Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers.

Novel 'super-binary' vectors that carried two separate T-DNAs were constructed. One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for beta-glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tumefaciens LBA4404 that carried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47% GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants. Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs. Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants. Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens was also tested, but frequency of co-transformation was relatively low.