Modified from Frame et al. (2002) Plant Physiology 129:13-22

N6E30 (callus initiation and maintenance):

N6 salts, N6 vitamins, 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 2.76 g L-1 proline, 100 mg/L casein hydrolysate, 30 g L-1 sucrose, 3 g L-1 phytagel, pH 5.8. Microwave-sterilize for about 5 minutes at high power followed by adding silver nitrate (1.7 mg L-1), then microwave again for 1 more minutes or until boiling.

Infection Medium:

N6 salts, N6 vitamins, 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.7g L-1 proline, 68.4 g L-1 sucrose, and 36 g L-1 glucose (pH 5.2). Filter-sterilized acetosyringone (AS, 100 mM) is added prior to use. 200mM AS stocks of AS (dissolved in 100% ethanol) are stored at –20ºC for use as needed.

Co-cultivation Medium (make fresh – use within 2 weeks):

N6 salts and vitamins, 1.5 mg L-1 2,4-D, 0.7 g L-1 L-proline, 30 g L-1 sucrose, 0.5 g L-1 MES and 3 g L-1 phytagel (pH 5.2). Filter sterilized silver nitrate (1.7 mg L-1), AS (100 mM), and cysteine (300 mg L-1) are added to this medium after microwave-sterilizing.

Resting Medium:

N6 salts and vitamins, 1.5 mg L-1 2,4-D, 0.7 g L-1 L-proline, 30 g L-1 sucrose, 0.5 g L-1 MES, and 8 g L-1 purified agar (pH 5.8). Filter sterilized cefotaxime (250 mg L-1), and silver nitrate (1.7 mg L-1) are added to this medium after microwave-sterilizing.

Selection Medium:

N6 salts and vitamins, 1.5 mg L-1 2,4-D, 0.7 g L-1 L-proline, 30 g L-1 sucrose, 0.5 g L-1

MES, and 8 g L-1 purified agar (pH 5.8). Filter sterilized cefotaxime (250 mg L-1), silver nitrate (1.7 mg L-1), and hygromycin (30 mg L-1, Sigma) are added to this medium after microwave-sterilizing.

Regeneration Medium I:

MS salts and N6 vitamins, 60 g L-1 sucrose, no hormones, 0.7 g L-1 L-proline, and 4.5 g L-1 phytagel (pH 5.8). Filter sterilized cefotaxime (250 mg L-1) and hygromycin (20 mg L-1) are added to this medium after microwave-sterilizing.

Regeneration Medium II:

MS Salts and N6 vitamins, 30 g L-1 sucrose, 3 g L-1 phytagel, (pH 5.8). Filter sterilized cefotaxime (250 mg L-1) is added to this medium after microwave-sterilizing.

 Methods:

Agrobacterium Preparation

The vector system, pTOK233 in LBA4404, is maintained on YEP medium containing 50 mg L-1 kanamycin and 50 mg L-1 hygromycin. Bacteria cultures for weekly experiments are initiated from stock plates that are stored for up to one month at 4ºC before being refreshed from long-term, –80ºC glycerol stocks.

Agrobacterium Infection

1. Agrobacterium cultures are grown for 3 days at 23°C on YEP medium amended with 50 mg L-1 hygromycin and 50 mg L-1 kanamycin.

2. One and half loops (3 mm) of bacteria culture is scraped from the 3-day old plate and suspended in 5 ml of liquid infection medium (Inf) supplemented with 100 µM AS (Inf +AS) in a 50 ml falcon tube. The tube is shaken on low speed (~75 rpm) for 30 min at 27°C.

3. For infection, 10-d-old Hi-II calli after subculture are harvested to bacteria-free Inf + AS medium (1.2 ml) in 2 ml eppendorf tubes and washed once with this medium. The wash is removed and 1 to 1.5 ml of Agrobacterium suspension (OD550 = 0.35 to 0.45) is added to the calli. Infection is accomplished by gently inverting the tube 30 times before resting it upright for 5 min. (A short sonication for 30 seconds after inverting may help increase transformation efficiency.)

Co-cultivation

1. After infection, calli are transferred to the co-cultivation medium and excess Agrobacterium suspension is pipetted off the medium surface.

2. Plates are wrapped and incubated in the dark at 23ºC for 3 days.

Resting

1. After 3 days for co-cultivation, embryos are transferred to resting medium at 27ºC in dark for 7 days.

Selection for stable transformation events

1. After 7 days on resting medium, calli, responding or not, are transferred to selection medium containing 20 mg L-1 hygromycin for 2 weeks. They are then sub-cultured for one more 2-week passages on selection medium.

2. Putatively transformed events are visible as rapidly growing Type II callus as early as five weeks after infection.

Regeneration of transgenic plants

1. Regeneration of R0 transgenic plants from Type II embryogenic callus is accomplished

by a two to three-week maturation step on Regeneration Medium I followed by germination in the light on Regeneration Medium II. Acclimatization of regenerated plants to soil is accomplished as described in Frame et al., 2000.