Creating the transgenic plant - Week 4-5
The following procedure will be explained during your lab period.
After the transformed shoots have developed it is necessary to transfer them to a hormone medium which will induce root formation (high auxin:cytokinin).
- Clean the hood with 70% ETOH. Flame sterilize all tools three times.
- Pick off shoots with your sterile tools. They might break away easily, but they might not. If not, cut them off at the base. Take care not to damage the shoot apex and do not include a lot of callus tissue. The shoot apex is the region where leaves are initiated; if damaged, new leaves will not be produced. It is best to pull the shoots by a single leaf or to cut carefully without touching the shoot.
- Transfer 1 genetically modified shoot to each of the 4 corners of a PlantCon box containing MSP2 media with 100 µg/ml ticarcillin and 25 µg/ml kanamycin. Put one control shoot in the center of your Plant Con. Be sure to force the shoots deep into the media.
- Seal the PlantCons with parafilm, label them completely and incubate at 25° C in continuous light.
- Observe your plants regularly. Roots may appear in the first 1-2 weeks but don't be dismayed if it takes longer.