Somatic hybridization

SOMATIC HYBRIDIZATION
ALI AHMAD BAJWA
M-PHIL BOTANY
THE UNIVERSITY OF LAHORE

• DEFINITION & INTRODUCTION
• PROCEDURE/STEPS OF TECHNIQUE
• ISOLATION OF PROTOPLASTS
• Importance of Protoplast Isolation
• Source of Protoplast
• Methods of Isolation of Protoplasts
• Purification of Protoplasts
• Testing the Viability of Isolated Protoplast
• FUSION OF PROTOPLASTS OF DESIRED SPECIES/VARIETIES
• IDENTIFICATION AND SELECTION OF SOMATIC HYBRID
CELLS
• CULTURE OF HYBRID CELLS
• REGENERATION OF HYBRID PLANTS
• PRACTICAL APPLICATIONS
• ADVANTAGES
• DISADVANTAGES
• CONCLUSION REMARKS
SYNOPSIS

DEFINITION & INTRODUCTION
Development of hybrid plants through the fusion of somatic protoplasts of two different plant
species/varieties is called “Somatic Hybridization”.
The term “Protoplast” was introduced in 1880 by Hanstein. A
cell with its cell wall removed either mechanically or
enzymatically is named as protoplast. First isolation of
protoplast was achieved by Klercker in 1892 by using
mechanical method. Küster in 1909 described the process of
random fusion in mechanically isolated protoplasts. The real
beginning in protoplast research was made by Cocking in 1960
who used enzymatic method for cell wall removal. Takebe et al
(1971) were successful to achieve the regeneration of whole
tobacco plant from protoplasts. Somatic hybridization (fusion
of protoplasts) is relatively a new versatile technique to induce
or promote genetic recombination in a variety of prokaryotic
and eukaryotic cells (Bhojwani S.S. et al 1977).

PROCEDURE/STEPS OF TECHNIQUE
ISOLATION OF PROTOPLASTS
The term “Protoplast” refers to the spherical plasmolysed
content of the plant cell enclosed by plasma membrane or
naked cell without cell wall. Before culturing protoplast, it
is important to isolate viable and uninjured protoplasts.
Production of hybrid plants through the fusion of
protoplasts of two different plant species/varieties is called
Somatic Hybridization, and such hybrids are called
Somatic Hybrids. Therefore, somatic hybridization can be
resorted to only when the following two criteria are
satisfied:
i) Isolation of protoplast in large quantity, and
ii) Totipotency of the isolated protoplasts.
Importance of Protoplast Isolation

Protoplast can be isolated from almost all plant parts:
 Roots, leaves, fruits, tubers, root nodules, endosperm, pollen mother cell, callus and
suspension culture
 Spongy and palisade mesophyll tissue obtained from mature leaves of Nicotiana and
Petunia.
 Anthers of Pelargonium (Abo El-Nil and Hilderbrandt, 1971)
 Callus of Gossypium hirsutum (Bhojwani, Cocking, and Power, 1977)
 Crassulacean acid metabolism (CAM) plants (Doddds, 1980)
 C3 and C4 plants (Kanai and Edwards, 1973)
 Solanum tuberosum (Upadhya, 1975)
Source of Protoplasts

ENZYMATIC METHOD: The plant cell wall is mainly composed of cellulose,
hemicellulose and pectin which are respectively degraded by the enzymes cellulase,
hemicellulase and pectinase. In plant cells we mainly uses these enzymes (cellulase,
hemicellulase and pectinase) at pH 4.5-6.0 & temperature 25-300C with incubation
period of half an hour to 20 hrs.
 Credit of developing High Yield Protoplast Isolation Technique from higher plant
protoplasts goes to Cocking (1960).
Methods of Isolation of Protoplasts

