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CHAPTER "
Protoplast Culture and Somatic
Hybridization
U.K. Tomar1* and P. K. Dantu 2
1
Arid Forest Research Institute, New Pali Road, Jodhpur–342005
2
Depts. of Botany, Dayalbagh Educational Institute, Dayalbagh, Agra–282 005
E-mail: uktomar@afri.res.in

In plants, where fairly distant species could be crossed, it has always not been possible to
obtain full hybrids between desired individuals because of sexual incompatibility barriers.
This has often proved to be a serious handicap in crop improvement programs through
hybridization. With the ability of isolating protoplasts from plant cells using enzymes in the
early 1960 by E. C. Cocking, the interest in genetic modification of somatic cells in higher plants
has developed. Within a decade, Takebe et al. (1971) regenerated completes plants from leaf
protoplasts of tabacco increased the potential of prtoplast culture techniques.
Not only isolated protoplasts but their fusion product, a somatic hybrid, can also be
regenerated into whole plants. Plant protoplasts can also take up foreign DNA, through their
naked plasma membrane, under specific chemical and physical treatments. Protoplasts also
provide an experimental system for a wide range of biochemical and molecular studies ranging
from investigations into the growth properties of individual cells to membrane transport.
Present chapter describes the methods of protoplast isolation, purification, culture
techniques as also protoplast fusion techniques and somatic hybrid selection procedures.

1. PROTOPLAST ISOLATION
The term protoplast refers to the spherical plasmolysed content of plant cell enclosed by
plasma membranes or naked cell without cell wall. Before going into the details about

*
Corresponding author
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protoplasts (Rose, 1980). The enzyme solution is filter sterilized through a 0.45µ filter
membrane and sterilized tissue cut into small pieces is added. Incubation for some time
releases protoplasts from the tissue.

PROTOPLAST PURIFICATION
The successful culture of protoplasts requires a pure population of intact and viable
protoplasts at a high yield. So the protoplasts require to be purified by removing the
undigested material (debris), burst protoplasts and enzymes.

Removal of Debris and Enzymes

Debris can be removed from the protoplast suspension by filtering the preparation through a
steel or nylone mesh of 100µ pore size. Enzyme is removed by centrifuging the protoplast
suspension at 600 rpm for 5 minutes. The protoplasts settle to the bottom of the centrifuge tube
while the supernatant is removed with the help of a pipette. The protoplasts are then re-
suspended in a washing medium containing an osmoticum only or osmoticum with nutrient
medium or hydrated calcium chloride. The suspension is centrifuged again to settle the
protoplasts and the washing medium is decanted. Traces of enzyme are removed by washing
the protoplasts twice or thrice with the medium.

Removal of Broken Protoplasts

Intact protoplasts are separated from the broken debries by suspending the protoplast
preparation in 20-40% sucrose solution and centrifugation at 350 rpm for three minutes. Intact
protoplasts collect at the top of the sucrose solution and are carefully removed with a pipette
(Gregory and Cocking, 1965; Power and Cocking, 1970; Evans et al., 1972).
Schenk and Hilderbrandt (1971) used ficoll solution while Larkin (1976) used density
buffer for purification of protoplast. In this method 0.5-3.0 volumes of crude protoplast
preparation after filteration through sterile muslin cloth is layered on lymphoprep in the
centrifuge tube and spun at 50-200 g for about 10 minutes. The protoplasts collect as a ring
between the enzyme solution and lymphoprep and debris settle to the bottom (Fig. 2A). The
protoplasts are removed from the interphase with a pasture pipette (Fig. 2B).

Protoplast Viability and Cell Wall Formation Tests

To a small volume of the protoplast suspension add equal volume of 0.1% calcoflour solution,
incubate for 5 minutes and then observe under fluorescent microscope. The cell wall will
fluoresce and protoplasts remain dark.

