In this article we will discuss about Protoplast Fusion:- 1. Meaning of Protoplast Fusion 2. Methods of Protoplast Fusion 3. Mechanisms.
Meaning of Protoplast Fusion:
Protoplast fusion is a physical phenomenon. During fusion, two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing chemicals. After adhesion, membranes of protoplasts fuse in some localised areas and, eventually, the cytoplasm of the two protoplasts intermingle.
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.
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.
Perhaps spontaneous fusion agents which are commonly known as chemical has some practical importance for chromosome fusogens. Generally, chemo fusion techniques doubling are followed in most of induced fusion experiments.
Chemical fusogens cause the isolated protoplasts to adhere to one another and leads to tight agglutination followed by fusion of protoplast (Fig. 6.14). The adhesion of isolated protoplast takes place either due to reduction of negative charges of protoplast or due to attraction of protoplast by electrostatic forces caused by chemical fusogens.
Chemo Fusion Procedures:
Several chemo fusion procedures have been proposed time to time to improve the fusion frequency and reproducibility of the fused product. Each and every method has its own merits and limitations.
Some chemo fusion methods are described below:
(a) Fusion Induced by Sodium or Potassium Nitrate:
Fusion of isolated onion sub-protoplasts plasmolysed with sodium salts was achieved for the first time by Kiister (1909). Subsequently, Michel (1937) demonstrated fusion between protoplasts using potassium nitrate as plasmolyticum. Power etal (1970) reported sodium nitrate induced fusion of cereal root protoplasts.
By this method, equal densities of protoplasts from two different sources are mixed and then centrifuged at 100 g for 5 minutes to get a dense pellet. This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to re-suspend the protoplast pellet. The suspended protoplasts are kept in water-bath at 35° C for 5 minutes and again centrifuged at 200 g for 5 minutes. The pellet is once again kept in water-bath at 30°C for 30 minutes.
Fusion of protoplast takes place at the time of incubation. The, pellet is again suspended by 0.1% sodium nitrate for 5-10 minutes. The protoplasts are washed twice with liquid culture medium by repeated centrifugation. Finally, the protoplasts are plated in semisolid culture medium.
Using the above principle, intra-and interspecific fusions have been achieved by several workers. However, sodium nitrate is toxic to cell at fusogenic concentration. The frequency of fusion is not very high in this method. Yet it is useful only for the protoplasts derived from meristematic cells.
(b) Fusion Induced by Calcium Ions at High pH:
In 1973, Keller and Melcher (from Germany) developed a method to effectively induce, fusion of tobacco protoplast at high temperature (37° C) in media containing high concentration of Ca2+ ions, (i.e., calcium chloride) at a highly alkaline condition (pH 10.5).
Equal densities of protoplasts are taken in a centrifuge tube and the protoplasts are spun at 100 g for 5 minutes. The pellet is suspended in 0.5 ml of medium. 4ml of 0.05M CaCl22H20 in 0.4M mannitol at pH 10.5 is mixed to the protoplast suspension.
The centrifuge tube containing protoplasts at high pH/Ca2+ is placed in the water bath at 30° C for 10 minutes and is spun at 50 g for 3-4 minutes. This is followed by keeping the tubes in water bath (37°C) for 40-50 minutes. About 20-30% protoplasts are involved in this fusion experiment.
(c) Fusion Induced by PEG:
In 1974, Kao and Michayluk from Canada discovered another fusion inducing chemical polyethylene glycol (PEG) which is the most effective agent discovered so far. Many fusion experiments are performed by a polyethylene glycol.
PEG induces protoplast aggregation and subsequent fusion. But the concentration and molecular weight of PEG are important with respect to fusion. A solution of 37.5% w/v PEG of molecular weight 1,540 or 6,000 aggregates mesophyll and cultured cell protoplasts during a 45 minutes incubation period at room temperature.
