Role of Biotechnology in Improvement of Banana


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Production of transgenic bananas resistant to Xanthomonas wilt disease,Genetic Transformation of Bananas,Development of nematode resistant plantain,Genetic Transformation of Plantain

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  • Dear Tripathi,
    I am Francis Mwatuni working with KEPHIS in Kenya.
    Quite informative slides on banana transformation.
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  • Dear Tripathi,

    I am Anuj Kr. Pandey,student of M.Sc. Biotechnology, is doing six month training on 'In-vitro micropropagation & agrobacterium mediated genetic transformatio in banana cultivars. By your slide I understand many things.

    Thanks & Regards

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Role of Biotechnology in Improvement of Banana

  1. 1. Role of Biotechnology in Improvement of Banana Leena TripathiInternational Institute of Tropical Agriculture, Uganda Contract Review Seminar 16th April 2009
  2. 2. Outline• Introduction – Banana and Plantain – Biotechnology - transgenic• Achievements – Transformation system – BXW resistance – Nematodes resistance – BSV resistance – Capacity Building• Future Plans
  3. 3. Banana and Plantain• World’s 4th most important food crop.• World Musa production is 104 million tonnes.• A third of the bananas produced globally are grown in Sub-Saharan Africa.• East Africa is the largest banana producing and consuming region in Africa.• Uganda is the world’s second largest producer.• Production is threatened by various constraints – declining soil fertility – pests and diseases
  4. 4. Why Biotechnology?
  5. 5. Global Area of Biotech Crops, 1996 to 2006: By Crop (Million Hectares)7060 Soybean Maize50 Cotton Canola40302010 0 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006Source: Clive James, 2006
  6. 6. Benefits and opportunities• Conventional breeding of banana is difficult and time consuming.• Gene technologies for improvement are becoming available.• Transformation technologies are available.• Biotechnology and Biosafety Policy are in place in many countries.
  7. 7. Transgenic Research in Banana• Transformation systems• Pest resistance - Nematodes• Disease resistance - Black sigatoka, bacterial wilt, viruses• Edible vaccines - Hepatitis B, cholera• Biofortification - pro-vitamin A, vitamin E, iron and zinc• Delayed Ripening/Prolonged shelf life• Yield Enhancement and plant architecture - Early maturing - Drought tolerance Tripathi et al. 2007 Tripathi et al. 2008
  8. 8. Production of transgenic bananasresistant to Xanthomonas wilt disease (supported by Gatsby & AATF)
  9. 9. Banana Xanthomonas wilt• BXW caused by Xanthomonas campestris pv. musacearum endangers thelivelihood of millions of farmers in East Africa.• First reported in Uganda in 2001.• The disease has also been reported in DR Congo, Rwanda, Tanzania, Kenyaand Burundi.Source: Tushemereirwe et al. 2006 Source: Bouwmeester et al. 2008
  10. 10. Xanthomonas Wilt• The disease affects almost all commonly grown banana cultivars.• The impacts of BXW are both extreme and rapid. Biruma et al. 2007 Tripathi et al. 2009
  11. 11. Rapid Technique for Screening Banana Cultivars for Resistance to Xanthomonas Wilt• An in vitro screening method was developed using small tissue culture grown plantlets.• Significant differences was observed in susceptibility among the various banana cultivars.• No significant difference in pathogenicity was observed between the pathogen isolates. Tripathi et al. 2008 Odipio 2008 M.Sc. Thesis
  12. 12. Relative Susceptibility of Banana Cultivars• Ten cultivars were tested.• There were significant differences insusceptibility among the variousbanana cultivars.• Beer banana cultivar ‘Pisang Awak’was found to be highly susceptible.• Dessert banana cultivars ‘DwarfCavendish’ and ‘Giant Cavendish’ werealso found to be highly susceptible.• Diploid parent ‘Musa balbisiana’ (BB)was found to be resistant.• EAHB cultivar ‘Nakitembe’ was found Tripathi and Tripathi 2008to be moderately resistant.
