This protocol was adapted from Clough and Bent (1998). Materials:
YEP medium (optional, see Step 2) (per liter)
10 g of yeast extract 10 g of Bacto-peptone 5 g of NaCl
Adjust pH to 7.0 and autoclave for 30 minutes on liquid cycle.
Infiltration medium (for 1 liter)
l/2x Murashige and Skoog salts (Sigma) lx Gamborg's B5 vitamins (GIBCO) 5% (w/v) sucrose
0.044 \iM benzylamino purine (10 [il/liter of 1 mg/ml stock in DMSO) 50 ^1/liter Silwet L-77 (Lehle Seeds, also known as "Vac-in-Stuff")
CAUTION: DMSO (see Appendix 3) Procedure
1. Three days prior to plant transformation, inoculate a 5-ml liquid culture of Agrobacterium carrying a suitable binary vector and incubate at 28°C with vigorous agitation. Use LB medium containing antibiotics that select for both the Ti and the T-DNA plasmids.
2. After 2 days, inoculate 200 ml of LB medium with 1 ml of the preculture and incubate again with vigorous agitation for a further 24 hours at 28°C.
Use YEP medium for higher Agrobacterium density.
3. Stop watering the plants and allow the soil to dry out a little, so that it will be less prone to falling out of the pots during dipping.
4. Pellet Agrobacterium by centrifuging at 6000 rpm in a GSA rotor (or equivalent) for 10 minutes. If possible, centrifuge at room temperature. Note that the cell pellet is pink.
5. Resuspend the cell pellet in 400 ml of infiltration medium.
6. Transfer the Agrobacterium suspension to a convenient vessel for dipping plants, e.g., a lid from a box of disposable pipette tips or a 400-ml beaker.
7. Invert a pot of plants and dip the inflorescence shoots into the suspension. Rest the pot on the edge of the beaker and allow the plants to soak for -30 seconds. The same suspension can be used for ten or more pots.
Try to avoid contamination of the soil with Agrobacterium, which produces a rather unpleasant smell.
8. After dipping, lay the pots on their sides in a plastic nursery flat, and place a transparent cover over them for the next 24 hours.
9. After 24 hours, remove the cover, rinse the plants with water, and return them to their normal growing conditions.
10. After about 3 weeks, collect seeds.
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