The permeability of electroporated cells and protoplasts of sugar beet.
A simple method has been developed to determine the changes in permeability of protoplasts and intact cells when electroporated. Cells and protoplasts of sugar beet, Beta vulgaris L., were subjected to electric pulse treatments of different field strengths, pulse number and pulse duration, and the ability to accumulate and retain the hydrophilic dye phenosafranine was determined spectrophotometrically. Results of timecourse studies of phenosafranine accumulation and retention indicated that pores are formed or enlarged rapidly in the plasmamembrane and remain permeable to phenosafranine for relatively long periods; the half-life of the 'pores" was temperaturedependent. Both cells and protoplasts retained the highest levels of phenosafranine when supplied with a series of five rectangular pulses of 50 μs duration and of field strength 2500 V·cm(-1). If these parameters were exceeded, The phenosafranine content was reduced, concomitant with a decline in viability as indicated by fluorescein-diacetate staining, indicating the loss of the integrity of the plasmamembrane. The pattern of accumulation and retention by protoplasts of radioactivity from [(3)H]pABD1, a modified bacterial plasmid, was similar to that of phenosafranine, but uptake of the plasmid by cells was not demonstrated. The mothod can be used to determine conditions for the optimum permeabilization of protoplasts for direct gene transfer.
Department of Biochemistry, Rothamsted Experimental Station, AFRC Institute of Arable Crops Research, AL5 2JQ, Harpenden, Herts, UK.
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Lindsey
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Jones