Overview of VIRCA Project          Douglas Miano  Biotech Communication Stakeholders Workshop      Hilton Hotel, Nairobi, ...
VIRCA PARTNERSDonald Danforth Plant Science Center         St. Louis, Missouri                                       Natio...
Cassava production in the worldBasic food for > 600 million people in the world Continent    Area (ha)   Production   Yiel...
Industrial uses of Cassava                     Food and Beverage       Paper                                          Star...
Cassava – Strategic Priority for East & Central Africa                                        Cassava: 2nd most           ...
Challenges to cassava production• Diseases and pests      - CMD, CBSD, CBB, whiteflies, mites, etc• Nutritional quality  -...
Disease constraints  • Cassava mosaic disease (CMD) has been the main disease  constrain                        -1st repor...
Cassava susceptible to CMDCMD resistant TME204
Challenge to management of the two diseases • Continuous presence of the virus and   the vector throughout the year • Atta...
Virus resistant cassava for Africa (VIRCA) Project  Goals       Deliver farmer-preferred transgenic cassava       resistan...
Cassava-infecting begomoviruses• 9 distinct species, 7 in Africa• ACMV and EACMV types most prevalent                     ...
Two Distinct Viruses                                                 Mal71                                                ...
Phase I: Gene discovery and proof of concept  Main target was CMD  • Coat protein based  • Replication mediated  • G5 prot...
Cloning in sense & anti-sense into pILTAB 0588                                 Anti Sense                                B...
• Transgenic cassava lines showing resistance to EACMV;   the effect of RNA-silencing constructs targeting the virus   rep...
Entries being evaluated in the CFTEntry   Line        Vector          Gene of interest Comments1       P670-007    pILTAB6...
Transgenic trials for CMD resistanceNBA Permit No.: BA/KARI/G22/6(2)            CMDK-1
Transgenic Virus-Resistant                         Incineration Pit          CassavaConfined Field Trial Plot Sketch    KA...
• Planting   of CMDK-1 at KARI Alupe: 11th May, 2011
CMD incidence                                           CMD Resistance to Transgenic Events                    120%       ...
Disease Severity                                    CMD Resistance to transgenic events                     4.50          ...
CMD severity (C-terminal)                                    CMD Resistance to transgenic events                     4.00 ...
CMD1a: Virus Infection 7 MAP                                     ACMV Positive         EACMV-UG Posotive                  ...
4 lines have been selected and are being tested for stability in the field
CMD1a: Storage Root Yield (Tons/Ha)                                                          31                           ...
Cassava brown streak disease (CBSD) CFT• To be conducted in KARI-Mtwapa, Coast  region• Application approved by NBA
IncinerationTransgenic Virus-Resistant Cassava                   Pit  Confined Field Trial Plot Sketch       KARI-Mtwapa S...
Root severity of CBSD in selected lines
Phase II: Product developmentInitial target of two products  – TME204 – Natural resistant to CMD but         highly suscep...
Product 1 –TME204 modified for resistance to CBSDTarget pathogens - cassava brown streak virus & Uganda cassava brown stre...
CBSD symptom scoring scaleScore Foliar Symptom Description  1   no symptoms on leaves or stems      mild/slight vein yello...
2. Broad spectrum VR in Ebwanateraka• Background - Farmer preferred cultivar Ebwanateraka:  • Ebwanateraka – previously mo...
Product #2 DescriptionCMD and CBSD Resistant EbwanaterakaResistance to CMD & CBSD – Engineered resistance to CMD & CBSD ba...
VIRCA Management and Oversight Structure                                                             PI and Director of   ...
VIRCA Communication Team                           KARI   NARO/                                  NACRRI       ISAAA- AfriC...
VIRCA DONORS
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
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Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project

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Dr. Douglas Miano provides an overview of the Virus Resistant Cassava (VIRCA) project. VIRCA is an ongoing biotech research project in Kenya and Uganda seeking to develop a virus resistant cassava variety.The cassava variety being developed is resistant to Cassava Brown Streak Disease (CBSD) and Cassava Mosaic Disease (CMD).

