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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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Outline
• Introduction– Banana and Plantain
– Biotechnology - transgenic
• Achievements– Transformation system
– BXW resistance
– Nematodes resistance
– BSV resistance
– Capacity Building
• Future Plans
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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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Why Biotechnology?
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Global Area of Biotech Crops, 1996 to 2006:
By Crop (Million Hectares)
0
10
20
30
40
50
60
70
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
Soybean
Maize
Cotton
Canola
Source: Clive James, 2006
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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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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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Production of transgenic bananas
resistant to Xanthomonas wilt disease
(supported by Gatsby & AATF)
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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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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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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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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 foundto be moderately resistant. Tripathi and Tripathi 2008
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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
pandemic.R4D Review 2008
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Genetic Transformation of Bananas
Transformation efficiency is
high but time consuming &
cultivar specific
Fast, cultivar independent
but transformation efficiency
is low and chances of
chimeras
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Genetic Transformation using shoot tips
Tripathi et al. 2005
• Binary vector pCAMBIA2301 containing the gusA reporter gene and nptII as
selectable marker.
• Transformation efficiency using shoot tip was low (1-2%).
Agrobacterium-mediated
transformation
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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 total from quarter pieces of each section.
Tripathi & Tripathi 2008
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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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Potential strategies to develop plants resistant
to Bacterial Wilt
• Several transgenic
technologies are available to
develop disease resistant
plants through
– Using R genes
– defense mechanism or
– antimicrobial proteins
Tripathi 2005
Gene Transgenic Plants
Magainin
analogs
Tobacco
Cecropins Tobacco, potato, apple
Attacins Pear, apple
Lysozymes Tobacco, potato, apple,
rice, tomato
Pepper Bs2 Tomato
Rice Xa1,
Xa21
Rice
Tomato Pto Tomato
Pepper pflp
& hrap
Tobacco, tomato,
broccoli, orchid, rice,
Arabidopsis
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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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Bacterial Pathogen: Type III protein secretion
system
HrpJ: transcription
regulator
HrpC: pili structure
protein
HrpZ: harpin
Erwinia ,
Pseudomonas
Ralstonia
Xanthomonas
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• 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.
Pepper pflp & hrap genes
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What happen in the PFLP or HRAP transgenic plant ?
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Mode of Action of pflp gene
HR
iron-depletion (antibiotic action) + HR enhancement
AOS
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Crops Transgene Disease resistance Pathogen
Tobacco hrap
pflp
Wild fire
Soft rot
Gray mold
Pseudomonas
Erwinia
Botrytis
Arabidopsis hrap
pflp
Soft rot Erwinia
Broccoli pflp Soft rot Erwinia
Orchids, Calla pflp Soft rot Erwinia
Rice pflp Leaf Blight Xanthomonas
Tomato
Potato
pflp
hrap
Soft rot
Bacterial wilt
Erwinia
Ralstonia
Enhanced resistance against virulent
pathogens in the transgenic crops
Source: TY Feng
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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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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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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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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
– 2/17- no symptoms
– 8/17- delayed symptoms
• 60 lines transformed with pBI-SAP1 were screened using in vitro plantlets
• Some promising lines showing no symptom or delayed symptoms have been obtained.
In vitro screening of transgenic plants for
resistance against Xanthomonas wilt; A-
inoculated control plant, B-F- inoculated
transgenic plants
Screening of potted
transgenic plants
Namukwaya et al. 2008
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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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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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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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Additive defence for BXW resistance
• Co-transformation
• Construct with hrap-pflp stacked
together
PCR analysis of the construct pBI-HRAP-PFLP,
amplifying 600bp of pflp gene and 1Kb of hrap
gene.
NOSP NPT II 35P hrap
NOSPNPT II 35P pflp
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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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• Semi-selective medium for isolation of Xcm from
infected plants
• YTS-CC Medium
– Yeast Extract (0.5%)
– Tryptone (0.5%)
– Sucrose (1%)
– Cycloheximide (150 mg/l)
– Cephalexin or Cefazolin (50 mg/l)
Tripathi et al. 2007
Diagnostics for BXW
Non-selective YPGA
Semi-selective media
Selective YTS-CC Tripathi et al. 2007
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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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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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Effect of Phytotoxic Factors and
Potassium Nutrition on BXW• Phytotoxic factors for banana were shown to be produced by Xcm in
culture filtrate.
• Increased potassium availability for banana reduced disease incidence.
c d e
f g h i j
ba
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)
and 2K (i). Controls (e) and (j) were
inoculated with sterile distilled water.
a c db
Control waterControl YPGB
Culture filtrate Culture filtrate
Atim et al. 2008
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Development of nematode resistant plantain
(supported by DFID/BBSRC)
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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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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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Additive Strategies
• Proteinase Inhibitors
– Cysteine proteinase inhibitor (cystatin)
– Potato tuber serine/aspartic proteinase inhibitor (PDI)
• Peptide repellent
– Developed to target nicotinic acetylcholine receptors and disrupt chemoreception
• RNAi to target nematode genes
– Essential housekeeping genes
– Genes involved in parasitism
Acetylcholine released into synapse
Nicotinic acetylcholine receptorAcetylcholine released into synapse
Nicotinic acetylcholine receptor
Bound peptide prevents acetylcholine function
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• 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
Nematode Resistance Plantain
R4D review 2009
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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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• Develop resistance to Banana streak virus
– In collaboration with JIC
– Gatsby Charitable Foundation
Genetic Transformation of Plantain for
BSV Resistance
The BSV sequence of
approx. 600bp of the viral
reverse transcriptase-
RNase H domain
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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
gene in transgenic plants; D- Amplification of gusA
gene in microbes from rhizoshere.
Fungal inoculated plant; B: Transgenic
plants in pots with inoculated soil; C:
Bacterial colonies on selective
medium; D: Re-isolated fungi.Kabuye et al. 2008
Kabuye 2008, M.Sc. Thesis
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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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Future Activities
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
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
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• 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
Acknowledgements