Experimental study on breakdown behavior and vacuole isolation of protoplasts under electrical pulses

Abstract

This paper presents the experiments on the breakdown behavior of plant protoplasts under electrical pulses. A microfabricated device was utilized for facilitating single-cell observation and for patterning the electric field distribution that enhanced the efficiency of membrane charging. The objectives of the work are to study the effects of pulse parameters on the breakdown behavior and to investigate the use of electric pulses for vacuole isolation. We observed the breakdown behavior of the protoplasts, prepared from butterfly pea, by applying sinusoidal electric pulses to activate the membrane breakdown. The pulse frequency was varied from 20 kHz to 1 MHz and the pulsing duration was between 1 and 100 ms. The experimental results exhibited two kinds of the breakdown behavior under electric field. That is, the breakdown took place on both cell membrane and tonoplast (vacuolar membrane), or the breakdown was limited only to the cell membrane. The probability of cell-membrane breakdown increased with longer pulsing duration or lower pulse frequency. The probability of vacuole isolation also varied with the pulse frequency and duration. Our results show that efficient vacuole isolation can be achieved by using a pulse frequency close to the characteristic frequency of membrane charging for an appropriate pulsing duration.