Agrobacterium-mediated genetic transformation of Cattleya with an Odontoglossum ringspot virus replicase gene sequence

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Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 cultured either under constant illumination with cool-white-fluorescent lamps or in the dark were cocultivated with Agrobacterium tumefaciens strain EHA105 carrying plasmid pSMAHdN627-ORSV harboring genes coding for Odontoglossum ringspot virus (ORSV) replicase and hygromycin phosphotransferase. PLBs were maintained in liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD600≈0.6) yielding medium dilution ratio of 1 : 10 and incubated for either 30 minutes or 3 h. Hygromycin-resistant secondary PLBs were induced after 4 weeks of culture on 2.5 g l−1 gellan gum-solidified ND medium containing 1 mg l−1 naphthaleneacetic acid (NAA), 0.1 mg l−1 benzyladenine (BA), 10 mg l−1 hygromycin, 20 mg l−1 meropenem, 10 g l−1 sucrose in both light- and dark-cultured PLBs. The number of resistant PLBs generated using dark-cultured PLBs was higher than those cultured under constant illumination. Presence of acetosyringone (AS) in the pre-culture medium was also effective for the transformation. The highest frequency of transformation was obtained when dark-cultured PLBs were pre-cultured with 100 μM AS for 3 days and inoculated with Agrobacterium liquid culture for 3 h. Transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by Southern blot hybridization. Transcription of ORSV replicase gene in transgenic lines was successfully confirmed by Northern blot hybridization.

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