TYPES OF ENZYMATIC METHOD: There are two types of enzymatic method. Both
methods have certain advantages and disadvantages (Evans and Cocking, 1977; Bajaj, 1977).
Generally 50 mM CaCl2 is added to increase the stability of released protoplasts (Rose, 1980).
1. One Step Method (Direct/Mixed Method): In this method protoplasts are isolated
from plant tissues directly by using two enzymes, cellulase and pectinase, simultaneously.
Power and Cocking (1968) used this method for isolation of protoplasts.
2. Two Step method (Sequential Method): This method was first used Takebe and
others in 1968 in two steps. In this method, cells are first isolated from callus or tissues by
using pectinase and to this cell suspension cellulase is added to digest the cell walls and
release protoplasts

 MECHANICAL METHOD
HISTORY: Klercher (1892) was the first to isolate
protoplast from plasmolyzed cell of Stratiates
aloides. The studies were later extended for
protoplast isolation from tissues of Onion bulbs.
PROCEDURE: Onion Scales were immersed in 1.0 M Sucrose until the protoplast shrunk
away from their enclosing wall and then the plasmolysed tissue was cut into small strips. The
protoplasts were released by Osmotic Swelling when these strips of the tissue were placed in
Low Concentration Sucrose Solution.
This method is suitable for isolation of protoplasts from higher plant tissue such as leaf, bulb
scale, fruit epidermis, radish roots
Figure: When tissue is cut at the dotted lines (A) with a sharp razor blade, some cells release uncut complete protoplast
and rest of the cells produced broken dead protoplasts as shown in Figure 1B marked with stars (*).

Commonly used methods include:-
1. Filtration (For Removal of Debris):- Debris (undigested material) can be removed from
protoplast suspension by filtering the preparation through a steel or nylon mesh of 100µ pore size.
2. Sedimentation & Washing (For Removal of Enzymes):- Enzyme is removed by
centrifuging 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 resuspended 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 enzymes are
removed by washing the protoplasts twice or thrice with the medium.
Purification of Protoplasts

FIGURE: Protoplasts purification by filterartion
PurificationofProtoplastsPROCEDURE/STEPS OF TECHNIQUE

3. Flotation (Separation of Protoplasts):- In this method intact
protoplasts are separated from the broken debris by suspending the
protoplast preparation in 20-40% Sucrose Solution and centrifuging at
350rpm for three (3) 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, 1971; Evans et al., 1972).
Schenk and Hildebrandt (1969) used Ficoll Solution.

4. Density Buffer Method: Larkin (1976) used this
method for purification of protoplasts. In this method 0.5-
3.0 volumes of crude protoplast preparation after filtration
through sterile muslin cloth is layered on LymphoPrep
(LymphoPrep™ is a ready-made, sterile and endotoxin
tested solution suitable for the purification of human
mononuclear cells) in the centrifuge tube and then 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 (See Figure).
FIGURE: Protoplasts in ring

Testing Viability of Protoplasts And Cell Wall Formation
Cell Wall Formation Test: To the
small volume of the protoplast
suspension add equal volume of 0.1%
Calcofluor solution, incubate for 5
minutes and then observe under
fluorescent microscope. The cell wall
will fluoresce and protoplast remain
dark.
FIGURE: Cell Wall is fluorescing while protoplasts remain dark

Testing Viability of Protoplasts And Cell Wall Formation
Protoplast Viability Test:
Fluorescein Diacetate (FDA)
solution in acetone (5mg/l) is added
to protoplast 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.
FIGURE: Viable Protoplasts visible in green color

As a thumb rule, low enzyme concentration at low temperature and high pH (5-8)
for short incubation period prove to 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 the presence of Ca++ or Mg++ showed a greater
capacity for the cell wall regeneration as compared to protoplasts in the absence of
these ions (Rose, 1980).
Factors Affecting Protoplast Isolation And Its Viability

FUSION OF PROTOPLASTS OF DESIRED SPECIES/VARIETIES
Methods of Protoplast Fusion
FIGURE: Fusing Protoplasts
Different methods of protoplast fusion are described by Bengochea and Dodds (1986).
Protoplast fusion can be broadly classified into
two categories:
1. Spontaneous fusion (fuse through their
plasmodesmata)
2. Induced fusion (needs fusion inducing
chemicals/Fusogens)
a) Mechanical fusion
b) Chemo fusion
c) Electro fusion

