.1 50.7 500 - 72 8.7 12.8 17.0 25.8 36.6 49.4 1000 - 24 9.8 13.5 16.7 23.9 23.6 44.8 1000 - 48 9.3 11.6 15.6 20.5 28.3 40.1 1000 - 72 9.8 13.3 16.8 23.8 33.2 44.7 1500 - 24 ' 10.3 12.2 17.2 24.2 35.3 44.9 1500 - 48 9.1 12.5 16.8 26.3 38.8 50.4 1500 - 72 8.9 12.1 15.2 23.6 33.7 46.0
F - Test ns ns ns ns ns ns C.V. (5) 13.0 16.0 23.3 23.3 21.1 46.3
Leaf production was monitored by counting the number of leaves every month. These data are presented in Table XI.
72 EFFECE TablTH . COLCHICINF TeXI O E TREATMEN NUMBEE TH N TF O R O MATURE LEAVE . KHAI'K F S1O
Colchicine treatment March April May June July August
0 - 0 6.0 11.0 16.5 22.2 27.8 33.0 500 - 24 6.3 11.3 16.0 20.8 26.2 31.2 500 - 48 5.6 12.0 17.2 22.8 27.8 33.3 500 - 72 5.0 9.8 15.8 21.3 27.0 32.0 1000 - 24 6.2 11.8 15.7 21.2 26.5 31.0 1000 - 48 5.8 10.3 15.7 20.5 25.8 31.3 1000 - 72 5.6 11.3 16.5 21.7 26.7 32.0 1500 - 24 6.0 11.0 15.3 20.8 26.7 32.2 1500 - 48 6.5 11.7 16.3 21.7 27.7 33.0 1500 - 72 5.4 10.7 15.8 21.5 27.2 32.0
Tes- F t ns ns ns ns ns ns C.V. (5) 14.0 6.7 7.2 5.4 4.2 3.7
The rat leaf eo f emergenc abous 6 eleaves/montwa 5- t days/leaf 6 stables - wa 5 n I r d .ho an , contrast, the height and circumference of the pseudostem increased more rapidly during the fourth, fifth and sixth months (Figure 2).
200 T T 40 is * — Number of leaves r °n "ie 35 ^ ° *"' Circumference o * - - Height 1 30 § 140 §> "e 12° ° 10 3 •a 20 * '3 80 (D _&Sn £!B so
Figure 2. Growth of pseudostem increase and number the in of leaves.
Bunch emergence occured in September. Time to shooting was about 234.7 - 296.3 days, as show Tabln ni e XIIe colchicine-treateTh . d plants were slowe shooto t rcontrole Th . s were eth quickest to shoot. The number of leaves at shooting was about 43 in all treatments (Table XIII). The fruits were harvested 50 - 56 days after flowering.
73 Table XII.EFFEC COLCHICINF TO E TREATMEN AVERAGN TO E NUMBEF RO DAYS TO SHOOTING OF 'K. KHAI1
Colchicine Colchicine treatment period (h) concentration (ppm) 24 48 72 Average
0 234.7 234.7 234.7 234* b .7 500 255.7 289.5 273.3 272. 8a 1000 263.8 275.7 276.5 272. Oa 1500 260.3 290.8 296.3 282. 5a
Average Trt. 259.9b 285.3a 282.Oa
C.V. (%) 22.8 * : Numbers followed by the same alphabetical superscript are not significantly different.
Table XIII. EFFECT OF COLCHICINE TREATMENT ON LEAF PRODUCTION AND BUNCH WEIGHT
Colchicine Totalf o . No remaining Bunch. treatment leaves leaf at shooting weight (g)
0 - 0 42.5 14.2 7 525 .0 500 - 24 43.8 13.8 5 716 .0 500 - 48 42.3 13.7 5 608 .3 500 - 72 41 .8 13.5 6 208 .3 1000 - 24 42.5 13.2 6 200 .6 1000 - 48 44.0 14 .0 5 650 .0 1000 - 72 43.2 12.8 5 558 .3 1500 - 24 42.5 13.5 5 800 .0 1500 - 48 44 .3 13.7 5 791 .7 1500 - 72 43.3 13.3 5 425 .0
F - Test ns ns C.V. (%) 8 6.2
e effec f Th colchicino t e treatmen buncn o t h weigh e furthear t r summarize Tabln i d e XIV. Higher concentratio colchicinf no e resulte lowen di r bunch weight numbee . Khaith 'K t f f so bu , o r fruits, number of hands per bunch, number of fruit per 1second hand, were not significantly different as shown in Table XV.
