Protoplast Isolation

Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape.

Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension the protoplasts can be cultured on media which induce cell division and differentiation. A large number of plants can be regenerated from a single experiment – a gram of potato leaf tissue can produce more than a million protoplasts, for example.

Protoplasts can be isolated from a range of plant tissues: leaves, stems, roots, flowers, anthers and even pollen. The isolation and culture media used vary with the species and with the tissue from which the protoplasts were isolated. Protoplasts are used in a number of ways for research and for plant improvement. They can be treated in a variety of ways (electroporation, incubation with bacteria, heat shock, high pH treatment) to induce them to take up DNA. The protoplasts can then be cultured and plants regenerated. In this way genetically engineered plants can be produced more easily than is possible using intact cells/plants.

Plants from distantly related or unrelated species are unable to reproduce sexually as their genomes/modes of reproduction etc. are incompatible. Protoplasts from unrelated species can be fused to produce plants combining desirable characteristics such as disease resistance, good flavour and cold tolerance. Fusion is carried out by application of an electric current or by treatment with chemicals such as Polyethylene Glycol (PEG). Fusion products can be selected for on media containing antibiotics or herbicides. These can then be induced to form shoots and roots and hybrid plants can be tested for desirable characteristics.