Poly--ornithine greatly increased infection of protoplasts by tobacco rattle virus (TRV), and had the largest effect when incubated with virus particles for at least 10 min before inoculation. Using a final concentration of 1 µg/ml TRV particles and 1 to 1.5 µg/ml poly--ornithine in 0.025 -phosphate buffer, pH 6.0, to inoculate mesophyll protoplasts of tobacco cv. Xanthi by the ‘indirect’ method, up to 98% of the intact protoplasts became infected. When the protoplasts were stored overnight at 5 °C before inoculation, 95% became infected. In protoplasts kept at 22 °C after inoculation, about half the yield of infective particles was produced during the first day and almost all the remainder during the second. The final yield was about 2 × 10 long virus particles and 6 × 10 short particles per infected protoplast. Fluorescent antibody staining showed that TRV coat protein antigen accumulated throughout the cytoplasm. In electron micrographs, the long TRV particles were associated with mitochondria whereas the short particles were generally dispersed in the cytoplasm. Infective RNA was produced after inoculation with long particles but TRV coat protein antigen, and long and short TRV particles, were made only in protoplasts inoculated with both kinds of particle; infection was not detected in protoplasts inoculated with short particles alone.


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