A binary Agrobacterium vector was developed for efficient plant transformation. This vector utilizes the trans-acting functions of the vir region of a Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene conferring high resistance to kanamycin and a lac alpha-complementing region from M13mpl9 with unique restriction sites for positive selection of inserted DNA. The vector was constructed by modifying the T-DNA region to include only border repeats and a chimaeric nopaline synthase-neomycin phosphotransferase gene. The T-DNA array of left and right borders and selectable marker was ligated into a derivative of the wide host range plasmid pRK252 containing a type III aminoglycoside phosphotransferase for selection in Agrobacterium. The prototype binary vector Bin 6 (15 kb) was obtained, which contains restriction sites within T-DNA for Sal I and EcoRI, an efficient selectable marker gene, and a screenable gene for identifying putative transformants. However, this vector was too large for routine blunt end ligations. It was modified by deleting unwanted sequences, resulting in the truncated T region, which was cloned into a pRK252 derivative. The binary vector Bin 19 was constructed by replacing a 1.6 kb EcoRI T-DNA fragment with a 440 bp Hae II fragment from M13mpl9 containing the alpha-complementary region of beta-galactosidase and an array of restriction enzyme sites. This binary vector is approximately 10 kb in length and is efficiently mobilized by the helper plasmid pRK2013 into A. tumefaciens LBA4404. The vector was tested for its ability to transform plant tissue, resulting in the production of kanamycin-resistant callus. Southern blot analysis confirmed the presence of T-DNA in transformed cells, with tandem or multimeric insertions in plant DNA being common. The efficiency of DNA transfer mediated by a trans configuration of T-DNA and virulence functions was compared to the normal cis configuration. The experiments showed no qualitative difference in the ease of transformation using explanted leaf tissues with Binary vectors compared with "cis" vectors, as transformed tissues were obtainable within two weeks of culture on kanamycin. The vector has several novel advantages, including a large number of restriction sites for positive identification of foreign DNA inserted in the T-region and a relatively small size allowing for efficient ligation of passenger DNA into the multiple cloning site. The vector requires no recombinational steps for integration into the Ti plasmid, making it useful in situations where intrinsically unstable sequences are to be transformed.A binary Agrobacterium vector was developed for efficient plant transformation. This vector utilizes the trans-acting functions of the vir region of a Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene conferring high resistance to kanamycin and a lac alpha-complementing region from M13mpl9 with unique restriction sites for positive selection of inserted DNA. The vector was constructed by modifying the T-DNA region to include only border repeats and a chimaeric nopaline synthase-neomycin phosphotransferase gene. The T-DNA array of left and right borders and selectable marker was ligated into a derivative of the wide host range plasmid pRK252 containing a type III aminoglycoside phosphotransferase for selection in Agrobacterium. The prototype binary vector Bin 6 (15 kb) was obtained, which contains restriction sites within T-DNA for Sal I and EcoRI, an efficient selectable marker gene, and a screenable gene for identifying putative transformants. However, this vector was too large for routine blunt end ligations. It was modified by deleting unwanted sequences, resulting in the truncated T region, which was cloned into a pRK252 derivative. The binary vector Bin 19 was constructed by replacing a 1.6 kb EcoRI T-DNA fragment with a 440 bp Hae II fragment from M13mpl9 containing the alpha-complementary region of beta-galactosidase and an array of restriction enzyme sites. This binary vector is approximately 10 kb in length and is efficiently mobilized by the helper plasmid pRK2013 into A. tumefaciens LBA4404. The vector was tested for its ability to transform plant tissue, resulting in the production of kanamycin-resistant callus. Southern blot analysis confirmed the presence of T-DNA in transformed cells, with tandem or multimeric insertions in plant DNA being common. The efficiency of DNA transfer mediated by a trans configuration of T-DNA and virulence functions was compared to the normal cis configuration. The experiments showed no qualitative difference in the ease of transformation using explanted leaf tissues with Binary vectors compared with "cis" vectors, as transformed tissues were obtainable within two weeks of culture on kanamycin. The vector has several novel advantages, including a large number of restriction sites for positive identification of foreign DNA inserted in the T-region and a relatively small size allowing for efficient ligation of passenger DNA into the multiple cloning site. The vector requires no recombinational steps for integration into the Ti plasmid, making it useful in situations where intrinsically unstable sequences are to be transformed.