Fusion of protoplast takes place during slow elution of PEG with liquid culture medium. Carrot protoplast can be fused by 28% PEG 1540 and the fusion can be promoted by Ca2+ ion at the concentration of 3.5 mM. But higher concentration of Ca2+ ion (10 or 50 mM) has been considered beneficial.
In some studies, high pH/Ca2+ and PEG method have been combined. By this method, the agglutination of protoplasts can be brought about using sufficient quantities (0.1-5 ml) of protoplast in centrifuge tube or micro densities (150 µ l) of protoplast on a coverslip. The PEG method has been modified slightly to fuse higher plant protoplast.
The modifications are given below:
1. PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide (DMSO).
2. Addition of concanavalin A (Con A) to PEG increases protoplast fusion frequency.
3. Sea water has been used alone or in combination with PEG to fuse protoplasts.
(d) Fusion Induced by Other Chemicals:
Some other chemicals have also been observed to promote protoplast fusion:
1. 15% solution of Polyvinyl alcohol (PVP) in combination with 0.05 CaCl2 and 0.3 M mannitol are used to fuse plant protoplasts.
2. Lectins are also known to agglutinate protoplasts.
3. Various proteins are also used for agglutination of protoplast.
(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.
This alignment is known as pearl chain arrangement [Fig. 6.16(a) and (b)]. It leads to cell-to-cell contacts which are a prerequisite of fusion. In the second step, once alignment is complete, fusion is induced by application of one to two brief high voltage DC pulse (800 v/cm, 15 µsec duration).
These DC pulse creates a reversible breakdown of the plasma membrane’s structure. When membrane breakdown occurs at sites of the cell contact, the ensuring membrane reorganization leads to cell fusion.
Thus, AC field-induced alignment followed by DC pulse-induced fusion leads to highly efficient protoplast fusion. After fusion, the protoplasts are transferred to culture media. This entire process from the introduction of protoplasts into the fusion chamber to their transfer to culture media can be completed in 5 min or less. In this method the fusion efficiencies are higher than the efficiencies of chemo fusion.
The alignment of cells into pearl chains in response to AC fields in known as mutual di-electrophoresis and is due to a field-induced separation of cell-surface charges. As a result, cells that are close together are attracted to each other and form pairs and chains of cells that radiate outward perpendicularly from the electrodes.
The formation of cell to cell contact depends on several factors:
(i) The frequencies, and voltage of AC field;
(ii) The shape of the electrodes and
(iii) The composition of the medium.
Generally, AC field frequencies in the range of 0.5-1 MHz (megahertz or million cycles per second) are used for bringing the protoplasts into a line. The voltage needed for cell alignment depends on the shape of the electrode and the distance separating them. With needle electrodes spaced 0.5 mm apart, a 5-10 V AC field produces good di-electrophoresis.
If the electrodes are kept further apart, the voltages must be correspondingly higher. To account for the electrode spacing, voltage used for di-electrophoresis and cell fusion are usually expressed as field strengths, i.e., V/cm.
In the above example, the AC field strength would be 100-200 V/cm. Generally, the electrodes used for di-electrophoresis are wire or needle because they result in a non-uniform electrical field. The more non-uniform the field, the lower the AC voltage required to produce cell alignment by di-electrophoresis. It should be pointed out that plate electrodes can also be used (Fig. 6.17).
The di-electrophoresis force is inversely proportionate to the conductivity of the media in which the protoplasts are suspended for fusion. If the conductivity of the medium is very high, the di-electrophoretic force will be very low.
However, the di-electrophoresis force is greatest in medium of low conductivity. Therefore, electro fusion is carried out in media containing an inert osmoticum such as mannitol or sucrose added with very little amount of salts, if required.
Sometimes a small amount of CaCl2 (0.1-0.5 mM) is added to improve fusion and to reduce cell lysis. But if the concentration of Ca++ is increased more than 0.5 mM, the medium will acquire higher conductivity and, ultimately, it greatly reduces di-electrophoresis.
This effect can be nullified if the AC voltage is increased. But it leads to heating of the medium and the resulting effect ultimately prevents the cell contacts and also reduces the viability of the protoplasts in the long run.
