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

Agrobacterium tumefaciens-mediated transformation of the legume Astragalus sinicus using... 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 pBINm-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 T1 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Cell, Tissue and Organ Culture Springer Journals

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

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References (28)

Publisher
Springer Journals
Copyright
Copyright © 2002 by Kluwer Academic Publishers
Subject
Life Sciences; Plant Sciences; Plant Physiology
ISSN
0167-6857
eISSN
1573-5044
DOI
10.1023/A:1015668132503
Publisher site
See Article on Publisher Site

Abstract

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 pBINm-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 T1 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.

Journal

Plant Cell, Tissue and Organ Culture – Springer Journals

Published: Oct 16, 2004

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