tobacco transformation - (May/04/2012 )
I have been doing this tobacco transformation using leaf disc as explant for quite some time, but i am facing so many problems. Please tissue culture experts help me out.
I have always marked that after washing the leaves cocultivated with agrobacterium, I grow them in selection medium in MS+ BAP+NAA medium with kanamycin (100 mg/l)and cefatoxime (500 mg/l) as antibiotics but I find constant agrobacterium overgrowth for weeks together even if i transfer to fresh selection media.
Please help me as i am not able to understand the situation.
Do you measure the OD of Agrobacterium?
Some points that you have to concerned with are: Adjust the OD of Agrobacterium just on exactly written in the protocol, place the cocultivation dishes in the temperature not over than 25-26 degree Celcius, increase the antibiotic concentration if it still didnt work (but, it could poise to the explant)