Electrically Stimulated Fusion of Different Plant Cell Protoplasts

Abstract

Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cels (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form "pearl chains" which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cel and mesophyll ceUl protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.The study of chemical-and virus-induced fusion is an area of research that has attracted an increasing amount of interest in the last decade (4, 11-13). Needless to say, the elucidation of the mechanisms involved in cell and membrane fusion would provide information on the dynamic properties of cell membranes and lead to an understanding of many biological phenomena in which membrane fusion occurs (e.g. fertilization, endo-and exocytosis, etc.). However, it is the possibility of somatic hybridization induced by cell fusion that really demonstrates the enormous potential of this phenomenon in the fields of biology, agriculture, and medicine (15).Despite the great efforts on behalfofa large number of scientists and the numerous publications dealing with various fusogenic substances, such as lipids, polyethyleneglycol, dimethyl sulfoxide, and fusogenic virus (7)(8)(11)(12)(13) Recently, Zimmermann and Scheurich (22) developed a quite different approach which they applied to mesophyll cell protoplasts of Vicia faba. The technique they used is based on the reversible electrical breakdown of cell membranes in response to a high external electrical field pulse of very short duration (a couple of,As) (9,19,20).Close membrane contact between two cells, a prerequisite for fusion, is achieved by performing breakdown experiments in the presence of an inhomogeneous, alternating electrical field of low field strength. By simple physical laws, the cells are drawn into regions of high field intensity within the inhomogeneous field and, in the process, they form "pearl chains." This phenomenon is termed dielectrophoresis (10). The fusion process is initiated as soon as electrical breakdown occurs in the membranes of two attached cells. This method of fusion yields a high proportion of synchronously fused cells. The process of fusion can be trig...

Electric field-induced cell-to-cell fusion

“…fusion between stomatal protoplasts and mesophyll protoplasts of V. faba) is also reported in the literature [59,69,86,87]. We can thus conclude that this technique works efficiently as far as the fusion of plant protoplasts is concerned.…”

Electrofusion ofNeurospora crassaslime cells

“…We have demonstrated that N. crassa slime cells can be subjected to dielectrophoresis and electrofusion under conditions comparable to those applied to other plant protoplasts [10]. Both processes are related to the electric properties of cell membranes, and, according to our expectations, all 3 slime mutants responded equally to external electric fields.…”

Electromanipulation of plant protoplasts

“…Two types of wave form of the pore ptilse exist. The initial wave form used by Zimmermann and Scheurich (1981) is a square ptilse and is the wave form that is used most. The benefits of using sqtiare pulses are that the exact parameters for pore formation, that is, external field strength and pulse length, can be determined precisely.…”