Judged by the yield of infective virus, good infection of cowpea mesophyll protoplasts with alfalfa mosaic virus is obtained when protoplasts are resuspended at 0 °C in 0.5 -mannitol, 0.01 -potassium phosphate, pH 5.6, containing 1 µg/ml poly--ornithine and about 2 µg/ml virus. At 25°C, virus infectivity is first detectable 12 h after inoculation and increases exponentially in the next 12 h. Forty hours after inoculation about 1 to 5 × 10 virus particles are produced per living protoplast. As with intact leaves, a mixture of bottom component, middle component and top component nucleoprotein is required for the infection of protoplasts.

Virus multiplication is not sensitive to chloramphenicol (200 µg/ml) but is completely inhibited by cycloheximide (10 µg/ml) applied at any point in the growth cycle. Actinomycin D (10 µg/ml) interferes with an early step in virus replication.


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