Table XIV. EFFEC COLCHICINF TO E TREATMEN AVERAGN TO E BUNCH WEIGHT
Duratio f treatmenno ) (h t Concentration Average (ppm) •24 48 72 (g)
0 7 525 .0 7 525 .0 7 525.0 7 525. Oa* 500 5 716 .7 6 508 .3 6 208.3 6 144. 4b 1000 6 200 .0 5 650 .0 5 558. 3 5 802.8bc 1500 5 800 .0 5 791 .7 5 425.0 5 672.2°
Average Trt. 5 905.6ns 5 983.3ns 5 730.5ns significant No 22.8) : C.Vs (% n ,. t * : Numbers followed by the same alphabetical superscript are not significantly different.
74 Treatment with higher concentrations of colchicine resulted in lower bunch weights of 'K. Khai'. However e numbeth , numbee th f fruit ro d f handro an s buncr pe s h wer t significantleno y different from the control (Table XV).
Table XV. FRUITING CHARACTERISTIC COLCHICINE-TREATEF SO . KHAID'K 1 No. Colchicine No No. fruits/second Weighf o t treatment fruit/bunch hands /bunch hand second hand(g)
0 - 0 119 .2 6.8 19.5 1 216 .7 500 - 24 122 .2 6.5 19.5 1 008 .3 500 - 48 119 .7 7.0 19.5 1 258 .3 500 - 72 119 .5 6.7 18.7 1 275 .0 1000 - 24 114 .5 6.5 19.8 1 133 .3 1000 - 48 116 .2 7.0 20.3 1 183 .3 1000 - 72 135 .7 7.2 19.7 1 300 .0 1500 - 24 112 .5 7.0 20.3 1 225 .0 1500 - 48 119 .7 6.3 19.7 1 100 .0 1500 - 72 129 .0 7.0 20.3 1 150 .0
Tes- F t ns ns ns ns C.V. (%) 13 .6 7.8 5.6 13
e effec Th f colchicino t e treatmen n fruio t t s sizexaminewa e y measurinb d e fruith g t circumference (Table XVI), length, peel thicknes solubld an s e solids content (Table XVII). Fruit firmnes s alsowa s examined (Table XVIII).
Table XVI. EFFEC COLCHICINF TO E TREATMENT DURATIOD NAN COLCHICINE CONCENTRATION ON AVERAGE CIRCUMFERENCE (cm) OF 'K. KHAI'FRUITS
Colchicine Colchicine treatment duratio) (h n concentran tio (ppm) 24 48 72 Average
0 10.0 10.0 10.0 10.0ab* 500 9.8 10.2 10.3 10. la 1000 10.0 9.3 10.2 9.8^ 1500 9.8 9.7 9.5 9.7b
Average Trt. 9.9na 9.7ns 10.0ns
C.V. (%) 5.0 significant nNo s: t * : Numbers followed by the same alphabetical superscript are not SIGNIFICANTLY different.
High concentrations of colchicine reduced the circumference of the fruit. Fruit length, peel thickness, and soluble solids content were not significantly different from the control (Table XVII).
