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 examine your putative transgenic plants. You should have single thriving shoots in the four corners, we hope. Some of your plantlets may not be transgenic and will be selected out by lack of kanamycin resistance (shown as failure to thrive or to develop roots in this medium.) Non-transgenics may have a brown spot where the shoot meets the medium. By now, the truly transgenic shoots should have formed adequate roots (white, fuzzy projections in the medium) and leaves to survive outside of their sterile world and be separated from the non-transgenics so that the non-transgenics are not being protected from selection by the secretion of the NPTII gene product from the transgenics. The control in the center (if you have a control--most already died before transfer to rooting medium) should not have developed roots but should, instead, have a brown spot where the shoot meets the medium showing it's lack of transgenic status. You will make notes about the appearance of the non-transgenics vs. the putative transgenics and evaluate the success of your selection process in your lab notebook. Then discard the non-transgenic plantlets and your plant con in the autoclave bag just outside or inside the potting room.

You will transfer ALL well developed putative transgenic plantlets (transformed with LBA4404/pBI121) found in your plant con. (Well developed means adequate leaves and roots for survival outside of tissue culture.) You will plant ONE plant per 4-inch pot. Don't throw away any thriving experimentals! If you have more than two, check with your classmates to see in case someone else in the class did not get 2 good plantlets and will need to adopt one or more of yours. If no one needs your spares, then pot each of them to soil yourself.

You may wear gloves if you don't want to get your hands dirty but it is not necessary. Grasp the plantlet gently with your fingers and pull using light force. The plantlets should come out easily, but if not, it is ok to dig out with your fingers a small piece of the media around the plantlet. Do not try to get the media off the roots unless the media is moldy. If there is mold on the plantlets you want to use, rinse it after you remove it from the plant con 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 labeled flask. Tamp the soil around the plant with your fingers, pressing down, 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 TG (transgenic). 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 soil in the sink, please use a paper towel to remove it.

Place your plants on a black tray for our lab section. The tray will go back to the growth chamber and be placed on the shelf designated for our lab day.

Please discard your plant con into the orange autoclave bag in (or just outside) 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.