A Large-scale Isolation of Vacuoles from Protoplasts of Mature Carrot Taproots

Abstract

Summary A simple large-scale method was developed for the isolation of viable protoplasts and then vacuoles from mature taproots of carrot ( Daucus carota L.). Digestion of phloem parenchyma with Cellulase Y-C and Pectolyase Y-23 for 3 h yielded 2 to 3 × 10 6 protoplasts/g fwt. Vacuoles were obtained from these protoplasts by a combination of osmotic and pH shock. Distribution of marker enzymes showed a yield of 50 % of the vacuoles with extravacuolar contamination of less than 1 % malate dehydrogenase and 4 % glucose-6-phosphate isomerase. In an application of the method it was demonstrated that the sugars accumulated by mature carrot taproots (fructose, glucose, and sucrose) are stored in the vacuole.