|
|
|
|
Fusion
of
Protoplasts - Fusion of protoplasts facilitates mixing of two whole genomes and could be exploited in crosses at interspecific, intergeneric, or even interkingdom levels, which are not possible by conventional techniques due to incompatibility. The fact that isolated protoplasts are devoid of walls makes them easy tools for undergoing fusion in vitro.
Mixing two genomes opens the door to gene transfer and a study of gene expression, stability of several traits, and cell genetic changes. Protoplast fusion may be spontaneous or induced. Induced fusion is brought about by mechanical, chemical or physical means.
|
Spontaneous Fusion: When the callus cultures or cells grown in suspension are subjected to enzymatic degradation of the cell wall, the plasmodesmata of adjoining cells expand instead of breaking down, facilitating spontaneous fusion of protoplasts to form homokaryons (homokaryocytes). These plurinucleate cells sometimes contain 2-40 nuclei.
This type of protoplast fusion is usually observed when protoplasts are isolated from tissue cultures grown in vitro and rare when they are isolated from mesophyll tissue. The spontaneous fusion products do not regenerate into whole plants except for undergoing a few divisions. Similar spontaneous fusion is like wise common during preparation of protoplasts from meocytes.
|
However, the sequential method of protoplast isolation or exposure of the cells to a strong plasmolyticum would break the plasmodesmatal connections and consequently reduce the frequency of spontaneous fusion. Spontaneous fusion is nonreproducible and rare.
Induced Fusion: Here the protoplasts are made to fuse with .the help of an external agent such as mechanical force or fusing chemicals or electric charges. Induced fusion is reproducible and efficient.
|
|
In fusion through mechanical means, the protoplasts of two different species or the same species are passed through a micropipette, the tip of which is partially blocked. During this the protoplasts come into intimate contact, are retained, and remain compressed by the flow of the liquid.This fusion does not depend upon the presence of a fusion inducing agent (fusogen). In" this procedure the protoplasts are likely to be injured.
In the fusion of protoplasts through chemical means, freshly isolated protoplasts are induced to undergo fusion by means of a range of fusogens, e.g. NaN03, artificial sea water, lysozyme, high pH/Ca++, polyethylene glycol (PEG), antibodies, concanavalin A, polyvinyl alcohol, text ran and dextran sulphate, fatty acids, lectins and esters. Of these, NaN03, high pH/Ca++ and PEG treatments are used with great success.
|
|
|
Induced fusion by NaNO3 was first demonstrated by Power et al. (1970). Isolated protoplasts were cleaned by floating in sucrose osmoticum. Transfer of the protoplasts in 0.25 M NaNO3 solution and subsequent centrifugation promoted fusion process. Carlson et al.(1972) used this method for producing the first somatic hybrid plant by fusing protoplasts of Nicotiana glauca and N. langsdorffii.
Even though NaNO3-induced fusion of protoplasts is reproducible, it promotes a very low incidence of fusion and only results in a small increase over spontaneous fusion frequencies. This could be due to the requirements of nearly identical osmotic characteristics of the protoplasts under fusion and the poor effect on protoplast viability.
To improve the incidence of fusion, Keller and Melchers (1973) used high alkaline pH (10.5), a medium containing high Ca++ ions and induced fusion of protoplasts at high temperature (370 C). Isolated protoplasts were incubated in a solution of 0.4 M mannitol containing 0.05 M CaCl2, with pH at 10.5 (0.05 M glycine-NaOH buffer) and temperature 370 C.
|
Aggregation of protoplasts generally took place at once and fusion occurred within 10 min.
It is believed that under these conditions the negative charge present on the protoplast membrane is lost, facilitating agglutination and subsequent fusion of protoplasts.
Many intraspecific and interspecific somatic hybrids have been produced using this procedure. Fusion frequencies of greater than 25% are reported with this method.
The polyethylene glycol (PEG) method is more often used today to achieve fusion of protoplasts. It was first proposed by Kao and Michayluk (1974).
This method envisages the incubation of protoplasts in a high molecular weight PEG and Ca++ ions in lower concentrations. About 0.6 ml of PEG solution (dissolve 1 g of PEG, mol wt 1500 in 2 ml of 0.1 M glucose, 10 mM CaCl2, and 0.7 mM KH2PO4) is added in drops to a pellet of protoplasts in a tube.
After having capped the tube, protoplasts in PEG are incubated at room temperature for 40 minutes. Occasional shaking of the tube helps to bring the protoplasts in contact. This is followed by elution of PEG by the addition of 0.5-1 ml of protoplast culture medium in the tube after every 10 minutes.
Preparations are now washed free of fusogen by centrifugation and the protoplasts resuspended in the culture medium. Both the molecular weight and the concentration of PEG are critical in inducing successful fusions. For inducing fusion high molecular weight PEG 1540-6000 is generally used at 25-30% concentrations.
The incidence of PEG induced fusion of protoplasts is very high, usually above 75 to 100%. The PEG-induced fusion is nonspecific and therefore useful for intra, interspecific, intergenetic, or even interkingdom fusion, involving plant and animal cells. The mechanism of action of PEG is not known clearly. However, it has been suggested that PEG acts as a molecular bridge, thereby dissociating the plasmalemma.
Also, the high polar nature as well as weak ionic charge of PEG facilitate the integration of groupings on the proteins and lipids of opposite membranes. PEG at low molecular weight is not able to produce tight adhesions.
Some modifications are suggested to the PEG method of Kao and Michayluk. The addition of concanavalin A to the PEG solution for the purpose of increasing the incidence of fusion, since this strengthens the attachment of protoplasts, is recommended. The addition of dimethyl sulphoxide (DNSO4) to make protoplasts more susceptible to PEG treatment is also recommended.
After treatment with fusogen, protoplasts are cultured following the usual procedures depicts the fusion of protoplasts using fusogen. The various products which may result as a consequence of nuclear and protoplast fusion.
In the fusion of protoplasts through an electric charge, they are brought into close contact by applying a non-uniform ac field of low intensity to the suspension followed by a brief intense dc pulse. The dc pulse induces remarkable, reversible breakdown of cell membranes in contact areas of the adjacent cells, resulting in fusion and consequent membrane reorganization.
This procedure is called electrofusion and has been found to be simpler, quicker and more efficient than other methods. More importantly, cells after electrofusion do not show cytotoxic responses as generally found in protoplasts or heterokaryons subjected to PEG treatment.
Senda et al. (1979) were the first to attempt fusion by positioning two microelectrodes with the help of a micromanipulator at the ends of adjoining Rauwolfia protoplasts. Success in inducing fusion was achieved with brief 5-12 amp dc pulses restricted to single protoplast pairs only.
Subsequently, 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 fusion chamber containing parallel wires or plates which serve as electrodes. Second, a low-voltage, rapidly oscillating ac field is applied, which causes protoplasts to become aligned into chains of cells (pearl chains) between the electrodes.
This creates a 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. The entire process, starting from the introduction of the protoplasts inside the chamber and their transfer to culture media, can be completed in 5 minutes or less.
The electrofusion apparatus is named the Zimmermann cellfusion TM system. Heterokaryons produced by electrical fusion divide in the culture medium and have demonstrated the capability of regenerating somatic hybrid plants.
| |
