Development of Tomato Non-Host Tomato Virus through ... - Galvez.pdf* Dolores & Bajet...
Transcript of Development of Tomato Non-Host Tomato Virus through ... - Galvez.pdf* Dolores & Bajet...
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CSC-IPB, College of Agriculture, UP Los Baños
Development of Tomato Non-Host to Tomato
Virus through Targeted Mutagenomics
and Bioinformatics Approaches
HF Galvez, R Schafleitner, AO Canama, RB Quilloy,
DV Lantican, RV Cortes, LL Tandang and BA Kebasen
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CSC-IPB, College of Agriculture, UP Los Baños
(2011 – 2014)
Philippines (DOST)-Taiwan (NSC)Cooperative R&D Project
Philippines: HF Galvez, AO Canama, RB Quilloy, DV Lantican, RV Cortes Crop Science Cluster-Institute of Plant Breeding
College of Agriculture, UP Los Baños
LL Tandang, BA Kebasen IPB-UPLB-BSU Highland Crop Research Station, Benguet State University
TILLING of tomato for multiple virus resistance
Taiwan Counterpart:
R Schafleitner, L Kenyon, P Hanson AVRDC – The World Vegetable Center
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CSC-IPB, College of Agriculture, UP Los Baños
Targeting Induced Local Lesions IN Genome
(TILLING)
Till BJ, SH Reynolds, EA Greene, CA Codomo, LC Enns, JE Johnsons, C Burtner, AR Odden,
K Young, NE Taylor, JG Henikoff, L Comai and S Henikoff. 2003. Large-scale discovery of
induced point mutations with high-throughput TILLING. Gen Res 13:524-530.
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CSC-IPB, College of Agriculture, UP Los Baños
Project Framework EMS- tomato mutants • 600+ M1:2 families (UPLB)
TILLING
Screening against Tomato Viruses
Induced Virus Resistance • Tomato mutants • DNA markers
DNA Marker Development through BIOINFORMATICS • Host R-genes • Virus avirulence gene sequence • Host factors for virus life cycle
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CSC-IPB, College of Agriculture, UP Los Baños
• Amplify mutant + reference DNA • Hybridize DNA hetero- and homoduplex formation
• Digest hetero- with single-strand DNA nuclease
• Fragment analysis
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M1:2 plants
Mutational (molecular) analysis SNPs detection
M2:3 generation of confirmed mutants
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M2:3 disease screening against ToLCV and CMV
Plant Virus
Resistance
and TILLING
Targeting Induced Local Lesions IN
Genome (TILLING)
Generate mutants as series of
variants (allele) for any target gene
Identify plants with point-mutation
at virus resistance gene sequences
Screen confirmed mutants for
resistance to target virus diseases
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CSC-IPB, College of Agriculture, UP Los Baños
• Amplify mutant + reference DNA • Hybridize DNA hetero- and homoduplex formation
• Digest hetero- with single-strand DNA nuclease
• Fragment analysis
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M1:2 plants
Mutational (molecular) analysis SNPs detection
M2:3 generation of confirmed mutants
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M0 generation seed
(Hawaii 7996)
Heterozygous
M1 plants
M2 plants
BWR screening
Collect seeds
Molecular analysis
Mutagen (EMS and gamma ray)Mutagen (EMS and gamma ray)
Phenotyping ofsegregating M2 plants
“Knock-outs” Interesting mutants
M2:3 disease screening against ToLCV and CMV
Year 1 to 2
Grow M1:2 plants; harvest leaf samples for DNA (UPLB)
Develop DNA markers & mutation screen of M1:2 for target R-genome sequences (UPLB & AVRDC)
Year 3
Select confirmed M1:2 mutants; grow M3 families (UPLB & AVRDC)
Screen M3 tomato mutants against ToLCV/CMV strains (UPLB & AVRDC)
UPLB/IAEA Research Project
(Galvez et al., 2009)
600+ M1:2 - EMS chemical mutagen
400 M1:2 – Gamma ray irradiation
1,000+ M1:2 families generated
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CSC-IPB, College of Agriculture, UP Los Baños
Expected Output
Allelic mutants of tomato with induced resistance against ToLCV/CMV (Year 3)
Point-mutations/SNPs as DNA markers tagging the induced virus resistance (Year 3)
Functional genomics characterization of virus resistance mechanism in tomato (Year 2)
Induced vast germplasm of tomato for virus resistance
DNA MARKERS tagging the induced resistance for MAS
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CSC-IPB, College of Agriculture, UP Los Baños
Workplan
Y1 Y2 Y3
Objectives Expected Output Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4
X X X X X
X XX X X X X X
X X X X XX X X X XTo screen for loss-of-
susceptibility mutants of
tomato against
TYLCV/TolCV, CMV
To develop DNA markers
tagging the viral resistance
gene sequences
Allelic variants of tomato
with induced virus
resistance
Molecular characterization
of induced resistance gene
sequences
Functional genomics
characterization of virus
resistance in tomato
Point-mutations and SNPs
as DNA markers tagging the
induced virus resistance
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CSC-IPB, College of Agriculture, UP Los Baños
Just completed generation advance (M3 seeds) of
large tomato M2 families.
