(i) Some visual markers, e.g., pigmentation, of the parental protoplasts may be used for the identification of hybrid cells under a microscope; these are then mechanically isolated and cultured. For example, the protoplasts of one species may be green and vacuolated (from mesophyll cells), while those of the other may be nonvacuolated and nongreen (from cell cultures). Where such features are not available, the protoplasts of two parental species may be separately labelled with different fluorescent agents.
This approach is time consuming, and requires considerable skill and effort. Several workers have attempted to devise systems, which specifically select for hybrid cells. In simple words
(ii) these systems, exploit some properties (usually, deficiencies) of the parental species, which are not expressed in the hybrid cells due to complementarily between their genetic systems. These properties may be sensitivity to culture medium constituents, antimetabolites, temperature, etc. inability to produce an essential biochemical (auxotrophic mutants), etc. These properties may be naturally present in the parental species or may be artificially induced through mutagenesis/genetic engineering.

These strategies are simple, highly effective and the least demanding. Recently, genetic engineering has been used to transfer resistance to an antibiotic/herbicide in one fusion parent and that to another one into the other parent; the hybrid cells are selected using a medium containing both the concerned antibiotics/herbicides.
A more general and widely applicable strategy is
(iii) to culture the entire protoplast population without applying any selection for the hybrid cells. All the types of protoplasts form calli; the hybrid calli are later identified on the basis of callus morphology, chromosome constitution, protein and enzyme banding patterns, etc.
In some cases, the identification may be delayed till plants are regenerated. In such a case, the protoplasts should