Protoplasts were isolated from leaves of three tomato cultivars and inoculated with citrus exocortis viroid (CEV), potato spindle tuber viroid (PSTV) and cucumber pale fruit viroid (CPFV), respectively, using glycine-KOH-buffered mannitol at pH 9. CPFV multiplied detectably in protoplasts from the tomato cultivar Hilda 72 but replication of the other two viroids was rarely detectable. The minimum viroid concentration for infection was 10 µg RNA/ml using 10 protoplasts/ml. Newly synthesized viroid RNA was detected 36 h after inoculation by bioassay, and 48 h after inoculation by H-uridine incorporation into the viroid RNA band obtained by polyacrylamide gel electrophoresis. Incorporation of radioactive uridine into CPFV in inoculated protoplasts reached about 0.6% of that into tRNA during 72 h after inoculation. In protoplasts isolated from systemically infected leaves, however, incorporation of H-uridine into CPFV was only 0.1% of that into tRNA. In inoculated protoplasts, incorporation of H-uridine into CPFV was much more rapid at 35 °C than at 25 °C but incorporation into tRNA was similar at the two temperatures.


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