Genetic Engineering of Potato

1. Genetic Engineering of Potato PlSc 490 – Potato Science Lecture Joe Kuhl March 27, 2014

2. Overview • Traditional potato modification • Define Genetically Modified • Transformation methods • Agrobacterium, ballistic, advanced methods • Transformation variables • Potato genetic engineering, examples • GM Testing

3. Potato Genetic Manipulation • Wide crosses • Ploidy manipulation • Mutatgenesis • Somaclonal variants • Somatic fusion • Embryo rescue

4. Solanum L. • ~1400 species (largest genus in the Solanaceae) • “Wild” potatoes: ~160 species • Section Petota (Potato) • Subsection Estolonifera (2 Series) • Subsection Potatoe (19 Series) • Includes tomato • Solanum lycopersicum • Diploid to hexaploid (x = 12) • 2x, 3x, 4x, 5x, 6x

5. Wide Crosses - Origin of ABPT 4X – S. acaule (A) x 2x – S. bulbocastanum (B) 3x – AB (4x – AB not useful) Doubling 6x – AB x 2x – S. phureja (P) Bottle next! ±4x – ABP x 4x S. tuberosum (T) 4x – ABPT 5.5x – ABPT x 4x – S. tuberosum 6x – ABPT Hermsen 1985

6. Somatic Fusion • Combining of somatic cells of uncrossable species • Single hybrid cells regenerated in culture Doubled ploidy Subject to somaclonal variation Expensive process

7. GMO = Genetically Modified Organism (also GM)Also genetically engineered (GE), transgenic, cisgenic, or intragenic

8. Genetic Engineering • Transgenic/Cisgenic Crops A transgenic crop plant contains a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination. Define by how a new variety is generated, not by what the variety is.

9. Genetic Engineering • Transgene The inserted gene sequence may come from related or unrelated plant, or from a completely different species. Example: transgenic papaya produces the PRSV coat protein Example: transgenic Bt cotton contains a gene from a bacterium

10. Genes Dr. Joe Kuhl- University of Idaho

11. Gene X

14. Traditional vs. Genetic Engineering • Only genes from closely related species are involved with traditional methods • Traditional methods mixes large sets of genes of mostly unknown function, as opposed to one or a few well-characterized genes with genetic engineering Ronald and Adamchak 2008

15. Organisms Traditional Breeding Genes • Plants • Animal • Microbes • Plants • Animal • Microbes Genetic Engineering Organisms Genes • Plants • Animal • Microbes • Plants • Animal • Microbes

16. Cisgenics Simplot Company: “All potato in potato” • Agrobacterium-based methods that utilize a plant-derived transfer DNA and a novel transient selection system to insert only native DNA into plants • Marker free approach • Selection against backbone incorporation

17. Percentage of respondents that would eat: Extra gene/same vegetable Extra gene/different vegetable Multi genes/different vegetable Animal gene Fungal gene Bacterial gene Viral gene 0 20 40 60 80 100 Lusk and Sullivan (2002) Food Technology

18. Hunter 2014

19. Modification Methods • Biological (DNA transfer) • Agrobacterium (stable vs. transient) • Physical (DNA transfer) – gene gun • Targeted genetic modification

20. Modification Methods • Gene(s) Transfer • Biological • Agrobacterium tumefaciens mediated T-DNA transfer Crown Gall

21. Agrobacterium-mediated Transfer

22. Modification Methods • Gene(s) Transfer • Physical • Particle Bombardment • Microprojectile-mediated

23. Gene Silencing • Exploits plant regulatory mechanism • RNA Interference (RNAi) • Targets specific plant gene(s) • Decrease or eliminate expression • May use siRNA or miRNA • Short-interfering RNA (siRNA) • Micro-RNA (miRNA)

24. Gene Silencing • Exploits plant resistance mechanism • RNA-mediated anti-viral defense • Modify virus vector to carry specific plant gene targets • RNAi (interfering RNA from dsRNA) • siRNA-mediated (~22bp oligonucleotidedimers) • Post-transcriptional gene silencing • Decrease or eliminate expression

25. Silencing of PolyphenolOxidase (PPO) Rommens et al. 2004

26. Targeted Genetic Modification • Engineered nucleases or meganucleases • Create DNA double-stranded breaks at specific genomic locations • This activates DNA repair mechanisms • With or without homologous template • Modify native plant genes in directed and targeted ways • modify endogenous genes

27. Targeted Genetic Modification Novel Restriction Enzymes: • Homing endonucleases • Zinc finger nucleases (ZFNs) • TALE nucleases (TALENs) • Transcription Activator-Like Effectors (TALEs) • CRISPR • Clustered regularly interspaced short palindromic repeats

