Additional Transformation Topics

1. Additional Transformation Topics

2. Chloroplast Transformation

3. For “containing” transgenes (plastids not transferred through pollen?) • Chloroplast transformation involves homologous recombination • Leaf discs are bombarded with plasmid constructs containing a selectable antibiotic resistance marker physically linked to the gene of interest, flanked by DNA for inserting into the correct site of the chloroplast genome

5. The antibiotic resistance marker most frequently used is the aadA gene encoding resistance for spectinomycin and streptomycin, driven by the promoter of the chloroplast encoded 16S rRNA gene. • This transformation procedure applied to tobacco, Arabidopsis or oil seed rape, generates plants in which all the chloroplast genomes are uniformly transformed (a condition referred to as homoplasmic), despite the fact that tobacco leaf cells may contain 100 chloroplasts, each containing 100 copies of the chloroplast genome.

7. Another advantage of chloroplast transformation is that foreign genes can be over-expressed, due to the high gene copy number, up to 100 000 compared with single-copy nuclear genes. And there does not seem to be gene-silencing and other instability that plague nuclear transformation. The gene product is retained inside the chloroplasts or can in principle be targeted to a specific compartment in the chloroplast.

9. Viruses as a tool in genetic engineering • Enter plant cells via insect carriers, wounding sites, and seeds. • Use host transcription, translation, and replication machinery to express viral genes. • mostly transient, occasionally DNA can integrate into the host genome to become stable transformation • Widely used in research laboratories to study gene function but less applicable in plant biotechnology 9

15. Antisense technology • Transient expression

16. Analysis of Transgenic Plants

17. How do you identify gene of interest?

22. Regeneration on Selective Medium Selectable Marker Gene

28. Bar gene

29. Scorable Marker Gene

30. GUS

34. GFP • Cloned from the jellyfish Aequoria victoria

35. GFP • 27 kDa monomer that fluorescences green under UV (365nm) or blue (490) light

36. GFP • Must be highly expressed

39. Luciferase

40. Phenotype

47. Southern Analysis