Agrobacterium-mediated transformation of protocorm-like bodies in Cattleya

Abstract

Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 were cocultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harboring genes coding for neomycin phosphotransferase II, hygromycin phosphotransferase, and β-glucuronidase (GUS) or plasmid pBBRacdS harboring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. PLBs were maintained in a liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD600 ≈ 0.6) yielding dilution ratios of either 1:10 or 1:40, and these were incubated for either 30 min, 3 h, or 6 h. Hygromycin-resistant secondary PLBs were induced after 4 weeks of culture on an ND medium containing 1.0 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, and solidified with 2.5 g L−1 gellan gum. The highest frequency of transformation was obtained when PLBs were inoculated with 1:10 Agrobacterium liquid culture for 3 h. The transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by histochemical GUS assay, polymerase chain reaction (PCR) analysis, and Southern blot hybridization.