Inoculation of protoplasts with viruses by electroporation

Optimal conditions for electroporation have been determined using inoculation of brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) and its RNA into protoplasts of Nicotiana tabacum and N. plumbaginifolia. The most satisfactory medium was 0.5-0.7 M mannitol; calcium ions were toxic and other electrolytes were not helpful during electroporation. Brief pulses (ca. 10 microsec) were less destructive to the protoplasts than longer ones (ca. 10 msec) and gave high percentage infections with CCMV RNA. RNA entered the protoplasts only if present during the voltage pulse. Optimal voltage depended on the sample size, interelectrode distance, and pulse duration. A 50-nF capacitor discharging a 5- to 10-microsec pulse through a 1-ml sample in 0.7 M mannitol with a 4-mm interelectrode distance gave maximum infection with minimal protoplast damage at 2.5 kV/cm. A single pulse was sufficient; multiple pulses slightly increased infection. Electroporation of viral RNA was at least as effective as inoculation in the presence of polyethylene glycol. Positively charged BMV also infected readily but negatively charged CCMV only poorly.