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).

1. Overview of VIRCA Project
Douglas Miano
Biotech Communication Stakeholders Workshop
Hilton Hotel, Nairobi, September 14, 2012

2. VIRCA PARTNERS
Donald 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. Cassava production in the world
Basic 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 10.94 100
Source: FAO (2005)

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. Cassava – Strategic Priority for East & Central Africa
Cassava: 2nd most
important subsector
for growth-inducing
development
Omamo et al. 2006.
IFPRI Report 150

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. 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; $6-25 billion/year)
• Cassava brown streak
disease (CBSV) also
becoming a major threat
Combined annual economic loss of
US$ 180 million in East Africa Necrotic rot of roots

8. Cassava susceptible to CMD
CMD resistant TME204

11. 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 farmers
VIRCA project was initiated to combat these virus
problems

12. 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)

13. 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)

14. 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 Kilifi20-3
HAM1h
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

15. 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)

16. Cloning in sense & anti-sense into pILTAB 0588
Anti Sense
BamH I BstB I
Kpn I
Sense
Xba I
All the constructs were initially made in pILTAB-0588 vector carrying
the Cassava Vein Mosaic Virus & 35S promoters and NOS poly A
sequence using Xba I & Kpn I and BamH I & BstB I restriction enzymes

17. • Transgenic cassava lines showing resistance to EACMV;
the effect of RNA-silencing constructs targeting the virus
rep gene
Non-transgenic cassava variety Transgenic cassava variety
60444 infected with K201 60444 infected with K201
• Clone K201 92% homology to EACMV-UG, 80-100% infectivity,
faster progression of symptoms

18. Entries being evaluated in the CFT
Entry Line Vector Gene of interest Comments
1 P670-007 pILTAB670 AC2 AC2 from EACMV-UG
2 P670-010 pILTAB670 AC2 AC2 from EACMV-UG
3 P560-001 pILTAB560 Full Length AC1 AC1 from EACMV-UG
4 P560-008 pILTAB560 Full Length AC1 AC1 from EACMV-UG
5 P560-011 pILTAB560 Full Length AC1 AC1 from EACMV-UG
6 P561-009 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG
7 P561-013 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG
8 P561-021 pILTAB561 C-Terminal AC1 AC1 from EACMV-UG
9 P506-027 pILTAB506 Cry-G5 Nuclear targeted
10 P506-036 pILTAB506 Cry-G5 Nuclear targeted
11 P505-027 pILTAB505 Cry-G5 Nuclear targeted
12 TME14 Nontransgenic None CMD resistant
13 TMS 60444 Nontransgenic None Susceptible control
14 TMS 30572 Nontransgenic None Resistant control

19. Transgenic trials for CMD resistance
NBA Permit No.: BA/KARI/G22/6(2)
CMDK-1

21. Transgenic Virus-Resistant Incineration Pit
Cassava
Confined 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

22. • Planting of CMDK-1 at KARI Alupe: 11th May, 2011

29. CMD incidence
CMD Resistance to Transgenic Events
120%
100% p505-027
p506-027
p506-036
80% p560-001
CMD 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

30. Disease Severity
CMD Resistance to transgenic events
4.50
4.00
p505-027
p506-027
3.50 p506-036
p560-001
CMD 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

31. CMD severity (C-terminal)
CMD Resistance to transgenic events
4.00
3.50
3.00
CMD 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

32. CMD1a: Virus Infection 7 MAP
ACMV Positive EACMV-UG Posotive
100
with 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

33. 4 lines have been selected and are being tested for stability in the field

34. 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

35. Cassava brown streak disease (CBSD) CFT
• To be conducted in KARI-Mtwapa, Coast
region
• Application approved by NBA

36. Incineration
Transgenic 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

37. Root severity of CBSD in selected lines

38. Phase II: Product development
Initial 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

39. Product 1 –TME204 modified for resistance to CBSD
Target 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

40. CBSD symptom scoring scale
Score 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

41. 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

42. Product #2 Description
CMD and CBSD Resistant Ebwanateraka
Resistance to CMD & CBSD – Engineered resistance to CMD & CBSD based
on siRNA technologies
Genetic insert – Single copy insert with no vector backbone
Disease resistance – Less than 20% CMD incidence, maximum root CBSD
severity score 2
Yield – At least 50% yield improvement over non-transgenic control, usable
storage root yield of al least 20t/ha
Duration of effect – resistance to both CMD and CBSD maintained over at
least 3 vegetative cycles
Response to other pathogens – efficacy against CMD & CBSD not
compromised by co-infection with CBB
Response to other stresses – virus resistance not adversely affected by
drought, temperature extremes, plant nutrition or insect attack
Agronomic characteristics – no negative pleiotropic effects on plant vigour,
morphology, quantity and quality of storage roots & vegetative
propagation
Mammalian toxicity - no increased toxicity or allergenicity to humans or
animals

44. 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 Africa
Implementation VIRCA Product Development Team
Monitoring VIRCA Steering Committee
Communication VIRCA Stake Holders Meeting

45. VIRCA Communication Team
KARI NARO/
NACRRI
ISAAA- AfriCentre SCIFODE
(Champions & (Champions &
working groups) working groups)

46. VIRCA DONORS