  13. 13. Why Transgenic Banana?• East Africa is the largest banana producing and consuming region in Africa.• BXW is causing an annual loss of over US$ 200 million in Uganda.• BXW attacks all banana varieties resulting in absolute crop loss.• Farmers prefer resistant varieties.• No source of germplasm exhibiting resistance against Xcm has been identified.• Transgenic technologies for banana may provide a timely alternative solution to control the BXW R4D Review 2008 pandemic.
  14. 14. Genetic Transformation of BananasTransformation efficiency is Fast, cultivar independenthigh but time consuming & but transformation efficiencycultivar specific is low and chances of chimeras
  15. 15. Genetic Transformation using shoot tips• Binary vector pCAMBIA2301 containing the gusA reporter gene and nptII asselectable marker.• Transformation efficiency using shoot tip was low (1-2%). Tripathi et al. 2005 Agrobacterium-mediated transformation
  16. 16. Regeneration of Banana• Regeneration system was established using sections of corm containing intercalary meristematic tissues .• Six different cultivars of banana were regenerated. – Mpologoma – Nakitembe – Mbwazirume – Pisang awak – Sukali ndiizi – FHIA-17• Regeneration efficiency was 93-97%.• 12-13 shoots were produced from whole section and 16-19 shoots in Tripathi & Tripathi 2008 total from quarter pieces of each section.
  17. 17. Genetic Transformation of East African Highland Bananas• A transformation system using intercalary meristematic tissues was developed. – Transformation efficiency - 10-12% – Cultivar independent – Rapid• Chimeric ?• First report of EAHB transformation Tripathi et al. 2008
  18. 18. Potential strategies to develop plants resistant to Bacterial Wilt Gene Transgenic Plants• Several transgenic Magainin Tobacco analogs technologies are available to Cecropins Tobacco, potato, apple develop disease resistant Attacins Pear, apple plants through Lysozymes Tobacco, potato, apple, – Using R genes rice, tomato Pepper Bs2 Tomato – defense mechanism or Rice Xa1, Rice – antimicrobial proteins Xa21 Tomato Pto Tomato Pepper pflp Tobacco, tomato, & hrap broccoli, orchid, rice, Tripathi 2005 Arabidopsis
  19. 19. Defense genes inducing hypersensitive response• HR is an induced resistance mechanism, characterized by rapid, localized cell death upon pathogen attack.• Several defense genes have been shown to enhance HR induced by the release of the proteinaceous elicitor.• Elicitor-induced resistance is not specific against particular pathogens.
  20. 20. Bacterial Pathogen: Type III protein secretion system HrpJ: transcription regulator HrpC: pili structure protein HrpZ: harpin Erwinia , Pseudomonas Ralstonia Xanthomonas
  21. 21. Pepper pflp & hrap genes• PFLP (plant ferredoxin-like protein) and HRAP (HR assisting protein) are cloned from sweet pepper , Capsicum annuum.• Intensify the HR caused by harpin (a proteineous elicitor secreted from bacterial pathogen).• These genes are effective against many bacterial pathogens, such as, Erwinia, Pseudomona, Ralstonia and Xanthomonas spp.
  22. 22. What happen in the PFLP or HRAP transgenic plant ?
  23. 23. Mode of Action of pflp geneiron-depletion (antibiotic action) + HR enhancement HR AOS
  24. 24. Enhanced resistance against virulent pathogens in the transgenic cropsCrops Transgene Disease resistance PathogenTobacco hrap Wild fire Pseudomonas pflp Soft rot Erwinia Gray mold BotrytisArabidopsis hrap Soft rot Erwinia pflpBroccoli pflp Soft rot ErwiniaOrchids, Calla pflp Soft rot ErwiniaRice pflp Leaf Blight XanthomonasTomato pflp Soft rot ErwiniaPotato hrap Bacterial wilt RalstoniaSource: TY Feng
  25. 25. Access of technology pflp and hrap gene• Established collaboration with Academia Sinica and received the construct in 2005.• Approach AATF for negotiating licensing.• AATF signed licence with Academia Sinica and provided sub-licensing to IITA in 2006.• Transformation is in progress at IITA in collaboration with NARO.
  26. 26.
  27. 27. Exit or Transfer strategy• The project also involves the capacity building of NARS for genetic transformation, biosafety regulations, risk assessment and management.
