The application of genetic transformation at ARC -VOPI to ...Tomato Melon Tolerance to: Fungus Virus...
Transcript of The application of genetic transformation at ARC -VOPI to ...Tomato Melon Tolerance to: Fungus Virus...
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The application of genetic transformation at ARC-VOPI
to improve plant traits
Dr. Inge Gazendam Regional Plant biotechnology forum
30 October 2014 ARC-Roodeplaat, Vegetable and Ornamental Plant Institute, Pretoria
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Overview • Background • History of projects at the institute • Recent projects
– Virus tolerant Ornithogalum – Drought tolerant potato
• Personal comments
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Background • Discovery of tumor inducing principle in Agrobacterium
– Smith and Townsend 1907
• Ti plasmid development – Schell 1974
• Application on model system A. thaliana – Somerville 1994
• Requirements – Tissue culture – Genes – Methods of transfer
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Biolistics
DNA adhered to tungsten/gold particles
Particle inflow gun
Target tissue: Callus, embryos, meristems,
cell suspensions
GUS staining of transformed cells
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Agrobacterium Target tissue: wounded explant Cut into small pieces & pre-culture
Plant regeneration
Infect with Agrobacterium
= co-culture stage
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History of projects Crops Traits Genes
Tobacco Potato Ornithogalum Soybean A. thaliana Sweetpotato Tomato Melon
Tolerance to: Fungus Virus Insects (PTM) Drought Herbicide Delayed ripening
GUS PGIP, Peroxidase PLRV, PVY, TSWV, SPFM, OrMV CryIa1 (Bt) LEA5, P5CR, SOD BASTA Inducible promoters: lupin, GST1
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Recent projects • Drought tolerant potato • Virus tolerant Ornithogalum
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A transgenic approach to improve the drought tolerance of potato
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Objective • Create a more drought tolerant potato through genetic
transformation – Enhance the transcription of drought-protective genes – Use potato’s own TF gene (StMYB1R-1) – Cis-genic approach more readily accepted
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Strategy
rd29A promoter StMYB1R-1 TF gene
Stress-inducible promoter
Transcription factor gene
Gene 1
Gene 2
Gene 3
Gene 4
Gene …
Downstream drought protective genes
activate
activate Desiccation stress
A. thaliana S. tuberosum
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Methodology 1. Gene isolation and cloning 2. Plant transformation 3. Molecular characterisation
1. PCR 2. GUS activity assays 3. RT-qPCR 4. Southern blot
4. Greenhouse drought trial
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1. Gene isolation and cloning
RT-PCR BP1 potato
PCR A. thaliana
StMYB1R-1 rd29A prom
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Plant transformation constructs A
pBI121
B
pBI121-rd29Ap:GUS
C
pBI121-CaMV:StMYB1R-1
D
pBI121-rd29Ap:StMYB1R-1
E
pBI121-neg
rd29Ap
StMYB1R-1
CaMV 35S GUS
rd29Ap StMYB1R-1
CaMV 35S
GUS
GUS
1. Gene isolation and cloning
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2. Plant transformation
Transform BP1 potato by Agrobacterium infection A:Transformed stem explants on selective medium B: Regeneration of shoots from transformed potato callus C: Shoots transferred into rooting medium
A B C
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3. Molecular analysis PCR with 6 different primer combinations • StMYB1R-1, rd29Ap, GUS, vector-specific • 92 plants selected for genomic DNA isolations • 83 lines were found to contain the expected inserted genes
M + 13 14 15 16 17 18 19 20 21 22 23 24
M + 1 2 3 4 5 6 7 8 9 10 11 12 Constructs Correct genes
A CaMV prom GUS vector 10 9
B rd29A prom GUS vector 24 24
C CaMV prom StMYB1R-1 vector 24 19
D rd29A prom StMYB1R-1 vector 24 21
E - GUS vector 10 10
92 83
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3. Molecular analysis
D: rd29Ap:StMYB1R-1
Transgenic StMYB1R-1 expression levels D lines: inducible transgenic StMYB1R-1 expression
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2 5 2 1 4 3 2 1 1 3 2 7 copies
M BP1 C2 C3 C9 C11 C16 C22 BP1 M BP1 D6 D16 D18 D19 D21 D23 BP1 +1 10 copies + 1 10 copies
C: CaMV:StMYB1R-1 D: rd29Ap:StMYB1R-1
904 bp
Southern blot of 12 selected lines
3. Molecular analysis
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4. Greenhouse drought trial • Measure:
a) Relative water content (RWC) b) Visual appearance c) Survival after drought stress d) Yield of biomass (tubers & leaves)
Control Stress 8 dwow
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4. Greenhouse drought trial Visual appearance – Trial 1 • Foliar tissue drooping after 11 days
BP1 C3 D6 BP1 C3 D6
Stress Control
One representative of each line
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Results • Greenhouse trials for improved drought tolerance
– First greenhouse trial: • Three transgenic lines (D6, C3 and D16) perform better under drought
stress than wild-type BP1 • RWC, visual appearance and survival
– Second greenhouse trial: • Confirm RWC% results for only line D6 • Biomass yield difference under drought (fresh and dried leaf and
tubers) between transgenic lines and BP1 was not significant
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Conclusion • Successfully transformed a local cultivar (BP1) with
a potato TF gene – Enhance the transcription of drought-protective genes – Stable insertion into genome and expression of transcript
• Greenhouse trials for evaluating improved drought tolerance – Differences in responses between transgenic lines and ‘BP1’
under drought conditions was not significant
• Same strategy not necessarily successful when applied to different organism and using other gene
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Transformation of Ornithogalum
for virus resistance
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Background: Ornithogalum • Indigenous flower species • Popular for pot plants and cut flowers • Important for the South African flower industry • Problem: highly susceptible to viruses, especially