Protoplast Viability
Fluorescein diacetate (FDA) solution in acetone (5 mg/l) is added to protoplasts suspension to
give a final concentration of 0.01%. After 5 minutes at room temperature the protoplasts are
examined using fluorescent microscope. Only viable protoplasts can be seen.
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Enzyme solution

Purified proplasts

Lymphprep

Debris
A
B
Fig. 2A. Purified Protoplast ring formation and debris
get settled at the bottom Fig. 2B. Purified protoplast population

FACTORS AFFECTING THE PROTOPLASTS ISOLATION AND ITS VIABILITY

To achieve a high yield of viable protoplasts, it is necessary to standardize the type and
concentration of osmoticum (e.g., sucrose, mannitol, sorbitol, glucose and other sugars).
Mannitol is the most commonly used osmoticum. The other condition to be standardized is
that of enzymes (cellulase, pectinase, hemicellulase, meicellase, pectinol, or dresilase) whether
they are to be used singly, in combination or sequentially with the osmoticum.As a thumb rule,
low enzyme concentration at low temperature and high pH (5-8) for short incubation period
proved to be better than longer incubation periods with high enzyme concentration, high
temperature and low pH value.Though the ionic salts when used with osmoticum degrade the
enzymes but increase the stability of protoplasts. Protoplasts isolated in presence of Ca++ or
Mg++ ions showed a greater capacity for cell wall regeneration as compared to protoplasts
isolated in the absence of these ions (Rose, 1980).

PROTOPLAST CULTURE
After viability test the protoplasts are cultured at a known density. Different methods have
been used for culturing the isolated protoplasts.

Suspension Culture
In this method protoplasts are suspended in a liquid medium with a suitable concentration of
osmoticum. Protoplasts at a density of 105/ml is generally plated on 25-50 ml of medium in an
Erlenmeyer flask. The cultures are shaken slowly to provide sufficient aeration for growth.
Sometimes shaking can cause bursting of protoplasts, so rpm of shaking required for a given
species should be standardized. Evans and Cocking (1975) suggested that 2 ml suspension of
protoplasts could be cultured in 25 ml flasks to facilitate aeration. Vasil (1976) suggested the
addition of ficoll to the medium to keep protoplasts floating and thereby allowing better
growth.
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Hanging Drop Method

Kao and his group developed the hanging drop method in 1970 which was subsequently used
by others (Bawa and Torrey, 1971). In this method, a suspension of protoplasts at a density of
104/ml to 105/ml is placed as 50 µl drops in plastic Petri plates, sealed with parafilm and
incubated in an inverted position at 25-30°C under lw light intensity (100-500 lux) or even in
dark. After cell wall regeneration and the initiation of cell division, fresh medium is added to
make cell suspension. The small size of drop helps in providing enough aeration to protoplasts
(Vasil, 1976).

Agar Plating Method

The method is almost similar to the one developed by Bergmann (1960) to grow callus cells of
tobacco and beans and was first employed for the protoplast culture by Nagata and Takabe
(1971). The major advantages of this technique are: (i) a large number of protocols can be
handled simultaneously, and (ii) plating efficiency can be determined easily. At the same time
the major drawback of this technique is that after regeneration of cell wall and induction of cell
division, osmotic potential of the medium can not be altered by addition of fresh medium
lacking osmotic stabilizer, since the initial medium is semi-solid. However, this problem can be
solved by transferring blocks of agar (in which protoplasts are cultured initially) to a fresh
medium with lesser concentration of osmoticum or altogether in its absence. This technique has
been modified in different ways viz., as feeder technique to support division of cells plated at
low density (Raveh et al., 1973), as micro vessel (Button, 1978), and as multiple drop arrays
(Potrykus et al., 1979).

Micro Culture Technique

This technique developed by Jones et al. (1960) for culturing isolated cells was used by Vasil
and Hilderbrandt (1965) to raise tobacco plants from isolated cells. Micro culture technique has
been successfully used for culturing protoplasts of tobacco and Petunia (Durand et al., 1973). A
drop of culture medium containing one or more protoplasts is put on a microscopic slide. On
either sides of this microscopic slide are kept two cover slips. A third one is put over these two
cover slips to shield the protoplasts suspensions.

Multidrop Array (MDA) Technique

This is a further improvement of hanging drop method and allows screening of large number
of hormonal and nutritional factors (up to 4900) using small amount of plant material
(Potrykus et al., 1979)

FACTORS AFFECTING PROTOPLASTS CULTURE

Successful growth and regeneration of protoplasts is dependent on a number of factors ranging
from the status of the donor plant to the culture conditions.
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Plant Species and Varieties

It is a well documented fact that even very small genetic difference leads to varying protoplast
responses to culture conditions.

