VECTORS CARRYING 2 SEPARATE T-DNAS FOR COTRANSFORMATION 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 con structed. 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.) tr ansformants were produced by Agrobacterium tumefaciens LBA4404 that ca rried the vectors. Frequency of co-transformation with the two T-DNAs was greater than 47%. GUS-positive, drug-sensitive progeny were obtain ed from more than half of the co-transformants, Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integr ation and segregation of the T-DNAs. Thus, the non-selectable T-DNA th at 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 f or the production of marker-free transformants of diverse plant specie s. Delivery of two T-DNAs to plants from mixtures of A. tumefaciens wa s also tested, but frequency of cotransformation was relatively low.