A method for the isolation of protoplasts from grape berry mesocarp tissue.

As single cell systems, protoplasts have been used in physiological, biochemical and molecular studies aiming towards the investigation, improvement or modification of plants. In grapevine, protoplasts have been isolated from several source tissues but not from grape berry, a major challenge given the uniqueness of grape fruit for human diet and wine production. Also, as the ripe grape berry has long been considered a 'small bag of sugary water' without cell compartmentation and/or membrane integrity, the isolation of intact cells from the mesocarp is of special scientific significance. Protoplasting from grape berry mesocarp cells was achieved with cellulase and pectolyase digestion, followed by differential and gradient centrifugations; however, given the special characteristics of berry tissue, cell wall digestion and protoplast purification were performed in a special environment to maintain their integrity and viability. Light and epifluorescence microscopy revealed the spatial organization of the cytoplasm, where an intricate acidic vacuolar apparatus predominates supporting the idea that berry softening during ripening is not strictly associated with loss in compartmentation and/or membrane integrity. Following the worldwide economical and social importance of wine in modern days, grape berry protoplasts are a major advance for both basic research of fruit ripening and biotechnological applications.