Plant Age and Organ

A second important factor for the successful culture is the age of the donor plant and
developmental stage of the donor part used for isolation of the protoplasts. The stages with
respect to their responses are germinating embryos, followed by seedling of one week,
plantlets, leaves, juvenile tissue from mature plant and mature tissue. Though having the same
genetic information protoplasts isolated from these parts behave differently in culture
conditions. Plant regeneration has been most successful from leaf protoplasts of herbaceous
species.

Pre Culture Conditions

Besides the factors mentioned above the behaviour of protoplasts in culture is highly
influenced by climatic factors and pre culture conditions. Different culture conditions of
seedling yields protoplasts having different responses when cultured.

Pretreatment to the Tissue, Before Isolation of Protoplasts

Pretreatments such as cold treatment, plasmolysis, and hormone treatment to the tissue
increases the chances of recovery of viable protoplasts and their plating efficiency.

Nutritional Requirement for Protoplasts

Nutritional requirement for the growth of protoplasts differ from the nutritional requirement
for tissues and cell culture. Protoplasts leak in cultures as they are devoid of cell walls, and
moreover, they have a greater surface area for the diffusion of metabolites than the tissues. So
the concentration of different metabolites in protoplasts is less than that in the tissues and cells.
To compensate for these loses from protoplasts the number of metabolites provided in their
culture media is more (Kao and Meichyaluk 1975).

Protoplasts Density
Protoplasts density is very crucial as it influences the plating efficiency and better surviving of
protoplasts. When plated at higher density protoplasts compete with one another while at
lower density losses of metabolites from protoplasts is more. The later situation of protoplast
leakiness can be circumvented by addition of required metabolites to make medium isotonic.
Alternatively, one can use nurse callus or feeder layer technique to provide essential organic
molecules to protoplast for survival and their subsequent growth (Menczel et al., 1978).

PROTOPLAST FUSION OR SOMATIC HYBRIDIZATION

As plant cells have an inhibiting cell wall it is very difficult to fuse them. But isolated
protoplasts were observed to fuse spontaneously because now the only barrier between the
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cytoplasm of two cells is the plasma membrane. After lot of refinements the techniques for
protoplast fusion became important to produce hybrids from sexually incompatible species.
Protoplast fusion is possible even between a plant cell and an animal cell and thus can have
wider application. The process of somatic hybridization involves: (i) protoplast isolation,
(ii) protoplast fusion, (iii) selection of somatic hybrids, and (iv) culture of somatic hybrids to
regenerate complete plants. Different methods of protoplast fusion are described by Bengochea
and Dodds, (1986). Three important methods are generally used for protoplast fusion: (a) high
Ca++ and high pH, (b) polyethylene glycol (PEG), and (c) electric field. Literature survey
indicates that the second and third methods are most commonly used due to better results. The
three methods are discussed below.Sexual hybridization occurs when haploid cells generated
in a previous meiosis fuse. The fusion of somatic diploid cells should generate a tetraploid
fusion product provided that the nuclei fuse, too. If this is the case, it is spoken of a synkaryon.
A fusion product where the nuclei stay separate is called a heterokaryon.

High Ca++ and High pH Induced Protoplast Fusion

Physical contact of two protoplasts is essential for their fusion. However, protoplasts do not
fuse easily due to two main reasons: (i) they have a net negative charge on their membrane
surfaces and force of repulsion works between protoplasts, and (ii) it is difficult to remove
water from hydrophilic surfaces of protoplasts which also create a repulsive force between two
protoplasts. It was observed that positively charged ions reduce the net negative charges of
membranes reducing the repulsive force considerably. Keller and Melcher (1973) found
calciumions were suitable for such purposes and developed this method of protoplast fusion
using high Ca++ ions in a high pH solution. Later the method was improved by Melcher and
Labib (1974). The technique involves the following steps: Freshly isolated protoplasts of
selected parents are mixed in a ratio of 1:1 with a final density of 2.5x105 protoplasts per ml. The
protoplasts are collected as a pellet by centrifuging at 50 g for 3 to 5 minutes. The supernatant is
removed and 2 ml of fusion mixture containing 50 mM CaCl2.2H2O, 50 mM Glycine-NaOH
buffer and 400 mM Mannitol is added. pH of the mixture is adjusted to 10.5. Protoplasts are re-
suspended in solution by gently shaking the centrifuge tubes.The protoplasts are collected by
centrifuging at 50 g for 3 to 5 minutes. The centrifuge tubes with protoplasts are incubated in a
water bath at 37°C for 10 to 30 minutes. The fusion mixture is replaced by washing medium
(600 mM Mannitol and 50 mM CaCl2.2H2O) and protoplasts are left for 30 minutes.The
protoplasts are washed twice with the washing medium.The protoplasts are re-suspended in
culture medium. Protoplast fusion products can be observed under microscope. These
protoplasts can be cultured on selection medium, which allows only somatic hybrids to grow.

