BISC 219/2009: Mod 3 Experiment 1 Creating the transgenic plant Week 8

Creating the transgenic plant - Week 7-8

In approximately 3-4 weeks after the transfer to rooting media the transformed plants will be large enough for transfer to soil and for assay of GUS activity and PCR detection of the introduced DNA.

Directions for Planting Plantlets to Soil

Take your plant con to the potting room. This little room is off to the left as you enter from the main hallway into the instrumentation room (SC Rm 308).

Take two 4 inch green plastic pots found on the bench and fill them with moistened grow mix found in the white buckets on the floor. If the soil appears dry you may add deionized water from the flask on the bench. Do not use tap water and do not saturate the soil with water.

Open your Plant Con and plant the two most developed tobacco plantlets of the four experimentals (LBA4404/pBI121) found in each of the four corners of your plant con. You will plant ONE plant per 4-inch pot. Before you throw away any extra experimentals check with your instructor in case someone else in the class did not get 2 good plantlets and will need to “borrow”.

Your control plant (in the center of your plant con) should not have developed and will be discarded today. Make notes about its appearance. If you do have a nicely developed control with no browning around the root area, you may plant it too in a separate well-labeled pot.

Grasp the plants gently with your fingers and pull using light force. The plantlets should come out easily, but if not, it is ok to break off a small piece of the media and plant that with the plant. Do not try to get the media off the roots unless the media is moldy. If there is mold or heavy bacterial growth on the plantlets you want to use, rinse the whole surface of the plant con and the plant well with tap water before planting.

Make a depression in the center of the soil with your thumb and place your plantlet into the depression. Pack soil around the plant, place the pot on a tray over the sink, and water your newly planted plantlets with deionized water from the flask. Tamp the soil around the plant gently with your fingers, pressing down slightly, so that the root hairs get good soil contact and the plant does NOT float to the top when you water it.

Place the top of your plant con over one pot and use another top (found on the bench) to cover your other plant. DO NOT seal with parafilm. It is time for the plants to come out of their sterile world.

Label your pots with colored tape that is clearly marked with your initials, lab day, team color, date, and the designation experimental or control. Do not label the clear plastic tops as we will remove these as soon as we wean the plants off high humidity.

Please leave the potting area as clean as you found it, or cleaner. Do not allow soil to go down the sink. If there is a lot of soil in the sink, please use a paper towel to remove it.

Take your plants back to the growth chamber and place them on the shelf designated for our lab day.

Please discard your original MSP2 plates and your plant con into the orange autoclave bag in the potting room. Do not put green plastic pots or soil in the autoclave bag. If the bag seems full, use one of the spares on the bench.