3.  Selection of fusion products

The somatic hybridization by electrofusion of protoplasts allow one-to-one fusion of desired pairs of protoplasts and, therefore, it is easy to know the fate of fusion products. However, protoplast suspension recovered after chemical treatments (fusogen) consists of the following cell types:

i.  unfused protoplasts of the two species/strains

ii.  products of fusion between two or more protoplasts of the same species (homokaryons), and

iii.  ‘hybrid' protoplasts produced by fusion between one (or more) protoplasts of each of the two species (heterokaryons)

The heterokaryons which are the potential source of future hybrids constitute of a very small (0.5-10%) proportion of the mixture. Therefore, an effective strategy has to be employed for their identification and isolation. Various protocols have been proposed and practiced for the effective selection of hybrids, including morphological basis, complementation of biochemical and genetic traits of the fusing partners, and manual or electronic sorting of heterokaryons/hybrid cells.

3.1. Morpho-physiological basis: The whole mixture of the protoplasts are cultured after fusion treatment and the resulting calli or regenerants are screened for their hybrid characteristics. Occasionally the hybrid calli outgrow the parental cell colonies and are identified by their intermediate morphology, i.e. green with purple coloured cells. However, the process is labour intensive and requires glasshouse facilities. It is limited to certain combinations showing differences in their regeneration potential under specific culture conditions.

 

3.2. Complementation: In this case complementation or genetic or metabolic deficiencies of the two fusion partners are utilized to select the hybrid component. When protoplasts of two parents, (one parent bearing cytoplasmic albino trait and the other parent bearing green trait) each parent carrying a non-allelic genetic or metabolic defect are fused, it reconstitutes a viable hybrid cell, of wild type in which both defects are mutually abolished by complementation, and the hybrid cells are able to grow on minimal medium non-permissive to the growth of the parental cells bearing green trait. Later, the calli of hybrid nature could be easily distinguished from the parental type tissue (albino trait) by their green color. The complementation selection can also be applied to dominant characters, such as dominant resistance to antibiotics, herbicides or amino acid analogues.

3.3. Isolation of heterokaryons or hybrid cells: The manual or electronic isolation of heterokaryons or hybrid cells is the most reliable method. Manual isolation requires that the two parental type protoplasts have distinct morphological markers and are easily distinguishable. For example, green vacuolated, mesophyll protoplasts from one parent and richly cytoplasmic, non green protoplasts from cultured cells of another parent. The dual fluorescence method also helps easy identification of fusion products. In this case, the protoplast labeled green by treatment with fluorescein diacetate (FDA, 1-20 mgl^-1) are fused with protoplasts emitting a red fluorescence, either from chlorophyll autofluorescence or from exogenously applied rhodamine isothiocyanate (10-20 mgl^-1). The labeling can be achieved by adding the compound into the enzyme mixture. This can be applied even for morphologically indistinguishable protoplasts from two parents.