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

Overview • Background • History of projects at the institute • Recent projects

– Virus tolerant Ornithogalum – Drought tolerant potato

• Personal comments

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

Biolistics

DNA adhered to tungsten/gold particles

Particle inflow gun

Target tissue: Callus, embryos, meristems,

cell suspensions

GUS staining of transformed cells

Agrobacterium Target tissue: wounded explant Cut into small pieces & pre-culture

Plant regeneration

Infect with Agrobacterium

= co-culture stage

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

Recent projects • Drought tolerant potato • Virus tolerant Ornithogalum

A transgenic approach to improve the drought tolerance of potato

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

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

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

1. Gene isolation and cloning

RT-PCR BP1 potato

PCR A. thaliana

StMYB1R-1 rd29A prom

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

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

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

3. Molecular analysis

D: rd29Ap:StMYB1R-1

Transgenic StMYB1R-1 expression levels D lines: inducible transgenic StMYB1R-1 expression

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

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

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

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

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

Transformation of Ornithogalum

for virus resistance

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

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

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)

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

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

Results Callus

Root Excise

Shoots

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

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

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

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

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

Personal comments 29th International Horticultural Congress (IHC2014), 17-22 August 2014, Brisbane, Australia

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

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

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