Genomic DNAs processed from pooled M2 families;
individual plant DNA extraction on-going.
Initial set of DNA markers available to mine candidate
virus-host factors in tomato. These were designed
using relevant bioinformatics tools.
Accomplishment Highlights
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CSC-IPB, College of Agriculture, UP Los Baños
Next generation sequencing (NGS) of amplified candidate
genes/loci is on-going with 15 DNA markers and using pooled
M2 families
Analysis of NGS results and search for interesting SNPs using
appropriate bioinformatics facility will proceed after NGS.
High-throughput phenotyping identifies interesting visible
mutations.
o High frequency of mutation observed in leaf morphology
and plant habit.
Accomplishment Highlights
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CSC-IPB, College of Agriculture, UP Los Baños
Theoretical Background/
Rationale
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CSC-IPB, College of Agriculture, UP Los Baños
TOMATO (Philippines)
High-value commodity - everyday ingredient of many dishes
Fresh or processed: rich source of vitamins, nutrients & Lycopene
2010 (BAS, Philippines) Tomato
Prod Area (Ha) 17,663 (2nd)
Prod Volume (MT) 204,272 (3rd)
Yield (MT/Ha) 11.56
Value of Production (USD) ($1 = P 42)
50 M
Background and Rationale
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CSC-IPB, College of Agriculture, UP Los Baños
Major Production Constraint : VIRUS DISEASES
Mainly Tomato Leaf Curl Virus (ToLCV) (Sta. Cruz et al. 2008)
ToLCV infection can reach 90-100%, with 50-100% yield loss
(DA-BAR Chronicle, 2010)
Philippine varieties : susceptible to tolerant (Sta. Cruz et al. 2008)
Production USD
($1 = P 42)
Value (BAS, 2010) 50 M
Ave. Yield Reduction (%) 50
Value of Potential Yield 100 M
Losses (virus disease) 50 M
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CSC-IPB, College of Agriculture, UP Los Baños
Monopartite Begomovirus (family Geminiviridae)
DNA-A like genome (circular ssDNA)
C4 and C2 proteins - suppressor of RNA silencing
(PVX-mediated transient assay)
Whitefly vector (Bemisia tabaci Genn.)