PEG Induced Fusion

Polyethylene glycol (PEG) induced protoplast fusion was developed by Kao and Michayluk
(1974). This technique is relatively more efficient than the previous one. Polyethylene glycol
molecules have polarity like membrane phoshopholipid molecules and get attached with
membrane proteins. When the attached PEG between two protoplasts is removed it results in
breakdown of membranes at the contact points causing protoplasts to fuse with subsequent
rejoining of plasmamembarnes of the adjacent protoplasts. The following steps are involved
for protoplast fusion through polyethylene glycol. Mix the freshly isolated protoplasts (while
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still in enzyme solution) of selected parents in a ratio of 1:1. Pass the suspension through a 62
µm pore size filter and collect the filtrate in a centrifuge tube. Seal the mouth of the tube with
screw cap. Centrifuge the filtrate at 50 g for 60 minutes to sediment the protoplasts. Remove the
supernatant with pasture pipette. Wash the protoplasts with 10 ml of solution I (solution I: 500
mM Glucose, 0.7 mM KH2PO4.H2O and 3.5 mM CaCl2.2H2O and pH 5.5). Remove and wash
the protoplasts in solution I and make a suspension with 4-5% v/v protoplasts per ml. Put a 2-
3 ml drop of silicon 200 fluid (100 cs) in a 60 x 15 mm sterile Petri dishes.Place a 22x22 mm cover
slip on the drop. Pipette 150 µl of protoplast suspension onto the cover slip with a pasture
pipette. Allow about 5 minutes for the protoplasts to settle on the cover slip forming thin layer.
Add drop-by-drop 450 µl of PEG solution (50% PEG-1540, 10.5 mM CaCl2.2H2O, 0.7 mM
KH2PO4.H2O) to the protoplast suspension and observe the protoplast adhesion under
inverted microscope. Inoculate the protoplast in PEG solution and keep at room temperature
(24°C) for 10 to 20 minutes. Gently add 0.5 ml aliquots of solution II (50 mM Glycine, 50 mM
CaCl2.2H2O, 300 mM Glucose, pH 9-10.5) at 10 minutes intervals. After another 10 minutes add
1 ml of protoplasts culture medium. Wash the protoplasts five times at five minutes intervals
with 10 ml of the fresh protoplasts culture medium. At the end of each washing do not remove
entire medium from the cover slip but leave behind a thin layer of the old medium adding fresh
medium to it. If parent protoplasts are distinguishable visually, it may be possible to assess the
frequency of heterokaryon formation at this stage. Culture the fused protoplasts together with
the unfused protoplasts on the same cover slip in a thin layer of 500 µl of culture medium. Put
additional 500-1000 µl medium in the front of droplets around the cover slip to maintain
humidity inside the Petri plates.

