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 | upside down8 Agrobacterium soln.ο 5
Sterile filter paper δ MSO1 48??e 1
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 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 on above solid MS media
5. Subculture:
After 2 weeks, onto [MS + BAP 1.0mg/L + NAA 0.1mg/L + Kanamycin 100mg/L]