Studies on gene transfer of shoot apical meristems by Agrobacterium-mediated genetic transformation in a progeny of Chinese wild <i>Vitis pseudoreticulata</i>

Abstract

Shoot tips and/or single-bud-internodes, containing shoot apical meristems (SAM) were presented, of Vitis '6-12-2', derived from a Chinese wild V. pseudoreticulata 'Baihe-35-1' × V. vinifera 'Carignane' cross, were used for Agrobacterium-mediated genetic transformation. In order to achieve Glyoxal oxidase (VpGLOX) overexpressing plants, the propagation and gene transfer system of shoot tips and/or single-bud-internodes undergoing either micropropagated (starting with microshoot- tips) or callus induced (starting with stems with single bud) in vitro plants were optimized. The results show that the most effective way to gain shoot tips and/or single-bud-internodes undergoing micropropagation procedure was to keep micro-shoot-tips in liquid C₂D4B medium at 80 rpm constant orbital shaking with light, then placed on solidified C₂D4B medium with 2.9 μM Gibberellic acid 3 (GA3) for elongation. In vitro stems with single buds gave best results for callus formation and adventitious buds induction on half-strength MS medium with 9.0 μM Thidiazuron (TDZ) and 2.9 μM mg·L^-1 α-Naphthaleneacetic acid (NAA). The highest gene transfer frequency was obtained when explants were infected for 10 min with the concentration of Agrobacterium tumefaciens with an optical density at 600 nm of 0.4, and then co-cultivated for 3 days. Incubation of shoot tips and/or single-bud-internodes in darkness for 3 days is helpful for enhancing gene transfer efficiency. Polymerase chain reaction (PCR) and PCR-Southern blot analyses were utilized to confirm putative transgenic plants. Up to 45 clones have proven to be transformed, and one of them has been planted out. This method opens a door for the gene transfer of recalcitrant Chinese wild V. pseudoreticulata.