An improved method for direct gene transfer and subsequent regeneration ofArabidopsis thaliana Landsbergerecta and two marker lines |
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Authors: | M Torres J Siemens M Meixner M D Sacristán |
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Institution: | 1. Institut für Angewandte Genetik, Freie Universit?t Berlin Albrecht-Thaer-Weg 6, 14195, Berlin, Germany
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Abstract: | A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 µg plasmid DNA and 28 µg carrier DNA per 6 x 105 protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10?4, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations. |
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Keywords: | PEG-mediated transformation protoplasts transgene inheritance |
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