Design of a novel system for the construction of vectors for Agrobacterium-mediated plant transformation |
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Authors: | Teresa Mozo and Paul J. J. Hooykaas |
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Affiliation: | (1) Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, NL-2333 AL Leiden, The Netherlands |
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Abstract: | Summary The loxP-Cre site-specific recombination system of phage P1 was used to develop a novel strategy to construct cointegrate vectors for Agrobacterium-mediated plant transformation. A pTi disarmed helper plasmid (pAL1166) was constructed by replacing the oncogenic T-DNA by a loxP sequence and a spectinomycin resistance marker in the octopine-type pTiB6 plasmid. The cre gene was cloned into an unstable incP plasmid. A third plasmid, which did not replicate in Agrobacterium and contained another loxP sequence together with a kanamycin resistance marker, was used to test the system. Electroporation of this third plasmid into an Agrobacterium strain harbouring both pAL1166 and the Cre-encoding plasmid resulted in kanamycin-resistant cells containing a cointegrate between pAL1166 and the incoming plasmid. Cointegration occurred by Cre-mediated recombination at the loxP sites, and the cointegrate was stabilized in the Agrobacterium cells by the loss of the Cre-encoding plasmid shortly after the recombination event had taken place. |
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Keywords: | Cointegrates loxP-Cre Plant Vectors Site-specific recombination |
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