Agrobacterium-mediated Tobacco Transformation

Thursday, December 04, 2003

Description
Agrobacterium-mediated Tobacco Transformation

Procedure
1. Agrobacterium culture and Harvest:

Agrobacterium overnight culture in YEP
cfg 5000pm, washing in MS, dilution (1/10 of original volum) with MS

2. Tobacco leaf sterilizaion:

70% EtOH 45 sec, 2.5% NaOCl 12 min, Sterile DDW washing 3 times

3. Agrobacterium inocculation:

0.5cm2 -cut Tobacco leaf 竱 upside down栏肺 Agrobacterium soln.俊 5?ETH>埃,
Sterile filter paper肺篡篮饶 MSO1枇隹?48矫埃鞠1杈?br>
4-1. Agrobacterium washing and Shoot induciton:

MS + BAP 1.0mg/L + NAA 0.1mg/L + Carbenicillin 500mg/L + Kanamycin 200mg/L
3 time washing in above liquid MS media (or wipe with filter paper)
Upside down 摹惑 on above solid MS media

4-2. Agrobacterium washing and Callus induciton:

MS + BAP 0.1mg/L + NAA 1.0mg/L + Carbenicillin 500mg/L + Kanamycin 200mg/L
3 time washing in above liquid MS media (or wipe with filter paper)
Upside down 摹惑 on above solid MS media

5. Subculture:

After 2 weeks, onto [MS + BAP 1.0mg/L + NAA 0.1mg/L + Kanamycin 100mg/L]

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Agrobacterium-mediated Tobacco Transformation