1. Spontaneous Fusion
Protoplast during isolation
often fuse spontaneously and
this phenomenon is called
spontaneous fusion .During
the enzyme treatment,
protoplast from adjoining
cells fuse through their
plasmodesmata to form
multinucleate (2-40)
protoplasts. Development and Characterization of Somatic Hybrids of Ulva
reticulata Forsskål (×) Monostroma oxyspermum (Kutz.)Doty

2. Induced Fusion
Fusion of freely isolated protoplasts from different sources with the
help of fusion inducing chemicals agents is known as Induced
Fusion. Normally isolated protoplast do not fuse with each other
because the surface of isolated protoplast carries negative charges
(-10mV to -30mV) around the outside of the plasma membrane.
And thus there is a strong tendency in the protoplast to repel each
other due to their same charges. So this type of fusion needs a
fusion inducing chemicals (Fusogens) which actually reduce the
electronegativity of the isolated protoplast and allow them to fuse
with each other (Narayanswamy S. 1994).

The isolated protoplast can be induced to fuse by three ways;
A) MECHANICAL FUSION: In this method the isolated
protoplast are brought into intimate physical contact mechanically
under microscope and using Micromanipulator or Perfusion
Micropipette.
Micromanipulator
B) CHEMO FUSION (CHEMICAL FUSION): Several chemicals have been used to
induce protoplast fusion such as NaNO3, Polyethylene Glycol (PEG) and Calcium
ions (Ca++). Chemical fusogens cause the isolated protoplast to adhere (stick) each
other and leads to tight agglutination followed by fusion of protoplast (Pasha C.R et al
2007; Jogdand S.N.2001).

NaNO3 Treatment:-Isolated protoplasts exposed to a mixture of 5.5% NaNO3 in 10% Sucrose
Solution. Incubation carried out for 5 mins at 350C followed by centrifugation. Protoplast
pellet kept in water bath at 300C for 30 mins during which fusion occurs. Induced fusion by
NaNO3 was first demonstrated by Power et al (1970).

Treatment With Calcium Ions (Ca++) At High pH:-This method involves spinning
(centrifugation) the protoplasts in a Fusion Inducing Solution (0.05M CaCl2, 0.4M mannitol at
pH 10.5, Glycine-NaOH buffer) for 30 minutes at 50g, after which the tubes are placed in a water
bath (37°C) for 40-50 minutes. This leads to fusion of 20-50% of the protoplasts. This method
was developed by Keller and Melchers (1973) for fusing two different lines of tobacco
protoplasts. The details of the protocol are described by Bhojwani and Razdan (1983).
Polyethylene Glycol (PEG) Treatment:-Isolated protoplasts in culture medium (1ml) are mixed
with equal volume (1ml) of 28-56% PEG (Mol. Wt. 1500-6000 dalton) in a tube. Tube is
shaken and then allowed to settle and settled protoplasts are washed several times with culture
medium during which fusion occurs.

Electro Fusion:- In this method an electric
field of low strength (10Kv/m) gives rise to
dielectrophoretic dipole generation within
the protoplast suspension and a high
strength of electric field (100Kv/m) for
some micro seconds are applied this lead to
fusion.
Pearl Chains of protoplasts

Fusion Products
Fusion of cytoplasm of two protoplasts results in coalescence of
cytoplasms. The nuclei of two protoplasts may or may not fuse
together even after fusion of cytoplasms. Cells containing non-
identical nuclei are referred to as Heterokaryons or
Heterokaryocytes (Mastrangelo, 1979). The fusion nuclei in a
nucleate heterokaryon results in the formation of a true Hybrid
Protoplast or Synkaryocyte (Constabel, 1978). The fusion of two
protoplasts from the same culture results in a Homokaryon.
Frequently genetic information is lost from one of the two nuclei. If
one nucleus completely disappears, the cytoplasms of the two
parental protoplasts are still hybridized (see Figure) and the fusion
product is known as “Cybrid” (Cytoplasmic Hybrid or
Heteroplast).

