Infection of brassica leaf protoplasts by turnip yellow mosaic virus

Abstract

Summary. Conditions optimal for the infection of Brassica leaf protoplasts by turnip yellow mosaic virus (TYMV) were found to be: pH 5.4-5.8 with citrate buffer; concentration of poly-L-ornithine, Izg/ml; concentration of TYMV, 0.2-1.0 lzg/ml. More than 90% of Brassica rapa and B. sinensis protoplasts were infected under these conditions. TYMV replication in Brassica protoplasts was followed by quantitating the virus extracted from protoplasts and separated by sucrose density gradient centrifugation. Brassica protoplasts produced I to 2 × 106 TYMV particles per cell within 48 hrs. Infection by TYMV induced the formation of polyplasts (aggregates of chloroplasts) in Brassica protoplasts. Polyplast formation paralleled the appearance of TYMV-specific immunofluorescence and could thus be used conveniently to determine the number of infected protoplasts. TYMV replication in Brassica protoplasts was resistant to actinomycin D and chloramphenicol, but was completely inhibited by cycloheximide.