The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.

1. PRESENTATION
ON
“PROTOPLAST
ISOLATION,CULTURE & FUSION”
PRESENTED BY-
ANISHA MUKHERJEE
M.Sc SECOND
SEMESTER
BIOTECHNOLOGY
GUIDED BY-
Dr. BHAWANA PANDEY
MA’AM

2. CONTENTS
 INTRODUCTION- The Protoplast
 HISTORY
 GENERAL PROCEDURE
 PROTOPLAST ISOLATION
 METHODS OF PROTOPLAST ISOLATION
MECHANICAL METHOD
ENZYMATIC METHOD
 PURIFICATION OF PROTOPLASTS
 VIABILITY OF PROTOPLASTS
 PROTOPLAST CULTURE
 OPTIMUM CULTURE CONDITION
 GENERAL METHODOLOGY
 CULTURE MEDIUM
 CULTURE METHODS
 IMPORTANCE OF PROTOPLAST CULTURE
 FACTORS AFFECTING PROTOPLAST CULTURE
 PROTOPLAST FUSION
 METHODS
 SPONTANEOUS FUSION
 MECHANICAL FUSION
INDUCED FUSION- Electrofusion, PEG fusion
 MECHANISM OF FUSION
 FUSION PRODUCTS – The Hybrids & Cybrids
 APPLICATION & SIGNIFICANCE OF PROTOPLAST FUSION
 APPLICATION OF PROTOPLAST & IT’S CULTURE
 REFERENCE

3. INTRODUCTION – The
Protoplast
Protoplast is a naked cell (without cell wall) surrounded
by a plasma membrane.
Protoplast = cell - cell wall
They are potentially capable of cell wall regeneration ,
growth and division.
It is fragile but can be cultured and grow into a whole plant.
Leaf mesophyll cells are the most suitable material for the
isolation of protoplasts.
The production of hybrid plants through fusion of two
different plant protoplasts (wall less naked cells) is known
as SOMATIC HYBRIDISATION and such hybrids are called
SOMATIC HYBRIDS.

4. HISTORY
 Hanstein in 1880 introduced the term
“Protoplast”.
In 1892, J.Klercker first isolated protoplast
mechanically by plasmolysing and
subsequently slicing the tissue of water warrior
(Stratiotes aloides).
 In 1960, E.C.Cocking first reported the the
enzymatic method for isolation of a large
number of protoplast from cells of higher
plants (by using concentrated solution of
cellulase enzyme).
 Rakabe and his associates (1971) were
successful to achieve the regeneration of whole
tobacco plant from protoplasts.

5. GENERAL PROCEDURE

6. PROTOPLAST ISOLATION
 It refers to the separation of protoplast from plant
tissue.
It is very important to isolate viable and uninjured
protoplast as gently and as quickly as possible.
 METHODS OF PROTOPLAST ISOLATION :-
 There are two methods of protoplast
isolation :-
1. Mechanical method
2. Enzymatic method

7. 1. MECHANICAL METHOD
 Tissue is immersed in 1.0 M sucrose until protoplasm
shrunk away from their enclosing cell wall (Plasmolysis).
 Plasmolysed tissue is cut with a sharp knife at such a
thickness that only cell walls are cut.
 Then these pieces are de-plasmolysed by using dilute
solution to release the protoplasts.
 Generally protoplasts were isolated from highly vacuolated
cells of storage tissues (onion bulbs, scales, radish root,
beet root).
 DISADVANTAGES :-
 Tedious process.
 Low protoplast viability.
 Low yield of protoplast.
 Unsuitable for the isolation of protoplasts from
less vacuolated cells.

8. MECHANICAL METHOD (cont.)

9. 2. ENZYMATIC METHOD
 Refers to the use of enzymes to dissolve the
cell wall for releasing protoplasts.
 Sources of protoplasts :-
 Protoplasts can be isolated from a wide variety of tissues and
organs that include leaves, roots, shoot apices, fruits, embryos
and microspores.
 Among these, the mesophyll tissue of fully expanded leaves of
young plants or new shoots are most frequently used.
 In addition, callus and suspension cultures also serve as good
sources for protoplast isolation.

