Agrobacterium Tumefaciens -Mediated Transformation Of Populus Deltoides Leaf Sections

Several factors, including Agrobacterium tumefaciens (At) exposure times and concentrations, were varied in efforts to increase transformation efficiency. Leaf sections of Populus deltoides clone C175, collected from shoot cultures, were inoculated with At strain LBA4404 carrying binary vector pBI121. Included in the vector were the selectable marker gene (NPTII) for kanamycin (K) resistance and the reporter gene (uidA) for betaglucuronidase production. Transformants were identified by selection on medium containing 50 mgK/L and confirmed by histochemical staining for uidA expression. Exposure to At for 120 min proved more effective than shorter times, and elevated concentrations gave more transformants than lower ones. Long exposure times and high concentrations, however, tended to reduce shoot formation. Selection of putative transformants with 50 mgK/L proved workable, but this level clearly inhibited regeneration. The selection process was therefore modified to include culture on nonselective medium for 14 days before transfer to selective medium. This gave higher transformation frequencies than otherwise obtained, apparently a result of transformed calli enlarging and organizing sufficiently to develop on selective medium.

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Author(s): Ronald J. Dinus, Camille J. Stephens, Shujun Chang

Publication: Tree Improvement and Genetics - Southern Forest Tree Improvement Conference - 1995