OPTIMIZATION OF ELECTROPORATION CONDITIONS FOR EXPRESSION OF GUS ACTIVITY IN ELECTROPORATED PROTOPLASTS AND INTACT PLANT-CELLS

Optimized electroporation conditions for beta-glucuronidase (GUS) expr ession in protoplasts from a dicot (tomato, Lycopersicon esculentum L. cv. Known You 301) and a monocot (creeping bentgrass, Agrostis palust ric Huds) were determined using an exponential pulse-wave generator Th e optimum field strength of the electroporation pulse for highest effi ciency of GUS expression was shown to vary with species used, but coul d be predicted based on simple staining techniques using trypan blue f or DNA uptake and fluorescein diacetate to measure viability. Addition al parameters, including plasmid DNA concentration, the protoplast or cell density, the ionic concentration of the electroporation media, an d the cell type used for electroporation all had an effect on the effi ciency of GUS expression. Comparative analysis of GUS expression in el ectroporated protoplasts and undigested mesophyll suspension cells dem onstrated that electroporation procedures can incorporate plasmid DNA through the cell wall. The size of the plasmid was not a significant f actor in GUS expression within the range of the plasmid sizes used for electroporation into protoplasts, however linearization of the plasmi d by restriction enzyme digestion facilitated the uptake of the DNA in to the cells. These results demonstrate that after optimization of ele ctroporation conditions a variety of plant protoplasts or cells can ef fectively take up and express DNA.