Electric Field Induced Fusion

In this method, developed by U. Zimmerman, protoplasts are placed in an electric field and are
exposed to high intensity electric pulse for a short duration (nano to micro second). This
exposure to electric field reversibly increases permeability of cell membrane. Local electrical
charge break down occurs in the plasma membranes resulting in fusion of adjacent protoplasts.
The original properties of membrane are restored within micro second to minutes depending
on the experimental conditions and membrane properties (Zimmerman and Scheurich, 1981;
Zimmermann and Vienken, 1982). An instrument designed by U. Zimmerman is used for
protoplast fusion and for genetic transformation through electroporation. The process of
electric field induced protoplast fusion can be explained in the following five steps:
Dielectrophoresis
Mutual dielectrophorasis
Membrane contact
Electric break down of membranes, and
Protoplast Fusion
(i) Dielectrophoresis: Close membrane contact of two protoplasts is one of the prerequisite
for protoplast fusion. This step is achieved by dielectrophoresis. Dielectrophoresis is concerned
with motion of neutral particles in a non uniform electric field. Neutral particles become
polarized in the presence of electric field with negative side towards anode and positive charge
of the same magnitude on the opposite side, i.e. towards cathode.In uniform electric field the
field strength is equal on both the sides. And in such a field these induced bipolar particles will
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not move to either side due to equal electric force on both sides (Fig. 3A). However, under non
uniform field (field strength on the two sides of particles is unequal) a net force acting upon the
particle results in linear motion towards the region of highest field intensity (Fig. 3B). This is
termed as dielectrophoresis.
(ii) Mutual Dielectrophoresis: One protoplast approaching another polarized protoplast
during the movement towards the region of highest field intensity, will encounter an
enhancement of local field divergence and will tend to move towards that protoplast since the
field strength is higher near that cell. As a result protoplasts in non uniform field form chain
like aggregates (so called pearl chain) with point to point membrane contact (Fig. 3C). This
effect is termed as mutual dielectrophoresis.

A. Uniform field developed equal force on both side of charged protoplasts B. Unequal electrical field developed net force
towards higher field strengthC. Non uniform electric field thus forms pearl chain of protoplasts
Fig. 3. Movement of protoplast in non uniform electric field towards higher strength electric field and forms
protoplast chain.

The attraction forces arising from dipole generation within protoplasts overcome both the
electrostatic repulsive forces between membrane surfaces of neighboring protoplasts bearing
net negative charges and repulsive hydration forces. Repulsive hydration force is assumed to
be a consequence of work required to remove water from hydrophilic surface as the protoplasts
approach one another. Dielectrophoresis and pearl chain formation usually have to be
performed in virtually non conductive solution (conductivity less than 10–4 cm–1).
(iii) Electric Break Down: Reversible electric breakdown in the zone of membrane contact is
the primary process responsible for the initiation of fusion (Fig. 4).Phospholipids are arranged
in a planer bi-layer into which peripheral or integral structural carrier proteins are embedded
in mosaic like fashion. The lateral fluidity of phospholipids is very high, while the movement
vertical to the membrane surface is severely limited. A flip-flop movement of lipids in this
direction is, therefore, highly difficult.
In an equivalent electrical circuit the membranes can be represented by a plate capacitor of
specific capacitance (Cm) and resistance (Rm) connected in parallel (Fig. 4A). The aqueous
external solution and polar heads of liquids represent the plate of capacitor, while membrane
interior forms a dielectric with relative dielectric constant of 2 to 3. The resistance of aqueous
external solution RE is in series with the membrane. The specific resistance of cell membrane is
in the order of 102 to 104 Wcm–2, while that of specific membrane bilayer is three to four orders
of magnitude higher. The specific capacitances of artificial and biological membranes on the
other hand are comparable of having values of 0.3 to 0.7 mFcm–2 and 1 mFcm–2, respectively.
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hybrids, subsequent division of fusion products and morphogenesis giving rise to hybrid
plants.
Different methods have been developed for the selection of somatic hybrids. These
methods can broadly be divided as follows:
Group 1 Selection of somatic hybrids by culturing them on such a medium on which only
somatic hybrid protoplast can grow (Power et al., 1977).
Group 2 Complementary selection of somatic hybrids on specific culture medium.
Group 3 Mechanical isolation by visual means and knowledge of identification of somatic
hybrids.
Group 4 Morphology of the plant after regeneration.
Group 1 - Selection Medium: Smith et al. (1976) used a model system involving the knowledge
from sexual interspecific Nicotiana hybrids that can be cultured and selected on media that fails
to support parental species. Chupeau et al. (1978) were the first to demonstrate the successful
use of this technique for selecting somatic hybrids. Fusion of protoplasts from Nicotiana
longsdorfii and N. gluca were induced using PEG. Parasexual hybrid colonies were selected for
their ability to grow without growth substances (Fig. 5).
Similarly protoplasts of sexual hybrid plants of Petunia parodii and P. hybrida can grow and
regenerate complete plants on specific medium containing Actinomycin D, where as
protoplasts of parents can grow and form colonies on this medium but can not regenerate
complete plants. Power et al. (1977) successfully used this medium for selecting somatic
hybrids.
Group 2 - Complementary selection: This method developed by Melcher and Labib (1974)
utilizes biochemical markers such as albino and chlorophyll deficient mutants (fig. 6). Somatic
hybrids can be selected on the basis of complementation of characters expressed by the
hybrids, which are not present in any of the parents. Sometimes, hybrid is selected on the basis
of characters of parent protoplasts present in somatic hybrid.
Melcher and Labib (1974) took two mutants of Nicotiana tabacum, one of which was
sublethal, that did not grow more than few inches (2-3 inches) in low light intensity (800 lux),
but at higher light intensities (10,000 lux) was able to grow into complete plants. The other

Fig. 5. Flow chart showing selection of hybrids using selection medium on which only hybrid can grow.
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Fig. 6. Flow Chart showing complementary selection of somatic hybrids on specific culture medium.

mutant was chlorophyll deficient that could not grow more than few inches. When protoplasts
of these two mutants were fused the hybrid produced green colonies in culture and later on
regenerated into full plants in low light intensity (800 lux).
Gleba et al. (1975) developed a similar complementation selection procedure that involves
complementation between plastome and genome chlorophyll mutant and does not utilize light
sensitivity as a selection marker. Cocking et al. (1977) used albino protoplasts of P. hybrida and
green leaf mesophyll protoplasts of P. parodii (Fig. 6). These somatic hybrids included strict
amphidiploids.
Group 3 - Mechanical Isolation: In this method heterokaryons are selected and isolated visually
under microscope. This method is particularly useful for those interspecies fusions where the
post zygotic incompatibility prevents sexual hybridization. In such cases of interspecies fusion
chromosome elimination may result in failure to select the somatic hybrids, while other
interspecies fusion may result in somatic hybrid protoplasts with no evidence of specific
chromosome elimination. In these instances the stable conditions may fail to be maintained on
organogenesis. Through this selection approach such barriers to whole somatic hybrid plant
formation can be identified and perhaps overcome.
Automatic separator of somatic hybrids from mixed population of protoplasts after fusion
process has been demonstrated. In this method parent protoplasts are stained with two
different colour dyes and somatic hybrids exhibiting mixed colour are sorted.
Group 4 - Morphology of the plant after regeneration: In Jhis method, the somatic hybrids are
regenerated into whole plants. The morphological characters of the hybrid plants are
compared with those of the two parental lines to establish the similarities. This method is time
consuming and is possible where regeneration of plants and hybrids is well established.

CONCLUSIONS
Protoplast technology has various applications other than regeneration of complete plants and
production of hybrids of sexually incompatible species. In fact, it has not produced any
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wonderful research results so far as it has promised in early 1960s. There is not even a single
successful production of agronomically useful hybrid plant through this technology.
Nevertheless, these techniques have been instrumental in generating basic scientific
information on cell biology, plant incompatibility, membrane functions, cell organelle studies
and cell wall regeneration. Ultrastructure and molecular architecture of plant cells. These
techniques are now being used for transfer of cytoplasmic male sterility. Protoplast can take up
macromolecules (nucleic acids and proteins), viruses, cell components like chromosomes and
chloroplasts by phagocytosis.

