INTEGRATION OF T-DNA BINARY VECTOR BACKBONE SEQUENCES INTO THE TOBACCO GENOME - EVIDENCE FOR MULTIPLE COMPLEX PATTERNS OF INTEGRATION

During the process of crown gall tumorigenesis, Agrobacterium tumefaci ens transfers part of the tumor-inducing (Ti) plasmid, the T-DNA, to a plant cell where it eventually becomes stably integrated into the pla nt genome. Directly repeated DNA sequences, called T-DNA borders, defi ne the left and the right ends of the T-DNA. The T-DNA can be physical ly separated from the remainder of the Ti-plasmid, creating a 'binary vector' system; this system is frequently used to generate transgenic plants. Scientists initially thought that only those sequences located between T-DNA left and right borders transferred to the plant. More r ecently, however, several reports have appeared describing the integra tion of the non-T-DNA binary vector 'backbone' sequences into the geno me of transgenic plants. In order to investigate this phenomenon, we c onstructed T-DNA binary vectors containing a nosnptll gene within the T-DNA and a mas2'-gusA (beta-glucuronidase) gene outside the T-DNA bor ders. We regenerated kanamycin-resistant transgenic tobacco plants and analyzed these plants for the expression of the vector-localized gosA gene and for the presence of binary vector backbone sequences. Approx imately one-fifth of the plants expressed detectable GUS activity. PCR analysis indicated that approximately 75% of the plants contained the gusA gene. Southern blot analysis indicated that the vector backbone sequences could integrate into the tobacco genome linked either to the left or to the right T-DNA border. The vector backbone sequences coul d also integrate into the plant genome independently of (unlinked to) the T-DNA. Although we could readily detect T-strands containing the T -DNA within the bacterium, we could not detect T-strands containing on ly the vector backbone sequences or these vector sequences linked to t he T-DNA.