Abstract
A protocol was developed to obtain stable transgenic orchids (Dendrobium nobile) via Agrobacterium-mediated transformation of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strains AGL1 and EHA105 were used, with each containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. PLBs were co-cultivated with A. tumefaciens, which had been activated with 100 μM acetosyringone (AS), for 2–3 days until the growth of A. tumefaciens was observed on co-cultivation medium containing 100 μM AS. Following co-cultivation, PLBs were cultured on selective medium containing 30 mg l−1 hygromycin and 250 mg l−1 cefotaxime. Proliferating PLBs were repeatedly selected for hygromycin resistance. A high efficiency of transformation (18%) was obtained with a total of 73 stably transformed lines produced. Incorporation and expression of the transgenes were confirmed by Southern blot analysis and GUS histochemical assay.
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