Introduction of Odontoglossum ringspot virus Coat Protein Gene into Cymbidium niveo-marginatum mediated by Agrobacterium tumefaciens to Produce Transgenic Plants

We cloned the Odontoglossum ringspot virus (ORSV) coat protein (CP) gene from ORSV-infected Epidendrum and constructed a pHPUNOJE-1 vector by ligating this CP gene, green fluorescence protein (GFP) gene, and hygromycin resistant (Hygr) gene into the plasmid pGreenII. This chimeric vector was mediated into A. tumefaciens LBA 4404. By infecting and co-culturing the rhizomes of Cym. niveo-marginatum with A. tumefaciens LBA 4404, ORSV CP gene transgenic shoots were obtained. GFP was observed in 64% of the 81 surviving rhizomes and in 39% of 215 regenerated shoots. The presence of ORSV CP, GFP, and Hygr genes was confirmed in the transgenic shoots by using polymerase chain reaction (PCR) analysis, whereas that of ORSV CP was confirmed by enzyme-linked immunosorbent assay (ELISA).