TRANSFORMATION OF DENDROBIUM PRIMULINUM LINDL. WITH ANTISENSE ACC OXIDASE

The objectives of this research were to improve regeneration of Dendrobium primulinum Lindl., to select a suitable antibiotic for elimination of Agrobacterium tumefaciens strain LBA4404 (pCAMBIA1305.1) while maintaining high levels of shoot regeneration, and to establish optimal condition for transformation of D. primulinum using A. tumefaciens and particle bombardment. The suitable medium for protocorm proliferation was New Dogashima Medium (NDM). The protocorms were successfully regenerated on NDM supplemented with BA and NAA. It was found that these protocorms were completely inhibited by hygromycin at a concentration of 15 mg/L. The optimal concentration of cefotaxime for elimination of A. tumefaciens strain LBA4404 (pCAMBIA1305.1) was 300 mg/L. Transformation of D. primulinum was investigated using A. tumefaciens and particle bombardment. The plasmid pCAMBIA1305.1 containing antisense ACC oxidase, hygromycin resistant (hpt) and ß-glucuronidase (gus) was used. The transformation percentage using A. tumefaciens was 62%. Particle bombardment was also used to deliver antisense ACC oxidase gene into the orchid species. Tungsten particles (0.7 µm) were coated with plasmid DNA and introduced into the protocorms. The result showed that the optimal distance to obtain the highest transformation efficiency was 12 cm. The highest transformation percentage using particle bombardment was 66%. The transformed protocorms and plantlets were verified by GUS assay. A GUS assay revealed the GUS activity while PCR method indicated the successful integration of the transgenes.