Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation.
Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.
D000123:Acetyltransferases / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004351:Drug Resistance; D004558:Electric Stimulation; D010303:Paromomycin / Q000494:pharmacology; D010947:Plants, Toxic; D010957:Plasmids; D011523:Protoplasts / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D010303:Paromomycin; D004247:DNA; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase