The infection of cucumber mesophyll protoplasts with tobacco mosaic virus

Protoplasts from the first leaf mesophyll of cucumber plants were isolated by an 18 hour combined petinase/cellulase treatment. Conditions favouring the infection of these protoplasts with tobacco mosaic virus (TMV), and the accumulation of infective virus up to 96 hours after inoculation have been studied. Infection of approximately 5-10 percent of the protoplasts, revealed by indirect fluorescent antibody staining, was achieved by pre treatment of the cells in 0.01 M citrate buffered mannitol (CBM), pH 5.2 with 2 μg/ml poly L ornithine followed by centrifugation and direct resuspension of the cells in the same mixture together with 2 to 4 μg/ml TMV. Higher concentrations of the polycation and buffer were toxic to the protoplasts. Under the best conditions, virus yields of approximately 10-20 μg TMV/10 protoplasts were attained, while after 72 hours incubation, significant amounts of virus could often be recovered from the incubation medium. Addition of actinomycin D to cultures of protoplasts 2 hours post inoculation partially inhibited development of infectivity.