for Crop Improvement: Case Studies from African Crops Leena Tripathi - - PowerPoint PPT Presentation

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Application of Genetic Modification for Crop Improvement: Case Studies from African Crops

Leena Tripathi International Institute of Tropical Agriculture, Nairobi, Kenya

5th Annual Biosafety Conference, Nairobi, 17th August 2016

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Agriculture Innovations

• Nearly a billion people going hungry in the world everyday.
• Agricultural output needs to be doubled to feed the world

population in 2050.

• World can only meet its future food needs through

harnessing scientific innovation and technology in agriculture.

• It's also critical to meet the global challenges of producing

more food with less land and water, improving nutrition, and helping farmers adapt to climate change.

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Genetic Engineering

• Potential to transform agriculture in Africa.
• To date, only three African countries (South Africa,

Burkina Faso and Sudan) have adopted GM crops.

• Other countries such as Uganda, Kenya, Nigeria,

Cameroon, Malawi and Ghana are at the forefront of leveraging biotechnology to address local agricultural challenges.

• They are focusing on problems such as pests, disease,

drought and low nutritional content of staple crops.

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Global Area of Biotech Crops

• In 20 years, GM crops have almost 100-fold increase since commercialization.

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Staple Crops for Africa

• Most of the staple crops in Africa receive little attention from the

private sector.

– Banana and Plantain – Cassava – Cowpea – Yam – Enset

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Banana and Plantain

• Rank 4th most important crop in Africa.
• World Musa production is 140 million

tonnes.

• A third of the bananas produced

globally are grown in 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

– pests like nematodes and weevils – diseases like fungal, bacterial and viral

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Transgenic Banana for Africa

• Disease resistance
• Bacterial wilt
• Black sigatoka
• Fusarium wilt
• BBTV
• Pest resistance
• Nematodes
• Biofortification
• Pro-vitamin A and iron

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Transformation System for Banana & Plantain

• Robust transformation system using cell

suspensions

• Gonja Manjaya , Gros Michael, Williams

Cavendish, Sukali Ndiizi

Tripathi et al. 2012 In Vitro Cell Dev Biol- Plant Tripathi et al. 2015 Frontiers in Plant Science

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• BXW caused by

Xanthomonas campestris pv. musacearum.

• First reported more than 45

years ago in Ethiopia on enset and then on banana.

• Outside of Ethiopia, it was

reported in Uganda in 2001.

• The disease has also been

reported in east Africa.

Tripathi et al. 2009, Plant Disease

Banana Xanthomonas wilt (BXW)

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Tripathi et al. 2009, Plant Disease

• The impacts of BXW are both extreme and rapid.
• All the varieties are susceptible.
• Overall economic losses were estimated at $2-8 billion over a decade.

Banana Xanthomonas Wilt

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Biruma et al. 2007, Af. J. Biotech. Tripathi et al. 2009, Plant Disease

• Insect vectors
• Infected planting materials
• Contaminated farming tools
• Traded banana products

Xanthomonas Wilt Disease: Spread

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Control of BXW

• Uprooting of diseased plants
• Chemical destruction of diseased mats
• Use of clean farming tools
• Debudding of male buds
• Use of clean planting materials
• Improving soil fertility
• Use of varieties that escape insect-

mediated transmission

Atim et al. 2013 Plant Disease Adikini et al 2013 Can J Plant Pathol Blomme et al. 2014 Eur J Plant Pathol

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• Resistance has been the best and most cost-effective

method of managing bacterial diseases.

• 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).

• Elicitor-induced resistance is not specific against particular

pathogens.

• USAID Project: IITA, NARO, AATF, KALRO

Transgenic Technology

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• Over expression of sweet pepper

Hrap or Pflp in transgenic banana.

• Sukali Ndiizi, Nakinyika, Gonja

Manjaya.

Generation of Transgenic Plants

Tripathi et al. 2010 Mol. Plant Pathology Namukwaya et al. 2012 Transgenic Research Tripathi et al. 2010 Acta Hort.

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Evaluation of Transgenic Plants

• All the PCR +ve lines

were evaluated

– Using in vitro plants – Potted plants in screen house

• No bacteria was found

at the point of inoculation in healthy transgenic lines after 6 weeks – PCR – Selective medium

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

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Molecular Characterization

M P 1 2 3 4 5 6 7 8 C Southern blot analysis of transgenic plants RT-PCR analysis of transgenic plants using gene specific primers; Northern blot analysis of transgenic plants

Tripathi et al. 2010 Mol Plant Pathol Namukwaya et al. 2012 Transgenic Research

Western blot analysis of transgenic plants

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• 65 transgenic lines (45 lines with Hrap gene and 20 lines

with Pflp gene) were planted in CFT

• BXW disease evaluation
• Agronomic performance

Confined Field Trial

Nature News 2010; Nature Biotech News 2011

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Evaluation for BXW Resistance

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Disease Evaluation

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Second ratoon crop

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External BXW Symptoms on Fruits

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Internal BXW Symtoms

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BXW Resistant lines

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

resistance in field for 3 generations.

Tripathi et al. 2014, Nature Biotechnology

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Agronomic Performance of Best Transgenic Lines

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Yield Parameters of Best Transgenic Lines

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2nd Confined Field Trial

• 10 lines (7 Hrap and 3 Pflp) with 12 replicates were further

evaluated in 2nd CFT in Uganda from Sept 2013-Dec 2015.

