Agrobacterium-mediated transformation of Vanda using protocorm-like bodies
Bipna Rani Shrestha1, Dong Poh Chin1, Ken Tokuhara2, Masahiro Mii1*
1Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan
2Research and Development Center, Orchid Santuary Dogashima, Nishina, Nishiizu-cho, Kamo-gun, Shizuoka 410-3514, Japan
*Author for Correspondence.
Laboratory of Plant Cell Technology, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan, E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
Abstract.
Transgenic plants of Vanda were successfully regenerated after co-cultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of ‘Tokyo Blue’ maintained in liquid New Dogashima medium (NDM) supplemented with 0.1 mg l-1 NAA, 1 mg l-1 BA and 30 g l-1 maltose under dark condition, were subjected to transformation experiments. The PLBs infected with Agrobacterium produced secondary PLB 4 weeks after transfer onto 3 g l-1 gellan gum-solidified NDM containing 10 mg l-1 meropenem and 10 mg l-1 hygromycin. Transformation efficiency was increased by prolonged period of inoculation (4 h) with Agrobacterium suspension culture 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.
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