1. Genetic Engineering of Plants BIT 220 End of Chapter 22

3. Why Create Transgenic Plants? 1. Improve agricultural value of plant increase yield (herbicide-resistance, pest-resistance) enhance nutrition enhance taste 2. Plants can produce proteins for human needs (pharmaceutics) 3. Modified plants can be used to study effects of genes An entire plant can be regenerated from a single cell ***TOTIPOTENCY*** No separation of germ and somatic cells

4. How to get a gene into a plant TRANSFORMATION 1. Agrobacterium tumefaciens Figure 22.15 2. Microprojectile Bombardment (biolistics) 3. Electroporation burst of electricity

5. Agrobacterium tumefaciens Soil Plant pathogen Causes tumors- crown galls Contain Ti plasmid Only infects Dicots 1. Wounded plant 2. A. tumerfaciens attached to plant at wound site (often at base of stem -Crown) 3. Wounded plant secretes phenolic compounds 4. These compounds activate virulence genes of A. tumefaciens

6. Fig 22.17 Transformation of plant cells by Agrobacterium tumefaciens harboring a wild-type Ti plasmid. © 2003 John Wiley and Sons Publishers

7. Fig 22.16 Structure of the nopaline Ti plasmid pTi C58, showing selected components. © 2003 John Wiley and Sons Publishers

8. Ti Plasmid FIGURE 22.16 1. T-DNA (Transferred DNA) transferred and expressed into plant causing tumor (crown gall) formation 2. Virulence Genes essential for the transfer and integration of the T-DNA 3. ori 4. Noc (Opine Catabolism) genes (other microbes do NOT have)

9. Genes within T-DNA 1. Enzymes to produce Auxin (iaa) 2. Enzyme to produce cytokinins 3. Genes for synthesis of Opines - carbon source for bacteria no use in plants 4. Tum -genes responsible for tumor formation

11. Engineered Ti plasmid 1. Clone foreign gene into T-DNA 2. Delete genes responsible for tumor formation 3. Add selectable marker

12. Infection of Plant Cell Continued Figure 22.17 1. Bacteria attaches to host 2. Virulence gene expression activated by compounds secreted by wounds 3. T-DNA is transferred 4. T-DNA integrates into host (plant) cell T-DNA is cut out of plasmid at left and right border Right border- responsible for integration 5. Other T-DNA genes are activated causing tumor formation

13. Biolistics Microprojectile Bombardment Coat gold or tungsten spheres with DNA Shot with particle gun through cell wall of plant cells Best to use small plasmids (<10kb) linear DNA (integration more efficient) Advantage: large range of plant species can be engineered

14. Delivery of Gene to the Chloroplast Can also integrate foreign DNA into the smaller chloroplast DNA Use Selectable marker Plus Foreign Gene on A. Single vector B. Dual Vector Advantage: Two genes integrate at separate portions of chloroplast DNA Does NOT interrupt essential chloroplast genes

15. Single Vector Double Vector

16. Reporter Genes/Selectable Markers Neomycin Phosphotransferase gene Resistance to Kanamycin GUS B-glucuronidase enzyme which converts colorless substrate to blue product Luciferase (firefly) ‘glows in dark’

17. Agricultural Biotechnology $67 billion/year industry FOOD LOSS One-third of world’s crops are lost to insects, disease and weeds HERBICIDE- RESISTANT PLANTS Reduce Cost, Labor, Environmental Stress 606 million pounds of herbicide/yr in US

18. Herbicide-Resistance Roundup Monsanto Glyphosate - inhibits the enzyme necessary for amino acid production KILLING WEEDS By modifying the ‘crop’ gene for amino acid production ROUNDUP has less affinity for enzyme Crops are resistance Disadvantage: Kills spiders, earthworms and fish Other Considerations: Cross Pollination -Resistant weeds? Will herbicide application really be reduced?

19. Other Apps of Plant Biotech 1. Vaccinations for Fungal infections 2. Freezing Tolerance 2 o C change in Canada can double output of grapes Wild-type Bacteria protein coat stimulates ice crystal formation on infected plants Ice-Minus Bacteria NO gene for this coat protein; lowers freezing pt Coat protein is used for snow production on ski slopes 3. Canola oil source of hirudin- anticoagulant