AN IMPROVED METHOD FOR DIRECT GENE-TRANSFER AND SUBSEQUENT REGENERATION OF ARABIDOPSIS-THALIANA LANDSBERG ERECTA AND 2 MARKER LINES

A protocol for PEG-mediated transformation of protoplasts is described for A. thaliana ecotype Landsberg erecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 m u g plasmid DNA and 28 mu g carrier DNA per 6 x 10(5) protoplasts in 1 5% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5-3.2% and ab solute transformation frequencies of 1-2 x 10(-4), were obtained. Shoo t regeneration frequencies of 40-60% were achieved, and fertile transg enic plants of the three tested lines were obtained. Southern blot hyb ridizations demonstrated multi-copy integration patterns in most cases . Hygromycin resistance segregation patterns of 3:1 and 15:1 were foun d, as well as unexpected segregation patterns, suggesting that modific ations in gene expression took place and that these can progressively occur over successive generations.