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Agrobacterium tumefaciens -mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression



To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBIN m-gfp5 -ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII . The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l −1 α-naphthaleneacetic acid (NAA) and 1.0 mg l −1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 μg ml −1 Km, 200 μg ml −1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T 1 seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.



Plant Cell, Tissue and Organ CultureSpringer Journals

Published: Jun 1, 2002

DOI: 10.1023/A:1015668132503

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