Figure 13.3: Sequential stages in protoplast fusion. (A) two separate protoplasts, (B) agglutination of two protoplasts, (C and D) Membrane fusion at localized site, and (E and F) development of spherical heterokaryon.

 

iv. Electrofusion: The chemical fusion of plant protoplast has many disadvantages – (1) The fusogen are toxic to some cell systems, (2) it produces random, multiple cell aggregates, and (3) must be removed before culture. Compare to this, electrofusion is rapid, simple, synchronous and more easily controlled. Moreover, the somatic hybrids produced by this method show much higher fertility than those produced by PEG-induced fusion.

Zimmermann and Scheurich (1981) demonstrated that batches of protoplasts could be fused by electric fields by devising a protocol which is now widely used. This protocol involves a two-step process. First, the protoplasts are introduced into a small fusion chamber containing parallel wires or plates which serve as electrodes. Second, a low-voltage and rapidly oscillating AC field is applied, which causes protoplasts to become aligned into chains of cells between electrodes. This creates complete cell-to-cell contact within a few minutes. Once alignment is complete, the fusion is induced by application of a brief spell of high-voltage DC pulses (o.125-1 kVcm^-1). A high voltage DC pulses induces a reversible breakdown of the plasma membrane at the site of cell contact, leading to fusion and consequent membrane reorganization. The entire process can be completed within 15 min.