Protoplast fusion/Somatic fusion:
- Protoplast fusion is a physical method of fusion of somatic cells from different plant to form hybrid.
- Mixing of protoplasts of two different genomes and can be achieved by either spontaneous or induced fusion methods.
Methods of Protoplast fusion:
1. Spontaneous fusion:
- Cell fusion is a process integral to plant development.
- The most prominent process is egg fertilization.
- The breakdown of cell wall during protoplast isolation led people to believe that there would be spontaneous fusion leading to the formation of homokaryons because multinucleate cells were detected as soon as enzymatic protoplast isolation techniques were applied.
- The argument that cell wall degradation would permit dilation of plasmodesmata, fusion and complete mixing of cells was supported by electron micrographic studies.
- Spontaneous fusion of protoplasts is observed when protoplasts are isolated from callus cultures.
- However, spontaneous fusion products do not regenerate in to whole plants except for undergoing a few divisions.
- Later studies revealed that isolated protoplasts are usually characterized by smooth surfaces and fusion has to be induced by one of a variety of treatments.
2. Induced fusion methods:
- Spontaneous fusion is of no value as fusion of protoplasts of different origins is required in somatic hybridization.
- To achieve this, a suitable agent (fusogen) is added to fuse the plant protoplasts of different origins.
- The different fusogens employed are: NaNO3, artificial sea water, lysozyme, high pH/Ca++, polyethylene glycol, antibodies, concavalin A, polyvinyl alcohol, electrofusion dextran and dextran sulphate, fatty acids and esters.
- Some of the methods that have been employed are explained below.
i. Treatment with sodium nitrate:
- Power et al. first reported induced fusion by NaNO3 (1970).
- Isolated protoplasts are suspended in a mixture of 5.5% sodium nitrate in a 10% sucrose solution.
- The solution containing the protoplasts is incubated in a water bath at 35°C for 5 min and then centrifuged for 5 min at 200x g.
- Following centrifugation, most of the supernatant is decanted and the protoplast pellet is transferred to a water bath at 30°C for 30 min.
- During this period, most of the protoplasts undergo cell fusion.
- The remaining aggregation mixture is gently decanted and replaced with the culture medium containing 0.1% NaNO3.
- The protoplasts are left undisturbed for sometimes after which they are washed twice with the culture medium and plated.
- This technique results in low frequency of heterokaryon formation, especially when mesophyll protoplasts are involved.
ii. Calcium ions at high pH:
- The effect of high pH and calcium ions on the fusion of tobacco protoplasts was first studied by Keller and Melchers (1973) .
- In their method, isolated protoplasts are centrifuged for 3 min at 50x g in a fusion-inducing solution of 0.5 M mannitol containing 0.05 M CaCl2·2H2O at a pH of 10.5.
- The centrifuge tubes containing the protoplasts are then incubated in a water bath at 37°C for 40–50 min.
- After this treatment, 20–50% of the protoplasts were involved in fusion.
iii. Polyethylene glycol (PEG) method:
- Kao and Michayluk (1974) and Wallin et al. (1974) developed PEG method of fusion of protoplasts.
- This is one of the most successful techniques for fusing protoplasts.
- The protoplasts are suspended in a solution containing high molecular weight PEG, which improves agglutination and fusion of protoplasts in several species.
- When adequate amount of protoplasts are available, 1 ml of the protoplasts suspended in a culture medium are mixed with 1 ml of 28–56% PEG (1500 –6000 MW) solution.
- The tube is then shaken for 5 sec and allowed to settle for 10 min.
- To remove PEG, the protoplasts are then washed several times by the addition of protoplast culture medium.
- The protoplast preparation is again suspended in the culture medium.
- The PEG method is popular for protoplast fusion as it yields in reproducible high-frequency heterokaryon formation, low cytotoxicity to most cell types and the formation of binucleate heterokaryons.
- PEG-induced fusion is non- specific and is thus applicable for interspecific, intergeneric or interkingdom fusions.
- Both the molecular weight and the concentration of PEG are critical in inducing successful fusions.
- PEG less than 100 molecular weight is not able to produce tight adhesions while that ranging up to 6000 molecular weight can be more effective per mole in inducing fusions.
- At higher molecular weight PEG produces too viscous a solution which cannot be handled properly.
- Treatment with PEG in the presence of/or by high pH/Ca++ is reported to be most effective in enhancing the fusion frequency and survivability of protoplasts.
iv. Electrofusion:
- Protoplasts are placed in to a small culture cell containing electrodes, and a potential difference is applied due to which protoplasts line up between the electrodes.
- If now an extremely short wave electric shock is applied, protoplasts can be induced to fuse.
- In this fusion method, two-step procedure is followed beginning with application of an alternating current (AC) of low intensity to protoplast suspension.
- Dielectrophoretic collectors adjusted to 1.5 V and 1 MHz and an electrical conductivity of the suspension medium less than 10–5 sec/cm generate an electrophoresis effect that make the cells attach to each other along the field lines.
- The second step of injection of an electric direct current (DC) field pulse of high intensity (750– 1000 V/cm) for a short duration of 20–50 μsec leads to breakdown of membranes in contact areas of adjacent cells resulting in fusion and consequent membrane reorganization.
- This electrofusion technique has been found to be simple, quick and efficient. Cells after electrofusion do not show cytotoxic response.
- However, this method did not receive much acceptance because specialized equipment is required.
Mechanism of Protoplast fusion
Protoplast fusion consists of three main phases:
Phase I: Agglutination or adhesion:
- Two or more protoplasts are brought into close proximity.
- The adhesion can be induced by a variety of treatments, e.g. concanavalin A, PEG, high pH and high Ca++ ions.
Phase II: Plasma membrane fusion at localized sites:
- Membranes of protoplasts are stuck together by fusogen get fused at the point of adhesion resulting in the formation of cytoplasmic bridges between the protoplasts.
- Plant protoplasts carry a negative charge from –10 to –30 mV.
- Due to common charge, the plasma membranes of two agglutinated protoplasts do not come close enough to fuse.
- Fusion requires that membranes must be first brought close together at a distance of 10Å or less.
- The high pH–high Ca++ ions treatment has shown to neutralize the normal surface charge so that agglutinated protoplasts can come in intimate contact.
- High temperature promotes membrane fusion due to perturbance of lipid molecules in plasma membrane and fusion occurs due to intermingling of lipid molecules in membranes of agglutinated protoplasts.
- PEG agglutinates to form clumps of protoplasts.
- Tight adhesion may occur over a large or small localized area.
- Localized fusion of closely attached plasma membranes occurs in the regions of tight adhesion and results in the formation of cytoplasmic bridges.
- It has been further suggested that PEG, which is slightly negative in polarity can form hydrogen bonds with water, protein, carbohydrates, etc. which are positive in polarity.
- When the PEG molecule chain is large enough it plays role as a molecular bridge between the surface of adjacent protoplasts and adhesion occurs.
Phase III: Formation of heterokaryon:
- Rounding off of the fused protoplasts due to the expansion of cytoplasmic bridges forming spherical heterokaryon or homokaryon.