A COMPARATIVE-STUDY OF DNA-SYNTHESIS IN COTYLEDON PROTOPLAST CULTURESOF BRASSICA-NAPUS, BRASSICA-CAMPESTRIS AND BRASSICA-OLERACEA USING ANIMMUNOCYTOCHEMICAL TECHNIQUE

The present study was designed (1) to observe the characterization of 5-bromo-2'-dexoyuridine (BrdU) incorporation into cultured Brassica co tyledon protoplasts and (2) to investigate the genetic differences in the levels of nuclear DNA synthesis (expressed by the percentage of nu clei labelled with BrdU) in cotyledon protoplast cultures from 12 cult ivars of three Brassica species (Brassica napus, B. campestris and B. oleracea) at an early stage using immunocytochemistry. Nuclei labelled with BrdU were different from those showing only staining with 4'-6'- diamidino-2-phenylindole (DAPI) under fluorescence and light microscop y. Two to 5% of nuclei were labelled with BrdU after 1 h of culture, i ndicating that nuclear DNA synthesis occurred at a very early stage of culture. The percentage of nuclei labelled with BrdU increased with t ime over the length of the culture period. The mean percentage of nucl ei labelled with BrdU in the 12 cultivars was about 25% at 24 h after culture initiation. The curve of the increase in percentage of nuclei labelled with BrdU exhibited an S-shape from 1 to 24 h. However, culti var differences in percentages of nuclei labelled with BrdU were very significant over the time course of 1-24 h from initial culture, with cultivars Eureka (B. napus), Global (B. napus), Narc 82 (B. napus), Bu nyip (B. campestris) and Sugar Loaf (B. oleracea) having a consistentl y higher percentage of nuclei labelled with BrdU than the other cultiv ars. Species differences were also significant, with cultivars of B. n apus showing much higher percentages than the tested cultivars of B. c ampestris and B. oleracea. The results indicate that the differences i n nuclear DNA synthesis in Brassica cotyledon protoplast cultures were most likely at both intra- and interspecies levels.