Binary vectors which allow the exchange of plant selectable markers and reporter genes

With the binary vectors pBIG and pBIB it is possible to: i) exchange the plant selectable marker/promoter fusion and the reporter gene, ii) use one vector and Agrobacterium strain system, thus reducing variables associated with using different vectors and strains, iii) allow for divergent transcription of the selectable marker gene and of promoter/reporter gene fusions. The two plasmids are derivatives of the binary vectors pBI 101 and pBIN 19 (1, 2). DNA fragments can be inserted into the T-DNA by the unique restriction sites of a pUC 19 polylinker. The RK2 functions of the plasmids allow the stable maintenance both in E. coli and A. tumefaciens. The two plasmids were constructed by removing the Bam HI site in pBI 101 (1) by filling in with Klenow-polymerase. The resulting plasmid was partially digested with Sst II, blunt ended and subsequently digested with Hind III and isolated on a low melting agarose gel. An Eco RI blunt ended/Bam HI fragment containing the polyadenylation site of gene 7 (3) and a Hind IWBam HI fragment which contains a Nos-promoter/kanamycin and a Nos-promoter/hygromycin gene fusion, respectively, were inserted into the vector, thus producing the vectors pBIG-KAN and pBIG-HYG. The Nos-promoter was taken from pPCV5013 Hyg (4), the neomycin-phosphotransferase (NPT II) gene from pBI 101 (1), and a modified hygromycin B phosphotransferase (HPT) gene was taken from pGDW 11 (5). Subsequently, the GUS-gene was exchanged by the pUC 19 polylinker {Hind WSst I) without a Bam HI site to create pBEB-KAN and pBIB-HYG, respectively. The vectors have been tested by leaf disk transformation of Nicotiana tabacum cv. Petit Havana SRI and were effective in producing resistant plants which produce shoots and roots on MSmedium containing the appropriate antibiotics (kanamycin sulfate 200 /tg/ml; hygromycin B 45 /ig/ml).