genetic transformation of rtb crops · • rtb crops contribute currently to 14% of global food...

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Genetic Transformation of RTB Crops

Leena Tripathi

International Institute of Tropical Agriculture, Nairobi, Kenya

World Congress on Root & Tuber Crops, 22nd Jan 2016, Nanning, China


Agriculture Innovations

• Food production needs to be increased by 70% by 2050.

• RTB crops contribute currently to 14% of global food supply.

• RTB crops are highly vulnerable to pests, diseases, and drought.

• New pests and diseases are emerging and rapidly spreading.

• GM has proven to be a useful alternative method for the introduction of new desirable traits.


Global Area of Transgenic Crops

• In 19 years, GM crops have almost 100-fold increase since commercialization.


Global Area of Transgenic Crops

Source: Clive James, 2014


GM Product Development Pathway

Commercialization

Gene Discovery Gene delivery

Transgenic plants Molecular testing

Glasshouse testing Confined field trial

10-15 years

Variety development Field trials

Multi-location field trials Food safety analysis Environmental safety

analysis

Open field trials Deregulation

Variety registration Variety release

Product Development

Research Proof of Concept

Yr 0 3-5 8-10 12-15


Plant Transformation Platform at IITA

• Plant transformation platform established at IITA – Banana and Plantain

– Cassava

– Yam

– Enset

http://upload.wikimedia.org/wikipedia/commons/e/eb/YamsatBrixtonMarket.jpg


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


Cassava Transformation Platform

• Genetic transformation of 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 • PPD

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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


Regeneration through somatic embryogenesis

• Protocol established for somatic embryogenesis and regenration of D. rotundata cv.2436.

• It takes 7-8 months for complete plant formation.

• Additional cultivars are under testing.


Agrobacterium mediated transformation of yam using embryogenic callus

a

e

c

• Regeneration through somatic

embryogenesis of yam has

been established.

• Embryogenic calli were

transformed with

Agrobacterium using gusA

reporter gene.

• Transformed calli are currently

under selection and

regeneration.


Genetic Transformation of Enset

• Enset is staple food for more than 20 million people in Ethiopia.

• Agrobacterium-mediated transformation system established.

• Developing bacterial wilt resistant varieties.


Transgenic Banana for Africa

• Disease resistance - Bacterial wilt - Black sigatoka - Fusarium wilt - BBTV

• Pest resistance - Nematodes • Biofortification - Pro-vitamin A and iron


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

• IITA in collaboration with University of Leeds

• Started under SARID-DFID/BBSRC project


Resistance Technologies

• Maize cystatin that inhibits nematode digestive cysteine proteinases

• Synthetic peptide that disrupts nematode chemoreception


• 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

PC

R

Wes

tern

Roderick et al. 2012 Mol Plant Pathol


Glasshouse Trials

Establishing the trial Harvest

Roderick et al. 2012 Mol Plant Pathol


Confined Field Trial

• Twelve promising lines were planted in CFT in Uganda for further evaluation.


Nematode Densities

Vegetative growth At flowering

At harvest Tripathi et al. 2015 Scientific Reports


Nematode Resistance

0%

20%

40%

60%

80%

100%

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

% R

esis

tanc

e to

nem

atod

es

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

Tripathi et al. 2015 Scientific Reports


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 will be used for delivery of the peptide to the rhizosphere via root border cells

Onyango et al. 2016 J Bio Res (under review)

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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. • Stacking nematode resistance with

anthracnose disease can enhance yam production.

Nematode Resistance Yam

http://www.cabi.org/portfolio/0x0/68573.img




Fast breeding of slow crops: Doubled haploid

• BREAD Project: UC Davis, CIAT, IITA

• Banana and cassava • Generated haploid inducer

banana using altered CenH3 (GFP_Tailswap, Tailswap and GFP_CENH3) in a cenh3-/- background.

• Transgenic banana crossed with wild type.


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.

• BMGF Project- QUT, IITA and DARS • Developing transgenic bananas

resistant to- • BBTV • Aphids


• 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)



• The impacts of BXW are both extreme and rapid. • Overall economic losses were estimated at $2 billion to $8 billion over

a decade.

