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.