High efficiency of stable genetic transformation in Dendrobium via microprojectile bombardment

Several parameters affecting transient beta-glucuronidase (GUS) expression in protocorms of Dendrobium cv. Jaquelyn Thomas were examined using bombardment technique with pActin-1D plasmid. The GUS activity in Dendrobium protocorm was not significantly affected by size of the target, type of particles, and helium gas pressure. However, the numbers of surviving tissues after bombardment were different. Transgenic orchids were established by bombardment of actively dividing protocorms with gold particles coated with pMNK1005 plasmid containing hygromycin phosphotransferase (hgh) and green fluorescent protein (gfp) genes driven by a ubiquitin promoter. A high efficiency of orchid transformation was established using three selection steps. The bombarded protocorms were screened on medium supplemented with 5 mg dm(-3) and 25 mg dm(-3) hygromycin and surviving protocorms were stringently selected on medium containing 30 mg dm(-3) hygromycin. The transformation efficiency was 19.87 % and GFP expressing protocorms were not chimeras. The integration of the transgene into genomic DNA of transgenic plantlets was confirmed by PCR and Southern blot hybridization. All but one of the transgenic lines contained multiple copies of the transgene.