III. Transformation of plants.
24. Once the cultures have grown up, take 1/2 mL from each culture and add that to 500 mL of LB liquid media with all antibiotics used in previous step. Grow the cultures in a floor shaker at 28°C overnight.
25. Transfer the contents of the cultures to centrifuge bottles and spin them down. NOTE: When using a centrifuge, balance the volume in the bottles to prevent the rotor from warping.
26. Decant and pippette off the supernatant.
27. Resuspend the pellets in 500mL of Plant Transformation Medium by swirling the medium in the bottles or letting them sit. DO NOT pipette, shake, or vortex to resuspend. Use the following recipe for Plant Transformation Medium:
|
Sucrose
MS Salt
MES
Benzylaminopurine
MS Vitamin
SILWET
ddH2O
|
1L
50 grams
2.2 grams
0.5 grams
10µL
1mL
200µL
1 Liter |
2L
100 grams
4.4 grams
1 gram
20µL
2mL
400µL
2 Liter | **Adjust the final pH of the medium to 5.8
NOTE: This solution does not ned to be autoclaved. It should be prepared just before it is used.
28. Once the transformed Agro has been resuspended, in the Plant Transformation Medium, it can be stored at 4°C for an extended period of time. The Agro is now ready to transform plants. Choosing the plants at the right developmental stage is important for increasing transformation efficiency. For Arabidopsis, allow the plants to bolt. As they begin to flower, cut the main inflorescence. This will cause the plant to produce more inflorescences which means more buds that can be transformed. The plants are ready for transformation with Agro once the plant has many buds, but before it has started flowering.
29. There are two ways to transform Arabidopsis plants. 1) Dip the plant in the Plant Transformation Medium. Simply take the enture pot, use one hand to prevent soil from falling out of the pot, and submerge the entire plant in a container filled with the transformed Agro Plant Transformation Medium. Leave the plant submerged for approximately 1 minute. Let the plants dry for a few hours before returning them to the growth chamber. Dip the plants every 3 days, do this twice more so that the plants are dipped a total of 3 times. 2) Spray the plants with the Plant Transformation Medium. Fill a bottle with the transformed Agro Plant Transformation Medium and attach a spray nozzle. Spray the plants copiously and return them to the growth chamber. Spray the plants every 3 days; do this twice more so that the plants are sprated a total of 3 times.
30. Let the transformed plants set seeds. Avoid over watering as mold will grow due to the sugar-rich transformation medium. Collect seeds, dessicate and plate them on appropriate selection medium to obtain transformants. |
