Antisense ACC Oxidase Gene Transformation of Dendrobium delacourii Guill Protocorm Mediated by Agrobacterium tumefaciens

Abstract

The objectives of this research were to investigate the efficiency of transformation of Dendrobium delacourii. by using Agrobacterium tumefaciens with antisense ACC oxidase gene. The effect of cefotaxime on the growth of Agrobacterium tumefaciens which harboured the plasmid pCAMBIA 1305.1 containing antisense ACC oxidase gene, hptll gene as a selectable marker and Gus gene as a reporter gene were found that A. tumefaciens could grow on LB medium without cefotaxime and supplement with 25 mg/l. cefotaxime and could not grow on 50, 75, 100, 125, 150, and 175 mg/l. The culture of Dendrobium delacourii on VW medium supplement with 100, 200, 300, 400, and 500 mg/l., cefotaxime concentration, were found that D. delacourii protocorms could grow in every concentration and tolerate up tp 500 mg/l cefotaxime. The study of hygromycin concentration on the growth of D. delacourii was found that 10, 15, 20, 25, and 30 mg/l., hygromycin, orchid protocorms could not tolerate, the  protocorms turned brown and could not grow. The genetic transformation of D. delacourii was mediated by A. tumefaciens and be the optimal co-cultivation time was 20 minutes. The transformation protocorrms had Gus gene expression, shown by histochemical Gus assay.