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Transgenic plants

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Genetics

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Transgenic plants

  1. 1. Transgenic plants Mohd bin Mahmud @ Mansor, FIAT
  2. 2. Introduction • 25,000 field safety and production trials • 60 genetically engineered plants and animals • 45 countries • 6500 in the USA alone (18,000 sites) • 74% of the soybeans • 71% of the cotton • 32% of the corn
  3. 3. Introduction • Has been practiced for hundreds of years • Slow and uncertain • Sexual cross between two lines, back-cross with offspring and one of the parents until a plant with desired characteristics is obtained. • Limited to only plants that can sexually hybridized
  4. 4. Introduction • Recombinant DNA technology • Offers unlimited possibility • Greater specificity • Genes from incompatible plants, animals, bacteria or insects • Sexual compatibility becomes irrelevant • Process is faster since the gene can be directly selected
  5. 5. Introduction • We will together discuss how foreign genes can be introduced into plant • Using Ti-plasmid system (Agrobacterium tumefaciens) • "Gene gun" • Also some major products of genetic engineering in plants • Production of insect-tolerant and herbicide-tolerant crops
  6. 6. Ti-plasmid System • Ti-plasmid as vector • Agrobacterium tumefaciens is a plant pathogenic soil bacterium • Causes crown gall disease • The key is 200kb circular DNA plasmid - Ti plasmid • During infection, T-DNA is transferred and inserted randomly into the genome of the host plant
  7. 7. Ti-plasmid System • The T-DNA contains genes for the synthesis of: 1. Food for the bacterium (amino acids - opines) 2. Plant hormones (auxin and cytokinin) 3. Vir genes • Both are expressed at high level • Overexpression of hormones leads to?
  8. 8. Ti-plasmid System • Virulence genes: Located at 35kb region on the Ti-plasmid Needed for the production of trans-acting proteins that essential for plant cell transformation 25 genes in 7 operons Switched on by chemicals (acetosyringone) produced by wounded plant cells
  9. 9. Ti-plasmid System • The signals perceived by virA protein, transduced to virG protein • Activating upstream region of other vir genes and activates the expression of the other vir genes • The activation of vir genes will excised T-DNA from the plasmid
  10. 10. Ti-plasmid System
  11. 11. Ti-plasmid System • Flanked by two borders • Sequence called RB and LB • Involve in excision of the T-DNA sequence • Starts at RB follows by a nick at LB as a single strand • Coated by virE proteins (coded by virE gene in vir-regions • Transported into plant cells via membrane channel formed by virB protein
  12. 12. Ti-plasmid System
  13. 13. Ti-plasmid System
  14. 14. Ti-plasmid System
  15. 15. Ti-plasmid System
  16. 16. Ti-plasmid System
  17. 17. The binary vector system • Developed from the concept of Ti-plasmid system • Essential functions for transfer are supplied separately. • Namely? A. ? B. ?
  18. 18. The binary vector system • Neomycin phototransferase II resistance to kanamycin • Contains polylinker Facilitate insertion of the DNA Contains series of unique restriction sites
  19. 19. The binary vector system
  20. 20. The Gene Gun • Using standard bacterial vectors • 1 micrometer in diameter • Tungsten or gold • Coated with DNA (gene of interest + selectable marker) • Fired with a gun with velocities over 400 mps directly into plant tissue of interest • Then transferred to a nurse cell culture plate
  21. 21. The Gene Gun • Major advantages: Possibility of adding a transgene to organelles that have their own DNA genome Need special markers: 1. Spectinomycin 2. Streptomycin
  22. 22. The Gene Gun
  23. 23. Insect resistance: Bt-crops • Bacillus thuringiensis - apore forming soil bacterium • Crystals consists of one or more delta-endotoxins or cry proteins of 130kDal • Upon digestion by larvae, proteases in the insect's gut cleave the protein into an active N-terminal 65-79 kDal • The toxin acts by binding to receptors on the cell membrane of the midgut epithelial cells • Inserts itself into the membrane, forms pores that kill the epithelial cells • Eventually kills the insect by colloid osmotic lysis
  24. 24. Insect resistance: Bt-crops • We have the information, so now what can we do? Discuss.

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