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AGROBACTERIUM VIR GENE INDUCTION PROTOCOL

1.    Grow Agrobacterium at 30^oC overnight in 5 ml rich medium (such as LB or YEP) containing the appropriate antibiotics.

2.     Dilute approximately 0.5 ml of the culture into 50 ml AB-sucrose minimal medium containing the appropriate antibiotics. Grow overnight at 30^oC until the bacteria are in late-log phase (Klett=100 on a Klett-Somerson Spectrophotometer, red filter).

3.    Spin down the bacteria. Resuspend in two volumes of induction medium containing 100 uM acetosyringone. Shake (very gently) 14-24 hours at room temperature (not at 30^oC).

4.    Spin down the bacteria. Resuspend in MS plant tissue culture medium. Inoculate plants.

5.    After two days, rinse the plant tissue in medium containing 100 ug/ml timentin. Continue incubating the tissue on solidified medium containing timentin.

6.    Stain the tissues after various periods of time (2-10 days) in X-gluc.

AB Medium:

   20x AB Buffer:

K₂HPO₄               60 g/l

NaH₂PO₄             20 g/l

Autoclave separately

20x AB Salts:

NH₄Cl                   20 g/l

MgSO₄.7H₂O         6 g/l

KCl                        3 g/l

CaCl₂                   0.2 g/l

FeSO₄.7H₂O         50 mg/l

pH to 7 before autoclaving

Combine 50 ml AB Buffer and 50 ml AB Salts with 900 ml sucrose-water (final concentration of sucrose in one liter is 0.5%).

Induction medium:

1x AB salts

2 mM NaPO₄

50 mM MES, pH 5.6

0.5% glucose

100 uM acetosyringone

This work is made possible by a grant from the National Science Foundation's Plant Genome Research Program.

Questions or Comments regarding this web site should be addressed to Dr. Stanton Gelvin