– circulative manner
At least 4 strains of ToLCPV
2 in Luzon and all 4 in Mindanao
Tomato leaf curl Philippine virus (ToLCPV)*
* Dolores & Bajet 1995-Philippine J Phytopathol 31; Kon et al. 2002 -J Phytopathol 150; Matsuda et al. 2008 -
Arch. Virol 153; Tsai et al. 2010 –Ann Appl Biol; Sharma et al. 2011–Arch Virol 156
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CSC-IPB, College of Agriculture, UP Los Baños
V1 = CP; V2 = Pre-CP
C1 = Rep
C2 = TrAP
C3 = Ren
C4 = small ORF in Rep, different reading frame
http://www.ncbi.nlm.nih.gov
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CSC-IPB, College of Agriculture, UP Los Baños
Tomato leaf curl Philippines betasatellite (ToLCPHB)*
DNAβ (betasatellite)
Requires helper begomovirus DNA (eg. ToLCPV)
- Replication, Encapsidation
- Insect transmission, Movement in plants
Pathogenecity determinant
- Induce severe symptom – co-inoculated with
ToLCPV (PVX-mediated transient assay)
~ half size & little sequence similarity with ToLCPV DNA
Conserved hairpin structure and a TAATATTAC loop
βC1 protein – suppressor of RNA silencing
* Sharma et al. 2011–Arch Virol 156
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CSC-IPB, College of Agriculture, UP Los Baños
http://www.ncbi.nlm.nih.gov
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CSC-IPB, College of Agriculture, UP Los Baños
Phytosanitation - rouging
Insect vector control- insecticide sprays
Cultural methods
Host plant resistance
Conventional breeding
Induced resistance
Genetic engineering
Virus Management
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CSC-IPB, College of Agriculture, UP Los Baños
Status of Host Plant Resistance and Breeding
Resistance to CMV is not available in tomato germplasm
No resistant lines have been developed yet by conventional
approaches (AVRDC Publication 04-610, 2004)
CMV resistant transgenic lines available but not effective to
Philippine strains of CMV (Galvez et al., unpublished data).
TYLCV resistant or tolerant tomatoes commercially available
BUT NOT EFFECTIVE with Philippine ToLCV
TYLCV and ToLCV – different virus species
(Zitter, 1991; Sta. Cruz et al., 2008; Tsai et al. 2010)
Tolerant tomato varieties for Philippine ToLCV (ToLCPV)
Fresh market-tomato hybrids available in private seed co.
Processing tomato: tolerant variety being bred by NFC-DA
(DA-BAR Chronicle, 2010)
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CSC-IPB, College of Agriculture, UP Los Baños
Characterized resistance genes (Ty loci) – Ty1 to Ty5
Reyes et al. unpublished data:
o Ty1 or Ty2 tomato lines – very susceptible to Phil ToLCV
o Ty3 + unknown R-gene(s) – moderate resistance
AVRDC - different combinations of Ty-1 to Ty-3 (Hanson 2009)
o Reduced symptom severity (TOLERANCE)
o DNA markers and MAS breeding established
o Best Ty2 + Ty3 tomato lines recommended (Hanson 2011)
Efforts continuing to identify and map other resistance
sources/loci (Hanson 2009)
Breeding for broad TYLCV/ToLCV resistance
Also resistance to insect vector
Status of Host Plant Resistance and Breeding
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CSC-IPB, College of Agriculture, UP Los Baños
Development of Tomato Non-Host to
Tomato leaf curl (Philippine) virus
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CSC-IPB, College of Agriculture, UP Los Baños
Virus Resistance Mechanisms in Plants
Kang BC et al., Annu. Rev. Phytopathol. (2005)
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CSC-IPB, College of Agriculture, UP Los Baños
• Viruses require host factors to complete their life cycle
• Absent or mutated host factors may cause recessive virus resistance
Kang et al., 2005
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CSC-IPB, College of Agriculture, UP Los Baños
Whitham S.A. and Wang Y. Curr Opin Plant Biol. (2004)
Summary of host proteins implicated in plant virus infection cycles
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CSC-IPB, College of Agriculture, UP Los Baños
Recessive Virus Resistance Genes
Maule A.J. et al., Molecular plant pathology (2007)
eIF4E eIF(iso)4E
eIF4E eIF4E
eIF4E
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CSC-IPB, College of Agriculture, UP Los Baños
TILLING for Crop Improvement
Crop/Plant sp. Trait Gene Reference Remarks
For Virus Resistance
Tomato potyviruses i.e. PVY, TEV and PepMoV eIF4E1, eIF4E2, eIF(iso)4E, eIF4G, eIF(iso)4G Piron et al. 2010 Immunity to two potyvirues; publish
the different primers designed to
amplify target genes in tomato
Melon MNSV and CVYV eIF4E, eIF4G Nieto et al. 2007
Watermelon potyviruses i.e. ZYMV, PRSV, WMV, SqVYV eIF4E Levi et al. (USDA) 2010 -
http://www.nationalwatermelonassociation.
