Induced Protoplast Fusion,Spontaneous Fusion,Plant Protoplasts,Polyethylene Glycol Treatment,Electrical Fusion

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	Induced Protoplast Fusion - Somatic hybridization is generally used for fusion of protoplasts either from two different species (interspecific fusion) or from two diverse sources belonging to the same species. To achieve this objective, spontaneous fusion may be of no value, and induced fusion requiring a suitable agent (fusogen) is necessary. In animals, inactivated Sendai virus is needed to induce fusion. In plants, however, the inducing agent first brings the protoplasts together and then causes them to adhere to one another for bringing about fusion. During the last two decades, a variety of treatments have been successfully utilized for fusion of plant protoplasts. These treatments particularly include the following: NaNO₃, high pH with high Ca⁺⁺ ion concentration and polyehtylene glycol (PFG).
(a) NaNO₃ treatment. This method was successfully utilized for fusion of protoplasts from root tips of oat and maize seedlings but is not preferred due to low frequency of fusion, particularly when highly vacuolated mesophyll protoplasts are used. The method involves the following steps: (i) isolated protoplasts are suspended in an aggregation mixture (5' 5% sodium nitrate in 10% sucrose solution). This mixture works as a fusion inducing mixture and causes fusion on incubation (water bath maintained at 35°C). In order to obtain a higher frequency of fused protoplasts, the mixture may be centrifuged and the pellet resuspended and incubated for one or more additional cycles.
(ii) Finally the mixture is replaced by a liquid medium and' the protoplasts in this mixture are incubated again; the cycle may be' repeated once or twice before plating the protoplasts on a solid medium. The fusion of protoplasts may be monitored at different steps through examination under an inverted microscope. (b) Treatment with calcium ions (Ca++) al high pH. This method involves spinning (centrifugation) the protoplasts in a fusion inducing solution (0' 05M CaCl₂ 2H₂O in O' 4M mannitol at pH 10, 5) for 30 min. at 50g, after which the tubes are placed in a water bath (37°) for 40-50 min.
This leads to fusion of 20-50% of the protoplasts. The details of the protocol are described by Bhojwani and Razdan (1983). The method has been found superior to other methods in some cases, but high pH may be toxic in other cases. (c) Polyethylene glycol (PEG) treatment. Since 1974, protoplast fusion has been successfully achieved in several crops, using polyethylene glycol (PEG) as a fusogen. The technique gives high frequency of fusion with reproducible results and involves low cytotoxicity. The technique can be used for fusion of protoplasts from unrelated plant taxa (e.g. soybean tobacco, soybean , maize, and soybean - barley), from unrelated animal taxa and also between those from animal and plant cells.
	The agglutination of protoplasts, during PEG treatment, can be brought about by the following two different methods: (i) When protoplasts are available in sufficient quantities, 1ml of culture medium with suspended protoplasts is added to 1ml of 56% solution of PEG and the tube shaken for 5 sec. The protoplasts are allowed to sediment fqr 10 min, washed with growth medium and examined for successful agglutination and fusion. (ii) If protoplasts are available in microquantities, drop cultures can be used. Two types of protoplasts are mixed in equal quantities and 4-6 microdrops (100 μl each) are placed in a small Petri plate and allowed to settle for 5-10 min. at room temperature. Two to three microdrops (50 μ/ each) of PEG are added from the periphery in each Petri plate, which is incubated for 30 min at room temperature (24°C).

This leads to agglutination of protoplasts. Sometimes a cover glass is placed in the middle of Petri plate 'before protoplast suspension is poured. This avoids sticking of protoplasts to the floor of Petri plate and also makes it convenient to handle the protoplasts including their fixation, staining and examination.
After PEG treatment, protoplasts are gradually washed and during this process, most of the fusion is achieved. PEG is then replaced by culture medium to allow growth of fused protoplasts.

(d) Electrical fusion. If protoplasts are placed into a small culture cell containing electrodes and a potential difference is applied, then the protoplasts will line up between the electrodes. If now an extremely short, square, wave electric shock is applied, protoplasts can be induced to fuse.