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Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project

  1. 1. Overview of VIRCA Project Douglas Miano Biotech Communication Stakeholders Workshop Hilton Hotel, Nairobi, September 14, 2012
  2. 2. VIRCA PARTNERSDonald Danforth Plant Science Center St. Louis, Missouri National Crops Resources Research Institute Namulonge, Uganda Kenyan Agricultural Research Institute Nairobi, Kenya International Institute for Tropical Agriculture BecA, Nairobi, Kenya VIRCA – Virus Resistant Cassava for Africa
  3. 3. Cassava production in the worldBasic food for > 600 million people in the world Continent Area (ha) Production Yield % Global (‘000 MT) (t/ha) Production Africa 11,662,941 103,423 8.87 52.9 Asia 3,508,103 58,373 16.64 29.8 Americas 2,683,268 33,601 12.52 17.2 Oceania 16,314 177 10.87 0.1 World 17,870,626 195,574 10.94 100Source: FAO (2005)
  4. 4. Industrial uses of Cassava Food and Beverage Paper Starch Wood CASSAVA Deg. Plastics Animal Feed Textile Ethanol 1. Cassava flour – Bakery and Confectionery 2. Cassava chips/pellets & leaves – Livestock feeds 3. Sweeteners – Food industry 4. Starch – paper, wood, batteries, pharmaceuticals and textile industry 5. Ethanol – Biofuel, distilleries
  5. 5. Cassava – Strategic Priority for East & Central Africa Cassava: 2nd most important subsector for growth-inducing development Omamo et al. 2006. IFPRI Report 150
  6. 6. Challenges to cassava production• Diseases and pests - CMD, CBSD, CBB, whiteflies, mites, etc• Nutritional quality - protein, micronutrients (iron, Zinc & vitamin A) contents, trace minerals• Post-harvest physiological deterioration• Cyanogenic potential• Lengthy growing cycle and recalcitrant nature of cassava - results in slow breeding
  7. 7. Disease constraints • Cassava mosaic disease (CMD) has been the main disease constrain -1st reported 1894 in present day Tanzania -Caused an epidemic in the 1990s - Africa losses an average 30-40% (15-24 million tonnes; $6-25 billion/year) • Cassava brown streak disease (CBSV) also becoming a major threatCombined annual economic loss ofUS$ 180 million in East Africa Necrotic rot of roots
  8. 8. Cassava susceptible to CMDCMD resistant TME204
  9. 9. Challenge to management of the two diseases • Continuous presence of the virus and the vector throughout the year • Attachment of farmers to particular cultivars which are susceptible to the Way forward? disease • Lack of resistant / tolerant genotypes preferred by farmersVIRCA project was initiated to combat these virusproblems
  10. 10. Virus resistant cassava for Africa (VIRCA) Project Goals Deliver farmer-preferred transgenic cassava resistant to CMD and CBSD to farmers in East Africa Capacity building (human and institutional)
  11. 11. Cassava-infecting begomoviruses• 9 distinct species, 7 in Africa• ACMV and EACMV types most prevalent African cassava mosaic virus (ACMV) East African cassava mosaic virus (EACMV) East African cassava East African cassava mosaic Malawi virus (EACMMV) mosaic virus cluster East African cassava mosaic Cameroon virus (EACMCV)Africa East African cassava mosaic Zanzibar virus (EACMZV) East African cassava mosaic Kenya virus (EACMKV) South African cassava mosaic virus (SACMV) Indian cassava mosaic virus (ICMV)India Sri Lankan cassava mosaic virus (SLCMV)
  12. 12. Two Distinct Viruses Mal71 Mal65 Mal63 86 Mal57 Mal77 Ug46 Mal9 100 Mal25 88 UgNamEBW Mal58 96 NTG10 Ug45 89 Ug53 100 IGA8 99 Ug52 HMA9 Mal72 75 LWR2 PANG 98 ZANZ Ke54 94 Nampula1-1 CBSV Ke125 76 Zanzibar7-1 100 67 99 BSA2 86 Zanzibar11-1 Zanzibar6-2 99 BSA4 CHAKE 55 MLB9 WETE 100 TanT8 75 KOR9 99 MLB3 54 KOR1 100 Naliendele3-1 100 TanC9 100 Mal17 ZANZ Mal27 PANG Tan70 53 KOR9 Zanzibar8-2 HAND 100 KOR1 76 99 Kibaha10-2 58 WETE Mo83 99 83 CHAKE Zanzibar13-1 76 TanZ Tan70 100 MLB9 Mo83 MLB3 62 HAND Mal18 97 100 Kikonde11-5 50 KenE Kikonde10-1 96 TanAB30 57 59 Shirazi7-1 87 TanZ 99 Nyumbasita5-4 Chumani Mbandar12-1 96 BSA2 0.05 88 Ug BSA4 57 Kilifi20-1 61 Kilifi20-3HAM1h 99 Coat protein Mwalumba16-2 ‘UCBSV’ 100 95 Namulonge Ke54 Diani3-1 Denyenye1-2 Kilifi18-2 51 LWR2 76 70 U23 78 HMA9 96 UgNamEBW 65 Kabanyoro4-3 7768 IGA8 NTG10 Ug16 UG42 71 Mal10 0.02
