, Volume 10, Issue 1, pp 43-52

Transient gene expression in electroporated protoplasts and intact cells of sugar beet

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Abstract

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.