10.  Enzymes for protoplast isolation :-
 The enzymes that can digest the cell walls are required for
protoplast isolation.
Chemically, the plant cell wall is mainly composed of
cellulose, hemicellulose and pectin which can be
respectively degraded by the enzymes cellulase,
hemicellulase and pectinase.
The various enzymes for protoplast isolation are
commercially available. The enzymes are usually used at :-
 pH 4.5 to 6.0,
 temperature 25-30°C ,
 incubation period that may range from half an hour to 20
hours

11.  METHODS OF PROTOPLAST ISOLATION :-
 The enzymatic isolation of protoplasts can be carried
out by two approaches :-
1. Two step or sequential method:
 The tissue is first treated with pectinase (macerozyme) to separate
cells by degrading middle lamella. These free cells are then exposed
to cellulase to release protoplasts. Pectinase breaks up the cell
aggregates into individual cells while cellulase removes the cell wall
proper.
2. One step or simultaneous method:
 This is the preferred method for protoplast isolation. It involves the
simultaneous use of both the enzymes -macerozyme and cellulase.

13.  ADVANTAGES OF ENZYMATIC METHOD :-
o The cells are intact and are not injured.
o Used for variety of tissues and organs such as
fruits, roots, petioles, leaves.
o Osmotic shrinkage is minimum.
o Cells remain intact and not injured.
o Protoplast readily obtained.
o High yield of protoplast.

14. PURIFICATION OF
PROTOPLASTS Protoplasts are purified by removing :-
 Undigested material (debris)
 Bursts protoplasts
 Enzymes
 Debris are removed by filtering the
preparation through a nylon mesh.
 Enzymes are removed by centrifugation
whereby the protoplasts settle to the bottom
of the tube and the supernatant removed
with the help of a pipette.
Intact protoplasts are separated from
broken protoplasts through centrifugation
and removed by a pipette as they are
collected at the top of tube.

15. PROTOPLAST
PURIFICATION

16. VIABILITY OF
PROTOPLASTS It is essential to ensure that the isolated protoplasts are healthy and viable so
that they are capable of undergoing sustained cell divisions and regeneration.
 There are several methods to assess the protoplast viability :-
 Fluorescein diacetate (FDA) staining method –The dye accumulates inside
viable protoplasts which can be detected by fluorescence microscopy.
 Phenosafranine stain – It is selectively taken up by dead protoplasts (turn red)
while the viable cells remain unstained.
 Measurement of cell wall formation- Calcofluor white (CFW) stain binds to
the newly formed cell walls which emit fluorescence.
 Oxygen uptake by protoplasts can be measured by oxygen electrode.
 Photosynthetic activity of protoplasts.
 The ability of protoplasts to undergo continuous mitotic divisions (this is a
direct measure).

17. PROTOPLAST CULTURE
 Isolated protoplast can be cultured in an appropriate
medium to reform cell wall and generate callus.
 Optimal culture conditions :-
1. Optimal density to the culture.
2. Optimal auxin to cytokinin ratio, glucose and sucrose.
3. Maintain osmoprotectant in the medium
4. Temperature: 20-28°C
5. pH: 5.5-5.9.

18. PROTOPLAST CULTURE (Cont.)
 Protoplasts cultured in suitable nutrient media first generate a new
cell wall.
 The formation of a complete cell with a wall is followed by an
increase in size, number of cell organelles and induction of cell
division.
 The first cell division may occur within 2 to 7 days of culture
 Resulting in small clumps of cell, also known as micro colony,
within 1 to 3 weeks.
● From such clumps, there are two routes to generate a complete
plant (depending on the species) :-
1. Plants are regenerated through organogenesis from callus masses.
2. The micro calli can be made to develop into somatic embryos, which
are then converted into whole plant through germination.

19.  Protoplasts are cultured either in semi-solid agar or liquid medium.
 Sometimes, protoplasts are first allowed to develop cell wall in liquid medium, and
then transferred to agar medium.
CULTURE MEDIUM
 Nutritional Components –
 In general, the nutritional requirements of protoplasts are similar to those of cultured
plant cells(callus and suspension cultures).
 Mostly MS media and B5 media with suitable modifications are used.
 Special features of Protoplast culture media –
1. The medium should be devoid of ammonium, and the quantities of iron and zinc
should be less.
2. The concentration of calcium should be 2-4 times higher than used for cell cultures.
This is needed for membrane stability.
3. High auxin/kinetin ratio is suitable to induce cell divisions while high kinetin/auxin
ratio is required for regeneration.
4. Glucose is the preferred carbon source by protoplasts although a combination of
sugars (glucose & sucrose) can be used.
5. The vitamins used for protoplast cultures are the same as used in standard tissue
culture media.

20. CULTURE METHODS
 The culture techniques of protoplasts are almost the same that are used for cell culture
with suitable modifications.
1. FEEDER LAYER TECHNIQUE :-
 For culture of protoplasts at low density feeder layer technique is preferred.
 This method is also important for selection of specific mutant or hybrid cells on plates.
 The technique consists of exposing protoplast cell suspensions to X-rays (to inhibit cell
division with good metabolic activity) and then plating them on agar plates.
2. CO-CULTURE OF PROTOPLASTS :-
 Protoplasts of two different plant species(one slow growing and another fast
growing)can be co-cultured.
 This type of culture is advantageous since the growing species provide the growth
factors and other chemicals which helps in the generation of cell wall and cell division.
 The co-culture method is generally used if the two types of protoplasts are
morphologically distinct.
3. MICRODROP CULTURE :-
 Specially designed dishes namely cuprak dishes with outer and inner chambers are
used for microdrop culture.
 The inner chamber carries several wells wherein the individual protoplasts in droplets of
nutrient medium can be added.
 The outer chamber is filled with water to maintain humidity.
 This method allows the culture of fewer protoplasts for droplet of the medium.

21. IMPORTANCE OF PROTOPLAST CULTURE
● Study of Osmotic behaviour
● Study of IAA action
● Study of Plasmalemma
● Study of Cell wall formation
● Organelle isolation
● Study of Morphogenesis
● Virus uptake and replication
● Study of photosynthesis
● Gene Transfer

22. FACTORS AFFECTING PROTOPLAST CULTURE
1. Plant species and varieties-
● Small genetic difference leads to varying protoplast responses to culture
conditions
2. Plant age -
● Age of donor plant and its developmental stage (smaller better)
○ Stages are germinating embryos, plantlets, leaves.
3. Pre-culture conditions-
● Protoplast culture are highly influenced by climatic factors and different
culture of seedlings yields protoplast having different responses when cultured.
4. Pre-treatment to the tissue, before isolating protoplasts-
● Cold treatment,plasmolysis and hormone increases the chance of recovery of
viable protoplasts and their plating efficiency
5. Density-
● Crucial as it influences plating efficiency and surviving of protoplasts. At
higher density, protoplasts compete with one another while at lower density
losses of metabolites from protoplasts is more.

23. PROTOPLAST FUSION
 The fusion of protoplast from different plants to form somatic hybrid and the
subsequent regeneration of plant through the callus.
 This pathway is an important achievement and the ultimate aim of this
technique to produce hybrid which cannot be produced by normal sexual means
 Protoplast fusion can be used to make crosses within species(interspecific),
between species (interspecific), within genera(intergeneric) and between genera
(intergeneric).
 Number of methods have been used to induce fusion between protoplasts of
different strains.
 METHODS OF FUSION : -
 The protoplast fusion can be achieved by these 3 methods :-
1. Spontaneous fusion
2. Mechanical fusion
3. Induced fusion

24. PROTOPLAST FUSION

25.  SPONTANEOUS FUSION-
 In spontaneous fusion, the adjacent protoplasts in enzyme
mixture have tendency to fuse together to form homokaryons
(having same type of nucleus).
 MECHANICAL FUSION-
 Gentle tapping of protoplasts suspension in a depression slide
results in protoplasts fusion.
 The giant protoplasts of Acetabularia have been fused
mechanically by pushing together two protoplasts.
 This fusion does not depend upon the presence of fusion
inducing agents.

26.  INDUCED FUSION -
 Freshly isolated protoplasts can be induced to undergo
fusion, with the help of a range of fusogen .
 FUSOGENS – The agents that induce the fusion of
protoplasts from different organisms.
 Examples of Fusogen :-
1. Sendai Virus – a parainfluenza virus (earlier used
fusogen)
2. Poly ethylene glycol (PEG)- The most commonly
used.
3. Sodium nitrate
4. High Ca2+ , high pH
5. Polyvinyl alcohol
6. Electro fusion

27. ELECTROFUSION
 When the protoplasts are placed in
a culture vessel fitted with micro-
electrodes and an electric shock is
applied, protoplasts are induced to
fuse.
 This technique is simple, quick
and efficient.
 The cells formed do not show
cytotoxic responses as in case of
fusogens (including PEG).
 LIMITATIONS :-
 This method requires specialized
and costly equipment.

