SOMATIC HYBRIDIZATION AND CYBRIDS

Introduction

Protoplast fusion and somatic hybridization techniques provide the opportunity for bypassing reproductive isolation barriers, thus facilitating gene flow between species. Fusion of protoplasts is accomplished by the use of PEG [poly (ethylene glycol)]. Protoplast fusion has helped in the development of somatic hybrids or cybrids (cytoplasmic hybrids). Protoplasts offer the possibility of efficient and direct gene transfer to plant cells. DNA uptake has been found to be easier in protoplasts than into intact plant cells.
Purified protoplasts once obtained from any two different sources (can be different tissues, different plants or species or different genera), they can be fused together to form somatic hybrids. This non-conventional method of genetic recombination involving protoplast fusion under in vitro conditions and subsequent development of their product to a hybrid plant is known as somatic hybridization. First, somatic hybrid plant of Nicotiana glauca(+) N. langsdorfii was reported by Carlson in 1972. Protoplasts can be induced to fuse by variety of fusogens or electrical manipulations which induce membrane instability. Most commonly reported fusion inducing agents are sodium nitrate (used by Carlson), high pH/ Ca2+ concentration and Polyethylene glycol (PEG) treatment. Sodium nitrate treatment results in low frequency of heterokaryon formation, high pH and high Ca2+ concentration suits few plant species whereas PEG is the most favored fusogen for its reproducible high frequency of heterokaryon formation and low toxicity. However, treatment with PEG in presence of high pH/ Ca2+ is reported to be most effective in enhancing heterokaryon formation and their survivability.
A more selective, simpler, quick and non toxic approach is electrofusion which utilizes electric shock or short pulse of high voltage to promote membrane fusion between two cells. Many useful somatic hybrid plants produced by electrofusion have been reported like Nicotiana plumbaginifolia(+) N tobacum, Solanum tuberosum(+) S. charcoense (resistant to Colorado potato beetle).

Somatic Hybrids

Protoplast population following fusion treatment is heterogeneous mixture of unfused parents, homokaryons, heterokaryons, fused protoplasts with independent two nuclei etc thus necessitating selection of stable hybrids. Therefore, some identification and selection system should be incorporated into each parental cell line before fusion. Usually, two parental cell lines with differing requirements as selective screens make selection of hybrid convenient as only those fused cells that possess complementary traits of both parents will thrive. Resistance to antibiotics, herbicides and ability to grow on specific amino acid analogs are few of the selection methods used. Besides these, two different vital stains which do not affect viability of cells like fluorescein isothiocynate (FITC) and rhodomine isothiocyanate (RITC) have been successfully used. Under fluorescent microscope, FITC stained cells appear green and RITC stained protoplasts appear red whereas fused cells having both RITC and FITC fluoresce yellow.

Cybrids:

Cybrids or cytoplasmic hybrids are cells or plants containing nucleus of one species but cytoplasm from both the parents. Cybrids are produced in relatively high frequency by either irradiating (with X-rays or gamma rays) the protoplasts of one species prior to fusion in order to inactivate its nuclei or by preparing enucleate protoplasts of one species and fusing them with normal protoplasts of the other species.