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Genetically Modified Bananas Resistant to Xanthomonas Wilt

L.  Tripathit,  J. N. Tripathi‘,  A.  Muwonge2, K.  Mburu1,A. ...
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Genetically modified bananas resistant to Xanthomonas Wilt

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Presented by Leena Tripathi (IITA) at RTB Annual Review and Planning Meeting (Entebbe, Uganda, 29 Sep-3 Oct 2014)

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Genetically modified bananas resistant to Xanthomonas Wilt

  1. 1. Genetically Modified Bananas Resistant to Xanthomonas Wilt L. Tripathit, J. N. Tripathi‘, A. Muwonge2, K. Mburu1,A. Kiggundu3 and W. K. Tl. ishemereinMe3 ‘lntemational Institute of Tropical Agriculture, Clo BecAIILRl Hub, Nairobi, Kenya ll[SEAI1(M vnommm on Roots, Tubers and Bananas ear CGIAR flnternatiorial Institute of 1l'opicaIAgrii: ulture, Kampala, Uganda 3NationaIAgricuIture Research Laboratories, Kawanda, Uganda Introduction The banana Xanthomonas wilt (BXW). caused by the bacterium Xanthomonas campestlis pv_ musacearum, is the most devastating disease ofbanana in east and central Africa, where banana is a major staple crop produced mostly by smallholder subsistence farmers. The disease has caused estimated economic losses of about $28 billion over the last decade. In the absence of natural host plant resistance among cultivated banana cultivars, transgenic plants were developed expressing the Hypersensitive Response Assisting Protein (Hrap) or Plant Ferredoxin Like Protein (Pflp) gene originated from sweet pepper. Fig 1: Symptoms olxanthornonaswilt disease. K Banana plantation damaged bywill, B-D: Yellow ooze from cutpseudostem, fruits and rachis. E: wilting and shriveling ofmale bud F: Premature ripening offruits. Generation of Transgenic Plants Hundreds of transgenic lines of five banana cultivars (Sukali Ndiizi, Nakinyika, Gonja manjaya, Gros Michel and Cavendish Williams) were generated expressing either Pflp or Hrap or both genes. The transgenic lines were analyzed for presence and integration of gene using PCR and Southern blot analysis respectively. The gene expression of promising lines were further tested with qRT»PCR and Northern blot analysis. "s—y—v—r-4:‘ — _ . .. ‘. .r 1:‘! ‘ nnrtxlz _‘_'-. .- . : l-sur my i. ‘:. —(— J ' ' u " — —- ' . _ . V . u - —4-»-«--- : .{-am n : ;.—q. . . . _ -- no _. .. I - i » u _ r . V ‘ . __t 7 .4”. .. »; .§. : ~. .. ,.. _.. ... . ; _ . 4 r . . . . A Zola- C nvurannrsnwnrommmc u 9 nnnn Y6 rsrync Fig. 3A: Schematic representation of constructs. B: PCR analysis of transgenic lines using gene specific primers, C’ Southem blot analysis ofgenorric DNA D: Gene expression analysis. sup- Theme 2 Evaluation of Transgenic Lines The transgenic banana plants have exhibited strong resistance to BXW in the screen house tests. The best 65 resistant lines were evaluated in a confined field trial in Uganda. All the non-transgenic control plants developed symptoms and finally wilted completely; whereas majority of transgenic lines had significantly higher resistance in comparison to control non—transgenic plants. Eleven of these transgenic lines (7 Hrap lines and 4 Pflp lines) were found to be highly resistant showing 100% disease resistance with both mother and ratoon crops. These lines are under evaluation for durability of disease resistance and agronomic performance in the second trial in Uganda. Aside from complete resistance to BXW. the transgenic lines also showed yield characteristics comparable to non- transgenic plants, indicating there are no observable unintended impacts. I" . i"¢“ii*: -i. %rn'fi c‘ 7 . . -1 “’ %" , , ». ~ - Fig. 4: Evaluation oftransgenic lines for resisbnoe against Xcrn in confined field trial A: Asymptomatic transgenic plant showing no symptom after artificial inoculation, 8. Control non~transgenic plant show‘ng wilting of leaves post inoculation, C-E: Fruit pinch and pseudostem of transgenic plant showing no symptom F~G: Fruit bunch of control non-transgenic plant showingdisease symptoms. . ... ... ... i . ... ... .. . ... ... .u . ... .., .. . . ». ... ... .. ~!. ':' , 3;; ""‘"' """" 3 . ,- AI/ jsf~*(. e': ;"}*‘; »i. sfi. .0‘ ~"t~‘f, v~" a ' , «{. .-“_. .-'g, - ’ 9, wweerie. » L’. E/ ea» L’. “""“"‘ ‘“'“""' S¢‘DNOI. l no-ai-mu »-. ... . nu. .. §r. . Bu umabin mi“. wi " ' 2 . - 3.” W i ft . . y S If g , i ‘E in ! 2 i; 3 . E '2 ' . s . ‘ f 5- . .~ 3 ‘A: 5 3 5- “‘ ‘may c "(-9) .5‘. -‘. D .3 .5 3 5 if A .5 Q‘ s ’, ~;). ~’. «“; ~’~. , u; ‘z= r»r. »r»r»»2/. * Fig. 5: Yield pertormanm ol the best eleven BXW resistant transgenic Iina ‘n comparison to control non-transgenic plans. A: Mean bunch weight of each plant, B: Mean number of hands per bunch, 0: Mean number olfruits per bunch, D: Mean weight ofeach fruit. Conclusion This study provides the first field based evidence for transgenic control of a bacterial disease in banana and progress towards development and release of transgenic bananas resistant to BXW. Such resistant varieties would boost the available arsenal to fight this disease epidemic and save livelihoods in Africa. Acknowledgements We wish to express our appreciation to Dr Feng. Academia Sinica. Taiwan for providing gene constructs and AATF for negotiating license of genes. We also thank The Gatsby Charitable Foundation and USAID lor financial support References Tripall‘liL . NMNQIM , Node 5 . A.rituaV‘ Tisherraeiwe WK ‘ Balm/ opadhyay. R. 12009) PlantDisease 931440-451 Tnpslhi L , Muslra H . Trlpettn J N . TusherriaeIwe. W.K l20l0j- Mol Plant Palhol.11:T21-731 Namuzwaya E ‘ Tripdhi L. Tlipethi J N , Atiriamve G Jilucasa 5 E , Ti5herrBenveWK l20T2)TrarlsgerticRe's.12:855-865-. T1ip. alriL , TIimthI. l.N . KiggLl1duA. Kon. 5 . Sholiosui F. . Tushemaeiv-‘eWK £2014} Nat. BSOEG: 32 Bfi-870 www. iita. org

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