Fusion of protoplasts can be accomplished by two distinct methods like chemical fusion and electro-fusion (Fig. 10.1). Earliest method known as mechanical fusion, where protoplasts are forced to fuse under applied pressure within the pipette. Protoplast in suspension can undergo spontaneous fusion in cultural conditions.
Method # 1. Chemical Fusion of Protoplast:
Chemicals such as sodium nitrate, polyethylene glycol and calcium ion at high pH are used for protoplast fusion with different degrees of success. Sodium nitrate (5%) in 10% sucrose solution is mixed with protoplast suspension, incubated in test tube and maintained at 35°C in water bath for 10 min.
This would allow an efficient fusion of protoplast. Calcium ion could play a significant role in inducing protoplast fusion. Addition of calcium ion at high pH (10.5), alone or in combination with other chemical allows more efficient fusion. The process involves centrifugation of protoplast for 3 min at 100 g in presence of solution containing 0.05M CaCl2 in 0.4M mannitol at pH 0.5.
Following incubation in water bath for 40 min at 37°C, fusion of protoplast is accomplished. Since calcium ion acts as cation, it can alter Zeta potential of charged ratio on the protoplast membrane. Treating protoplast with calcium ion significantly affects p-potential of protoplast surface, i.e., from -10 mV to -50 mV. Besides, cell to cell contact is aided by calcium ion.
Polyethylene Glycol Mediated Fusion:
Polyethylene glycol (PEG) can initiate tight agglutination of protoplast. The protoplast in nutrient medium is mixed with 1 ml of PEG solution and is shaked for 5 s, resulting in agglutination followed by fusion of protoplasts. PEG at the concentration between 36 and 56% is employed for protoplast fusion.
Combined role of basic solution (pH 10.5) containing Ca, 0.05M CaCl2 and PEG treatment improves the efficiency of protoplast fusion. More frequently, protoplasts are treated with PEG solution after settlement on the glass cover slips. It has been suggested that combined use of PEG and Ca acts as a bridge between the two protoplasts and it is because of protoplasts’ slight negative polarity.
Method # 2. Electro-Fusion of Protoplast:
Electro-fusion is extremely a rapid method for protoplast fusion. When protoplasts are exposed to electric field, membrane fusion is achieved by close membrane contact, transient membrane breakdown and reorganization of membrane contact. Pioneer work on electro-fusion has been carried out by Zimmerman. Electro-fusion is a biophysical process that involves the application of alternative current (AC) and direct current (DC).
In the first step, protoplasts are exposed to a high frequency of AC field strength (0.5 to 1.5 MHz) that generates dipoles through a condition known as dielectrophoresis. Due to dielectrophoresis, protoplast moves in the increasing field strength and the mobilization of charge takes place within the protoplasts. As a consequence, protoplast aggregate or bound close together.
In the following step, a short pulse of direct current (DC) of sufficient voltage is able to cause membrane breakdown. Once DC is applied, cell membrane breaks down at the point of contact between the cells. This happens because the cell interior acts as a better conductor than surrounding medium. As a consequence, most of the current moves through the cells rather than through the medium.
Moving of DC pulse at the point of cell contact leads to cell fusion rather than cell lysis. Duration and nature of DC pulse is crucial in achieving protoplast fusion. The square wave DC pulse, with a rise and decay time of less than 2 µs is sufficient to disrupt the membrane junction. Generally a single square wave pulse of 600-700 V/cm will cause fusion. Long pulse duration of 100 µs and high voltage will cause cell lysis (Fig. 10.2).