28. Plant Transformation • Gene(s) Transfer • Integration of transgene(s) into the plant genome Trait

29. Plant Transformation • Variables (for each “event”) • Copy number • Location in the plant cell • Location in the plant genome • Content of transferred genetic information • Resulting phenotype

30. Plant Transformation • Gene(s) Transfer – Plant Breeding and Testing • Desired trait(s) • Activity of the introduced gene • Stable inheritance of the gene • Avoid unintended effects on plant growth, yield, and quality

31. Potato Transformation • Lengthy breeding programs, tetrasomic inheritance, asexually propagated • High in vitro regeneration capacity • Excellent host for Agrobacterium tumefaciens • One of the first crops to be successfully transformed (Ooms et al. 1986), A. rhizogenes • Stiekema et al. 1988, A. tumefaciens

32. Potato Transformation • Ultimate objective – transfer of a gene into an existing cultivar to produce an enhanced version • Silencing – interferes with the operation of the naturally occurring gene, to switch off, reduce activity, or delay natural operation

33. Potato TransformationAgrobacterium-mediated Transfer • Copy number, one or more copies • Diploid regenerates doubled their chromosome number • Some tetraploid regenerates were male-sterile • “Random” insertion • Create large populations of independent transformants

34. Potato TransformationPhenotypic Changes • Off-types: genotype (cultivar) dependent • 15-80% • Field grown • Generate sufficient material and trial under field conditions • “Generally, majority of transgenic material was phenotypically indistinguishable from control plants, and stable over several generations” (S. Millam)

35. Potato Transformation Trait

36. Potato - Commercial Lines • Commercial lines: 1995-2001 • ‘New Leaf’ – Baccillus thuringiensis (Bt) CryIIIA gene, Colorado potato beetle resistance • R. Burbank, Atlantic, Superior • ‘New Leaf Plus’ – Bt resistance plus PLRV resistance • ‘New Leaf Y’ – Bt resistance plus PVY resistance

37. Simplot Generation 1: 2014+ Low acrylamide, low bruise Generation 2: 2016-2017 Cold-sweetening resistance Generation 3: 2018+ Late blight resistance, PVY resistance Target cultivars (initially): Ranger Russet, Russet Burbank, Atlantic, Snowden

38. Current GM Crops

39. Gruskin 2012

40. Marshall 2012

41. Potato - Potential Traits • Disease and pest resistance • Colorado potato beetle (cryIIIA) • Potato tuber moth (cryV, cryIAc9) • Potato cyst nematodes (chicken egg white cystatin) • Viruses, e.g. PLRV and PVY (sense and antisense) • Bacteria and fungi • Erwinia and Phytophthorainfestans

42. Colorado Potato Beetle Resistance • Bt Insect-Resistance • “Bt” short for Bacillus thuringiensis, a soil bacterium whose spores contain a crystalline (Cry) protein • Cry breaks down in insect gut to release a toxin (delta-endotoxin) – toxic to some insects cryIIIA

43. Late Blight Resistance • Katahdin transformed with RB • late blight resistance R gene from S. bulbocastanum Song et al. 2003

44. Useful Traits • Tuber Quality • Anti-bruise (down-regulate PPO) • Reduced glycoalkaloid content (down-regulate Stg1) • Starch • High amylopectin and high amylose • Reducing sugars (over-express ADPglucose pyrophosphorylase)

45. Potato - GM Traits • Gene silencing of vacuolar acid invertase using RNAi Bhaskar et al. 2010

46. Useful Traits • Nutritional value • Inulin (express artichoke genes) • Carotenoids (down-regulate zeaxanthin epoxidase, express Erwinia phytoene synthase) • Pharmaceutical • Vaccines, others…

47. GM Testing GM crops are the most extensively tested crops ever added to the food supply. • GM plants must be shown to shown to be the same as the parent crop from which it was derived • If a new protein trait has been added, the protein must be neither toxic nor allergenic

48. GM Information • Source of the gene • Characterization of the insert • Compositional analysis • Plant toxins, anti-nutrients, and allergens • Unintended up- or down-regulation of critical molecules Of 129 transgenic crops submitted to FDA (1995-2012) - All failed to detect any significant differences, or any believed to have biological relevance (engineered vs. nonengineered or reference species) DeFrancesco 2013

49. NAS 2004

50. American Association for the Advancement of Science – October, 2012 “Indeed, the science is quite clear: crop improvement by the modern molecular techniques of biotechnology is safe.” • European Commission – 2010 report “The main conclusion to be drawn from the efforts of more than 130 research projects, covering a period of more than 25 years of research and involving more than 500 independent research groups, is that biotechnology, and in particular GMOs, are not per se more risky than e.g. conventional plant breeding technologies.”