Incubating tobacco leaf tissue, from which the lower epidermis was peeled, overnight with 0.3 to 0.4% Macerozyme and 0.6 to 1.2% cellulase, depending on leaf condition, produced a good yield of protoplasts that were susceptible to infection by TMV. Fluorescent antibody staining showed that 20 to 80% of protoplasts became infected, and infectivity tests indicated that an average of about 4 × 10 virus particles/infected protoplast, or 100 to 500 µg virus/10 protoplasts were produced by 2 days after inoculation. The production and infection of protoplasts depended less on the season when the plants were grown than with the ‘two-step’ method. Also, plentiful stable protoplasts were obtained without adding potassium dextran sulphate to the macerating medium. Calcium is required in the incubating medium for virus infection but can be partially replaced by Mg, which is not essential in the presence of Ca. Virus attained the greatest concentration when the protoplasts were inoculated as soon as they were washed free from the enzymes.


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