Electrofusion of Tobacco and Pea Protoplasts

전기장하에서의 담배 및 완두 원형질체 융합

Intra- and inter-specific protoplast fusion of tobacco (Nicotiana tabacum cv. Virginia 115) and pea (Pisum sativum cv. Sparkle) were carried out in highly inhomogeneous alternating electric fields. Under the electric field of alternating current (AC, sine wave), 600 V/cm and 800 kHz for tobacco protoplast, and 600 V/cm and 700 kHz for pea protoplasts, the protoplasts were aggregated in pearl chains. Intra-specific protoplast fusions were most effectively induced within the aggregates of tobacco and pea, respectively, by the additional application of a single high field pulse of direct current (DC, square wave) at 1 kV/cm for 50 $mutextrm{s}$. Inter-specific fusions between protoplasts of the two plants were most effectively induced in the electric field of 600 V/cm and 700 kHz, and square wave pulse at 1 kV/cm for 50 $mutextrm{s}$. The duration of the pulse over the electrical breakdown voltages was simulated from 1 to 100 $mutextrm{s}$ in both tobacco and pea protoplast. The yield of the electrofusion products was significantly high (above 60%), compared with that (20%) of the standard fusion method by polyethylene glycol (PEG) 4,000, and the viability of electrofused protoplasts was above 70%, but that of PEG-fused protoplasts 8~16%, when determined by Evan's blue staining method.