  28. 28. Genetic transformation using pflp gene • Five cultivars has been transformed (Kayinja, Sukali nidizi, Mpologoma, Naketimbe, Naykinika). • Transformation using meristems and also suspension cultures. • More than 300 lines has been developed. • Molecular characterization and efficacy trail is in progress.PCR analysis amplifying a 600bp fragment of pflp gene Tripathi et al. 2009
  29. 29. Transgenic plants challenged with Xanthomonas campestris pv. musacearum• 35 lines transformed with pCAMBIA1304-35S SAP1 were screened using in vitro plantlets.• 17 promising lines were further screened using potted plants In vitro screening of transgenic plants for resistance against Xanthomonas wilt; A- – 2/17- no symptoms inoculated control plant, B-F- inoculated – 8/17- delayed symptoms transgenic plants• 60 lines transformed with pBI- SAP1 were screened using in vitro plantlets• Some promising lines showing no symptom or delayed symptoms have been obtained. Namukwaya et al. 2008 Screening of potted transgenic plants
  30. 30. Genetic transformation using hrap gene• Four cultivars has been transformed (Sukali nidizi, Mpologoma, Naykinika, Kayinja).• More than 200 lines has been developed.• Molecular characterization of more than 60 lines has been done. PCR analysis amplifying a 1kb fragment of hrap geneSouthern Blot Analysis
  31. 31. Evaluation of transgenic plants• 20 lines transformed with pBI-HRAP were evaluated. – 16/20 – no symptoms – 4/20 - delayed symptoms• No bacteria was found at the point of inoculation after 6 weeks.
  32. 32. Research Design• Access of technology• Development of bananas with Xanthomonas wilt resistance• Molecular characterization of transgenic plants• Evaluation of transgenic banana plants in Laboratory conditions• Confined field trials of transgenic plants against BXW• Biosafety and impact analysis studies• Widescale deployment of transformed banana
  33. 33. Additive defence for BXW resistance• Co-transformation• Construct with hrap-pflp stacked NOSP NPT II 35P hrap together NOSPNPT II 35P pflp PCR analysis of the construct pBI-HRAP-PFLP, amplifying 600bp of pflp gene and 1Kb of hrap gene.
  34. 34. Allergenicity assessment of the protein encoded by pflp and hrap gene• Bioinformatics approach to identify any potential protein sequence matches with any allergenic proteins• Results of the FASTA3 search of the PFLP and HRAP protein against Allergen Online version 7.0 did not identify any significant alignment with an allergen.• There were no matches of greater than 35% identity over 80 amino acids.
  35. 35. Diagnostics for BXW Semi-selective media• Semi-selective medium for isolation of Xcm from infected plants• YTS-CC Medium – Yeast Extract (0.5%) Non-selective YPGA – Tryptone (0.5%) – Sucrose (1%) – Cycloheximide (150 mg/l) – Cephalexin or Cefazolin (50 mg/l) Selective YTS-CC Tripathi et al. 2007Tripathi et al. 2007
  36. 36. Molecular Diagnostics• PCR detection of Xanthomonas campestris pv. musacearum in banana.• PCR was used to monitor the movement of Xcm along banana pseudostem of a mother plant and its associated suckers. Adikini et al. 2008 Adikini 2009, Master Thesis
  37. 37. Characterization of diversity of Xcm• Genetic homogeneity among Ugandan isolates of Xanthomonas campestris pv. musacearum revealed by RAPD analysis.• No significant difference in pathogenicity. Odipio 2008, M.Sc. Thesis Odipio et al. 2009
  38. 38. Effect of Phytotoxic Factors and Potassium Nutrition on BXW• Phytotoxic factors for banana were shown to be produced by Xcm in culture filtrate. Culture filtrate Culture filtrate Control YPGB Control water a b c d• Increased potassium availability for banana reduced disease incidence. BXW symptoms on FHIA 17 grown on 0.1K (a), 0.5K (b), 1K (c) and 2K (d) and on Kayinja grown on 0.1K (f), 0.5K (g), 1K (h) a and 2K (i). Controls (e) and (j) were b c d e inoculated with sterile distilled water. Atim et al. 2008 f g h i j
  39. 39. Development of nematode resistant plantain (supported by DFID/BBSRC)
  40. 40. Nematodes• Nematodes pose severe production constraints.• Limited sources of nematode resistance and tolerance are present in the Musa gene pool.• Some resistance has been identified against Radopholus similis, but this needs to be combined with consumer-acceptable traits.• Several species of nematodes are often present together.• Biotechnology offers sustainable solutions to the problem of controlling plant parasitic nematodes.
