Long-term stability of root cultures of tomato transformed with Agrobacterium rhizogenes R1601
Lipp Joao, K.H. | Brown, T.A.
Tomato explants were transformed with Agrobacterium rhizogenes R1601 and root clones used to initiate long-term cultures in liquid and on solid media. The liquid cultures were maintained for 50 passages over 25 months in the presence and absence of kanamycin. During this time the clones retained their high growth rates and antibiotic resistance phenotypes. The nptII gene was detected by PCR and dot blot hybridization in DNA from clones at the 21st passage, and NPT-II enzyme activity was detected in cell extracts prepared at the 45th passage. The solid cultures were maintained for 12 passages over 12 months, either with continual selection, or with alternation during the last six passages between selective and non-selective media. The nptII gene was detected by PCR in DNA from cultures at the 5th passage and NPT-II enzyme activity was detected in cell extracts of vigorously growing clones from the 12th passage. Passages in the absence of selection had no discernible effect on the stability of the transformed state. The results indicate that the Ri-transformed state can be maintained in tomato root clones during long periods of culture.
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