protoplast fusion

Somatic fusion,

 also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid.

Methods of Protoplast Fusion:

Broadly speaking, protoplast fusion can be classified into two categories:

1. Spontaneous fusion;

2. Induced fusion.

In somatic hybridisation, spontaneous fusion is of little significance. The methods used for induced fusion can again be sub-categorized.

I. Spontaneous Fusion:

Protoplasts, during isolation, often fuse spon­taneously and this phenomenon is called spon­taneous fusion. Simple physical contact is sufficient to bring about the spontaneous fusion among the similar parental protoplasts. During the enzyme treatment for the isolation of pro­toplasts, it is found that protoplasts from ad­joining cells fuse through their plasmodesmata to form a multinucleate protoplast.

Electron microscopic studies have shown that as the cell walls are enzymatically degraded, the plasmodesmatal connection between the adjacent cells enlarge due to removal of its constriction and the enlargement of pit fields.

Eventually, the greater enlargement of plasmodesmata allow the entry of organelles into neighbouring cells. Finally a complete coalescence of adjacent cell takes place. Spontaneous fusion is strictly intraspecific and gives rise to homokaryon.

The protoplasts, once they are freely iso­lated, do not fuse spontaneously with each other. An exception is the protoplast from microsporocytes of some plants of lily family where the freely isolated protoplasts fuse spon­taneously. This type of spontaneous fusion has been used to produce inter-generic fusion, e.g., the spontaneous fusion of microsporocyte protoplast of Lolium longiflorum and Trillium kamtschaticum.

II. Induced Fusion:

Fusion of freely isolated protoplasts from dif­ferent sources with the help of fusion inducing chemical agents is known as induced fusion.

Normally, isolated protoplasts do not fuse with each other because the surface of the isolated protoplast carries negative charge (-10 to -30 mV) around the outside of plasma membrane and, thus, there is a strong tendency for pro­toplasts to repel one another due to their same charges.

So this type of fusion needs a fusion inducing chemical agent or system which actually reduces the electronegativity of the isolated protoplasts and allow them to fuse with each other.

Actually, induced fusion is a highly impor­tant and a valuable technique because the protoplast from widely different and sexually incompatible plants can be fused by this procedure. This technique has the possibility and ability to combine different genotypes beyond the limits imposed by sexual process. The fun­damental objectives of somatic hybridisation are mainly based on induced protoplast fusion.

The isolated plant protoplasts can be induced to fuse by three ways:

(i) Mechanical Fusion:

 

In this process, the isolated protoplasts are brought into intimate physical contact me­chanically under microscope using microma­nipulator and perfusion micropipette. This micropipette is partially blocked within 1 mm of the tip by a sealed glass rod. In this way the protoplasts are retained and compressed by the flow of liquid. By this technique occasional fusion of protoplast has been observed.

(ii) Chemo-Fusion:

Spontaneous fusion of two or more adjoining Several chemicals have been used to induce somatic protoplasts is of no practical use, but protoplast fusion. Sodium nitrate (NaN03), this may be important in studies of the nature polyethylene glycol (PEG), Calcium ions and function of plasmodesmata, the physiology (Ca2+), Polyvinyl alcohol etc. are the most and control of mitosis in multinucleated cells commonly used protoplast fusion inducing and nuclear fusion.

(iii) Electro Fusion:

Electro fusion is a modern technique of pro­toplast fusion which involves the use of mild electrical fields in protoplast suspension for inducing protoplast fusion. This technique is very easy, simple and fast. It is often more efficient than chemical induced fusion (chemo fusion).

Electro fusion is also applicable to those species whose protoplasts exhibit a severe toxic response to polyethylene glycol used for chemo fusion. The origin of electrofusion is based on biophysical studies of cell membrane.

The first report of protoplast electro fusion is that of Senda etal (1979). They placed two microelectrodes at the ends of pairs adhering Rauwolfia protoplasts and induced fusion with 5-12 µAmp DC pulse. In their experiment fusion yields were restricted to single protoplast pairs. However, Zimmermann and Scheurish (1981) improved this method for the large scale fusion of plant protoplasts.

In this protocol, fu­sion is a two-step process. First the protoplasts are put into a small fusion chamber (Fig. 6.15) containing parallel wires or plates which serve as electrodes. After that, a low-voltage rapidly oscillating AC field (100v/cm, 0.6 MHz) is applied. Within a few minutes, this AC field causes the protoplasts to become aligned into chains of cell between the electrodes.