REFERENCES
Bawa, S.B. and Torrey, J.G. (1971) “Budding” and Nuclear Division in Cultured Protoplasts of Corn,
Convolvulus, and Onion. The Bot. Gaz. 132: 240-245.
Bengochea, T. and Dodds, J.H. (1986). Plant Protoplasts: A biotechnological Tool for Crop Improvement.
Bergmann, L. (1960) Growth and division of single cells of higher plants in vitro. Journal of General
Physiology 43:841–851.
Button, J. (1978) The effect of some corbohydrate on growth and organization of Citrus ovular callus. Z.
Pflanzenphysiol. 88: 61-68.
Carlson, P. S., Smith, H. H. and Dearing, R. D. (1972)Parasexual interspecific plant hybridization. Proc.
Nat. Acad. Sci. US 69: 2292-2294.
Chupeau, Y.; Missonier, C.; Hommel, M-C; Goujaud, J. (1978): Somatic hybrids of plants by fusion of
protoplasts. Molecular and General Genetics 105 (3): 239-45.
Cocking E.C. (1960) A method for the isolation of plant protoplasts and vacuoles. Nature 187: 962-963.
Cocking, E. C ; George, D.; Price-Jones, J. J.; Power, J. B. (1977) Selection procedures for the production of
inter-species somatic hybrids of Petunia hybrida and P. parodii. II Albino complementation selection.
Plant Science Letters 10:7-12.
Durand, J. Potrykaus, I. and Donn G. (1973) Plantes isuues protoplastes de Petunia. Z. Pflanzenphysiol. 69:
26-34.
Evans P. K. and Cocking E. C. (1975) The techniques of plant cell culture and somatic cell hybridisation..
In: New Techniques in Biophysics and Cell Biology (R. H. Pain and B. J. Smith, Eds.), pp 127-158, John
Wiley and Sons Ltd.
Evans, P. K., Keates, A. G. and Cocking, E. C. (1972) Isolation of protoplasts from cereal leaves. Planta 104:
178-191.
Gleba, Y. Y. (1979) Nonchromosomal Inheritance in Higher Plants as Studied by Somatic Hybridisation.
In: Proceedings Fourth Annual College of Biological Sciences Colloquium, “Plant Cell and Tissue Culture-
Principles and Applications”, Pp.775-788. Ohio State Univ. Press, Columbus.
Gregory, D. W., and Cocking, E. C. (1965) The large-scale isolation of protoplasts from immature tomato
fruits. J. Cell Biol. 24:143-146.
Jones, L. Hildebrandt, A. Riker, A. Wu, J. (1960) Growth of somatic tobacco cells in microculture. Am. J.
Bot. 47: 468-475.
Kao, K. N. (1976) A method for fusion of plant protoplasts with polyethylene glycol. In: Cell Genetics in
Higher Plants (D. Duddits et al. Eds.). pp.233-237.Akad. Kiado, Budapest.
Kao, K.N. Keller, W.A. and Miller, R.A. (1970). Cell divisions in newly formed cells from protoplasts of
soybean. Exp. Cell Res. 62: 338-340.
Kao, K. N. and Michayluk, M. R. 1974) A method for high-frequency intergeneric fusion of plant
protoplasts. Planta 115:355-67.
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Kao, K.N., Constabel, F., Michayluk, M.K. and Gamborg, O.L. (1974)Plant protoplast fusion and growth
of intergeneric hybrid cells. Planta 120:215-27, 1974.
Keller, W.A. and Melchers, G. (1973) The effect of high pH and calcium on tobacco leaf protoplast fusion.
Z. Naturforsch. 28, 737-741.
Klercker, J. von (1892) Eine Methode zur isolier lebender protoplasten. Ofvers Vetensk Akad Forhandl 49:
463-474.
Larkin, P.J. (1976). Purification and viability determinations of plant protoplasts. Planta 128, 213–216.
Menczel, L., Lazar, G. and Maliga, P. (1978) Isolation of somatic hybrids by cloning Nicotiana
heterokaryons in nurse cultures. Planta 143: 29-32.
Power, J. B. and Cocking, E. C. (1971) Fusion of plant protoplasts. Science Progress (Oxford) 59: 181-98.
Power, J. B., Berry, S. F., Frearson, E. M. and Cocking, E. C. (1977) Selection procedures for the production
of inter-species somatic hybrids of Petunia hybrida and P. parodii. I Nutrient media and drug
sensitivitycomplementation selection. Plant Science Letters 10: 1-6.
Raveh, D., Huberman, E. and Galun, E. (1973) In vitro culture of tobacco protoplasts. Use of feeder
techniques to support division of cells plated at low densities. In Vitro 9: 216-22.
Melchers, G. and Labia, G. (1975) Somatic hybridization of plants by fusion of protoplasts. Selection of
light resistant hybrids of “haploid” light sensitive varieties of tobacco. Molec. Gen. Genet. 135:
277-294.
Nagata, T. and Takebe, I. (1971) Plating of isolated tabacco mesophyll protoplasts on agar. Planta Vol. 99:
12-20.
Potrykus, I. Harms, C.T. and Lorz, H. (1979) Multiple-drop-array (MDA) technique for the large scale
testing of culture media variations in hanging microdrop cultures of single cell systems. I. The
technique. Plant Sci. Lett. Vol. 14, 231-235.
Raveh, D. Huberman, E. and Galun, E. (1973) In vitro culture of tobacco protoplasts. Use of feeder
techniques to support divison of cells plated at low densities. In Vitro 9: 216-22.
Rose, R. J. (1980). Factors that influence the yield, stability in culture and cell wall regeneration of. spinach
mesophyll protoplasts. Aust. J. Plant Physiol. 7: 71-725.
Schenk, R. U., and Hildebrandt, A. C. (1969) Production of protoplasts from plant cells in liquid culture
using purified commercial cellulases. Crop. Sci. 9:629-631.
Smith, H.H. Kao, K.N. and Combatti, N.C. (1976) Interspecific Hybridization by protoplast Fusion in
Nicotiana. The J. Heredity 67: 123-128. 1976.
Takebe, I., Labib, G. and Melchers, G. (1971) Regeneration of whole plants from isolated mesophyll
protoplasts of tobacco. Naturwissenschaften 58: 318–320.
Vasil, I. K. (1976) The progress, problem, and prospects of plant protoplast research. Adv. Agron., 28: 119-
160.
Vasil, V. and Hildebrandt, A. C. (1965) Differentiation of Tobacco Plants from Single, Isolated Cells in
Microcultures. Science Vol. 150: 889-892.
Zimmerman, U. (1982) Electric field-mediated fusion and related electrical phenomena. Biochimica et
Biophysica Acta 694:227–277.
Zimmerman, U. and Scheurich, P. (1981) High frequency fusion of plant protoplasts by electric fields.
Planta151: 26-32.
Zimmermann, U. and Vienken, J. (1982) Electric field-induced cell-to-cell fusion. J Membr Biol 67: 165-182.

