Establishment of an Agrobacterium-mediated genetic transformation procedure for the experimental model orchid Erycina pusilla
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The establishment of a genetic transformation system for Erycina pusilla (E. pusilla) would be a major step for orchid research and help establish the species as a potential experimental model. Here we describe the development of an efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation of E. pusilla protocorms. Plants were transformed with a binary plasmid containing the Arabidopsis thaliana gene Methionine sulfoxide reductase B7 (MSRB7) using the A. tumefaciens strain EHA105. Explants were selected on hygromycin-containing New Dogashima medium (Tokuhara and Mii in Plant Cell Rep 13(1):7–11, 1993) with 6-benzylaminopurine and α-naphthaleneacetic acid for 5 months to diminish the ratio of false positive explants, and it was determined that 3 month-old E. pusilla protocorms represented the optimal stage for transformation. Positive transformants were confirmed by Southern blot and PCR analyses, and by methyl viologen tolerance assays. Since E. pusilla has a shorter life cycle than most other orchids, self-pollinated T1 progeny was obtained within only 18 months. In addition, the results indicated that overexpression of MSRB genes can be used to avoid the effects of abiotic stress in economically important plants such as orchids.
KeywordsMethionine sulfoxide reductase Methyl viologen Orchid
Cauliflower mosaic virus
Methionine sulfoxide reductase B
This work was supported by a grant from Academia Sinica, Grant (98-2324-B-001-003-CC1) from the Development Program of Industrialization for Agricultural Biotechnology of the Republic of China and Grant (99AS-1.1.1-FD-Z1) from the Agriculture and Food Agency, Council of Agriculture, Executive Yuan of the Republic of China. We thank Dr. Kah Wee Koh for discussion and manuscript editing. We thank PlantScribe (www.plantscribe.om) for editing this manuscript.
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