A simple method for the production of highly competent cells ofAgrobacterium for transformation via electroporation |
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Authors: | A C McCormac M C Elliott D F Chen |
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Institution: | (1) The Norman Building Institute for Plant Science Research, De Montfort University, Scraptoft, LE7 9SU Leicester, UK |
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Abstract: | The introduction of binary plasmids intoAgrobacterium hosts forAgrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies
are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular
combinations ofAgrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly
competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection
of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of
growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level
of transformation-competent cells to be maximized. We have observed that there is significant variation between transformedAgrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of threeArgobacterium tumefaciens (EHA101, LBA4404, C58) and twoArgobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycinres, kanamycinres, and gentamycinres). |
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Keywords: | Agrobacterium binary plasmids electroporation transformation competence selection |
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