  13. 13. Phase I: Gene discovery and proof of concept Main target was CMD • Coat protein based • Replication mediated • G5 protein based • Defective DNA based • RNAi (gene silencing)
  14. 14. Cloning in sense & anti-sense into pILTAB 0588 Anti Sense BamH I BstB I Kpn I Sense Xba IAll the constructs were initially made in pILTAB-0588 vector carryingthe Cassava Vein Mosaic Virus & 35S promoters and NOS poly Asequence using Xba I & Kpn I and BamH I & BstB I restriction enzymes
  15. 15. • Transgenic cassava lines showing resistance to EACMV; the effect of RNA-silencing constructs targeting the virus rep geneNon-transgenic cassava variety Transgenic cassava variety60444 infected with K201 60444 infected with K201 • Clone K201 92% homology to EACMV-UG, 80-100% infectivity, faster progression of symptoms
  16. 16. Entries being evaluated in the CFTEntry Line Vector Gene of interest Comments1 P670-007 pILTAB670 AC2 AC2 from EACMV-UG2 P670-010 pILTAB670 AC2 AC2 from EACMV-UG3 P560-001 pILTAB560 Full Length AC1 AC1 from EACMV-UG4 P560-008 pILTAB560 Full Length AC1 AC1 from EACMV-UG5 P560-011 pILTAB560 Full Length AC1 AC1 from EACMV-UG6 P561-009 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG7 P561-013 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG8 P561-021 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG9 P506-027 pILTAB506 Cry-G5 Nuclear targeted10 P506-036 pILTAB506 Cry-G5 Nuclear targeted11 P505-027 pILTAB505 Cry-G5 Nuclear targeted12 TME14 Nontransgenic None CMD resistant13 TMS 60444 Nontransgenic None Susceptible control14 TMS 30572 Nontransgenic None Resistant control
  17. 17. Transgenic trials for CMD resistanceNBA Permit No.: BA/KARI/G22/6(2) CMDK-1
  18. 18. Transgenic Virus-Resistant Incineration Pit CassavaConfined Field Trial Plot Sketch KARI-Alupe Substation Chain Length = 75m linked fence topped with barbed wire Foot Bath Transgenic and Control Cassava Plants CMD Infected Cassava Plants Gate Guard Row Cassava Plants Width = 60m Guard House Store
  19. 19. • Planting of CMDK-1 at KARI Alupe: 11th May, 2011
  20. 20. CMD incidence CMD Resistance to Transgenic Events 120% 100% p505-027 p506-027 p506-036 80% p560-001CMD Incidence (%) p560-008 p560-011 p561-009 60% p561-013 p561-021 p670-007 40% p670-010 TME14 TMS 30572 20% TMS 60444 0% 1 3 5 7 9 11 13 15 17 19 22 24 26 28 30 32 34 WAP
  21. 21. Disease Severity CMD Resistance to transgenic events 4.50 4.00 p505-027 p506-027 3.50 p506-036 p560-001CMD severity (1-5) p560-008 3.00 p560-011 p561-009 p561-013 2.50 p561-021 p670-007 p670-010 TME14 2.00 TMS 30572 TMS 60444 1.50 1.00 1 5 9 13 17 22 26 30 34 WAP
  22. 22. CMD severity (C-terminal) CMD Resistance to transgenic events 4.00 3.50 3.00CMD severity (1-5) p561-009 p561-013 p561-021 2.50 TME14 TMS 30572 TMS 60444 2.00 1.50 1.00 1 5 9 13 17 22 26 30 34 WAP
  23. 23. CMD1a: Virus Infection 7 MAP ACMV Positive EACMV-UG Posotive 100with ACMV and EACMV-% positive plants infected 90 80 70 60 50 UG 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Lines n = 621 Gene Silencing Lines
  24. 24. 4 lines have been selected and are being tested for stability in the field
  25. 25. CMD1a: Storage Root Yield (Tons/Ha) 31 26.9 7X 23.3 20.5 18.5 16.9 12.8 7.5 4.6 4.4 4 1.2 0.6 0.7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Lines
  26. 26. Cassava brown streak disease (CBSD) CFT• To be conducted in KARI-Mtwapa, Coast region• Application approved by NBA
  27. 27. IncinerationTransgenic Virus-Resistant Cassava Pit Confined Field Trial Plot Sketch KARI-Mtwapa Station Chain linked fence topped with Length = 87m barbed wire Store Guard House Gate Foot Bath Width = 80m
