Somatic hybridization is a fusion of two somatic cells for the production of hybrids.
The somatic hybrids are intra/inter-specific hybrids.
Here distant parental cells are fused to obtain somatic hybrids.
Plant cells have cell walls so it is not possible to fuse intact plant cells. Protoplasts are isolated before somatic hybridization
STEPS INVOLVED IN SOMATIC HYBRIDIZATION
A. Isolated protoplasts of two species.
B. Adhesion of protoplasts
C. Formation of a connection between two protoplasts.
D. Dissolution of the intervening membrane of the two protoplasts.
F. Fusion of the cytoplasm.
E. Formation of heterokaryon.
Different methods of protoplasts fusion
CHEMICAL FUSION
Calcium chloride (CaCl2)
is used for chemical fusion. Calcium ions form a bridge between the cells. So, calcium ions are used to fuse somatic
cells.
NaNO3 is used for the fusion of somatic
cells.
Polyethylene glycol
PEG forms a bridge between the somatic cells.
In the plant, PEG is a fusogen that forms a
bridge between the somatic cells. This ensures the adhesion of protoplast with each
other. Once the adhesion is complete, the intervening cell membrane of the protoplast
is dissolved. This results in the fusion of cytoplasm. The cell membrane is
regularized and the heterokaryon is produced. This heterokaryon then undergoes mixing
of the nucleus and results in the formation of somatic hybrids.
Electrofusion
Where the cells are fused under the current of
electricity electric current of 0.5-1.5 volt is passed through the suspension
medium that contains isolated protoplast. This allows charge separation of
isolated protoplast. These protoplasts now behave like a dipole. They line up
between the two poles. The charge is disturbed by passing a current of high
voltage for a few seconds. The intensity charge is passed between the electrodes,
which is 0.125- 1kVcm-1. The high-intensity charge causes
reversible membrane breakdown. This allows the formation of a contact area
between the two protoplasts. Fusion of protoplast takes less than 10 minutes in the electric field. The presence of calcium chloride 1mM in the fusion mixture
increases fusion frequency protoplast. The density should be 1x104 protoplasts per ml for
electro-fusion of protoplasts.
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| CHARGE SEPARATION OF PROTOPLASTS UNDER ELECTRIC FIELD |
In the microchamber or microdroplet method of
protoplast culture, protoplast can be fused in the microchambers. Platinum
wires are used as electrodes for electrofusion. Electrofusion is more suitable
for mesophyll cells than root or callus protoplasts (Pelletier 1993)
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| ELECTROFUSION OF PROTOPLASTS IN MICROCHAMBER |
Selection of fusion products
Morpho-physiological basis of selection
Fusion product can be selected by studying the
morphology of cellular hybrid callus, where the fusion product of Solanum
tuberosum to Solanum ceraceifolium where one parent forms a green color callus and
the other form brown yellow color callus, and the fusion product forms
intermediate callus where green color and purple color cells are present.
Hybrid vigor
Hybrid vigor where cells of Dianthus chinensis
and Dianthus barpatus show hybrid vigor. Hybridized cells of these parents divide and form shoots and roots vigorously.
Markar based selection or complement selection
Where metabolic deficiencies of two fusion parents are utilized to select a hybrid. The metabolic deficient parents are eliminated by themselves in the medium that doesn't contain the metabolic component. However, hybrids will survive in this medium.
The complement selection also utilizes
Herbicide resistance, antibiotic resistance, or amino acid analog genes as these can be markers.
Isolation of heterokaryon
In the low-density culture where use and products are cultured in the low-density medium so that callus of different colors and morphology are selected individually in this medium.
Morphologically isolated fusion products can be manually selected through a micropipette, where the two fused protoplasts have a different color to that of their parent. This process was first determined by Hoffmann in 1978-1979.
Fluorescent isolation of heterokaryons
Dual fluorescence labeling system where
protoplast are labeled by Green pigment the Fluorescein diacetate (1 to 20mgl-1
) which emit green color and the other set of protoplast are labeled with red
color using Rhodamine isothiocyanate (10 to 20mgl-1). This labeling is
achieved by adding enzymes and the fluorescent product to the culture mixture. Manual
isolation of product was done through Pasteur pipette.
The dual-labeled products can also be isolated
using FACS where cells are sorted on the basis of the wavelength they emit. Due to
their staining, different wavelengths are emitted by the cells. Thus the cells are selected into the different containers by FACS.
Verification of hybrid product
It is done to study morphology where flower color or expression of leaf variegation can be used to determine the hybridization of the fusion product.
Cytological analysis
Where the number of chromosomes is estimated for hybrid cells. The chromosomes number could be multiple of
the number of chromosomes that were present in parents.
Isozyme analysis
Here the isozymes that are present in two different
parents that reflect different band patterns are studied through molecular isolation. Isozymes studied for hybrid verification are Phosphatases, esterases, peroxidases, and phosphoglucomutase.
DNA analysis
Restriction enzyme polymorphism studied for
hybrid product establish the hybrid ability or DNA fingerprinting can be used
for hybrid products.
The fate of the genome of hybrid
The fate of the genome depends upon the number and type of
cells fused.
Genome segregation occurs during cell division, after fusion genome segregation during regeneration of the plant.
Inter-parent recombination of plastid-genome occurs rarely so plastids are selectively eliminated in hybrid products.
The nuclear chromosome may be
selectively eliminated from the hybrid products, so this may result in the formation
of hybrids and result in a novel combination of the plastid-mitochondrial genomes.
USES OF SOMATIC HYBRIDIZATION
Tomato hybrids have been developed that are resistant to TMV and spotted wilt virus.
Environment tolerance and stress-tolerant plants can be developed through hybridization.
The high-yielding plants can be developed through symmetric hybridization.
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