ISOLATION OF PROTOPLASTS AND VACUOLES FROM SUGARCANE SUSPENSION AND STEM PARENCHYMA CELLS

THE VACUOLE is a compartment within sugarcane cells in which most of the sucrose is stored. Despite its importance, the sugarcane vacuole is not well understood, because it has been very difficult to examine from sucroseaccumulating stem cells. It is only separated from other cell components by a single membrane which is generally broken during attempts to isolate the vacuole for study. One new approach to sugarcane improvement is to direct novel enzymes into the vacuole to convert sucrose into higher value biomaterials. Unfortunately, the vacuole is very acidic and it also contains proteolytic enzymes that seem to degrade most introduced enzymes before they can perform useful bioconversions. This new potential led us to revisit the challenge of vacuole isolation from mature sugarcane stems. Our immediate interest was to use the isolated vacuoles to understand how to stabilise introduced enzymes. They would also be very useful in studies to understand how sugarcane achieves its exceptional stored sugar concentrations. A first step to vacuole isolation is the removal of the cellulose walls surrounding sugarcane cells, yielding protoplasts. So we developed a method to isolate protoplasts from sugarcane immature and mature stem parenchyma cells by: (i) using an enzyme solution containing cellulase onozuka RS, macerozyme, pectinase and driselase; (ii) adjusting the solution to match the internode osmotic strength; (iii) pretreating internodes at 40C for 1-2 days before the enzyme treatment; (iv) vacuum infiltration of tissues in enzyme solution and changing the solution after 1-2 hours of incubation; (v) purification of protoplasts in Ficoll gradients. However, osmotic lysis of these protoplasts yielded less than 2% of vacuoles in most trials and polybase-induced lysis did not yield any vacuoles.