Suggested Readings
1. Bengochea, T. and Dodds, J.H. (1986). Plant Protoplasts: A biotechnological Tool for Crop
Improvement. Chapman & Hall.
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2. Dodds, D.J. and Bengochea, T. (1987) Plant Protoplasts: A Biotechnological Tool for Plant
Improvement, Chapman & Hall.
3. Davey, M.R. and Kumar, A. (1983) Higher plant protoplasts: retrospect and prospect. In: Plant
Protoplasts. International Review of Cytology, (Giles, K.L. Ed.) Supp. 16, 219-263. Academic Press,
New York.

REVIEW QUESTIONS
Long Questions
1. Write a note on five important applications of protoplast techniques.
2. Write short note on protoplast isolation techniques and which one is more efficient?
3. Write a note on cell wall and protoplast viability test.
4. What are the methods of protoplast fusion? Describe any one method in detail.
5. Describe three methods of somatic hybrid selection.

Multiple Choice Questions

1. Who discovered enzymatic method of protoplast isolation?
(a) Klercker (b) E.C. Cocking
(c) I. K. Vasil (d) U. Zimmermann
2. Which enzyme is used to dissolve middle lamella in plant cells?
(a) Cellulase (b) Pactinase
(c) Both of above (d) Non of above
3. Crude cellulase enzyme was isolated from which organism?
a) Bacteria (b) Snail
(c) Fungus (d) Algae
4 Which chemical is used in cell wall test?
(a) Saffranin (b) Calcoflour
(c) Fluorescence diacetate (d) Non of the above
5 Which chemical is used in protoplast viability test?
(a) Analine blue (b) Calcoflour
(c) Fluorescence diacetate (d) Saffranin
6 High Ca+ ions and high pH is used for protoplast fusion to
(a) Increase positive charge (b) Increase membrane fluidity
(c) Make protoplast neutral (d) All of above
7. What effect on protoplast can be observed under an electric field
(a) Bursting (b) Shrinking
(c) Dipole formation (d) Non of the above
8. Regeneration of completed plants through protoplasts was first reported in
(a) Datura (b) Petunia
(c) Nicociana tabcum (d) Daccus carrota
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Protoplast Culture and Somatic Hybridization &'

9. First successful regeneration of complete plants from somatic hybrid have been
reported by
(a) Takebe, I. et al (b) E.C. Cocking,
(c) I.K. Vasil (d) J.B. Power
10. Who developed electric induced protoplast fusion technique?
(a) P. S. Carlson (b) E.C. Cocking
(c) I. K. Vasil (d) U. Zimmermann

Answers
1. (b) 2. (b) 3. (c) 4. (b) 5. (c) 6. (d)
7. (c) 8. (c) 9. (a) 10. (d)

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