Transcript SOMATIC HYBRIDIZATION - GCG-42

SOMATIC HYBRIDIZATION
• Development of hybrid plants through the
fusion of somatic protoplasts of two different
plant species/varieties is called somatic
hybridization.
Somatic hybridization technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants
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.
• Plant cell wall acts as physical barrier and protects
cytoplasm
from
microbial
invasion
and
environmental stress.
• It consists of a complex mixture of cellulose,
hemicellulose, pectin, lignin, lipids, protein,
• For dissolution of different components of the cell
wall it is essential to have the respective enzymes.
History
• Hanstein introduced the term ‘Protoplast’.
• The isolation of protoplasts from was first achieved
through by Klercker (1892) on plasmolysed cells.
• Cooking (1960) for the first time isolated the
protoplasts of plant tissues by using cell wall
degrading enzymes viz., cellulase, hemicellulase,
pectinase, and protease extracted from a saprophytic
fungus Trichoderma viride & Myrothecium
verrucaria.
• First achievement in protoplast fusion by Power
(1970)
Isolation of Protoplast
(Separation of protoplasts from plant tissue)
1. Mechanical Method
2. Enzymatic Method
1. Mechanical Method
Cells Plasmolysis
Plant Tissue
Microscope Observation of cells
Cutting cell wall with knife
Release of protoplasm
Collection of protoplasm
1. 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
Leaf sterlization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase
Pectinase
Protoplasm
released
Release of
isolated cells
Protoplasm released
cellulase
Isolated
Protoplasm
Enzymatic Method
• Used for variety of tissues and organs including
leaves, petioles, fruits, roots, hypocotyls, stem, shoot
apices, embryo, microspores.
• Mesophyll tissue - most suitable source
• High yield of protoplast
• Easy to perform
• More protoplast viability
Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion
Intraspecific
Intergeneric
2. Induced Fusion
Chemofusion
Mechanical
Fusion
Electrofusion
Spontaneous Fusion
• Protoplast fuse spontaneously during
process mainly due to physical contact.
• Intraspecific produce homokaryones
• Intergeneric have no importance
isolation
Intraspecific protoplast fusion
• Intraspecific protoplast fusion is the cross between
the same species in an individual which involves the
isogenic strains or the non-isogenic ones.
• The true value of protoplast fusion as a mean for
establishing parasexual crosses has been realized so
far in a few fungi. For example, in Cephalosporium
acremonium.
• This technique offers the only way of carrying out
crosses and genetic analysis.
•
• Protoplast fusion technique made it possible to
produce a preliminary genetic map of 8 linkage
groups for C. acremonium. Genes which enhance the
production of antibiotics have been identified and
allied to specific linkage groups.
• The other examples are :
-Absidia glauca, Candida maltosa, Aspergillus niger,
Fusarium graminearum, Penicillium verruculosum,
T. reesei, etc.
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.
• Some of the examples where interspecific hybrids were
produced through protoplast fusion are:
- S. cerevisiae x S. fermentali,
- S. cerevisiae x S. lipolytica,
- P. chrysogenum x P. notatum,
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Induced Fusion
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Chemofusion- fusion induced by chemicals
• Types of fusogens
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PEG
NaNo3
Ca 2+ ions
Polyvinyl alcohol
Induced Fusion
• 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
– the fusion process
• 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
Calcofluor white (CFW) staining
• CFW binds to the β-lined glycosides in the newly
synthesized cell wall which is observed as a ring of
fluorescence around the plasma membrane.
Protoplast density
• Protoplasts have both maximum as wellas
minimum plating densities for growth.
• Published procedures suggest that protoplasts
should be cultured at a density of 5x103 to 106
cells/ml with an optimum of about 5x104
protoplasts/ml.
Identification and Selection of
somatic hybrid cells
• Hybrid identification- Based on difference between
the parental cells and hybrid cell with respect to
• Pigmentation
• Cytoplasmic markers
• Fluorochromes like FITC (fluoroscein
isothiocyanate) and RITC (Rhodamine
isothiocyanate) are used for labelling of
hybrid cells
• Presence of chloroplast
• Nuclear staining
• Heterokaryon is stained by carbol-fuschin,
aceto-carmine or aceto-orcein stain
Culture of the hybrid cells
• Hybrid cells are cultured on suitable medium
provided with the appropriate culture conditions.
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.
Advantages of somatic hybridization
• Production of novel interspecific and intergenic
hybrid
– Pomato (Hybrid of potato and tomato)
• 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.
Advantages of somatic
hybridization
• Production of heterozygous lines in the single species
which cannot be propagated by vegetative means
• Studies on the fate of plasma genes
• Production of unique hybrids of nucleus and
cytoplasm
Limitations of Somatic
hybridization
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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 hybrids
No confirmation of expression of particular trait in
somatic hybrids
Application of Somatic hybridization
– protoplast fusion to create somatic hybrids
• "wide crosses" where even embryo culture
won't work
–Citopsis gilletiana (wild) x Citrus sinensis
–citrus sexually incompatible spp.
–wild relative has disease/nematode resistance
–somatic hybrid used as a rootstock
– protoplast fusion to create somatic hybrids
• Solanum somatic hybrids
–S. tuberosum dihaploids fused with wild
diploid S. chacoense
–resulting somatic hybrid (4n) is backcrossed
to S. tuberosum cultivars (also 4n)
–overcomes sterility due to ploidy differences
between somatic and sexual hybrids
Protoplast culture and regeneration
• From the protoplast solution of known density (about
105 protoplast/ml) about 1 ml suspension is poured on
sterile and cooled down nutrient medium in Petri
dishes.
• The plates are incubated at 25°C in a dim white light.
Protoplast culture and regeneration
• The protoplasts regenerate a cell wall, undergo cell
division and form callus. The callus can also be
subcultured.
• Embryogenesis begins from callus when it is placed
on nutrient medium lacking mannitol and auxin. The
embryo develops into seedlings and finally mature
plants.