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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
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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.
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Global Area of Transgenic Crops
• In 19 years, GM crops have almost 100-fold increase since commercialization.
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Global Area of Transgenic Crops
Source: Clive James, 2014
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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
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Plant Transformation Platform at IITA
• Plant transformation platform established at IITA – Banana and Plantain
– Cassava
– Yam
– Enset
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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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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
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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.
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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.
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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.
• Developing bacterial wilt resistant varieties.
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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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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
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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
PC
R
Wes
tern
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 Densities
Vegetative growth At flowering
At harvest Tripathi et al. 2015 Scientific Reports
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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
% R
esis
tanc
e to
nem
atod
es
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 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
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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.
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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.
• BMGF Project- QUT, IITA and DARS • Developing transgenic bananas
resistant to- • BBTV • Aphids
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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. • Overall economic losses were estimated at $2 billion to $8 billion over
a decade.
Banana Xanthomonas Wilt
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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
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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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• 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
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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
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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Natural inoculation of 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.
• An additional five lines showed 85-93% resistance with mother plants and 100% resistance with ratoon plants.
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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Expression of Hrap or Pflp gene
Tripathi et al. 2014, Nature Biotech
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Gene Stability Analysis
PCR
RT- PCR
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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.
• All transgenic lines tested showed 100% resistance to BXW.
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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 2020
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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
• Analysis conducted by Richard Goodman, Nebraska University.
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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.
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• 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
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Ex-ante impact assessment of GM banana resistant to BXW
Banana production loss due to BXW incidence
Ainembabazi et al. 2015 Plos One
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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.
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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.
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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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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
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• 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
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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
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Plant Transformation Group at IITA-Nairobi
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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
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