Agrobacterium-mediated genetic transformation of a Dendrobium orchid |
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| Authors: | Men Shuzhen Ming Xiaotian Liu Rongwei Wei Chunhong Li Yi |
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| Affiliation: | (1) Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, The National Laboratory of Protein Engineering and Plant Genetic Engineering, Department of Biotechnology, College of Life Sciences, Peking University, Beijing, 100871, China;(2) Institute of Tissue Culture, Shenzhen Research Center of Agricultural Science, Shenzhen, 518040, China |
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| 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  -glucuronidase gene (gus-int) as a reporter gene. PLBs were co-cultivated with A. tumefaciens, which had been activated with 100  M acetosyringone (AS), for 2–3 days until the growth of A. tumefaciens was observed on co-cultivation medium containing 100 M 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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| Keywords: | Agrobacterium tumefaciens co-cultivation hygromycin protocorm-like bodies transgenic |
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