75 Table XVII. EFFECT OF COLCHICINE TREATMENT ON AVERAGE PEEL THICKNESS, LENGTH AND SOLUBLE SOLIDS CONTENTS OF . KHAI'K 1 FRUIT
Colchicine Peel thickness Fruit length Soluble solids treatment (mm) (cm) (% Brix)
0 - 0 1.8 13.5 28.7 500 - 24 1.5 12.2 28.7 500 - 48 2.0 13.3 28.8 500 - 72 1.7 13.7 28.7 1000 - 24 1.5 12.8 28.8 1000 - 48 2.0 12.7 28.7 1000 - 72 2.0 13.3 28.7 1500 - 24 2.0 12.8 28.7 1500 - 48 1.5 13.2 28.8 1500 - 72 2.0 12.5 28.5
F - Test ns ns ns C.V. (%) 7.8 6.9 1.7
Table XVIII. EFFECT OF COLCHICINE CONCENTRATION AND TREATMENT DURATION ON FRUIT FIRMNESS OF 'K. KHAI1
Colchicine Colchicine treatment duratio) (h n concentration (ppm) 24 48 72 Average
0 0.882 0.882 0.882 0.8823* 500 0.819 0.920 0.899 0.879a 1000 0.792 0.787 0.844 0.808b 1500 0.724 0.844 0.757 0.775b
Average Trt, 0.778b 0.850a 0.833e
C.V. (%) 9.3 ns : Not significant * : Numbers followed by the same alphabetical superscript are not significantly different.
Increased concentration f Colchicino s e reduced flesh firmness. High concentrationf o s colchicine affecte e developmenth d r maturatioo t e frui s th comparea tf e controlo nth o t d . However, such effects may have resulted from damage caused by the treatment. Measurement of stomatal size, and chromosome counts revealed that the ploidy (2n = 22) was not effected by the colchicine treatment. Recently, Orizaline has been shown to be effective in inducing polyploidy (Novak, personal communication). Protocol effectivr sfo e inductio polyploidf no banann yi a should be developed, especially in combination with in vitro tissue/cell culture techniques.
III.3.5. Leaf Spot Disease Symptom
Throughout the 'K. Khai' experiments, no leaf spot disease symptoms were observed under field conditions. However, this disease did infect another variety 'K. Leb Mu Nang' which was growing near the experimental plot. The absence of disease symptoms on the 'K. Khai' plants requires investigation.
76 BIBLIOGRAPHY AAOUINE, M. In vitro propagation of banana. Hort Abstr. 62(3) (1989) : 311. MURASHIGE, . SKOOF d reviseA an G . T d mediu r rapimfo d growt d bioassayhan s with tobacco tissue culture. Physiol. 15 (1962) : 473-479 . BABPRASERTSILAYOIC d an . B , H Banana Genetic Resources Exploratio n Thailandi n .
Kasetsart Univ. Bangkok. . (1983pp 1 13 ) SILAYOI, B, N. SINGBURAUDOM and K. WANICHALKUL Induced mutation of 'Kluai Khai1 through tissue culture. IAEA repor . 5427/R1/RNo t 3 (1991) 4pp. SILAYOI . B "Bananas, " Prachacho . LtdnCo . Bangkok Thain (i .. )(1991pp 0 29 ) SIMMONDS evolutioe Th Bananase th . f N n,o . Longman . Ltd, GreeCo . . d (1962pp nan 0 17 ) SIMMONDS, N. "Bananas" 2nd edition. Tropical Agriculture Series Longman. (1966) 512 pp.
VAKILI, N. G. Colchicine induced polyploidy in Musa. Nature. 194 (1962) : 453-454
Next page(s) left blank 77 CONCLUSIONS AND RECOMMENDATIONS
Breedin f bananago plantaind an s bees sha n hindere problemo t e ddu s associated with sterility and vegetative propagation. Although both banana plantaind san importane sar s subsistenca t d ean cash crops in tropical countries they have not fully benefited from the application of conventional breeding approaches, as have many other tropical crops. Improvement of cultivars by the introduction of desirable agronomic characters through genetic manipulation, has only recently been demonstrate thesn di e crops.
