Positive-negative selection and T-DNA stability in Arabidopsis transformation |
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Authors: | Gallego ME Sirand-Pugnet P White CI |
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Institution: | (1) Centre de Recherche sur les Plantes, CNRS ERS 569, Université de Paris XI, 91405 Orsay Cedex, France;(2) Present address: Laboratoire de Génétique et d'Amélioration des Champignons Cultivés, Université Victor Segalen Bordeaux 2-INRA, CRA de Bordeaux-, BP81, 33 883 Villenave d'Ornon Cedex, France |
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Abstract: | We have analysed the application of positive-negative selection for the selection of homologous recombination interactions between the chromosome and a T-DNA molecule after transformation of plant cells. Two different genomic loci in a cell suspension of Arabidopsis thaliana were chosen to study gene targeting events. One was the chalcone synthase (CHS) gene present as a single copy and the second an hemizygous chromosomally inserted T-DNA containing the hpt gene, conferring resistance to hygromycin, flanked by CHS sequences. The target lines were transformed with replacement-type T-DNA vectors which contained a positive selectable marker flanked by the regions of the CHS gene and a negative selectable marker to counter-select random insertions. As negative marker we used the Escherichia coli codA gene encoding cytosine deaminase, conferring upon the cells sensitivity to 5-flourocytosine (5-FC). Doubly selected transformants represent 1–4% of the primary transformed cells. Targeting events were not found at the chalcone synthase locus nor at the artificial hpt locus in a total of 4379 doubly selected calli, corresponding to at least 109475 individual primary transformants. We show by PCR and Southern analysis that the 5-FC resistance in the majority of these cells is associated with substantial deletions of the T-DNA molecule from the right-border end. |
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Keywords: | Arabidopsis Agrobacterium T-DNA CodA positive-negative selection recombination |
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