Behavior and Viability of Tobacco Protoplasts in Response to Electrofusion Parameters

Abstract

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per (22,24). For plant cells, typical procedures involve three steps; alignment of protoplasts with an AC field, disruption of contiguous membranes with a brief DC pulse, and fusion of the protoplasts (1,15,22,24). Much of the emphasis in previous research has been directed toward the modification of the DC pulse to achieve maximum fusion rates while little data has been presented on the effects of both the AC fields and the DC pulses on cell viability. Indeed, only recently have published reports appeared on the successful regeneration of shoots from hybrids of electrically fused protoplasts (2, 4). We report here the effects of AC and DC currents, separately and in combinaton, on the viability and integrity of protoplasts and on the movement of protoplasts.MATERIALS AND METHODS Plant Material. Nicotiana rustica var brasilia and N. tabacum cv MD 872 were grown under greenhouse conditions and harvested 70 to 90 d after germination. One week prior to harvest the plants were transferred to an environmental growth chamber that maintained a 12 h day length at 23°C. Shoots and cell suspensions of N. tabacum cv Connecticut 'John Williams Broadleaf' were cultured in vitro as described by Owens (11) and Uchimiya and Murashige (19), respectively.Preparation of Protoplasts. Protoplasts from greenhouse grown plants were isolated from mature mesophyll leaf tissue with 1% (w/v) Macerase3 (300 units/g) and 2% (w/v) Cellulysin (10,000 units/g) in 50 mm Mes-NaOH buffer (pH 5.7) with 0.6 M mannitol as described previously (16). Protoplasts were isolated from tissue culture by an adaptation of the methods of Owens (10) and Lin (6). One g of suspension cells in 40 ml of 0.15% Cellulysin, 1% rhozyme HP-150, and 0.05% Driselase was incubated for 4 h at 30°C with shaking at 50 rpm. All enzymes were desalted and dissolved in a solution of 0.7 M mannitol, 9.25 mM CaCl2, and 0.44 mm Ca(H2PO4)2 adjusted to pH 5.7. Protoplasts were harvested by centrifugation (100g for 3 min) and resuspended in 4 ml of 16% (w/v) Ficoll in 0.7 M mannitol. Four ml each of 14% (w/v) and 12% (w/v) Ficoll in 0.7 mannitol 3 Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture, and does not. imply its approval to the exclusion of other products or vendors that may also be suitable.

Electrically Induced Protoplast Fusion Using Pulse Electric Fields for Dielectrophoresis

“…The influence of the AC field frequency on dielectrophoresis has been studied in detail (11,16). Our data indicate a frequency dependence of protoplast chain formation similar to that established by Pohl and Crane (11).…”

Electroporation in Biological Cell and Tissue: An Overview

“…The electrofusion is a two-step process; it involves cell membrane electroporation and a close physical contact of two electroporated membranes in fusogenic state (Zimmermann 1982;Saunders et al 1986). The electric field parameters needed for introduction of small molecules and for electrofusion are similar.…”

“…For this application, an alternating electric field of low amplitude on the order of few hundred volts per centimeter and frequencies in the range of 10 kHz to 6 MHz is used (Zimmermann 1982;Vienken and Zimmermann 1985;Saunders et al 1986). During electrophoresis the polarized cells are attracted to the areas of high field strength (Oblak et al 2007).…”

“…Cells migrate toward each other and form pearl chains. The procedure is rapid and has negligible effect on cell viability (Saunders et al 1986). The alternating electric field is then switched off and an electroporation pulse is applied.…”

Effects of La3+ on Surface Charges, Dielectrophoresis, and Electrofusion of Barley Protoplasts

“…We used Ca2+ in order to stabilize and enhance electrofusion. The effectiveness of Ca2 + on electrofusion was recently confirmed with plant (7,14,17,20,21) and animal cells (15). On the other hand, a low fusion efficiency, compared with a weakly conductive pure osmoticum medium, has been reported because addition of Ca salts increases conductivity of media (25).…”