The effectiveness of mutation breeding as a means to improve vegetatively propagated crops has been demonstrated. However e cas th f Musa, eo n i , difficultie supplyinn i s handlind gan e gth large populations thae necessar ar e tapplicatio th r fo y f mutatioo n n breeding techniques, have inhibited the application of mutation breeding methods. Recent developments in biotechnology have provided new tools for the improvement of Musa. In vitro culture techniques enabl e productioeth d irradiationan f largo n e population f somatio s c embryos or plantlets. Using this approach, progress in the development of improved Musa cultivar s expectedi s . However, there remain some obstacle e morth eo t s wide spread an d immediate application of in vitro mutagenesis methods for Musa improvement. For example, defined protocols for mutation induction and screening are needed. Problems with chimerism in regenerated mutagenized material, and ploidy level variants, must be overcome. Additional research is required on the use of cell and protoplast culture methods for mutation induction. Developmen f cultivaro t s with improved disease resistance characteristics will remaia n priority. In mutation breeding programs, processes for screening of potentially useful mutants are as important as the mutation induction process itself. Appropriate screening techniques must be develope majoe r eacth dfo f rho disease organisms being investigated. Initia vitron i l screenins gi preferred in order to minimize the number of plants that will require further field testing. This Coordinated Research Progra initiates mwa studo dt solvd probleme yan eth s associated with the application of in vitro mutation breeding techniques for bananas and plantains. Biotechnology offers new and effective approaches to compliment conventional Musa breeding programs. Suggestion e applicatioth r fo s f biotechnologicano l technique r Musafo s improvement will be presented. There continue urgenn a e b t o neet s develoo dt p improved cultivar f bananaso s plantainsd an , since currently grown cultivars are susceptible to a number of serious diseases and insect pests. Developmen f improveo t d cultivar a difficul s i sd time-consumin an t g process. Howevers i t i , necessary in view of the enormous economic importance of bananas and plantains as a staple food an a currency-earnind g export crop n vitroI . mutation breeding approaches will complement conventional techniques and combination ,i n wit e novehth l techniques currently being developed, will spee e developmenth d f improveo t d varieties. Close cooperatio d collaboratioan n n among research groups involved with banana and plantain improvement is essential for exchange of research informatio r efficienfo d nan t transfe technologf o r developino yt g countries. The participants concluded the following:
. 1 Conventional Breedin Mutatiod gan n Breeding 1.1. Conventional and mutation breeding programs are both important in Musa improvement, and ca complementarye nb . Lac f adequatko e resistanc diseaseo et smose appearth e t b urgen o st t problem. However, numbea ther e ar ef othe o r r breeding objectives that coul e tackledb d using available germplas induced man d mutations. 1.2. Embryo rescue will be an important technique for the effective utilization of Musa genetic resources. Embryo rescue will be used in recovering important hybrid plants from breeding
79 programs. Techniques to further improve the recovery of plants are necessary. In addition, technique r embryfo s o cultur ef increasin o wil e b l g importanc effortn ei recoveo t s r cryo- preserved embryos used for long-term storage of important breeding stocks and Musa gerrnplasm. 1.3. Micropropagation from meristem apicad san l shoot tips continue importann a e b o st t technique for rapid multiplicatio f clonano l materials from conventiona non-conventionad an l l breeding programs. More knowledg mechanismn eo s effectin regulatioe gth f somaclonano l variatios ni urgently needed. Variation mus e minimizeb t d during clonal propagatio appropriatd nan e screening and selection techniques must be developed in order to identify somaclonal variants in micropropagated materials. Alternatively, method enhanco st e somaclonal variatioe b n nca utilize mutation di n breeding programs. 1.4. Anther culture technique e beinar s g develope e purposth r f producinfo do e g homozygous diploids from doubled haploids. This technique wil usefue b l supplyinn i l g haploid materials for mutation induction. Anther culture techniques require further development. In addition, developmen vitron i f o t colchicine treatment e productioth r fo s f doubleo n d haploidsd an , tetraploids from improved diploid materials, is required. advancemene th r 1.5Fo . f mutatioo t n breeding technology, researc somatin ho c embryogenesis i s of continued importance. Simplification of the regeneration procedures, in order to avoid or eliminate chimerism in mutagenized regenerated callus or plants, is required. Advances in somatic embryogenic techniques, and in techniques for plant regeneration from single cells/protoplasts alse ar ,o needed.
