Isolation and fusion of protoplasts in sugar beet (Beta vulgaris L.)

In this study, attempts were made to isolate, fuse and culture the leaves derived protoplasts in vitro from seedling of sugar beet cultivers, viz Francesca and Meghribel. Several combination and concentrations of hydrolytic enzymes for protoplast isolation were used. Protoplasts were successfully isolated from Francesca and Meghribel when incubated in an enzyme mixture containing 2% cellulase, 1%hemicellulase and 1% pectinase for 18 h. at 25°C in dark, with good yield and viability. However, the best response was obtained with genotype Meghribel, the protoplast yields were 4.85 x105. There was a significant difference between the genotypes and enzyme mixtures. PEG-mediated protoplast fusion between Francesca and Meghribel was successful. Combination of different PEG concentrations (15, 20, 25, 30 or 35%) and different treatment durations (15, 20, 25 and 30 min.) were tested. The fusion frequency (%) was determined my measuring heterokaryon frequency. In general, 20% PEG combined with 20 and 25 min treatment duration produced the highest fusion frequency, but the highest rate of viability was obtained at 20% PEG with 20 min treatment duration; the capacity of heterokaryon to form cell colonies was highest when cultured on liquid or agarose solidified MS medium. No micro colonies formed on agar solidified MS medium after two weeks of culture. Purified protoplasts cultured in liquid, agarose or agar solidified MS medium were tested. Best results were obtained when protoplasts were cultured in liquid MS medium which gave the highest formation of 5333 and 5767 microcolonies in both the genotypes Francesca and Meghribel, respectively.