COMPARATIVE EFFECTS OF FUSION FACILITATORS ON ELECTROFUSION ATTRIBUTES OF NICOTIANA-TABACUM MESOPHYLL PROTOPLASTS

Mesophyll protoplasts isolated from in vitro-grown Nicotiana tabacum L . shoots were subjected to electrofusion. Dielectrophoresis was induce d by an AC field of 50 V cm(-1) inter-electrode distance and 0.5 MHz o scillation frequency. Fusion was effected by two 0.7 kV cm(-1) DC puls es, each of 50 mu s duration, applied within one second of each other. Various chemical treatments were tested for their effects on dielectr ophoresis efficiencies (percentages of protoplasts that made contact w ith at least one other protoplast under the AC field), fusion efficien cies (percentages of protoplasts participating in fusion events), cell lysis (percentages of protoplasts bursting during the electrofusion p rocesses), overall viabilities of fusion products 24 h post-fusion and overall plating efficiencies 7 d post-fusion (percentages of fusion-d erived cells that had undergone division). The various attributes asse ssed on the electrofusion of protoplasts in the control treatment, 10% mannitol, differed considerably for experiments carried out on differ ent days. Relative to the control treatment, only the Ca2+ treatments, and to a lesser extent lipase treatment reduced dielectrophoresis eff iciencies. Polyamines, cytochalasins and Ca2+ treatments significantly reduced cell lysis percentages. All electrofusion facilitators tested (except for spermine at 150 mg l(-1), the cytochalasins B and D, and Ca2+ treatments) increased fusion efficiencies to more than 1.5 times those obtained with the standard 10% mannitol electrofusion medium. Ca 2+ treatments increased overall viabilities of fusion products by more than 1.5 times. With the exception of the prostaglandins, lecithin an d CaCl2 treatments, overall plating efficiencies were reduced by treat ment of protoplasts with fusion facilitators. Substantial increases in overall plating efficiencies over those observed in the control treat ment were obtained using prostaglandin F-2a, lecithin and CaCl2.2H(2)O treatments. The implications of the results are discussed.