1.6. Emphasis shoul e developmene placeb th d n o d f earlo t vitron i y screening techniquer fo s protoplasts, single cells, somatic embryos and regenerated plantlets. Final evaluation of clones resulting from in vitro selection should be performed in countries where the proper condition bananr sfo plantaid aan n cultivation exist. 1.7. Screening techniques, especiall thes yappliea e yar diseaso dt e resistance, developee neeb o dt d for in vitro (mass screening), glasshouse/growth cabinet (intermediate mass screening) and field conditions (final evaluation). Clones with resistanc racFusariuf o et o e4 w mno wile ar t available. Althoug t appropriathno r commerciaefo l cultivation, these usee cloneb s da n ca s resistant control genotypes for comparison. 1.8. Diploid material can be used to study host/pathogen interactions, their genetic control, and als r developmenofo f resistanco t e screening techniques. Biochemica r moleculao l r markers that correlate with resistance genes identifie diploin i d d materials, coul e usefub d o helt l p identify resistanc variouo et s disease mutagenizen si d triploids. 1.9. Additional knowledge of the response of materials to the effects of various mutagens (physical and chemical s neededi ) e responsTh . f variouo e s cultivars, unde n vitro i n vivoi rr o conditions, must be examined. The identification of easily discernible characteristics, that
reflect the effectiveness of the mutagen treatment, and that are correlated with induced mutation frequency e neededar , orden i 1 formulato t r e appropriate mutagen dose othed an s r treatment condition. l.lO.It is important to develop a greater understanding of the ontogenetic reactions of various types of materials (plantlets, explant tissues, callus, cell suspensions, etc.) to the mutation induction treatments e abln ordei b ,o effectivelt eo t r y manipulat e mutatioth e n induction process. Techniques to reduce the formation of non-chimeric plants will enhance the production of stabl usefud ean l mutants. Greater understandin origie th f f regenerategno o d shoots (axillary . adventitiousvs alss )i o required. 1.11.It is highly desirable to develop restriction fragment length polymorphism (RFLP), DNA amplification fingerprinting (DAF), random amplified polymorphi (RAPDA cDN othed an ) r Polymerase Chain Reaction-based molecular marker systems. These markers can be used with M. acuminata (AA) subspecie . balbisianaM d san (BB orde)n i identifo rt y molecular markers
80 correlated with morphologica agronomid an l c traits diseasd an , e resistance markeA DN r. system n als e usefuca sob r assessinfo l g genetic variation within pathogen species sucs a h Fusarium oxysporum Mycosphaerellae th d an complex. L12.lt also seems timely to examine cytoplasmic DNA variation using restriction endonuciease analysis of M. acuminata subspecies, M. balbisiana and the triploid cultivars in order to determin e contributioth e f cytoplasmio n c factor o agronomit s e c th characters o t e Du . probable influence of Mycosphaerella toxin(s) on chloroplast function during disease expression, RFLP mapping of the chloroplast genome may disclose chloroplast DNA-encoded genes effecting susceptibilit thio yt s disease. Protoplast fusion produco t , e cybrids carrying resistanc toxinso e t additiona n a s i , l are futurr afo e research. above 1.13.Ith f eo l areasnal muse t loosw , no t e nee e sigh th produc o dt f o t e cultivars with suitable agronomic characteristic thad adaptee san ar t regionao dt l environments.
2. IMPROVEMENT OF RESISTANCE TO DISEASES AND PESTS OF BANANA AND PLANTAIN 2.1. One of the main objectives of mutation breeding is to obtain resistance to diseases and pests. This can be achieved by following different strategies: (a) Changes in the degree of expression of resistance or tolerance of the host plant to the pathogen. (b) Changes in the phenology or physiology of the host plant, such as flowering habit or spee f growto d h (shortenin f productiogo no diminist cycle)s a o hs , damag o individuaet l plants. 2.2. To successfully implement these strategies a greater understanding of the pathogens, and of the host/pathogen interactions, is necessary. A better understanding of the stages and mechanisms of pathogen development in the host will facilitate the development and identification of resistant plants. A better understanding of the nature of susceptible vs. resistant host reactions is also necessary.
