Me. Kononov et al., INTEGRATION OF T-DNA BINARY VECTOR BACKBONE SEQUENCES INTO THE TOBACCO GENOME - EVIDENCE FOR MULTIPLE COMPLEX PATTERNS OF INTEGRATION, Plant journal, 11(5), 1997, pp. 945-957
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.