T-DNA integration site analysis in transformed barley (Hordeum vulgare L.) by Tail-PCR


Avrupa Biyoteknoloji Kongresi, İstanbul, Turkey, 1 - 04 September 2011

  • Publication Type: Conference Paper / Summary Text
  • City: İstanbul
  • Country: Turkey
  • Istanbul University Affiliated: Yes


Characterization of flanking sequences of genomic T-DNA insertions
is important not only for functional reverse genetics but also
to understand the nature of transformation by Agrobacterium. In
this study, we have transformed embryo-derived barley cultures
by using EHA105 strain carrying pCAMBIA2301 plasmid coding
for gusA and npt II genes. We have designed specific and arbitrary
degenerate primers in addition to previously reported primer
information. Total twenty-nine seedlings which were phenotypically
resistant to G418 and PCR positive for gusA gene were used
in Tail-PCR amplifications. DNA fragments produced by secondary
and tertiary reactions were evaluated for their size differences and
sequenced after gel purification. As a result, three of the analysed
sequences (total of 28) were found to include flanking chromosomal
barley DNA of T-DNA integrations. BLAST analyses showed
that these separate sites had homology with the ESTs of CV061251,
BU975041 and FD526384 corresponding to coding regions of barley
genome. In conclusion, Tail-PCR is efficient to study transgene integrations
in barley without the need of vector constructions required
for alternative methods such as plasmid rescue