Fusion of Plant Protoplasts by Electric Fields

Abstract

The electrical fusion technique of Zimmermann and Scheurich (1981 Planta 151: 26-32) has been used to fuse mesophyll protoplasts of Avena, Zea, Vigna, Petunia, and Amaranthus. Electrical fusion proves to be a simple, effective, and general fusion technique that can be controlled to form either dikaryons or large multinucleate fusion bodies. In addition, we show that Vigna mesophyll protoplasts that are subjected to the electrical fields used in this technique are viable in culture. The construction of the fusion chambers, necessary electrical equipment, and the fusion protocol are described in sufficient detail for reproduction of the technique.The fusion of protoplasts has become an important tool in plant somatic cell genetics, especially for the production of interspecific hybrids (10). Recently, the list of methods available for protoplast fusion has been expanded by the development of techniques for fusing cells electrically (8,14 MATERIALS AND METHODSFusion Chamber. We used the flow-through chamber diagrammed in Figure 1. It was built of two pieces of glass glued onto a glass microscope slide with epoxy cement. Two metal rods, which served as electrodes, were glued between these pieces of glass so that an even slot 500 ,um wide remained in the center of the chamber. Care was taken to keep this central slot free of glue. The ends of the chamber were fitted with polyethylene tubing to make an inlet and an outlet port. The entire chamber was covered with a cover slip and sealed with epoxy cement. Several different types of metal were tried as electrodes; gold, platinum-iridium, and silver all worked equally well. The crucial factor in making a successful chamber proved to be getting the electrodes as nearly parallel as possible.

Expression of genes transferred into monocot and dicot plant cells by electroporation.

“…Electrical impulses of high field strength reversibly permeabilize biomembranes (17) and thus have had two important applications: the introduction of macromolecules into cells (15,16) and the induction of cell fusions (18,19). Conditions required to electrically transfer DNA into mouse cells have been reported (15), and recently a simple apparatus was used to transfer DNA into mouse and human cells by electroporation (16).…”

Effects of Prostaglandins E2 and F2α on the Electrofusion of Pea Mesophyll Protoplasts

“…Zimmermann et al (16) avoided side effects by using nonelectrolyte solutions or solutions in which the electrolyte concentration did not exceed 1 mM. Bates et al (1) showed that dielectrophoresis of oat protoplasts was not prevented by the presence of 0.1 mM Ca2", however, higher concentrations of salt were inhibitory. Chapell et al (3) found that Ca2+ inhibits electrically mediated protoplast fusion events by 50% at 2 mM and completely at above 5 mm.…”

“…The electrofusion technique proposed by Zimmermann and Scheurich (13) has some advantages over the chemically induced fusion techniques previously used. Using electrofusion the number of cells to be fused can be preselected, fusion is rapid, the yield of fusion products can be very high, and the whole fusion process can be observed microscopically (1,15).…”

Electrically Induced Protoplast Fusion Using Pulse Electric Fields for Dielectrophoresis

“…Zimmermann and coworkers (21) avoided side effects by using nonelectrolyte solutions or solutions in which the electrolyte concentration did not exceed 1 m1. Bates et al (3) showed that dielectrophoresis of oat protoplasts was not prevented by 0.1 mm Ca2", but higher concentrations of salt were inhibitory. Chapel et al (6) found that Ca2" inhibits electrically mediated protoplast fusion events by 50% at 2 mm and completely at concentrations >5 mm.…”