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|標題:||Optimization of electroporation conditions for expression of GUS activity in electroporated protoplasts and intact plant cells|
|期刊/報告no：:||Plant Physiology and Biochemistry, Volume 35, Issue 12, Page(s) 959-968.|
|摘要:||Optimized electroporation conditions for beta-glucuronidase (GUS) expression in protoplasts from a dicot (tomato, Lycopersicon esculentum L. cv. Known You 301) and a monocot (creeping bentgrass, Agrostis palustric Huds) were determined using an exponential pulse-wave generator The optimum field strength of the electroporation pulse for highest efficiency of GUS expression was shown to vary with species used, but could be predicted based on simple staining techniques using trypan blue for DNA uptake and fluorescein diacetate to measure viability. Additional parameters, including plasmid DNA concentration, the protoplast or cell density, the ionic concentration of the electroporation media, and the cell type used for electroporation all had an effect on the efficiency of GUS expression. Comparative analysis of GUS expression in electroporated protoplasts and undigested mesophyll suspension cells demonstrated that electroporation procedures can incorporate plasmid DNA through the cell wall. The size of the plasmid was not a significant factor in GUS expression within the range of the plasmid sizes used for electroporation into protoplasts, however linearization of the plasmid by restriction enzyme digestion facilitated the uptake of the DNA into the cells. These results demonstrate that after optimization of electroporation conditions a variety of plant protoplasts or cells can effectively take up and express DNA.|
|Appears in Collections:||生命科學系所|
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