Ornithogalum mosaic virus (OrMV)
Virus symptoms on leaves Yellow flower of Ornithogalum hybrid A2
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Objective • The release of a transgenic Ornithogalum line with
effective resistance against OrMV • Benefit:
– Economic benefit to the South African cut flower industry
– Use this line to incorporate virus resistance into other susceptible Ornithogalum varieties in a breeding program
– Reduce yield losses of growers – Yield products of higher quality
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Methods • OrMV coat protein and OrMV replicase genes • Virus resistance through gene silencing (RNAi)
– Post-transcriptional silencing (PTGS) – Use OrMV coat protein gene to silence virus gene – Self-complementary hairpin RNA (hpRNA)
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Methods • Gene synthesis and cloning
– Coat protein gene of a South African isolate of OrMV
– Add two pairs of restriction sites during PCR
– pSTARLING-A vector from CSIRO • Commonwealth Scientific and Industrial
Research Organisation, Australia
pSTARLING Hairpin7521 bp
cre introntmL terminatorM13F(-20)
T7 primer
Ubi prom & intro
Amp resistance
OMVCP
OMVCP
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Methods • pCAMBIA1300 plant
transformation vector
• Agrobacterium-mediated transformation – leaf explants of Ornithogalum A2 – Regenerate putative transgenic plants from transformed callus – Hygromycin antibiotic selection
pCAM1300-RNAi OMVCP A13619 bp
kanamycin R
Hygromycin R
cre intron
CaMV 3'UTR (polyA signal)
pVS1 Sta
pBR322 bom site
T border (L)
T border (R)tmL terminator
Ubi prom & intron
CaMV35S
PlacZ
pVS1-REP
pBR322 ori
OMVCP
OMVCP
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Results Callus
Root Excise
Shoots
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Results • PCR screening with OrMV-CP primers
– 18 lines positive out of 20 screened
PCR screen results with OrMV-CP specific primers pl+: positive plasmid control pC: pCAMBIA1300 transgenic A2: Untransformed Ornithogalum line A2 - : Negative water control
M pl+ 1 2 3 4 5 6 7 8 9 10 pC A2 - M
M pl+ 11 12 13 14 15 16 17 18 19 20 pC A2 - M
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Results • Multiplication of the selected transgenic lines
– Between 37 and 154 in vitro plantlets each of 18 individual transgenic lines
Transgenic Ornithogalum plants that are being multiplied in vitro in tubs
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Greenhouse efficacy trials • Require pure OrMV source for virus infection trials
– Screen diseased plants from flower breeding program with RT-PCR – Electron microscopy of virus-infected plant samples – Sequencing of cloned coat protein RT-PCR products
• Mechanical infection method – Very low transmission rates – Symptoms visible only after 8 weeks
Virus symptoms on infected Ornithogalum plant
OrMV successfully transmitted to only 2 out of 30 healthy plants
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Greenhouse efficacy trials • Greenhouse trial planted on 30 July 2014
– 24 replicates of each transgenic line – Ready for infection as soon as successful infection method is
identified – Establishment rate 2 months later = 97%
Hardening off in vitro transgenic Ornithogalum
plants
Transgenic Ornithogalum plants after 2 months in the
greenhouse
Dripper irrigated pots before planting
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Way forward To perform virus resistance efficacy trial: Multiplied 18 transgenic events in vitro Yes Have OrMV source Yes Mechanical infection method Optimise
After virus infection: Track progression of infection with ELISA and visual assessments
Pending
Molecular characterisation of transgenic lines: gDNA isolation without polysaccharides Optimise Southern blot to verify stable integration of OrMV-CP DNA into plant genome
Pending
Northern blots of siRNA expression levels Pending
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Personal comments 29th International Horticultural Congress (IHC2014), 17-22 August 2014, Brisbane, Australia
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Personal comments • Regulatory issues • Red zone: deregulation and commercialization
– Scientists out of their comfort zone – Don’t get anywhere if you listen to what you hear
• Dennis Gonsalves
• Refine technology = new plant breeding techniques – Site-directed nucleases (SDN)
• Introduce foreign stretch of DNA into specific site – Oligonucleotide directed mutagenesis (ODM)
• Repair mismatched oligonucleotide = single mutation at defined site – Genome editing
• TALENs (Transcription activator-like effector nuclease) • CRISPR-Cas (Clustered regularly interspaced palindromic repeat associated proteins)
– Regulatory considerations = GMO or not? – Regulate product and not technology that produced it
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Personal comments • 1st generation GMO was to producer benefit • Good examples
– Bt toxin, little collateral damage • Alternative to toxic spraying
– Phytophtora resistant potato GMO • 35 genes, 10-12 years, durable • Classical breeding: 1 R gene, 45 years, cross with Andes potato lose qualities
– Transgenic papaya resistant to ringspot virus • Dennis Gonsalves (Hawaii) • Pathogen derived resistance,
– vaccinate with PRSV coat protein gene – started in 1991, demonstration to farmers 1997 – 1999 – present: Hawaii island Puna all papaya are transgenic
• Bad example – Roundup Ready
• Excessive spraying throughout cropping season • Residues in food
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Personal comments • Next generation should be to consumer benefit • Relative advantage must be obvious to consumer
– e.g. nutritional value (β-carotene, iron, folate, fatty acid composition) – health benefits (amylose) – sustainability – ornamental (flower and plant architecture) – pest and disease resistance
• Off-putting terms – Genetic, modified, engineered, TALENs, editing, Zinc fingers
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