Agrobacterium Culture and Agrobacterium — Mediated transformation

• Infiltration media selection plates
• 5X MS salts 1X MS salts
• 1X B5 vitamins .3% sucrose
• 5% sucrose 8 g TC agar
• 044 mM benzylamino purine
• 03% Silwet L-77
• Autoclave and add appropriate antibiotics (for pCGN add Kan 50 µg/mL; timentin 200 mg/µL).

1. Plant seeds of appropriate genotype on top of cheesecloth-covered soil, making sure the soil fills the pot. Use a rubber band to secure the cheesecloth.
2. After plants have bolted, clip off the primary bolt to encourage the growth of secondary bolts. Perform infiltration 4-8 days after clipping. Vacuum infiltration 1. Grow a large liquid culture of Agrobacterium (such as ASE strain) carrying the appropriate construct. Start a 25 mL overnight (LB+ antibiotics) 2 to 3 days ahead of infiltration. One day prior to infiltration, use this culture to inoculate a 400-mL culture.
3. After 24 hours of growth, cells are usually at a density of at least 1 OD. Harvest cells by centrifugation and resuspend to an OD of .8 in infiltration media.
4. Use a crystallization dish for the actual infiltration—but the pots do not fit exactly, so any ideas/improvements in this area are encouraged. Invert the pot and stick it into the infiltration solution. Put into the vacuum oven at RT. Infiltrate for 10–15 mins at 10–15 in³ Hg.
5. Release vacuum and remove pot, lay it on its side in the tray, and cover it with Saran wrap. Remove the cover the next day and stand upright.
6. The Agro infiltration solution can be reused for multiple pots.

1. Pour selection plates.
2. Sterilize seeds in 50% water/50% bleach/drop of Tween. Rinse 3–4 times with sterile water.
3. Plate seeds by resuspending in room temperature .1% agarose and pipetting onto selection plates. Dry plates in laminar flow hood until set. Use 1 mL agarose for every 500–1000 seeds. Plate 2000–4000 seeds per 150 × 15 mm plate. Put a small number of control seeds from a known transformed plant on one of the plates for comparison.
4. Vernalize plants for 2 nights in cold room. Move plates to growth chamber.
5. After about 7 days, transformants should be clearly visible as dark green plants with roots that extend over and into the selection media.
6. Transfer seedlings to soil.