Assessment of co-transformation technique for Malaysian orchid variety (Dendrobium Savin White) with cymbidium mosaic virus coat protein gene

S. Rogayah, K.H. Nasir, W.C. Yeong and V. Pillai

Abstract

Enzyme linked immunosorbent assay (ELISA) was conducted to test 300 samples of orchid plants, collected from commercial orchid nurseries around Malaysia. The orchid samples were either apparently healthy or exhibited virus-like symptoms. ELISA results showed that 88% of the samples were infected with cymbidium mosaic virus (CyMV) including the orchid samples that did not exhibit any viral symptoms and looked healthy in appearance. Shoot tips from virus free plants were used to induce protocorm-like bodies (PLBs). The generated PLBs were bombarded with gold particle coated with plasmid pRQ6 and pJIT.CyMVcp containing hygromycin phosphotransferase (hptII) selectable marker gene and CyMVcp gene respectively. A total of 500 PLBs were bombarded and after 5 months selection in hygromycin media, 63 putative transgenic lines were recovered. Based on polymerase chain reaction analysis (PCR) on putative transgenic lines, 53 showed positive for the presence of hptII and CyMVcp genes. This study revealed that cotransformation of hptII and CyMVcp genes in two different vectors manage to achieve 10.6% transformation efficiency. The transgenic orchid plants were established in pots and acclimatised in the greenhouse. The transgenic plants were found morphologically similar compared to untransformed plants.

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