the medium used in protoplast fusion experiments is

Two glass capillary microelectrode are placed in contact with the protoplasts. This technique is known as electrofusion of protoplasts. Recently, mild electrical stimulation is being used to fuse protoplasts. The PEG method has been modified slightly to fuse higher plant protoplast as indicated below:Ī) PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide.ī) Addition of concanvalin A to PEG increases protoplast fusion frequency.Ĭ) Sea water has been used alone or in combination with PEG to fuse protoplasts. In some studies, high PH/Ca++ and PEG method have been combined. But higher concentration of Ca++ ion has been considered beneficial. Carrot protoplast can be fused by 28% PEG 1500 and fusion can be promoted by ca++ ion at concentration of 3.5 mM. Fusion of protoplast takes place during slow elusion of PEG with liquid culture medium. A solution of 37.5% w/v PEG of molecular weight 1500 to 6000 aggregates mesophyll and cultured cell protoplasts during a 45 minutes incubation at room temperature. But the concentration and molecular weight of PEG are important with respect to fusion. PEG induces protoplast aggregation and subsequent fusion. About 20-30% protoplast are involved in this fusion experiment. This followed by keeping the tubes in water bath (37 0C) for 40- 50 minutes. The centrifuge tube containing protoplast at high PH or Ca++ is placed in water bath at 30 0C for 10 minutes and is spun at 50 g for 3 to 4 minutes. a 4 ml of 0.05 M CaCl2, 2H2O in 0.4 M mannitol at PH 10.5 is mixed to the protoplast suspension. The pellet is suspended in 0.5 ml of medium. Equal densities of protoplasts are taken in centrifuge tube and protoplasts are spun at 100 g for 5 minutes. Kelier and Melchers ( 1973) developed a method to effectively induce fusion of tobacco protoplasts at a high temperature ( 37 0C) in media containing high concentration of Ca++ ions at a highly alkaline condition ( PH 10.5). The frequency of fusion is not very high in this method. Finally, the protoplasts are plated in semisolid culture medium. The fusions of protoplast take place at the time of incubation. The pellet is once again kept in water bath at 30 0C for 30 minutes. The suspended protoplasts are kept in water – bath at 35 0C for 5 minutes and again centrifuged at 200 g for 5 minutes. This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to Resuspend the protoplast pellet. Normally, isolated protoplasts donot fuse with each other because the surface of the isolated protoplasts carries negative charge around the outside of plasma membrane and thus is a strong tendency for protoplasts to repel one another due to their same charges.Įqual densities of protoplasts from two different sources are mixed and then centrifuged at 100g for 5 minutes to get a dense pellet. Protoplast released from meiocytes in enzyme solutions readily fuse by gentle tapping in depression slide.įusion of freely isolated protoplasts from different sources with the help of fusion inducing chemical agents is known as induced fusion. However, in this procedure protoplasts are likely to get injury. This fusion does not upon the presence of fusion inducing agents. The giant protoplast of Acelabularia have been fused mechanically by pushing together two protoplast. The protoplasts once they are freely isolated, donot fuse spontaneously with each other. These plurinucleate cells sometimes contain 2-3 nuclei, a phenomenon attributed to expansion and subsequent coalescence of the plasmodesmatal connections between the cells. An important aspect has been that incompatibility barriers do not exist during the cell fusion process at Interspecific, Intergeneric or even inter kingdom level.ĭuring the enzymatic degradation of cell walls some of the adjacent protoplasts may fuse together to form homocaryons. During fusion, two or more protoplasts come in contact and adhere with one another spontaneously or in presence of fusion inducing chemicals. Protoplasts fusion is a physical phenomenon.

The medium used in protoplast fusion experiments is