28. PEG (Polyethylene glycol) Fusion
 This chemical has been identified as a potent fusogen.
 This has become the method of choice, due to its high
success rate, for the fusion of protoplasts from many plant
species.
 The isolated protoplasts in culture medium(1 ml) are mixed
with equal volume (1 ml) of 28-56% PEG (mol. wt. 1500-
6000 Daltons) in a tube.
 PEG enhances fusion of protoplasts in several species.
 This tube is shaken and then allowed to settle.
 The settled protoplasts are washed several times with culture
medium.

29. Fig. Protoplast fusion practical results

30. ADVANTAGES OF PEG METHOD :-
 PEG treatment method is widely used for
protoplast fusion as it has several
advantages:-
 It results in a reproducible high frequency of
heterokaryon formation.
 Low toxicity to cells.
 Reduced formation of binucleate heterokaryons.
 PEG induced fusion is non-specific and therefore can
be used for a wide range of plants.

31. MECHANISM OF FUSION
 The fusion of protoplasts involves three phases- agglutination,
plasma membrane fusion & formation of heterokaryons.
 AGGLUTINATION (adhesion) :-
 When two protoplasts are in close contact with each other, adhesion occurs. It
can be induced by fusogens, e.g. PEG, high pH and high Ca2+ .
 PLASMA MEMBRANE FUSION :-
 Protoplast membranes get fused at localized sites at the points of adhesion,
which leads to the formation of cytoplasmic bridges between protoplasts.
 The plasma membrane fusion can be increased by high pH and high Ca2+ ,high
temperature & PEG, as explained below –
a) High pH and high Ca2+ ions neutralise the surface charges on the
protoplasts.This allows closer contact and membrane fusion between
agglutinated protoplasts.

32. MECHANISM OF FUSION(Cont.)
b) High temperature helps in the intermingling of lipid
molecules of agglutinated protoplast membranes so that
membrane fusion occurs.
c) PEG causes rapid agglutination & formation of clumps of
protoplasts. This results in the formation of tight adhesions
of membranes & consequently their fusion.
 FORMATION OF HETEROKARYONS :- The fused protoplasts
get rounded as a result of cytoplasmic bridges leading to the
formation of spherical homokaryon or heterokaryon.

33. FUSION PRODUCTS – THE HYBRIDS AND CYBRIDS
 The nuclei of two protoplasts may or may not fuse together
even after fusion of cytoplasms.
 The binucleate cells are known as heterocyst.
 When nuclei of two different sources are fused, the cells are
known as hybrid.
 Only cytoplasms fuse and genetic information from one of
the two nuclei is lost is known as Cybrids i.e. cytoplasmic
hybrid.
 Some of the protoplasts of the same type may undergo
fusion to produce homocytes each with 2-40 nuclei.

34. Fig. Fusion products of two different protoplasts

35. APPLICATION & SIGNIFICANCE OF
PROTOPLAST FUSION
 Protoplast of sexually sterile (haploid, triploid,
aneuploid) can be fused to produce fertile diploid
plant.
 It helps to overcome the sexually incompatibility
barrier.
 It helps in production of disease resistant or insect
resistant plant.
It helps to enhance the productivity of crops.
It helps in monoclonal antibody production.

36. APPLICATION OF PROTOPLASTS
& THEIR CULTURE
The protoplast in culture can be regenerated into a whole plant.
Hybrids can be developed from protoplast fusion.
It is easy to perform single cell cloning with protoplasts.
Genetic transformations can be achieved through genetic engineering
of protoplast DNA.
Protoplast are excellent materials for ultra structural studies.
Isolation of cell organelles and chromosomes is easy from protoplasts.
Protoplasts are useful for membrane studies (transport & uptake
processes).
Isolation of mutants from protoplast cultures is easy.

37. REFERENCES
 Biotechnology By U.Satyanarayan
Biotechnology By B.D.Singh
 Plant Tissue Culture By Kalyan Kumar De