  41. 41. Additive Resistance against nematodes• Several species occur in the same soils – Radopholus similis, Pratylenchus spp, Helicotylenchus spp, Meloidogyne spp, Rotylenchulus reniformis – Combined losses 57% yield loss• Risk of single transgenic deployments – Variation in nematode resistance – Risk of virulence• Gene stacking the best way forward – Tested with potato – Provide upto 99% resistance
  42. 42. Additive Strategies• Proteinase Inhibitors – Cysteine proteinase inhibitor (cystatin) – Potato tuber serine/aspartic proteinase inhibitor (PDI) Acetylcholine Nicotinic acetylcholine receptor released into synapse• Peptide repellent – Developed to target nicotinic acetylcholine receptors and disrupt chemoreception Bound peptide prevents acetylcholine function• RNAi to target nematode genes – Essential housekeeping genes – Genes involved in parasitism
  43. 43. Nematode Resistance Plantain• Develop nematode resistant plantains – University of Leeds• Constructs A) Ubiquitin: zeacystatin B) Double 35S : Repellent C) Ubiquitin : potato serine/aspartic proteinase inhibitor (PDI) D) Ubiquitin : zeacystatin + Double 35S : PDI E) Ubiquitin : zeacystatin + Double 35S : Repellent F) Ubiquitin : zeacystatin + Double 35S : Repellent + Double 35S : PDI R4D review 2009
  44. 44. Genetic Transformation of Plantain• Regeneration and transformation system – Cultivar Gonja – Multiple buds • Direct organogenesis • Somatic Embryogenesis – Construct pCAMBIA 2301• Transformation for nematode resistance is in progress• Transformed Gonja with 4 different constructs and explants are on regeneration medium.
  45. 45. Genetic Transformation of Plantain for BSV Resistance• Develop resistance to Banana streak virus – In collaboration with JIC – Gatsby Charitable Foundation  The BSV sequence of approx. 600bp of the viral reverse transcriptase- RNase H domain
  46. 46. Horizontal geneflow from transgenic banana to micro-organisms PCR analysis using primers specific for hph or gusA gene; A- Amplification of hph gene in transgenic plants; B- Amplification of hph gene in microbes from rhizoshere; C- Amplification of gusAFungal inoculated plant; B: Transgenic gene in transgenic plants; D- Amplification of gusAplants in pots with inoculated soil; C: gene in microbes from rhizoshere.Bacterial colonies on selectivemedium; D: Re-isolated fungi. Kabuye et al. 2008 Kabuye 2008, M.Sc. Thesis
  47. 47. Capacity Building• Provide national partners with access to use of technologies and training of staffs• Trained staffs and students (11)• Trained more than 20 NARS in genetic transformation and tissue culture• Trained more than 70 NARS in Biosafety and GMO detection – UNIDO – FAO (Kenya, Tanzania, Uganda)
  48. 48. Future ActivitiesBased on technologies developed and application of technologies• Confined field trail of BXW resistant bananas• Development and evaluation of nematode resistance plantains• Improved diploid lines, which can expand the breeding scope• Evaluate transgenic bananas having hrap gene for fungal disease (Fusarium wilt and Black Sigatoka) resistanceGeneration of new technologies• Gene pyramiding for multiple traits – Bacterial + fungal resistance – Bacterial Disease + nematode resistance• Development of high yielding varieties using fd3 gene• Yam transformation for nematode resistance
  49. 49. Acknowledgements• Research Team • Staffs • Students• Partners/Collaborators • Academia Sinica, Taiwan • University of Leeds • NARO • AATF • IITA scientists• Funding support • Gatsby Charitable Foundation • BecA/CIDA • DFID/BBSRC • AATF • IITA