Agrobacterium-mediated transformation using the strain LB4404 harbouring the binary vector pBI121 with the P5CS gene under CaMV 35S promoter and the bar gene as a plant selectable marker was used for transforming grape explants.
Keywords: Abiotic stress; Grape; Genetic transformation; Gene expression; P5CS gene; Proline
For this study it was hypothesized that either it is possible to clone abiotic stress P5CS gene in grapes or P5CS gene is associated with proline accumulation and salt resistance?
Isolation and purification of recombinant plasmid DNA from transformed cells: Agrobacterium tumefaciens strain LBA4404, harbouring the binary vector pBI121, containing the P5CS gene which encodes D1-pyrroline-5-carboxylate synthetase (P5CS) and the bar gene (bialophos herbicide resistant gene) as a selectable marker under the control of the cauliflower mosaic virus 35S promoter (P35S) and NOS terminator were used for transforming grape explants (Fig.
tumefaciens culture preparation: Agrobactrium strain LBA 4404 containing the pB1121 with bar and P5CS genes were applied in this research.
Cloning the P5CS gene: DNA extracted from grapes explant was used to amplify the P5CS gene using specific primers.
Two pairs of specific primers were used to identify transformed plants, the first was designed to amplify 540 bp of the bar gene (namely PI, 5'-AAA AGC TTC CAC CAT GAG CCC AGA ACG ACG-3' and P2, 5'-AAG GAT CCT TAG ATC TCG GTG ACG G') the second pair was designed to amplify the P5CS gene (namely P3, 5'-TAC TGA GAC TGT GAA GTA GCC T-3' and P4, 5-' ATG GCA TTG CAG GCT GCC GTT A -3').
Leaf painting assays: As plasmid pBI121 contains the P5CS gene, which encodes D1-pyrroline-5-carboxylate synthetase (P5CS) and bar gene (herbicide resistant gene) as selectable marker, putative transgenic grape (Vitis vinifera) plants were treated with the recommended concentration of Basta herbicide (2.