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Factors Influencing the Infection of Cowpea Mesophyll Protoplasts by Alfalfa Mosaic Virus

Department of Biochemistry, State University, Wassenaarseweg 64, Leidne, The Netherlands

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 M-mannitol, 0.01 M-potassium phosphate, pH 5.6, containing 1 µg/ml poly-L-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 x 10⁶ virus particles are produced per living protoplast. As with intact leaves, a mixture of bottom component, middle component and top component b 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.

Received 20 December 1976; accepted 24 February 1977.