2.3. Research on direct interference of the host plant with the pathogen is important for development of resistant clones which involves screening and evaluation procedures : vitron I ) screenin(a g (b) Glasshouse screening (c) Field testing r economi2.4Fo . c reasons, preliminary screening shoul conductee db earls da possibls ya e during improvemene th t process. Field testin finae sees gth i ls na stag evaluatiof eo procesa n ni f so screenin selectiod gan n that commence vitro.n di 2.5. Of the diseases and pests identified as being of importance in the genus Musa, the following were given highest priority: (a) Black Sigatoka (Mycosphaerella fijiensis var. difformis) and Yellow Sigatoka (M. musicola) ) Fusariu(b m wilt (Fusarium oxysporum f.sp. cubense) ) (c Moko (Pseudomonas solaricearum) (d) Banana Bunch Virup yTo s (BBTV) ) (e Nematodes 2.6 vitron I . screenin f culturgo lende us e s e filtrateitselth o t f s (toxins f fungao )bacteria d an l l pathogens. Their functio importancd an n resistance/susceptibln ei e reactions requires further investigation. The use of peroxidases, and their increased production in cultivars resistant to
81 various fungi, also requires further study. These types of in vitro screening techniques will be of most use where resistance is based on an enzymatic or physiological reaction that operates cellulae ath t r level casen I . s where resistanc base s emorphologicai a n do r structurao l l factor this vitron typi f eo screening techniqu havy ema limiteea d application thesn I . e latter cases, a system providin gdireca t contact betwee hose nth fungae t th plan d l an tpathoge n (e.g- co . e requiredcultureb y ma ) . Nevertheless n vitroi , screening shoul e investigateb d t i s a d represent meana s o obtaist earln na y evaluation s economicai d an , r masfo l s screenine gth products fro vitromn i mutation breeding programs. 2.7. Screening of plantlets in a glasshouse requires the availability of standardized inoculum, reliable inoculation methods and reproducible evaluation procedures. Progress has been made to fulfill these requirements for Black Sigatoka and Yellow Sigatoka, and to partially fulfill these requirements for Fusarium wilt and Moko, although procedures should be further refined.
2.8. Preliminary experiments indicate that both environmental conditions virulence th e d th an ,f eo pathogen strain influencn ca , plante th e s respons pathogee th o et n pressure thin I .s regard technique e urgentlar s y needed that enabl e rapid accuratth e an d e identificatione th d an , characterization, of pathogen isolates. This may involve the use of Vegetative Compatibility Groups (VCGs volatiled e an cas) th f Fusariu eo n i s m wilt r moro , e sophisticated molecular genetic maps in the case of Black Sigatoka and Fusarium wilt.
2.9. Even when suitable laboratory and greenhouse facilities exist, testing for susceptibility using different pathogen strains must be conducted outside of banana and plantain producing countries. This restriction is necessary in order to avoid the risk of introducing new pathogens into production areas. 2.10.The ultimate evaluation of promising clones by field evaluation must be conducted in banana and plantain producing countries, preferably under several environmental conditions. Due to e resourceth s required (land, labor, etc.), field evaluation shoul e limiteb d o preselectet d d clones. Expertis d standardizean e d methodologie r resistancfo s e evaluation shoule b d established in banana and plantain producing countries to guarantee reliable and reproducible rating r pathogefo s n response assessmend an , f diseaso t e resistance. Standardizatio f thesno e methods coul achievebe d in collaboratiod n witInternationathe h l Networthe for k Improvemen f Banano t Plantaid aan n (INIBAP).
2.12.In some case physiologe sth phenologd yan hose th importane tf b yplano y ma t conferrinn i t g resistance. For instance, it has been implied that cold tolerance in Cavendish clones is correlated with resistanc o Fusariut e m e subtropics th wilt n i ,y screenin B . r colfo g d tolerance, greater resistanc e pathogeth o y resultt e shorteA ma n . r production cycle, especiall intervae yth l from shootin harvesto gt , would reduce host damage resulting from leaf spot diseases time f sheddinTh eo . f hermaphroditgo e flower bananan i s bees ha s n suggested as being related to resistance to Moko disease. 2.13.The observation of in v//ra-regenerated plants and plants arising from conventional breeding program e abovth f seo witcharactersy h an e importan b y ma ,effort n i t develoo t s p resistant varieties. Researchers and breeders need to be aware of these characters when evaluating progeny from their breeding programs.
