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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