Transient gene expression in electroporated Solanum protoplasts.
Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts--250 V/cm; Désirée mesophyll protoplasts--225 V/cm; Désirée suspension culture protoplasts--225 V/cm; and Désirée tuber protoplasts--150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36-48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.
Jones
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AFRC Institute of Arable Crops Research, Biochemistry Department, Harpenden, Herts, U.K.
Ooms
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Jones
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D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics; D004560:Electricity; D015870:Gene Expression; D009029:Mosaic Viruses / Q000235:genetics; D009154:Mutation; D010944:Plants / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011523:Protoplasts; D014162:Transfection
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase