A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.
The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-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 of Agrobacterium 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 transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).
McCormac
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AC
Norman Building Institute for Plant Science Research, De Montfort University, Scraptoft, Leicester, UK.
Elliott
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MC
Chen
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DF
D016960:Agrobacterium tumefaciens / Q000187:drug effects / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018274:Electroporation / Q000379:methods; D005819:Genetic Markers; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D012231:Rhizobium / Q000187:drug effects / Q000235:genetics*; D012641:Selection, Genetic; D000198:Spectinomycin / Q000494:pharmacology; D014169:Transformation, Bacterial / Q000187:drug effects / Q000235:genetics*
D000900:Anti-Bacterial Agents; D005819:Genetic Markers; D005839:Gentamicins; D007612:Kanamycin; D000198:Spectinomycin