Protoplast Culture (technique)

A protoplast is a plant cell that has had its rigid cell wall removed but still retains an intact plasma membrane, and because the cell wall is gone, protoplasts can be induced to fuse with one another in ways that walled cells cannot - making them the entry point both for regenerating whole plants from single cells and for creating somatic hybrids between different species (section 5.3.3.3). Protoplasts are isolated from small bits of plant tissue, most often leaf tissue, by immersing the tissue in a mixture of cell-wall-digesting enzymes - typically 0.5% Macrozyme and 2% Onozuka cellulase - dissolved in a 13% sorbitol or mannitol solution at pH 5.4, then incubating this overnight at 25 degC. Gently teasing the digested cells apart releases the protoplasts, which are then transferred into a 20% sucrose solution to keep them viable and centrifuged so that the pure protoplasts can be separated from leftover cell-wall debris. To fuse two protoplasts together, a suitable fusogen is used - most commonly polyethylene glycol (PEG). Isolated protopla …