Suppression of transfer of non-T-DNA 'vector backbone' sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens

Vectors for transformation of higher plants mediated by Agrobacterium tumefaciens were modified so that one, two or three additional copies of the left border (LB) sequences were inserted close to the original LB of the T-DNA. A gene for beta-glucuronidase (gusA) was placed outside the T-DNA to monitor the transfer to plants of 'vector backbone' sequences. The expression of GUS in immature embryos of rice that had been co-cultivated with A. tumefaciens carrying these constructs was around one tenth of that with A. tumefaciens carrying an unmodified control vector. Between 88 and 127 of independent transformants were regenerated from rice tissues infected with A. tumefaciens carrying each of these vectors. The GUS expressors among the rice transformed with the modified vectors were much less frequent than ones among the control transformants, and rate of reduction in the ratio of transgenic plants that expressed GUS was higher than 93%. Detection of a fragment across the LB region by the polymerase chain reaction and the gusA gene by Southern hybridization correlated well with GUS expression. These results indicate that transfer of the 'vector backbone' from the control vectors resulted mainly from inefficient termination of formation of the transfer intermediate of the T-DNA and additional LB sequences effectively suppressed such transfer. This approach is simpler than the strategy to place a 'lethal gene' outside the T-DNA and will likely help produce 'clean' transformants efficiently.