SOMATIC HYBERIDIZATION

Development of hybrid plants through the

fusion of somatic protoplasts of two different
plant species/varieties is called somatic
hybridization.
Main Points related to Somatic Hybridisation

• Involves Somatic Cells.

• Tissue Culture is essential.
• Equal contribution of cytoplasm.
• Overcomes the barrier of cross incompatibility.
• Novel combination of genes from unrelated
species.
TYPES OF SOMATIC HYBRIDS
 PROT0PLAST
Hanstein introduced the term ‘Protoplast’.

Protoplast also known as a naked

plant cell refers to all the components
of plant cell excluding the cell wall.

Protoplast is the biologically active

and most significant material of cells.
 PROTOPLAST ISOLATION
(Separation of protoplast from plant tissue)
 MECHANICAL METHOD
• Used for vacuolated cells like onion bulb scale,
radish and beet root tissues
• Low yield of protoplast
• Laborious and tedious process
• Low protoplast viability
 ENZYMATIC METHOD
• Used for vacuolated cells like onion bulb scale,
radish and beet root tissues.
• Low yield of protoplast.
• Laborious and tedious process.
• Low protoplast viability.
FUSION OF PROTOPLAST
 SPONTANEOUS FUSION
• Protoplast fuse spontaneously during isolation
process mainly due to physical contact.

• Intraspecific spontaneous fusion.

• Intergeneric spontaneous fusion.
 INTRASPECIFIC PROTOPLAST FUSION
• Intraspecific protoplast fusion is the cross between
the same species.
• Example – Datura
(Datura starmonium)
 INTERSPECIFIC PROTOPLAST FUSION

• Interspecific protoplast fusion is the crosses between two

different species.

• Interspecific protoplast fusions are of much importance in the

area where new products are to be produced. Due to new genetic
set up many noval secondary metabolites such as, antibiotics may
be produced.
 INDUCED FUSION
Chemofusion - fusion induced by chemical

Types of fusogens
• PEG
• NaNo3
• Ca 2+ ions
• Polyvinyl alcohol
Mechanical Fusion-
Physical fusion of protoplasts under microscope by
using micromanipulator and perfusion micropipette.

Electrofusion-
Fusion induced by electrical stimulation-
• Pearl chain of protoplasts is formed by low
strength electric field (10kv m-1)
• Fusion of protoplasts of pearl chain is induced
by the application of high strength electric field
(100kv m-1) for few microsecond.
Protoplast fusion and somatic hybrids
• Electrofusion –
protoplasts are aligned in
a special chamber,
electric current is
applied, opening
channels in cell
membrane.
• PEG fusion–
protoplasts are coated
with PEG, then
incubated together;
where cell membranes
fuse, channels begin to
form.
After fusion, "fusion
products" begin to
"round up"
 Protoplast viability

The most frequently used staining methods for

assessing protoplast viability are:

- Fluorescein diacetate (FDA) staining.

- Phenosafranine staining.
- Calcofluor white (CFW) staining.
Fluorescein diacetate (FDA) staining

• FDA, a dye that accumulates inside the plasma

membrane of viable protoplasts.
• Viable intact protoplasts fluoresce Yellow
green within 5 min.
• FDA is dissolved in CH3COCH3 & used at a
concentration of 0.01%.
 Phenosafranine staining
• It is specific for dead protoplasts that turn Red
in staining procedure.
• Viable cells remain unstained by
Phenosafranine
 SELECTION AND IDENTIFICATION OF
HYBRID CELLS
Hybrid identification- Based on difference between the
parental cells and hybrid cell with respect to
• Pigmentation
• Cytoplasmic markers
• Fluorochromes like FITC (fluoroscein
isothiocyanat).
• RITC (Rhodamine isothiocyanate) are used for
labelling of hybrid cells.
• Presence of chloroplast.
• Nuclear staining.
• Hetrokaryon is stained by carbol-fuschin, aceto-
carmine or aceto-orcein stain.
 CULTURE OF HYBRID CELLS
Hybrid cells are cultured on suitable
medium provided with the appropriate
culture conditions.
• Alpha MEM with nucleosoides.
• 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.
 CYBRIDS
The cytoplasmic hybrids where
the nucleus is derived from only
one parent and the cytoplasm is
derived from both the parents
are referred to as cybrids . The
phenomenon of formation of
cybrids regarded as cybridization
. Genetically cybrids are hybrids
only for cytoplasmic traits.
 POTENTIAL OF SOMATIC
HYBRIDIZATION
Production of novel
interspecific and
intergenic hybrid.
Pomato (Hybrid of
potato and
tomato).
POTENTIAL OF SOMATIC HYBRIDIZATION

• Production of fertile diploids and polypoids

from sexually sterile haploids, triploids and
aneuploids.
• Transfer gene for disease resistance, abiotic
stress resistance, herbicide resistance and
many other quality characters.
 PROBLEMS AND LIMITATION OF
SOMATIC HYBERIDIZATION
• Poor regeneration of hybrid plants.
• Non-viability of fused products.
• Not successful in all plants.
• Production of unfavorable hybrids.
• Lack of an efficient method for selection of
• hybrid.
• No confirmation of expression of particular
trait in somatic hybrids.
 CONCLUSION
Protoplast technology has various
applicatipons other than regeneration of
complete plant and production of hybrids of
sexually incompatible species. These
techniques have been instrumental in
generating basic scientific information on cell
biology, plant incompatibility, membrane
function , cell organelles studies, cell wall
regeneration, ultrastructure and molecular
architecture of plant cells.