Efficient production of transgenic plantls of Vanda through sonication-assisted Agrobacterium-mediated transformation of protocorm-like bodies

Abstract

In Vanda orchids, it is important to produce cultivars with economically important traits such as disease and pest resistances and novel flower colors, which are difficult to achieve by conventional cross breeding methods. To realize these breeding objectives, it is now expected to apply genetic transformation technology to introduce useful foreign genes into Vanda orchids. However, there has been almost no information on the genetic transformation of Vanda. Transgenic plants were successfully regenerated after co-cultivating protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLBs of ‘Tokyo Blue’ maintained in liquid New Dogashima medium (NDM) under dark condition, were subjected to transformation experiments. The PLBs inoculated with Agrobacterium produced secondary PLBs 4 weeks after transfer onto 3 g l−1 gellan gum-solidified NDM containing 30 g l−1 maltose, 10 mg l−1 meropenem and 10 mg l−1 hygromycin. Transformation efficiency was increased by prolonged period of infection (240 min) and wounding treatment of PLBs by sonication (5 min) during inoculation. Transformation of the hygromycin-resistant plantlets regenerated from different PLBs was confirmed by histochemical GUS assay, PCR analysis and Southern hybridization. With this transformation system, approximately 17 independent transgenic plants were obtained from 1 g PLBs.