Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
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Transcript of Dr. Douglas Miano - Overview of the Virus Resistant Cassava (VIRCA) Project
Overview of VIRCA Project
Douglas Miano
Biotech Communication Stakeholders Workshop
Hilton Hotel, Nairobi, September 14, 2012
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
Cassava production in the world
Continent Area (ha) Production (‘000 MT)
Yield (t/ha)
% Global 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)
Basic food for > 600 million people in the world
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
Food and Beverage
Starch
Ethanol
Animal Feed
Paper
Textile
CASSAVA Deg. Plastics Wood
Industrial uses of Cassava
Cassava – Strategic Priority for East & Central Africa
Omamo et al. 2006. IFPRI Report 150
Cassava: 2nd most important subsector for growth-inducing development
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
• Cassava brown streak
disease (CBSV) also
becoming a major threat
Disease constraints
Combined annual economic loss of
US$ 180 million in East Africa
Necrotic rot of roots
• 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 susceptible to CMD
CMD resistant TME204
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 disease
• Lack of resistant / tolerant genotypes preferred by farmers
VIRCA project was initiated to combat these virus problems
Way forward?
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)
African cassava mosaic virus (ACMV)
East African cassava mosaic virus (EACMV)
East African cassava mosaic Malawi virus (EACMMV)
East African cassava mosaic Cameroon virus (EACMCV)
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)
Sri Lankan cassava mosaic virus (SLCMV)
Africa
India
East African cassava
mosaic virus cluster
Cassava-infecting begomoviruses
• 9 distinct species, 7 in Africa
• ACMV and EACMV types most prevalent
Mal71 Mal65 Mal63 Mal57 Mal77 Ug46 Mal9 Mal25 Mal58 Ug45 Ug53 Ug52 Mal72 PANG ZANZ
Nampula1-1 Zanzibar7-1
Zanzibar11-1 Zanzibar6-2
CHAKE WETE KOR9 KOR1
Naliendele3-1 Mal17 Mal27
Tan70 Zanzibar8-2 HAND Kibaha10-2
Mo83 Zanzibar13-1
TanZ MLB9
MLB3 Mal18
Kikonde11-5 Kikonde10-1 Shirazi7-1
Nyumbasita5-4 Chumani Mbandar12-1
BSA2 Ug BSA4
Kilifi20-1 Kilifi20-3 Mwalumba16-2 Namulonge
Ke54 Diani3-1
Denyenye1-2 Kilifi18-2 LWR2
U23 HMA9
UgNamEBW Kabanyoro4-3 IGA8
NTG10 Ug16 UG42 Mal10
100
100
5475
100
99
86
100
5761
99100
100
99
99
8699
7694
89
76
67
100
5957
99
5170
78
71
656877
96
96
95
88
76
0.02
Coat protein
CBSV
‘UCBSV’
UgNamEBW NTG10 IGA8 HMA9 LWR2
Ke54 Ke125
BSA2 BSA4
MLB9 TanT8
MLB3 TanC9
ZANZ PANG
KOR9 KOR1
WETE CHAKE
Tan70 Mo83 HAND KenE
TanAB30 TanZ
100
98
8896
100
9955
99
75
100
83
53
100
8796
5097
58
76
62
0.05
HAM1h
Two Distinct Viruses
Main target was CMD
• Coat protein based
• Replication mediated
• G5 protein based
• Defective DNA based
• RNAi (gene silencing)
Phase I: Gene discovery and proof of concept
Xba I
Kpn I
BamH I BstB I
Sense
Anti Sense
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
Cloning in sense & anti-sense into pILTAB 0588
Non-transgenic cassava variety 60444 infected with K201
Transgenic cassava variety 60444 infected with K201
• Transgenic cassava lines showing resistance to EACMV; the effect of RNA-silencing constructs targeting the virus rep gene
• Clone K201 92% homology to EACMV-UG, 80-100% infectivity, faster progression of symptoms
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
Transgenic trials for CMD resistance
NBA Permit No.: BA/KARI/G22/6(2)
CMDK-1
Transgenic Virus-Resistant Cassava
Confined Field Trial Plot Sketch
KARI-Alupe Substation
Transgenic and Control Cassava Plants
CMD Infected Cassava Plants
Guard Row Cassava Plants
Incineration Pit
