genetic improvement of bananas with enhanced resistance...

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Genetic Improvement of

Bananas with Enhanced

Resistance to Pathogens and

Pests

Leena Tripathi

International Institute of Tropical Agriculture, Nairobi, Kenya

IPBO Conference, Ghent, 30-31 August 2018


Banana


Yield Gap in Banana Production

Matooke Plantain

30 t/ha/year

7.8 t/ha/year

70 t/ha/year

35.5 t/ha/year

Current Potential

Ranges

from 5 to

30t/ha/year

Source: Rony Swenen, Banana Breeder at IITA


Banana: Diseases & Pests

Black Sigatoka Fusarium wilt Nematode Weevil

Bacterial wilt Banana bunchy top Banana streak virus


we need better and more

efficient ways for crop

improvement

With increasing demand for

closing yield gaps...

one option is through

Biotechnology

Banana Improvement


• BXW caused by Xanthomonas campestris pv. musacearum.

Tripathi et al. 2009, Plant Disease

Banana Xanthomonas Wilt (BXW)

Wasukira et al. 2012, Genes



Symptoms of BXW Disease



Xanthomonas Wilt Disease: Spread


Banana Production Loss due to BXW

Ainembabazi et al. 2015 Plos One


• Overall economic losses were estimated at $2 billion to $8 billion over a decade.

• BXW attacks all banana varieties resulting in absolute crop loss.

• Resistance has been the best and most cost-effective method of managing diseases.

• No resistance source of germplasm.

Control of BXW


• Pflp (plant ferredoxin-like protein) and Hrap (HR assisting

protein) are defence genes from Capsicum annuum.

• Elicitor-induced resistance based on HR.

• These genes are effective against many bacterial

pathogens, such as, Erwinia, Pseudomona, Ralstonia

and Xanthomonas spp.

Transgenic Approach


Transgenic Banana

Tripathi et al. 2010 Mol Plant Pathol

Namukwaya et al. 2012 Transgenic Research

Tripathi et al 2007 Eur. J Plant Pathology

Adikini et al. 2011 Plant Pathology


• 65 transgenic lines were planted in CFT

• BXW disease evaluation

• Agronomic performance

Confined Field Trial

Nature News 2010; Nature Biotech News 2011


Disease Evaluation


BXW Resistant Lines

• 11 lines (7 Hrap lines and 4 Pflp lines) showed 100%

resistance in field for 3 generations.

• Aside from full resistance to BXW, the transgenic lines also

showed yield comparable to non-transgenic varieties.

Tripathi et al. 2014, Nature Biotech


Trait Durability

• 10 lines (7 Hrap and 3 Pflp) with more replicates were

further evaluated in 2nd CFT in Uganda.

• All transgenic lines tested showed resistance to BXW.


Product Development

• Single gene resistance can

break down

• Durable and enhance resistance

– Gene pyramiding

• Transformation – matooke varieties in Uganda

• Transgenic bananas with

stacked genes for durable

resistance to BXW

– Hundreds of lines developed

– Tested in glasshouse trial


Food Safety

No convincing evidence was

found to suggest that the HRAP

or PFLP proteins represent risks

of allergy or toxicity to humans.


Non-target Effect of PFLP and HRAP

• The expression of Pflp and Hrap genes did not show any

effect on non-target microorgansms in soil.


Potential Adoption of GMB-BXW

Willingness to adopt GMB-BXW

Projected adoption rate of GM BXW

resistant banana

Ainembabazi et al. 2015 Plos One

This study based on cost

estimates and benefits suggests

that investment in development of

GMB-BXW is not only essential

but also economically viable.


Transgenic expression of the rice Xa21

pattern-recognition receptor in banana


Generation and Evaluation of

Transgenic Plants

Tripathi et al. 2014 Plant Biotech J


BXW Resistant vs Susceptible Genotypes

Pis

an

g a

wak

Mu

sa b

alb

isia

na


• Vicilin-like antimicrobial peptide

• SWEET14

• Early nodulin-like protein

• R genes

• Pathogen-recognition receptors (PRR)

Validation of Musa Genes for

Resistance to BXW


Nematodes

• Nematode losses 57% yield to banana in

Africa

• Several species occur in the same soils

– Radopholus similis, Pratylenchus spp,

Helicotylenchus spp, Meloidogyne spp,

Rotylenchulus reniformis


Field Resistance of GM Plantain to

Nematodes

• Twelve promising lines were planted in CFT in Uganda

for evaluation.


Nematode Densities

Vegetative growth At flowering

At harvest Tripathi et al. 2015 Scientific Reports


Nematode Resistance

Tripathi et al. 2015 Scientific Reports

0%

20%

40%

60%

80%

100%

+nem D12 D30 D46 C15 P53 P46 C6 P78 D66 D14 P48 P77

% R

esis

tance

to

nem

ato

de

s

harvest ** *** *** *** *** *** *** *** *** *** *** ***

flowering * ** *** ** ** *** *** *** *** ***

growth * ** ** ** *** ** *** ***


• Developing transgenic bananas

resistant to-

• BBTV and

• Aphids

Adults collected

Banana Bunchy Top Virus


Genome Editing of Banana

Genome edited banana

• Clustered regulatory interspaced

short palindromic repeats

(CRISPR) is a useful tool for

genome editing.

• It could mutate several genes at

the same time in precise manner.

Control Plants

• Knockouts

• Replacement

• Insertion


Banana Streak Virus

• Banana streak virus (BSV) is a plant pathogenic virus of the family Caulimoviridae and genus Badnavirus.

• BSV is dsDNA Reverse Transcribing Viruses.

• Badnaviruses have shown to integrate into their host genomes.

• It has been shown that genomic sequences are integrated into the genomic DNA of Musa and Ensete.

• Traditional techniques for virus eradication, such as meristem tip culture, are not appropriate.


• Integration only occur in cultivars/ hybrids with the B

genome.

• BSV infection may arise from the activation of viral

sequences that are integrated into the banana genome.

• Tissue culture and hybridization through conventional

breeding may be triggers for the activation.

BSV present in two forms

Integrated Episomal Stress

Banana Streak Virus


CRISPR/Cas9 Construct

eBSOLV ( 23.6 Kb)

CRISPR/Cas9 Construct

BSV (7.39 Kb)


Analysis of Banana Mutant Lines

A) Banana mutants. B) PCR analysis for integration of T-DNA in transgenic lines using

primers designed from Cas9 sequence.

A B


PCR was performed with primers flanking two gRNAs in ORF1 and ORF2.

PCR analysis of GE Banana


• GE and control wild type plants were potted.

• Plants were stressed.

• No BSV symptoms were developed on several of the GE

events.

• Frame-shift mutations in all three ORFs should have

disrupted functions of several genes.

Phenotyping of GE Banana


Communication

Food

Evolution


• GM approaches shows enormous potential for development

of varieties resistant to diseases & pests.

• GE is emerging powerful tool for crop improvement.

• There are several GM products on horizon for Africa.

Conclusions


Acknowledgements

Partners

• NARO-Uganda

• KALRO- Kenya

• AATF- Kenya

• University of Leeds, UK

• QUT, Australia

• Academia Sinica, Taiwan

• University of Nebraska

Financial Support

• CGIAR Research Program

• Gatsby Charitable Foundation

• USAID

• Bill & Milinda Gates Foundation

Plant Transformation

Lab at IITA

genetic improvement of bananas with enhanced resistance...

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