IDENTIFICATION AND SELECTION OF SOMATIC HYBRID CELLS
Various protocols have been proposed and practiced for the effective selection of hybrids:
1. Genetic Complementation: Complimentary selection of somatic hybrids on specific culture
medium (Melscher & Labib 1974; Smith et al.1976). In this case complementation or genetic
or metabolic deficiencies of the two fusion partners are utilized to select the hybrid component.
When protoplasts of two parents, (one parent bearing cytoplasmic albino trait and the other
parent bearing green trait) each parent carrying a non-allelic genetic or metabolic defect are
fused, it reconstitutes a viable hybrid cell, of wild type in which both defects are mutually
abolished by complementation, and the hybrid cells are able to grow on minimal medium non-
permissive to the growth of the parental cells bearing green trait. Later, the calli of hybrid nature
could be easily distinguished from the parental type tissue (albino trait) by their green color. The
complementation selection can also be applied to dominant characters, such as dominant
resistance to antibiotics, herbicides or amino acid analogues.

2. Mechanical isolation by visual means and knowledge of identification of somatic
hybrids.
3. Morphology of the plant after regeneration. Somatic hybrids in most of the cases
show characters intermediate between the two parents such as, shape of leaves,
pigmentation of corolla, plant height, root morphology and other vegetative and floral
characters. The method is not much accurate as tissue culture conditions may also alter
some morphological characters or the hybrid may show entirely new traits not shown by
any of the parents.

FIGURE: Fused protoplast (left) with chloroplasts (from a leaf cell)
and colored vacuole (from a petal)
4. Pigmentation:-(Morpho-Physiological Basis of
Calli): The whole mixture of the protoplasts are
cultured after fusion treatment and the resulting
calli or regenerants are screened for their hybrid
characteristics. Occasionally the hybrid calli
outgrow the parental cell colonies and are
identified by their intermediate morphology, i.e.
green with purple coloured cells. However, the
process is labour intensive and requires glasshouse
facilities. It is limited to certain combinations
showing differences in their regeneration potential
under specific culture conditions.

5. Cytoplasmic Markers
• Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC (Rhodamine isothiocyanate) are
used for labelling of parental cells
• Manual isolation requires that the two parental type protoplasts have distinct morphological
markers and are easily distinguishable. For example, green vacuolated, mesophyll protoplasts
from one parent and richly cytoplasmic, non-green protoplasts from cultured cells of another
parent. The dual fluorescence method also helps easy identification of fusion products. In this
case, the protoplast labeled green by treatment with fluorescein diacetate (FDA, 1-20 mgl-1) are
fused with protoplasts emitting a red fluorescence, either from chlorophyll autofluorescence or
from exogenously applied rhodamine isothiocyanate (10-20 mgl-1). The labeling can be
achieved by adding the compound into the enzyme mixture. This can be applied even for
morphologically indistinguishable protoplasts from two parents.

6. Nuclear staining
• Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-orcein stain.
7. Phytotoxins (soybean resistant to Hm T toxin whereas Zea mays sensitive to it)
8. Molecular analysis: Specific restriction pattern of nuclear, mitochondrial and
chloroplast DNA characterizes the plastomes of hybrids and cybrids. Molecular markers
such as RFLP, RAPD, and ISSR can be employed to detect variation and similarity in
banding pattern of fused protoplasts to verify hybrid and cybrid.
9. Specific amino acid (Conavalin present in soybean but not in sweet clover, alfalafa)
10. Auxin autotrophy (Nicotiana glauca and Nicotiana langsdorffi)

