Creating the Transgenic Plants - Days 3-5
To be done outside of class but procedure will be demonstrated during your lab period.
After several days of incubation the Agrobacterium will have transferred the
T-DNA into the tobacco cells at the cut surface of the leaf pieces. The transferred DNA will be stably integrated into the tobacco cell chromosomes.
At this point it is necessary to:
a. kill the Agrobacteria with ticarcillin
b. select for growth of the transformed plant cells with kanamycin
c. promote the development of the transformed cells into shoots by growth on media with high levels of cytokinin to auxin.
- Clean the hood with 70% ETOH. Flame sterilize all tools three times.
- Transfer the leaf pieces into MS4 plates containing 100 µ g/ml ticarcillin and 50µ g/ml kanamycin.
- Seal with parafilm and incubate at 25°C under continuous light for approximately 1 month.
- Observe your plates weekly. You will initially see the formation of transformed callus along the edges of the leaf pieces. After 3-4 weeks shoots will be present.
Record your obsevations in your lab notebook. Note condition of each explant at least once or twice a week.