Protoplast isolation

This protocol describes the process of isolating protoplasts from plant cells and then fusing the protoplasts in order to carry out further investigations. 

Plants from distantly related or unrelated species are unable to reproduce sexually as their genomes 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.

Having isolated and fused the protoplasts, suggested investigations include:

*Investigate the effect of using different types of lettuce or other plants on the efficiency of protoplast production (number of intact or broken protoplasts; degree of roundness of protoplasts; time taken to produce protoplasts)

*Investigate the effect of using different plant organs (root, leaves, flowers) on efficiency of protoplast production

*Investigate the effect of varying enzyme concentrations or types (pectinase, cellulase, viscozyme) on the efficiency of protoplast production 

*Investigate the effect of duration of plasmolysis (in sorbitol solution) and centrifugation on the intactness or number of protoplasts produced

*Investigate the effect of protoplast concentration on the percentage of protoplasts which fuse

This resource was produced by Science & Plants for Schools (SAPS).