In electro fusion, cell contacts can also be improved simply by use of very high protoplast densities or by addition of small amount of PEG to the medium. In these alternative approaches, electro fusion is obtained by application of only the high voltage DC pulse.
It has been studied and observed that cell membranes undergo a dramatic increase in permeability when exposed to high voltage DC pulses. This phenomenon can be detected by using radio-labelled tracer molecules and their rate of movement across the membrane in comparison to control one.
On the basis of experimental evidences it has been suggested that increase in membrane permeability mainly happens due to formation of discrete, nanometer-sized transient pores in response to these high voltage pulses.
This phenomenon is termed as electroporation. It means the formation of transient pores in the cell membrane in response to electric impulse. The size and number of the pores increases with increasing voltage and pulse length.
DC pulses of 1,000 V/cm for 10-15 µsee. are usually effective for the electroporation and electro fusion of plant protoplasts. At room temperature the pores remain open only seconds, pore closing restores normal membrane properties. Actually protoplast fusion takes place when electroporation occurs at intercellular contact sites.
It has been also observed that there is definite relationship between the magnitude of an applied electric field (E) and the formation and distribution of pores in the plasma membrane. When the voltage of the applied field is just sufficient to induce pore formation, i.e., Vcr or critical voltage, the pores form only at the poles of the protoplast, i.e., area of the membrane orthogonal to the electrode (Fig. 6.18).
In di-electrophoretically aligned protoplast, such pulses lead to protoplast fusion because the poles are the sites of cell-to-cell contact. But the application of higher voltage pulses (E > Vcr) induces pore formation over more of the cell surface (Fig. 6.18). Excessive pore formation outside the cell-to-cell contact sites leads to cell lysis.
In case of electro fusion, two factors must be kept in mind. First, all protoplasts placed in fusion chamber are not aligned into chains by the AC field and, in chamber with wire or needle, electrode fusion occurs preferentially in chains located where the wires are closest together.
Thus, there is always a background of un-fused protoplasts. Second, high percentage of protoplast fusion sometimes favours the formation of multicellular fusion.
Table 6.3 provides an overview of experiments in which the viability of culture of electrically fused protoplasts has been demonstrated:
Recent work suggests that electro fusion has created new experimental opportunities in the field of somatic hybridisation. It is a speedy, easy and efficient method with which high percentage of fusion of protoplasts derived from different plant source can be achieved. Exploration and amplification of these opportunities should give electro fusion a permanent place among the techniques used by plant somatic cell genetics.
Mechanisms of Protoplast Fusion:
The mechanism of protoplast fusion is not fully known. Several explanations have been put forward to understand the mechanism of protoplast fusion.
Some important explanations are:
1. When the protoplasts are brought into close proximity, this is followed by an induction phase whereby changes induced in electrostatic potential of the membrane result in fusion. After fusion, the membrane stabilizes and the surface potential returns to their former state.
2. When the protoplasts are closely adhered, the external fusogens cause disturbance in the intramembranous proteins and glycoproteins. This increases membrane fluidity and creates a region where lipid molecule intermix, allowing coalescence of adjacent membranes.
3. The negative charge carried by protoplasts is mainly due to intramembranous phosphate groups. The addition of Ca2+ ions causes the zeta-potential of plasma membrane to be reduced and under that condition the protoplasts aggregate.
4. The high alkaline solution used in chemo fusion induces the intramembranous production of lysophospholipid which may be linked with membranous fusion.
5. The high molecular weight (1,000-6,000) polymer of PEG acts as a molecular bridge connecting the protoplasts and Ca2+ ions link the negatively charged PEG and membrane surface. On elution of the PEG, the surface potential are disturbed, leading to inter-membrane contact and subsequent fusion. Besides this, the strong affinity of PEG for water may cause local dehydration of the membrane and increase fluidity, thus inducing fusion.
6. PEG itself induces aggregation, but a-tocopherol present as an impurity in commercial grade PEG actually promotes membrane fusion.