11. Isozyme Analysis: Multiple molecular forms of same enzyme which catalyses similar or
identical reactions are known as “Isozymes”. Electrophoresis is performed to study banding pattern
as a check for hybridity. If the two parents exhibit different band patterns for a specific isozyme the
putative hybrid can be easily verified. The isozymes commonly used for hybrid identification include,
acid phosphatase, esterase, peroxidase.
12. Chromosomal Analysis (Cytological Analysis): Chromosome counting of the hybrid is
an easier and reliable method to ensure hybridity as it also provides the information of ploidy level.
Cytologically the chromosome count of the hybrid should be sum of number of chromosomes from
both the parents. Besides number of chromosomes, the size and structure of chromosomes can also be
monitored. However, the approach is not applicable to all species, particularly where fusion involves
closely related species or where the chromosomes are very small. Moreover, sometimes the
somaclonal variations may also give rise to different chromosome number.

13. Antibiotics: Drug sensitivity technique was originally developed by
Power et al (1976) for the selection of hybrids of Petunia sp. This method is
useful for the selection hybrids of two plant species, if one of them is sensitive
to a drug. Protoplasts of Petunia hybride (species A) can form macroscopic
callus on MS medium, but are sensitive to (inhibited by) actinomycin D.
Petunia parodii protoplasts (species B) form small colonies, but are resistant to
actinomycin D.
When these two species are fused, the fused protoplasts derive both the
characters-formation of macroscopic colonies and resistance to actinomycin D
on MS medium. This helps in the selection of hybrids (Fig. 44.6). The parental
protoplasts of both the species fail to grow. Protoplasts of P. parodii form very
small colonies while that of P. hybrida are inhibited by actinomycin D.

CULTURE OF HYBRID CELLS
Hybrid cells are cultured on suitable medium
provided with the appropriate culture conditions.
• Iscove's MDM with 25 mM Hepes.
• DMEM/F-12 with 15 mM HEPES.
• Alpha MEM with Nucleosides
• RPMI 1640 Medium

REGENERATION OF HYBRID PLANTS
Plants are induced to
regenerate from hybrid calli.
These hybrid plants must be at
least partially fertile, in
addition to having some useful
property, to be of any use in
breeding schemes.

REGENERATION OF HYBRID PLANTS
CELL WALL REGENERATION:
• May be completed in two to several days.
• Although protoplast in culture generally start regenerating a cell wall within a few
hours after isolation.
• Protoplast lose their characteristic spherical shape once the wall formation is complete.
• Regeneration of cell wall can be demonstrated using Calcalfluor White ST
fluoresecent Stain or Tinapol solution.

ADVANTAGES/BENEFITS/MERITS
PRACTICAL APPLICATIONS
SYMMETRIC HYBRIDS--PRODUCTION OF NEW SPECIES
Symmetric hybrids can be produced between species,
which cannot be hybridized sexually. These hybrids can be
readily used in breeding programs for transfer of useful
genes to crops or may be useful as new species. The first
symmetric somatic hybrid of Citrus was created by
protoplast fusion of C. sinensis and Poncirus trifoliata
(Ohgawara et al., 1985) and the production of hybrid
plants between two sexually incompatible Citrus genera
was first reported in 1988, where C. sinensis L. Osb. cv.
‘Hamlin’ protoplasts were fused with Severinia disticha
(Blanco) Swing protoplasts (Grosser et al., 1988).

CYTOPLASMIC TRANSFERS ARE TIME SAVING
Cytoplasm transfers can be affected in one year, while backcrossing may take 15-16
years. Even where backcrossing is not applicable, cytoplasm transfers can be made using
this approach.
MITOCHONDRION-CHLOROPLAST FUSION
(RECOMBINANT ORGANELLAR GENOMES)
Mitochondria of one species can be combined with chloroplasts of another species. This
may be very important in some cases, and is not achievable by sexual means even
between easily crossable species. Recombinant organellar genomes, especially of
mitochondria, are generated in somatic hybrids and cybrids. Some of these recombinant
genomes may possess useful features.