2.14.In the cases of Black Sigatoka and Fusarium wilt, susceptible and resistant clones are available as control differential s bein i theird e an sgus promote INIBAy db Internationan a n Pi l Musa Testing Program. They may be used as control materials for the screening of mutagenized populations, although they may be valid as control plants only for the mechanism of resistance unique to them. Screening methods should be capable of detecting new resistance mechanisms. 2.15.No natural resistance to BBTV has been observed in the genus Musa, therefore appropriate screening and selection techniques still need to be developed for this disease. Control of BBTV currently depends on the development of suitable indexing procedures and the
82 movement of only certified clean planting material. In addition to mutation breeding for resistance f plano e application t a us transformatio e e ,b th thery r nfo ema n techniques using virus coat protein gene(s) and/or inoculation of material using attenuated strains of virus. It is highly desirable that these research programs continue.
. 3 INTERNATIONAL CO-OPERATIO CO-ORDINATIOD NAN N dimensione 3.1Th . tase th k f o aheas d require considerable international cooperatione th o t e du , breadth of the knowledge of the materials and objectives involved. Cooperation with the International Network for the Improvement of Bananas and Plantains (INIBAP) is desirable as is collaboration with the international agricultural research centers such as the International Institut f Tropicao e l Agriculture (IITA r Centro ) o Agronomico Tropica e Investigaciod l ny Ensenanza (CATIE).
3.2. Various regional and national programmes are supported through FAO, IAEA, UNDP and various other donor agencies. Strong national programmes form a solid foundation for international coordination. Strengthening of national programmes should therefore be a high priorit internationan yi l programmes. 3.3. The Agriculture Laboratory of IAEA's Seibersdorf Laboratories near Vienna, has emphasis on the development and application of technology related to the use of mutagenesis for plant improvement, and on transferring such technology to breeders in member states. Particular attention is currently paid to bananas and plantains where, due to the difficulties associated with conventional breeding approaches, mutation breeding may play a crucial role in improvement efforts. Banana d plantaian s n breeders shoul e encourageb d o utilizt d e th e various services offered by the Laboratory, particularly its training programme and for assistanc mutagen ei n treatments.
Next page(s) lef3 t8 blank PARTICIPANTS IN THE CO-ORDINATED RESEARCH PROGRAMME
Gonzales, E.S. Universidad Nacional, Escuel Cienciae ad s Agrarias, Apartado Posta, 86 l Heredia, Costa Rica
Jarret, R.L. USDA-ARS, 1109 Experiment Street, Griffin, Georgia, 30223-1797, United States of America
Krikorian, A.D. State University of New York at Stony Brook, Departmen f Biochemistryo t , Stony Brook, New York 11794-5215, United States of America
Mendes, B.M.J. Universidad de Sao Paulo, Campus de Piracicaba, Avenida Centenario 303, Caixa Postal 96, CEP 13.400 Piracicaba, SP, Brazil
Perea-Dallos, M. Biology Department, Universidad Nacional de Colômbia, Bogotá, Colômbia
Siddiqui, S.H. Plant Genetics Division, Atomic Energy Agricultural Research Centre, Tandojam Sind, Pakistan
Silayoi. B , Department of Horticulture, Kasetsart University, Bangkhen, Bangkok, Thailand
Smith, M. Maroochy Horticultural Research Station, Queensland Departmen Primarf o t y Industries, P.O.Box 5083, S.C.M.C., Nambour 4560Q , , Australia
Valerin Aguilar, A.T. Universidad Nacional, Escuel Cienciae ad s Agrarias, Apartado Postal 86, Heredia, Costa Rica
Yang, P. Beijing Institute of Nuclear Engineering, P.O.Box 840, Beijing, 100840, China
Scientific Secretary
Amano. E , Plant Breeding and Genetics Section, Joint FAO/IAEA Division, IAEA, Wagramerstrasse 5, P.O. Box 100, A-1400, Vienna, Austria
85 Novak, F. Plant Breeding Unit, Joint FAO/IAEA Programme, IAEA Laboratories, Seibersdorf, Austria
Observers at the second Research Co-ordination Meeeting, San Jose, Costa Rica
Goto, J. National Atomic Energy Commission, Costa Rica
RepresentativeO DercksenFA , P.M, Cost. a Rica
Guendel, F. Universidad Nacional, Costa Rica
Rivera Goto, G. Escuela de Ciencias Agrarias, Universidad Nacional, Costa Rica
Salazar Androvetto, R.A. Universidad Nacional, Costa Rica
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