Agrobacterium-mediated transformation of protocorm-like bodies in Cattleya

作者：Zhang Lin; Chin Dong Poh; Mii Masahiro^*

来源：Plant Cell, Tissue and Organ Culture, 2010, 103(1): 41-47.

DOI：10.1007/s11240-010-9751-3

• Agrobacterium tumefaciens
• Cattleya
• Orchid
• Protocorm-like body
• Transformation

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 beta-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 (OD(600) a parts per thousand 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.

• 出版日期2010-10