PRODUCTION OF NOVEL INTERSPECIFIC
AND INTERGENIC HYBRID
• Pomato (Hybrid of potato and tomato)

PRODUCTION OF FERTILE DIPLOIDS AND POLYPLOIDS FROM
SEXUALLY STERILE HAPLOIDS, TRIPLOIDS AND ANEUPLOIDS
Anssour et al. (2009) examined both allo- and auto-tetraploid Nicotiana species, and showed that
there were substantial changes both in morphology and in the genome which are found in the
hybrid species compared with their ancestral diploids.
GENE TRANSFER
Gene Transfer for disease resistance (e.g. TMV, Potato Virus X, Club Root Disease), abiotic stress
resistance (cold tolerance gene in tomato), herbicide resistance and many other quality characters.
Asymmetric hybridization is very promising as it allows partial genome transfer (Derks et al., 1992;
Trick et al., 1994; Liu & Deng, 2002), which may be better tolerated than a whole-genome transfer
(Ramulu et al., 1996a,b). As in other agricultural species, trait introgression from ‘wild’ species of
the genus Nicotiana has been used to improve the cropped species, and characters from at least 13
different species have been transferred into tobacco (Lewis, 2011).

PRODUCTION OF HETEROZYGOUS LINES
Production of heterozygous lines in the single species which cannot be propagated by
vegetative means.
FATE OF PLASMA GENES
Somatic cell fusion is useful in the study of cytoplasmic genes (plasma genes) and their
activities and this information can be applied in plant-breeding experiments.
PRODUCTION OF UNIQUE HYBRIDS OF NUCLEUS AND
CYTOPLASM
Cybridization has made it possible to transfer cytoplasmic male sterility.

DISADVANTAGES/LIMITATIONS/DEMERITS
CEREALS & PULSES
Techniques for protoplast isolation, culture and fusion are not available for many important crop
species like many cereals and pulses.
CHROMOSOMAL ELIMINATION
In many cases, chromosome elimination occurs from somatic hybrids leading to asymmetric
hybrids. Such hybrids may be useful, but there is no control on chromosome elimination.
GENETIC INSTABILITY
Many somatic hybrids show genetic instability, which may be an inherent feature of some species
combinations.
ABNORMALITIES
Many somatic hybrids either do not regenerate or give rise to sterile regenerants. Such hybrids are
useless for crop improvement. All interfamily somatic hybrids are genetically unstable and/or
morphologically abnormal, while intergeneric and intertribal hybrids are genetically stable but
produce abnormal and/or sterile plants or only teratomata.

DISADVANTAGES/LIMITATIONS/DEMERITS
POOR REGENERATION
Poor regeneration of hybrid plants
NON-VIABILITY
Non-viability of fused products.
INEFFICIENT SELESCTION METHODS
There are limitations in the selection methods of hybrids, as many of them are not
efficient. Not very successful in all plants.
NO CONFIRMATION OF EXPRESSION OF PARTICULAR TRAIT
No confirmation of expression of particular trait in somatic hybrids. Techniques for
protoplast isolation, culture and fusion are very complicated.

CONCLUSION REMARKS
Protoplast technology has various applications other than regeneration of complete plants
and production of hybrids of sexually incompatible species. These techniques have been
instrumental in generating basic scientific information on cell biology, plant
incompatibility, membrane functions, cell organelle studies, cell wall regeneration,
ultrastructure and molecular architecture of plant cells. These techniques are now being
used for transfer of cytoplasmic male sterility. Protoplasts can take up macromolecules
(nucleic acids and proteins), viruses, cell components like chromosomes and chloroplasts
by phagocytosis. Somatic hybridization allows transfer of cytoplasmic organelle in a
single generation and offer unique opportunities for combining mitochondria of one
species and chloroplast of another species in a single hybrid.
This technique in the future will be one of the most frequently used research tools for
tissue culturists, molecular biologists, biochemical engineers and biotechnologists.

Somatic hybridization

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