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

1. Role of Biotechnology in
Improvement of Banana
Leena Tripathi
International Institute of Tropical Agriculture, Uganda
Contract Review Seminar
16th April 2009
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2. Outline
• Introduction
– Banana and Plantain
– Biotechnology - transgenic
• Achievements
– Transformation system
– BXW resistance
– Nematodes resistance
– BSV resistance
– Capacity Building
• Future Plans
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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
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4. Why Biotechnology?
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5. Global Area of Biotech Crops, 1996 to 2006:
By Crop (Million Hectares)
70
60 Soybean
Maize
50 Cotton
Canola
40
30
20
10
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
Source: Clive James, 2006
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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.
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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
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8. Production of transgenic bananas
resistant to Xanthomonas wilt disease
(supported by Gatsby & AATF)
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9. Banana Xanthomonas wilt
• BXW caused by Xanthomonas campestris pv. musacearum endangers the
livelihood of millions of farmers in East Africa.
• First reported in Uganda in 2001.
• The disease has also been reported in DR Congo, Rwanda, Tanzania, Kenya
and Burundi.
Source: Tushemereirwe et al. 2006 Source: Bouwmeester et al. 2008
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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
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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
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12. Relative Susceptibility of Banana Cultivars
• Ten cultivars were tested.
• There were significant differences in
susceptibility among the various
banana cultivars.
• Beer banana cultivar ‘Pisang Awak’
was found to be highly susceptible.
• Dessert banana cultivars ‘Dwarf
Cavendish’ and ‘Giant Cavendish’ were
also found to be highly susceptible.
• Diploid parent ‘Musa balbisiana’ (BB)
was found to be resistant.
• EAHB cultivar ‘Nakitembe’ was found
Tripathi and Tripathi 2008
to be moderately resistant.
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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.
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14. Genetic Transformation of Bananas
Transformation efficiency is Fast, cultivar independent
high but time consuming & but transformation efficiency
cultivar specific is low and chances of
chimeras
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15. Genetic Transformation using shoot tips
• Binary vector pCAMBIA2301 containing the gusA reporter gene and nptII as
selectable marker.
• Transformation efficiency using shoot tip was low (1-2%).
Tripathi et al. 2005
Agrobacterium-mediated
transformation
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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.
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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
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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
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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.
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20. Bacterial Pathogen: Type III protein secretion
system
HrpJ: transcription
regulator
HrpC: pili structure
protein
HrpZ: harpin
Erwinia ,
Pseudomonas
Ralstonia
Xanthomonas
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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.
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22. What happen in the PFLP or HRAP transgenic plant ?
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23. Mode of Action of pflp gene
iron-depletion (antibiotic action) + HR enhancement
HR
AOS
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24. Enhanced resistance against virulent
pathogens in the transgenic crops
Crops Transgene Disease resistance Pathogen
Tobacco hrap Wild fire Pseudomonas
pflp Soft rot Erwinia
Gray mold Botrytis
Arabidopsis hrap Soft rot Erwinia
pflp
Broccoli pflp Soft rot Erwinia
Orchids, Calla pflp Soft rot Erwinia
Rice pflp Leaf Blight Xanthomonas
Tomato pflp Soft rot Erwinia
Potato hrap Bacterial wilt Ralstonia
Source: TY Feng
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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.
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26. www.iita.org

27. Exit or Transfer strategy
• The project also involves the capacity building of
NARS for genetic transformation, biosafety
regulations, risk assessment and management.
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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
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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 www.iita.org

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 gene
Southern Blot Analysis
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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.
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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
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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.
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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.
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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. 2007
Tripathi et al. 2007
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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
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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
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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 www.iita.org

39. Development of nematode resistant plantain
(supported by DFID/BBSRC)
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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.
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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
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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
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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
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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.
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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
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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 gusA
Fungal inoculated plant; B: Transgenic gene in transgenic plants; D- Amplification of gusA
plants in pots with inoculated soil; C: gene in microbes from rhizoshere.
Bacterial colonies on selective
medium; D: Re-isolated fungi.
Kabuye et al. 2008
Kabuye 2008, M.Sc. Thesis
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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)
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48. Future Activities
Based 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) resistance
Generation 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
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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
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