Banana Xanthomonas Wilt


Xanthomonas campestris pv. musacearum

• Closly related to X. vasicola pv. vasculorum

• Previous work showed that Xcm is a highly monomorphic pathogen

• Genome-wide sequencing reveals two major sub-lineages

Studholme et al. 2010, FEMS Wasukira et al. 2012, Genes


Biruma et al. 2007, Af. J. Biotech.


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

Xanthomonas Wilt Disease: Spread


• All cultivars are susceptible.

• There were significant differences in susceptibility among the various cultivars.

• Brewing banana cultivar ‘Pisang Awak’ was found to be highly susceptible.

• Dessert banana cultivars ‘Cavendish’, ‘Gros Michel’, ‘Sukali Nidiizi’ were also found to be highly susceptible.

• All the EAHB cultivars tested were found to be susceptible.

• Wild type ‘Musa balbisiana’ (BB) was found to be resistant.

Tripathi and Tripathi 2008, Af. J. Biotech. Tripathi et al. 2008, Eur. J. Plant Pathol.

Relative Susceptibility of Banana Cultivars


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


• 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

Transgenic Technology


• 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.


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


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


• 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


Evaluation for BXW Resistance


Disease Evaluation


Natural inoculation of second ratoon crop


External BXW Symptoms on Fruits


Internal BXW Symtoms


BXW Resistant lines

• 11 lines (7 Hrap lines and 4 Pflp lines) showed 100% resistance in field for 3 generations.

• An additional five lines showed 85-93% resistance with mother plants and 100% resistance with ratoon plants.

Tripathi et al. 2014, Nature Biotechnology


Agronomic Performance of Best Transgenic Lines


Yield Parameters of Best Transgenic Lines


Expression of Hrap or Pflp gene

Tripathi et al. 2014, Nature Biotech


Gene Stability Analysis

PCR

RT- PCR


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.

• All transgenic lines tested showed 100% resistance to BXW.


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 2020


• 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

• Analysis conducted by Richard Goodman, Nebraska University.


Environmental Safety Analysis

• Community-level profile of rhizosphere microorganisms was performed using the PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) approach.

• No significant differences in bacterial community diversity was observed due to the transgenes.


• The Xoo raxSTAB operon is required for elicitation of Xa21-mediated immunity in rice.

• Xoo raxSTAB operon is highly conserved in diverse Xanthomonas species, including Xcm and that the Xa21 gene confers resistance to Xcm.

• Transgenic bananas expressing Xa21 were generated and evaluated in glasshouse.

Transgenic expression of the rice Xa21 pattern-recognition receptor in banana

Tripathi et al. 2014 Plant Biotech


Ex-ante impact assessment of GM banana resistant to BXW

Banana production loss due to BXW incidence

Ainembabazi et al. 2015 Plos One


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.


Commercialization of GM banana

• On release of GM banana, its expected initial adoption rate ranges from 21–70% while ceiling adoption rate is up to 100%.

• Aggregate benefits vary substantially across the target countries ranging from US$20–953 million, being highest in countries where disease incidence and production losses are high.


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.


Genome Editing

• CRISPR/Cas9 is a new tool for genome editing • Using CRISPR/Cas9 to knockout integrated sequence

of Banana Streak Virus (BSV) in banana genome - In collaboration with UC Davis

• BREAD Project: Iowa State University and IITA - Genome editing of yam


• GM approaches shows enormous potential for development of 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 2020.

Conclusions


Acknowledgements Partners • NARO-Uganda • EIAR- Ethiopia • KARI- Kenya • University of Leeds, UK • Iowa State University • University of California, Davis • Queensland University of

Technology, Australia • ETH, Zurich • DDPSC • AATF • Academia Sinica, Taiwan

Financial Support • Bill & Melinda Gates

Foundation • CGIAR Research Program • DFID/BBSRC • Gatsby Charitable

Foundation • USAID • NSF


Plant Transformation Group at IITA-Nairobi


CNN: Earth’s Frontiers

http://edition.cnn.com/video/#/video/international/2011/03/

24/ef.genetic.modification.bk.a.cnn

Genetically Modified bananas in Uganda

http://edition.cnn.com/2011/WORLD/africa/03/22/uganda.banana.gm/index.html

genetic transformation of rtb crops · • rtb crops contribute currently to 14% of global food...

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