  28. 28. Root severity of CBSD in selected lines
  29. 29. Phase II: Product developmentInitial target of two products – TME204 – Natural resistant to CMD but highly susceptible to CBSD – Ebwanatareka – Susceptible to both CMD and CBSD• Regeneration and transformation of other local popular varieties going on
  30. 30. Product 1 –TME204 modified for resistance to CBSDTarget pathogens - cassava brown streak virus & Uganda cassava brown streak virus Proven efficacious as pILTAB718 in GH grafts and in CFT Lead construct pILTAB5001 CsVMV antisense strand promoter sense strand T-nos 800 bp ΔFL CP 900 kb ΔFL CP 900 kbintron ΔFL CP 900 kb ΔFL CP against against CBSV CBSUV 5.2 kb
  31. 31. CBSD symptom scoring scaleScore Foliar Symptom Description 1 no symptoms on leaves or stems mild/slight vein yellowing or chlorotic blotches on leaves no brown 2 streaks/lesions on green stem portions mild/slight vein yellowing or chlorotic blotches on leaves mild brown 3 streaks/lesions on green stem portions severe/extensive vein yellowing or chlorotic blotches on leaves severe Score Root Symptom Description brown streaks/lesions on green stem 1 no symptoms on storage roots 4 portions no defoliation, stem dieback less than 5% of storage root tissue or stunting 2 is necrotic severe/extensive vein yellowing 5-10% of storage root tissue is 3 or chlorotic blotches on leaves severe necrotic 5 brown streaks/lesions on green stem 10-50% of storage root tissue is portions defoliation, stem dieback or 4 stunting necrotic More than 50% of storage root 5 tissue is necrotic
  32. 32. 2. Broad spectrum VR in Ebwanateraka• Background - Farmer preferred cultivar Ebwanateraka: • Ebwanateraka – previously most widely grown in Uganda – also good fit in Kenya, perhaps throughout Lake Victoria region • Outstanding public pull-through expected due to variety popularity • High yield (potential ~32t/ha when disease-free), sweet, mealy, soft, early maturing (12 months), long-lasting in the field >24 months, desirable upright architecture • Next step in complexity for technical and regulatory development • Broad spectrum control of virus disease complex (CMD & CBSD) • EACMV-UG • ACMV • CBSV • CBSUV • siRNA based resistance
  33. 33. Product #2 DescriptionCMD and CBSD Resistant EbwanaterakaResistance to CMD & CBSD – Engineered resistance to CMD & CBSD based on siRNA technologiesGenetic insert – Single copy insert with no vector backboneDisease resistance – Less than 20% CMD incidence, maximum root CBSD severity score 2Yield – At least 50% yield improvement over non-transgenic control, usable storage root yield of al least 20t/haDuration of effect – resistance to both CMD and CBSD maintained over at least 3 vegetative cyclesResponse to other pathogens – efficacy against CMD & CBSD not compromised by co-infection with CBBResponse to other stresses – virus resistance not adversely affected by drought, temperature extremes, plant nutrition or insect attackAgronomic characteristics – no negative pleiotropic effects on plant vigour, morphology, quantity and quality of storage roots & vegetative propagationMammalian toxicity - no increased toxicity or allergenicity to humans or animals
  34. 34. VIRCA Management and Oversight Structure PI and Director of VIRCA Scientific and Technical Executive Claude Executive Council Fauquet Affairs Director of Product Director of Development and Biosafety and Communication Affairs Anton Paul Regulatory Affairs Bua Anderson in East AfricaImplementation VIRCA Product Development Team Monitoring VIRCA Steering CommitteeCommunication VIRCA Stake Holders Meeting
  35. 35. VIRCA Communication Team KARI NARO/ NACRRI ISAAA- AfriCentre SCIFODE (Champions & (Champions & working groups) working groups)
  36. 36. VIRCA DONORS
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