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.
(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.
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.
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.
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.
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.
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
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
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.