Challenges and Opportunities in Plant Biotechnology

1. Challenges and Opportunities in Plant Biotechnology Dave A. Vadnais

2. Lecture Outline • Applications in Plant Biotechnology • How Plants are Transformed • Functional Genomics • Recombinant DNA Techniques • Plant Transformation • Tissue Culture • Screening and Isolation of Transformants • Field and Laboratory Testing

3. Lecture Outline (cont.) • Gene Transfer to Commercial Cultivars • Product Commercialization • Plant Transformation Issues • Regulatory and Safety Issues • Societal Issues

4. Applications in Plant Biotechnology • Agronomic trait improvement • improved winter hardiness and stand persistence • insect resistance • pathogen tolerance • herbicide tolerance • nitrogen fixation • drought tolerance

5. Applications in Plant Biotechnology • Nutritional Improvement • increasing genistein content (soybeans) • oil quality (soybeans & canola) • changing amino acid ratios (corn) • forage digestibility (alfalfa) • Improved Breeding • QTL cloning for grain productivity and plant height

6. Applications in Plant Biotechnology • Green Factories • biodegradable plastics from plants • pharmaceuticals from plants • Avian influenza vaccines (alfalfa) • Large scale pharmaceutical protein production • improved bioenergy sources • Biobutanol – closer to gasoline than ethanol

7. Genetically Modified Organisms(GMO’s) • All creatures are genetically modified • Genetic modification is natural • Viruses • Bacteria • Radiation

8. Genetically Modified Organisms(GMO’s) • Methods used by people to genetically modify organisms • Chemical mutagens (Methyl Bromide) • Radiation (gamma radiation) • Genetic engineering

9. DNA to Protein A Short Introduction

10. Primary/Secondary Structure Tertiary Structure Quaternary Structure Protein Structure

11. Three polypeptides make up one multimeric protein Protein Structure

12. GCT AAA CGT TGT TAT AAT TCT sense DNA Ala Lys Arg Cys Tyr Asn Ser Protein CGA TTT GCA ACA ATA TTA AGA antisense GCC AAG CGC UGC UAC AAC UCC mRNA GCU AAA CGU UGU UAU AAU UCU mRNA DNA Code is Degenerate

13. Promoter Exon Intron Exon DNA Transcription Exon Intron Exon RNA From DNA to Protein Poly A

14. Exon Intron Exon Poly A RNA Splicing Exon Exon Poly A mRNA Translation Transit Peptide Protein From DNA to Protein

15. The Plant Cell • Transit peptides direct proteins to organelles • mitochondria • chloroplast • vacuole • cell wall

16. How Plants are Transformed What are the requirements?

17. Functional Genomics • Three major areas of study • Metabolomics • Understanding of products and the metabolic pathways used to their creation • Proteomics • Understanding the role of proteins in the life and regulation of living organisms • Genomics • Understanding the genetic control of various metabolic and proteomic pathways

18. Functional Genomics • Gene identification and isolation • micro-array technologies • cloning • sequencing • knowledge of regulatory elements

19. DNA Micro-Array Technology • Complete organism genome on micro array • Used to determine gene activity • Helps understanding of genetic pathways

20. Recombinant DNA Technologies • Cut DNA from Donor • Paste DNA into plasmid • Insert plasmid into Host Bacterium • Transform Plants

21. Antibiotic Resistance Gene Antibiotic Resistance Gene pVINV Antibiotic Resistance Gene Viral 35S promoter Yeast Invertase Gene Patatin Transit Peptide The Binary Vector • Multiplied in E. coli and Agrobacterium • Modular structure • Parts can be cut and pasted with great precision • Composed of DNA from many sources

22. Bacterial Selection • Bacteria are plated onto selection media • antibiotics • E. coli • DNA isolated for Particle Bombardment • Agrobacterium • Used to infect plant material

23. Plant Transformation • Four methods used today: • Electroporation • Micro-injection • Particle Bombardment * • Agrobacterium *