com
Barley Bymovirus (BaMMV-BaYMV complex)
resistance
Hv-eIF4E (Rym 4/5 Locus) http://194.47.52.113/janlars/partnerskapAlna
rp/ekonf/20091022/SvenGottwalds.pdf
Other Traits
Tomato fruit quality traits, disease & stress response
i.e. necrosis, wilting
RIN, Gr, Rab11a, Exp1, PG, Lcy-b, Lcy-e Minoia et al. 2010 TILLING platform publicly available via
web for mutation screening services
Tomato various potentially all "The Genes That Make Tomatoes" Watanabe et al. 2007 TILLING platform in micro-Tom in SOL
wheat, soybean, maize,
lettuce, tomato, rice,
various various Slade and Knauf 2005
Soybean oil content, protein content, allergen-free various http://www.ars.usda.gov/is/AR/archive/jul0
5/genes0705.htm
Melon fruit shelf life ACC oxidase, L124F Dahmani-Mardas et al. 2010
Wheat waxiness waxy genes i.e. Wx-A1, Wx-D1 Dong et al. 2009
Wheat decreased or absent amylose fraction GBSSI or starch synthase I Slade et al. 2005 in commercial application
Wheat various various Parry et al. 2009
Barley semi-dwarfism/lodging resistance, powdery
mildew resistance, two-rowed spike
sdw1, mlo, HvHox1 (Vrs1 Locus) http://194.47.52.113/janlars/partnerskapAlna
rp/ekonf/20091022/SvenGottwalds.pdf
Barley powdery mildew resistance mlo, mla resistance genes Mejlhede et al. 2006
Africa orphan crop e.g. Tef semi-dwarfism/lodging resistance plant height genes, green revolution genes www.atdforum.org/journal/html/2009-34/5/
Arabidopsis, maize, lotus,
barley, wheat
various various Till et al. 2007 proof-of-concept to production in crop
improvement
various various various Till et al. 2007
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CSC-IPB, College of Agriculture, UP Los Baños
Piron F. et al., PLoS ONE 5(6) 2010
Piron F. et al., PLoS ONE 5(6) 2010
Immune to two potyviruses
Primers designed to
amplify eIF4E1, eIF4E2, eIF(iso)4E, eIF4G, eIF(iso)4G in tomato
Watanabe et al. Plant
Biotech 24(33:38) 2007
TILLING platform in micro-Tom in SOL database
Tomato TILLING
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CSC-IPB, College of Agriculture, UP Los Baños
Tomato leaf curl virus
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CSC-IPB, College of Agriculture, UP Los Baños
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CSC-IPB, College of Agriculture, UP Los Baños Lozano-Duran et al. 2011.
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CSC-IPB, College of Agriculture, UP Los Baños
Lozano-Duran et al. 2011.
Heat shock protein cognate 70 (HSC70)
70% reduced TYLCSV accumulation Coatomer delta subunit (deltaCOP)
100% abolished or 0% virus accumulation
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CSC-IPB, College of Agriculture, UP Los Baños
Candidate gene list for recessive resistance genes Criteria: Demonstrated reduced symptome expression after mutating or repressing the expression of a specific gene
Source: Literature Begomovirus: Lozano-Durán et al 2011, Xie et al 1999, McGarry et al 2003, Carvalho et al 2006, Singh et al 2006
CMV: Yoshi et al 1998, Huh et al 2011
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CSC-IPB, College of Agriculture, UP Los Baños
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CSC-IPB, College of Agriculture, UP Los Baños
DNA Markers through SGN Database and other Bioinformatics Tools
Hayde F. Galvez Genetics Laboratory Crop Science Cluster-Institute of Plant Breeding College of Agriculture University of the Philippines Los Baños
Fulbright Visiting Scholar October 17, 2011 to April 13, 2012
Boyce Thompson Institute for Plant Research
Host Supervisor: Dr. Lukas Mueller (SGN)
DNA Marker Development
(virus-host factors)
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CSC-IPB, College of Agriculture, UP Los Baños
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CSC-IPB, College of Agriculture, UP Los Baños
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CSC-IPB, College of Agriculture, UP Los Baños
AVRDC Training Grant (2 UPLB/Project Staff)
December 2012 (Part-I); February 2013 (Part-II)
AVRDC – The World Vegetable Center, Tainan, Taiwan
Host Supervisor: Dr. Roland Schafleitner
(TILLING in combination with NGS)
Pre-NGS at AVRDC
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Institute of Plant Breeding, College of Agriculture, UP Los Baños
Example:
Pool 1 = 97 M2families
Pool 2 = 50 M2 families
Pool 3 = 47 M2 families
I. PART 1 (December 2012)
M2 Family Genomic DNA Pooling
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Institute of Plant Breeding, College of Agriculture, UP Los Baños
PCR amplification of the target virus-host factor loci for high throughput DNA
sequencing. PCR amplification conditions of fifteen (15) TYLCV
and CMV candidate host factors were optimized.
