2. Protoplast fusion
Protoplast fusion could be spontaneous during isolation of protoplast or it can be induced by mechanical, chemical and physical means. During spontaneous process, the adjacent protoplasts fuse together as a result of enzymatic degradation of cell walls forming homokaryons or homokaryocytes, each with two to several nuclei. The occurrence of multinucleate fusion bodies is more frequent when the protoplasts are prepared from actively dividing callus cells or suspension cultures. Since the somatic hybridization or cybridization require fusion of protoplasts of different origin, the spontaneous fusion has no value. To achieve induced fusion, a suitable chemical agent (fusogen) like, NaNO3, high Ca2+, polyethylene glycol (PEG), or electric stimulus is needed.
i. Fusion by means of NaNO3: It was first demonstrated by Kuster in 1909 that the hypotonic solution of NaNO3 induces fusion of isolated protoplast forming heterokaryon (hybrid). This method was fully described by Evans and Cocking (1975), however this method has a limitation of generating few no of hybrids, especially when highly vacuolated mesophyll protoplasts are involved.
ii. High pH and Ca++ treatment: This technique lead to the development of intra- and interspecific hybrids. It was demonstrated by Keller and Melcher in 1973. The isolated protoplasts from two plant species are incubated in 0.4 M mannitol solution containing high Ca++(50 mM CaCl2.2H2O) with highly alkaline pH of 10.5 at 37°C for about 30 min. Aggregation of protoplasts takes place at once and fusion occurs within 10 min.
iii. Polyethylene glycol treatment: Polyethylene glycol (PEG) is the most popularly known fusogen due to ability of forming high frequency, binucleate heterokaryons with low cytotoxicity. With PEG the aggregation occurred mostly between two to three protoplasts unlike Ca++ induced fusion which involves large clump formation. The freshly isolated protoplasts from two selected parents are mixed in appropriate proportions and treated with 15-45% PEG (1500-6000MW) solution for 15-30 min followed by gradual washing of the protoplasts to remove PEG. Protoplast fusion occurs during washing. The washing medium may be alkaline (pH 9-10) and contain a high Ca++ ion concentration (50 mM). This combined approach of PEG and Ca++ is much more efficient than the either of the treatment alone. PEG is negatively charged and may bind to cation like Ca++, which in turn, may bind to the negatively charged molecules present in plasma lemma, they can also bind to cationic molecules of plasma membrane. During the washing process, PEG molecules may pull out the plasma lemma components bound to them. This would disturb plamalemma organization and may lead to the fusion of protoplasts located close to each other (Figure 13.3). The technique is nonselective thus, induce fusion between any two or more protoplasts.