Agrobacterium-mediated
Transformation of tobacco

Laboratory #4

Leaf tissues of tobacco will be exposed to Agrobacterium tumefaciens containing genes for GFP and hygromycin resistance for 2 days co-culture. Leaf tissues will then be placed on a medium containing Timentin to halt the growth of bacterium and 50 mg/l hygromycin to select for transformed shoots.

 Murashige and Skoog salts
Gamborg B5 vitamins
3% sucrose
1 mg/l BA

FOR 1.5 LITER
15 ml    MS FeEDTA  add first
15 ml    MS Sulfates
15 ml    Halides
15 ml    P, B, Mo
15 ml    B5 Vitamins
2.5 g    NH4NO3
2.85 g  KNO3
45 g sucrose
1.5 ml (1 mg/ml) BA
volume up to 1500 ml

pH to 5.7

Co-culture medium

500 ml
No addition
1 g Gelrite

Selection medium

1 L
No addition
2 g Gelrite

After Autoclaving, add
50 mg hygromycin N (1 ml of 100 mg/l stock)
400 mg Timentin (400 mg/l ticarcillin, 80 mg/l clavulanic acid, in 5-10 ml of water, filter sterilized directly into medium)

 Selection medium is the same as co-culture medium but containing 50 mg/l hygromycin and 400 mg/l Timentin (400 mg/l ticarcillin, 80 mg/l clavulanic acid).

Leaf tissues from tobacco plants grown in the greenhouses will be excised, surface sterilized (15% bleach for 15 minutes) and dipped into a prepared overnight culture of Agrobacterium grown in LB. Agrobacterium will be centrifuged and resuspended in OMS medium. Leaf tissues will be blotted on filter paper to remove the excess bacteria and plated on co-culture medium for 2 days. On Thursday, leaf tissue will be transferred to selection medium until shoots appear. Shoots will be periodically assayed for GFP expression. Control leaf tissues will not be dipped in Agrobacterium but will be treated as co-cultured tissues.


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