24. Plant Cell Cytosol Nucleus AgrobacteriumTransformation

25. Biolistic Plant Transformation • Biolistic gun • Patented by Dupont • One of the two main transformation methods used • Used highly compressed air

26. Biolistic Plant Transformation • Uses DNA coated gold or tungsten beads • Compressed air blasts beads into the cells or tissue • Cells take up the foreign DNA

27. Tissue Culture • The Big Picture • Regeneration from single cells

28. Plant Tissue Culture • Somatic Cell Embryogenesis • Cell Suspension Culture • Protoplast Regeneration • Anther Culture • Organogenesis

29. Somatic Embryogenesis • Donor plant and explants

30. Somatic Embryogenesis • Sterilization • Co-cultivation • Bombardment

31. Somatic Embryogenesis • Somatic Embryos Origin

32. Somatic Embryogenesis • Induction

33. Somatic Embryogenesis • Embryo Development

34. Somatic Embryogenesis • Germination

35. Somatic Embryogenesis • Plant Development

36. Screening and Isolation of Transformants • Polymerase Chain Reaction (PCR) • is the gene present? • Southern Analysis • gene copy number • Northern Analysis • gene expression patterns • Enzyme Assays • gene product activity

37. Non-Trans Plasmid Trans 1 Trans 2 Trans 3 Trans 4 Marker Polymerase Chain Reaction(PCR) • Only Foreign DNA targeted • Only specific regions of DNA amplified • Easy and quick to screen 100’s of plants

38. Southern Analysis • Determines gene copy number • Detects only foreign DNA

39. Laboratory Testing of Transgenic Plants • Laboratory and Greenhouse Testing • Is the plant producing the required material? • Is it healthy? • Will it survive?

40. Field Testing of Transgenic Plants • Field Testing • Allows evaluation of the new plant in the real world • Will the plant survive real world stresses? • Is the new trait inherited? • Will the trait transfer to commercial varieties?

41. Plant Biotech Products • Golden Rice • Gene insertion (ProVitamin A and Phytase) • Enhanced beta-carotene (vitamin A) and Iron absorption • Helps prevent Vitamin A deficiency (VAD) and Iron Deficiency Anemia (IDA)

42. Plant Biotech Products • Flavr savr tomato • Gene knockout (antisense expression)

43. Plant Biotech Products • Bt Corn • Gene overexpression (constitutive)

44. Plant Biotech Products • RR Soybean • Gene overexpression (constitutive)

45. Plant Biotech Products • Salt tolerant tomato • Gene insertion • Gives ability to grow on salt contaminated soils • Ability to accumulate salt from the soil Zhang and Blumwald (2001) Nature Biotechnology 19

46. Plant Biotech Products • High Energy Alfalfa • Gene targeting • Yeast invertase to vacuole • using patatin transit peptide

47. Commercialization Issues • Freedom To Operate • Many of the enabling technologies patented • May require permission and/or payment of licensing fees • Can cause many problems in getting the product to the market • Could force researchers to re-invent the wheel

48. Major Concerns About Plant Biotechnology • Imprecise technology • Food and product safety • Cross-species transfer (horizontal gene transfer) • Genetic pollution • New weeds

49. Imprecise Technology • Scientists can now place a single copy of a gene precisely in the genome • Matrix Attachment Regions • More stable expression of transgenes • Cre-Lox and FLP/FRT system • Enables precise placement and removal of transgenes • Antibiotic resistance genes • Herbicide resistance genes • Transgenic trait genes Ow and Srivastava, 2004 Trends in Biotech 22 (12): 627-629

50. Food and Product Safety • Canadian Food Inspection Agency Roles • Regulate transgenic field trials • Ensures field trials conducted in reproductive isolation • Environmental Protection • prevent gene pollution (gene transfer to non-target species) • prevent the environmental release of toxins • prevent the production of Super-weeds