The expected product sizes were amplified in all
candidate host factors except for AGO1-1, a
candidate host factor for CMV infection
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Institute of Plant Breeding, College of Agriculture, UP Los Baños
PCR amplification 15 TYLCV and CMV
candidate host factors
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Institute of Plant Breeding, College of Agriculture, UP Los Baños
PCR fragments standardized and precipitated
for NGS (lllumina Hi-Seq2000)
New pool 1
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Institute of Plant Breeding, College of Agriculture, UP Los Baños
Sequence data analysis will be done using
appropriate softwares /programs
TILLING experiments
High resolution melting experiments
II. PART 2 (February 2013)
POST NGS, TILLING and HRM
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CSC-IPB, College of Agriculture, UP Los Baños
Generation Advance (M3 Seeds)
of Tomato M2 Families
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CSC-IPB, College of Agriculture, UP Los Baños
Generation advance of tomato mutants
IPB Experimental Field
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CSC-IPB, College of Agriculture, UP Los Baños
Greenhouse - seedlings
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CSC-IPB, College of Agriculture, UP Los Baños
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CSC-IPB, College of Agriculture, UP Los Baños
EMS- tomato mutants • 600+ M1:2 families (UPLB)
TILLING
Screening against Tomato Viruses
Induced Virus Resistance • Tomato mutants • DNA markers
DNA Marker Development through BIOINFORMATICS • Host R-genes • Virus avirulence gene sequence • Host factors for virus life cycle
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CSC-IPB, College of Agriculture, UP Los Baños
Ultimate Outcome
OPTIMUM FRUIT QUALITY of harvest
Full-maturity of crop in the field
Critical especially for processing tomato
Stable supply and market price
Year-round production including dry months with high
population of insect-vector
Increase net income of farmers by ~P 76,600/Ha
Significant savings on pesticide use & associated costs.
Ex-ante economic analysis (Mamaril, 2009)
Potential of P 25.5M to P 180.5M total economic benefits
(evenly distributed among producers & consumers)
Ex-ante economic analysis (Mamaril, 2009)
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CSC-IPB, College of Agriculture, UP Los Baños
Molecular biologists/breeders
Professional and student researchers
Public & private tomato breeding
institutions/seed companies
Eventual COMMERCIALIZATION – release of
ToLCV/TYLCV resistant tomato variety(ies)
Ultimate beneficiaries – tomato farmers
and other industry stakeholders, including
in organic agricultural production
Target Beneficiary
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CSC-IPB, College of Agriculture, UP Los Baños
Thank you!
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CSC-IPB, College of Agriculture, UP Los Baños
Bulacan Ilocos
Pangasinan Nueva Ecija
Baguio
Los Baños
Tomato ToLCV (Tomato leaf curl virus)
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CSC-IPB, College of Agriculture, UP Los Baños
Tomato CMV (Cucumber mosaic virus)
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CSC-IPB, College of Agriculture, UP Los Baños
Fulbright Visiting Scholar Program ( No. G-1-00005)