Infection of Turnip Leaf Protoplasts with Turnip Rosette Virus

The optimum conditions for infection of turnip protoplasts with turnip rosette virus (TRosV) were 0.2 to 2 × 105 protoplasts/ml, 5 to 10 µg virus/ml, 1 µg poly-l-ornithine/ml in 0.6 m-mannitol, buffered with 5 to 10 mm-tris at pH 7.0 to 8.6. Under these conditions, 70% of protoplasts became infected, as indicated by staining with fluorescent antibody. The proportion of protoplasts infected was determined by factors such as pH and buffer ions and by the concentrations of protoplasts, virus and poly-l-ornithine during inoculation. The period of pre-inoculation incubation of virus and poly-l-ornithine was also critical, but mannitol concentration and the inoculation period and temperature had little effect on percentage protoplasts infected. Time course studies showed a single step multiplication of TRosV; the virus was detected on sucrose gradients at 24 h after inoculation and at 60 h reached its maximum concentration, 3 × 106 virions per infected protoplast.