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Product Development

• Durable and enhance resistance

– Gene pyramiding

• Transformation

– 2 dessert varieties for Kenya – 2 matooke varieties in Uganda

• Transgenic bananas with stacked

genes for durable resistance to BXW – Hundreds of lines developed – Tested in glasshouse trial

• CFT in Kenya and Uganda with

stacked lines in 2016.

• BXW resistant bananas will be ready

for release in 2021.

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• Bioinformatics sequence comparisons were performed

comparing the amino acid sequences of both proteins to known allergens and toxins (AllergenOnline.org and NCBI).

• No convincing evidence was found to suggest that the

HRAP or PFLP proteins represent risks of allergy or toxicity to humans.

• Peppers are commonly consumed with rare cases of

allergy and no reports of toxicity.

• Food safety studies will be done.
• Nutritional quality will be checked.

Biosafety of PFLP and HRAP

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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.

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Nematodes

• Nematode losses 30-50% yield to banana in

Africa.

• Limited sources of nematode resistance

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 occur in the same soils

– Radopholus similis, Pratylenchus spp, Helicotylenchus spp, Meloidogyne spp

• Gene stacking the best way forward

– Several approaches available

• Partners: IITA, University of Leeds, NARO

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Resistance Technologies

• Maize cystatin that inhibits nematode digestive cysteine proteinases
• Synthetic peptide that disrupts nematode chemoreception

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• Plantains transformed

– Cystatin – Peptide – Additive

• Generated about 250 transgenic

lines of Gonja manjaya

• Confirmed by

– PCR – Cystatin

• Western

– Peptide

• Bioassay

– Nematode challenge in screenhouse

Generating Transgenic Plantain

1 _ 0.5_ M - + lines 20 _ Kb Kda PCR Western

Roderick et al. 2012 Mol Plant Pathol

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Glasshouse Trials

Establishing the trial Harvest Roderick et al. 2012 Mol Plant Pathol

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Confined Field Trial

• Twelve promising lines were planted in CFT in Uganda

for further evaluation.

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Nematode Resistance

0% 20% 40% 60% 80% 100% +nem D12 D30 D46 C15 P53 P46 C6 P78 D66 D14 P48 P77 % Resistance to nematodes

harvest ** *** *** *** *** *** *** *** *** *** *** *** flowering * ** *** ** ** *** *** *** *** *** growth * ** ** ** *** ** *** ***

Tripathi et al. 2015 Scientific Reports

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Precision Delivery of Transgene

• Root-cap-specific promoter

identified for Arabidopsis (gene MDK4-20).

• Maize root cap promoter

(ZmRCP1) homologue identified and cloned.

• Tested the construct in

banana using reporter gene.

• It can be used for delivery of

the peptide to the rhizosphere via root border cells.

Onyango et al. (2016) J Biol Res

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Banana Bunchy Top Virus

• BBTV is a major limiting factor in

banana production.

• No known sources of resistance to

BBTV within banana germplasm.

• No opportunity to incorporate BBTV

resistance into any of the conventional banana breeding programs.

• Developing transgenic bananas

using RNAi targeting -

• BBTV
• Aphids
• BMGF Project- QUT, IITA and DARS

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Cassava Transformation Platform

• Genetic transformation
• f African farmer

preferred cultivars of cassava

• Using friable

embryogenic callus

• 7 east African cultivars
• 5 west African cultivars

Nyaboga et al. 2013 Frontiers in Plant Biotech Nyaboga et al. 2015 Frontiers in Plant Biotech

• Developing resistance
• CBSD
• CBB

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D E F A

Genetic Transformation of Yam

• Agrobacterium-mediated

transformation system established for yam using apical meristems.

• It takes 5–6 months from

transformation to regeneration of complete transgenic plant.

Nyaboga et al. 2014 Frontiers in Plant Sci

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• Yams are susceptible to a number of

species of endoparasitic nematodes.

• Yam nematodes reproduce and build

up large populations in stored tubers, causing severe damage.

• Nematodes also facilitate fungal and

bacterial attacks that cause anthracnose disease, dry rot, soft rot and wet rot.

• Technologies proven in plantain can be

applied to yam.

Nematode Resistant Yam

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Genetic Transformation of Enset

• Enset is staple

food for more than 20 million people in Ethiopia.

• Agrobacterium-

mediated transformation system established.

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Control of Bacterial Wilt in Enset

• Enset production has been

severely threatened by bacterial wilt disease.

• The technology (Pflp and Hrap)

mastered in the proof-of-concept for control of BXW can be extended to enset.

• BMGF Project- IITA and EIAR.

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• GM approaches shows enormous potential for development
• f varieties resistant to diseases & pests.
• The techniques mastered in these proof-of-concept states

can be extended to a wide range of other crops.

– Technology for nematode resistance can be applied to yams – BXW resistance technology can be applied to bacterial diseases of enset, cassava etc.

• There are several products on horizon for Africa.
• BXW resistant banana planned to be commercialized in

2021.

Conclusions

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Acknowledgements

Partners

• NARO-Uganda
• EIAR- Ethiopia
• KALRO- Kenya
• University of Leeds, UK
• Donald Danforth Plant Science

Centre

• Qeensland University of

Technology, Australia

• ETH
• AATF
• Academia Sinica, Taiwan

Financial Support

• Bill & Melinda Gates

Foundation

• CGIAR Research Program
• DFID/BBSRC
• Gatsby Charitable

Foundation

• USAID
• NSF