Protoplast Fusion: Meaning, Methods and Its Mechanisms

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:
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.

(ii) Chemo-Fusion:
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:
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:

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:

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:
 PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide (DMSO).
 Addition of concanavalin A (Con A) to PEG increases protoplast fusion frequency.
 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:
 15% solution of polyvinyl alcohol (PVP) in combination with 0.05 CaCl2 and 0.3 M mannitol are
used to fuse plant protoplasts.
 Lectins are also known to agglutinate protoplasts.
 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.

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 ar-
rangement [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:

 The frequencies, and voltage of AC field;
 The shape of the electrodes and
 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 > V cr)
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.

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:

 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.

 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.

 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.

 The high alkaline solution used in chemo fusion induces the intramembranous production of lysophospholipid which may

be linked with membranous fusion.

 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.

 PEG itself induces aggregation, but a-tocopherol present as an impurity in commercial grade PEG actually promotes

membrane fusion.