Somatic Hybridization

Somatic Hybridization
DEFINITION & INTRODUCTION

Development of hybrid plants through the fusion of somatic protoplasts of two different plant
species/varieties is called “Somatic Hybridization”.
PROCEDURE/STEPS OF TECHNIQUE
ISOLATION OF PROTOPLASTS
Importance of Protoplast Isolation
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 made possible
only when the following two criteria are satisfied:
i) Isolation of protoplast in large quantity, and
ii) Totipotency of the isolated protoplasts.
Source of Protoplasts
Protoplast can be isolated from almost all plant parts:
 Roots, leaves, fruits, tubers, root nodules, endosperm, pollen mother cell, callus
and suspension culture
Methods of Isolation 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.
TYPES OF ENZYMATIC METHOD: There are two types of enzymatic method. Both
methods have certain advantages and disadvantages
Generally 50 mM CaCl2 is added to increase the stability of released protoplasts.
One Step Method (Direct/Mixed Method): In this method protoplasts are isolated from plant
tissues directly by using two enzymes, cellulase and pectinase, simultaneously.
Two Step method (Sequential Method): 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
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 (*).
PROCEDURE: Cells are immersed in 1.0 M Sucrose until the protoplast shrunk away from
their enclosing wall and then the plasmolysed tissue are cutted into small strips. The
protoplasts are released by Osmotic Swelling when these strips of the tissue are 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
Purification of Protoplasts
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. 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.
FIGURE: Protoplasts purification by filterartion

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.
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
debris between
settle the enzyme
to the bottom (See solution and lymphoprep FIGURE: Protoplasts in ring
and
Figure).
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 FIGURE: Viable Protoplasts visible in green color
Factors Affecting Protoplast Isolation And Its Viability
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.
FUSION OF PROTOPLASTS OF DESIRED SPECIES/VARIETIES
Methods of Protoplast Fusion
Different methods of protoplast fusion are described:
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
FIGURE: Fusing Protoplasts
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.
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.
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.
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.
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
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.
The fusion nuclei in a nucleate heterokaryon results in the
formation of a true Hybrid Protoplast or Synkaryocyte. 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).

Somatic Hybridization

Uploaded by

Copyright:

Available Formats

Somatic Hybridization

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Somatic Hybridization

Uploaded by

Copyright:

Available Formats

Somatic Hybridization

DEFINITION & INTRODUCTION

Before culturing protoplast, it is important to isolate

Generally 50 mM CaCl2 is added to increase the stability of released protoplasts.

2.Sedimentation & Washing (For Removal of Enzymes):- Enzyme is removed by

FIGURE: Protoplasts purification by filterartion

3. Flotation (Separation of Protoplasts):- In this method intact

Cell Wall Formation Test: To the

FIGURE: Cell Wall is fluorescing while protoplasts remain dark

Electro Fusion:- In this method an electric

Pearl Chains of protoplasts

You might also like