Gate
Guard House
Store
Chain
linked fence
topped with
barbed wire
Foot
Bath
Width = 60m
Length
= 7
5m
• Planting of CMDK-1 at KARI Alupe: 11th May, 2011
CMD incidence
CMD Resistance to Transgenic Events
0%
20%
40%
60%
80%
100%
120%
1 3 5 7 9 11 13 15 17 19 22 24 26 28 30 32 34
WAP
CM
D In
cid
en
ce
(%
)
p505-027
p506-027
p506-036
p560-001
p560-008
p560-011
p561-009
p561-013
p561-021
p670-007
p670-010
TME14
TMS 30572
TMS 60444
Disease Severity
CMD Resistance to transgenic events
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
1 5 9 13 17 22 26 30 34
WAP
CM
D s
ev
eri
ty (
1-5
)
p505-027
p506-027
p506-036
p560-001
p560-008
p560-011
p561-009
p561-013
p561-021
p670-007
p670-010
TME14
TMS 30572
TMS 60444
CMD severity (C-terminal)
CMD Resistance to transgenic events
1.00
1.50
2.00
2.50
3.00
3.50
4.00
1 5 9 13 17 22 26 30 34
WAP
CM
D s
ev
eri
ty (
1-5
)
p561-009
p561-013
p561-021
TME14
TMS 30572
TMS 60444
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14% p
osi
tive
pla
nts
in
fect
ed
wit
h A
CM
V a
nd
EA
CM
V-
UG
Lines
ACMV Positive EACMV-UG Posotive
CMD1a: Virus Infection 7 MAP
Gene Silencing Lines n = 621
4 lines have been selected and are being tested for stability in the field
CMD1a: Storage Root Yield (Tons/Ha)
7.5
4.6
1.2
4.4
0.6 0.7
18.5 16.9
12.8
31
20.5
26.9
4
23.3
1 2 3 4 5 6 7 8 9 10 11 12 13 14
7X
Lines
Cassava brown streak disease (CBSD) CFT
• To be conducted in KARI-Mtwapa, Coast region
• Application approved by NBA
Transgenic Virus-Resistant Cassava
Confined Field Trial Plot Sketch
KARI-Mtwapa Station
Incineration
Pit
Gate
Guard
House
Store
Chain linked
fence topped with
barbed wire
Foot Bath
Width = 80m
Length
= 8
7m
Root severity of CBSD in selected lines
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
Product 1 –TME204 modified for resistance to CBSD
Target pathogens - cassava brown streak virus & Uganda cassava brown streak virus
against
CBSV
against
CBSUV
pILTAB5001
sense strand
800 bp
intron
CsVMV
promoter antisense strand
5.2 kb
ΔFL CP 900 kb ΔFL CP 900 kb ΔFL CP
Lead construct
T-nos
ΔFL CP 900 kb
Proven efficacious as pILTAB718 in GH grafts and in CFT
Score Foliar Symptom Description
1 no symptoms on leaves or stems
2
mild/slight vein yellowing or chlorotic
blotches on leaves no brown
streaks/lesions on green stem
portions
3
mild/slight vein yellowing or chlorotic
blotches on leaves mild brown
streaks/lesions on green stem
portions
4
severe/extensive vein yellowing
or chlorotic blotches on leaves severe
brown streaks/lesions on green stem
portions no defoliation, stem dieback
or stunting
5
severe/extensive vein yellowing
or chlorotic blotches on leaves severe
brown streaks/lesions on green stem
portions defoliation, stem dieback or
stunting
Score Root Symptom Description
1 no symptoms on storage roots
2 less than 5% of storage root tissue
is necrotic
3 5-10% of storage root tissue is
necrotic
4 10-50% of storage root tissue is
necrotic
5 More than 50% of storage root
tissue is necrotic
CBSD symptom scoring scale
• 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
2. Broad spectrum VR in Ebwanateraka
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
Product #2 Description
Implementation
Executive
VIRCA Management and Oversight Structure
Communication
Monitoring
Anton
Bua
Claude
Fauquet
Paul
Anderson
PI and Director of Scientific and Technical Affairs
Director of Biosafety and Regulatory Affairs
Director of Product Development and
Communication Affairs in East Africa
VIRCA Executive Council
VIRCA Product Development Team
VIRCA Stake Holders Meeting
VIRCA Steering Committee
KARI
SCIFODE
(Champions &
working groups)
NARO/
NACRRI
ISAAA- AfriCentre
(Champions &
working groups)
VIRCA Communication Team
VIRCA DONORS