The application of genetic transformation at ARC-VOPI to improve plant traits Dr. Inge Gazendam Regional Plant biotechnology forum 30 October 2014

Transcription

1 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

2 Overview Background History of projects at the institute Recent projects Virus tolerant Ornithogalum Drought tolerant potato Personal comments

3 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

4 Biolistics Target tissue: Callus, embryos, meristems, cell suspensions GUS staining of transformed cells Particle inflow gun DNA adhered to tungsten/gold particles

5 Agrobacterium Target tissue: wounded explant Cut into small pieces & pre-culture Plant regeneration Infect with Agrobacterium = co-culture stage

6 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

7 Drought tolerant potato Recent projects Virus tolerant Ornithogalum

8 A transgenic approach to improve the drought tolerance of potato

9 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

10 Strategy Desiccation stress activate rd29a promoter Stress-inducible promoter StMYB1R-1 TF gene Transcription factor gene activate Gene 1 Gene 2 Gene 3 Gene 4 Gene Downstream drought protective genes A. thaliana S. tuberosum

11 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

12 1. Gene isolation and cloning StMYB1R-1 RT-PCR BP1 potato rd29a prom PCR A. thaliana

13 1. Gene isolation and cloning Plant transformation constructs A pbi121 CaMV 35S GUS B pbi121-rd29ap:gus rd29ap GUS C pbi121-camv:stmyb1r-1 CaMV 35S StMYB1R-1 D pbi121-rd29ap:stmyb1r-1 rd29ap StMYB1R-1 E pbi121-neg GUS

14 2. Plant transformation A B C 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

15 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 Constructs Correct genes M A CaMV prom GUS vector 10 9 B rd29a prom GUS vector C CaMV prom StMYB1R-1 vector D rd29a prom StMYB1R-1 vector M E - GUS vector

16 3. Molecular analysis D: rd29ap:stmyb1r-1 Transgenic StMYB1R-1 expression levels D lines: inducible transgenic StMYB1R-1 expression

17 3. Molecular analysis C: CaMV:StMYB1R-1 D: rd29ap:stmyb1r-1 M BP1 C2 C3 C9 C11 C16 C22 BP1 M BP1 D6 D16 D18 D19 D21 D23 BP copies copies 904 bp copies Southern blot of 12 selected lines

18 Measure: 4. Greenhouse drought trial a) Relative water content (RWC) b) Visual appearance c) Survival after drought stress d) Yield of biomass (tubers & leaves) Control Stress 8 dwow

19 4. Greenhouse drought trial Visual appearance Trial 1 Foliar tissue drooping after 11 days Stress Control BP1 C3 D6 BP1 C3 D6 One representative of each line

20 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

21 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

22 Transformation of Ornithogalum for virus resistance

23 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

24 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

25 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)

26 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 Amp resistance M13F(-20) tml terminator T7 primer pstarling Hairpin 7521 bp OMVCP cre intron Ubi prom & intro OMVCP

27 Methods pcambia1300 plant transformation vector cre intron tml terminator OMVCP T border (R) pvs1 Sta OMVCP pcam1300-rnai OMVCP A bp pvs1-rep Ubi prom & intron pbr322 bom site pbr322 ori PlacZ kanamycin R CaMV35S T border (L) Hygromycin R Agrobacterium-mediated transformation CaMV 3'UTR (polya signal) leaf explants of Ornithogalum A2 Regenerate putative transgenic plants from transformed callus Hygromycin antibiotic selection

28 Results Callus Shoots Root Excise

29 Results PCR screening with OrMV-CP primers 18 lines positive out of 20 screened M pl pc A2 - M M pl pc A2 - M PCR screen results with OrMV-CP specific primers pl+: positive plasmid control pc: pcambia1300 transgenic A2: Untransformed Ornithogalum line A2 - : Negative water control

30 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

31 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

32 Greenhouse efficacy trials Greenhouse trial planted on 30 July replicates of each transgenic line Ready for infection as soon as successful infection method is identified Establishment rate 2 months later = 97% Dripper irrigated pots before planting Hardening off in vitro transgenic Ornithogalum plants Transgenic Ornithogalum plants after 2 months in the greenhouse

33 Way forward To perform virus resistance efficacy trial: Multiplied 18 transgenic events in vitro Have OrMV source Mechanical infection method After virus infection: Track progression of infection with ELISA and visual assessments Yes Yes Optimise Pending Molecular characterisation of transgenic lines: gdna isolation without polysaccharides Southern blot to verify stable integration of OrMV-CP DNA into plant genome Northern blots of sirna expression levels Optimise Pending Pending

34 Personal comments 29 th International Horticultural Congress (IHC2014), August 2014, Brisbane, Australia

35 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

36 Personal comments 1 st generation GMO was to producer benefit Good examples Bt toxin, little collateral damage Alternative to toxic spraying Phytophtora resistant potato GMO 35 genes, 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 Bad example started in 1991, demonstration to farmers present: Hawaii island Puna all papaya are transgenic Roundup Ready Excessive spraying throughout cropping season Residues in food

37 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

38