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

Abstract

Summary: 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 β‐glucuronidase (GUS). A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tume‐faciens 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.