WHAT ARE SOMATIC CELL ?

A somatic cell (from Ancient Greek sôma, meaning "body"), or vegetal

cell, is any biological cell forming the body of an organism; that is, in a
multicellular organism, any cell other than a gamete, germ cell,
gametocyte or undifferentiated stem cell.
 WHAT IS A PROTOPLAST?

• “The protoplast is a part of plant cell which lies within the cell wall
and can be plas­molysed and which can be isolated by removing the
cell wall by mechanical or enzymatic proce­dure”. Vasil (1980)

• Isolated protoplast is only a naked plant cell surrounded by plasma

membra­ne—which is potentially capable of cell wall re­generation,
cell division, growth and plant regen­eration in culture.
 WHO PROPOSED THE WORD PROTOPLAST ?

Protos= first ; plastos= formed

Protoplast-term was proposed by in Johannes Ludwig Emil Robert von

Hanstein (German Botanist)1880 to refer to the entire cell, excluding the cell
wall
 TIMELINE OF PROTOPLAST RESEARCH
J. Klercker (1892):
First isolated proto­plast mechanically from plasmolyzed cell of wa­ter warrior aquatic plant (Stratiotes
aloides). No attempt was made to culture them.
E. Kiister (1927):
In the fruits of several plants like Solatium nigram (Britain weed), Lycopersicon esculenium (tomato) etc. the
cell wall are hydrolysed during fruits ripening process so that free protoplasts and protoplasmic units were
utilized.
R. Chambers and K. Hofler (1931):
Isolated few protoplasts by using thin slices of epidermis of onion bulb
E. C. Cocking (I960):
First reported the enzymatic method for isolation of protoplast in a large number from root tip cells of
Lycopersicon esculentum by using a concentrated solution of cellulase enzyme, prepared from cultures of the
fungus Myrothecium verrucaria to degrade cell wall.
 SOMATIC HYBRIDIZATION

Development of hybrid plants through the fusion of somatic protoplasts of

two different plant species/varieties is called somatic hybridization, and such
hybrids are known as somatic hybrids.

This is a non conventional genetic procedure involving fusion between

isolated protoplast under in vitro condition and subsequent development of
their product (heterokaryon) to a hybrid plant.
 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

PROCEDURE OF SOMATIC HYBRIDIZATION
1. ISOLATION OF PROTOPLAST
 Method of mechanical isolation
of protoplasts.

• A small place of plant tissue

Plasmolysis of cells

• Cutting of cell wall by microscalpel

under microscope.

• stages of liberation of protoplasts.

• isolated protoplast and empty cells

 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

Pectinase + Cellulase
 ENZYMATIC METHOD

• Used for variety of tissues and organs including leaves, petioles, fruits,
roots, coleoptiles, hypocotyls, stem, shoot apices, embryo

• Mesophyll tissue - most suitable source

• High yield of protoplast

• Easy to perform

• More protoplast viability

COMMERCIAL ENZYMES , NAMES & SOURCE
 2. FUSION OF THE PROTOPLASTS OF DESIRED
SPECIES/VARIETIES
It involves mixing of two different genomes and can be achieved by
following method as;

 Spontaneous Fusion Method

 Induced Fusion Method.

 Mechanical Fusion Method

 SPONTANEOUS FUSION METHOD

• Cell fusion is natural process as is observed in case of egg fertilization.

• During the course of enzymatic degradation of cell walls, adjoining protoplasts

may fuse to form homokaryocytes (homocaryons).

• These fused cells may sometimes contain high number of nuclei (2-40).

• The frequency of homocaryons formation was found to be high in protoplasts

isolated from dividing cultured cells
EXAMPLE OF TOBBACCO PROTOPLAST FUSION
 MECHANICAL FUSION METHOD

• The protoplasts can be pushed together mechanically to fuse.

• Protoplasts of Lilium and Trillium in enzyme solutions can be fused by gentle

trapping in a depression slide.

• Mechanical fusion may damage protoplasts by causing injuries.

 INDUCED FUSION METHOD

• Isolated protoplasts can be fused by induction.

• There are several fusion inducing agents which are collectively referred to
as fusogen example are as: sodium nitrate ,high gylcol, polyvinyl alcohol,
lysozyme, concavalina, electrofusion dextran and dextran sulphate , fatty
acid and esters
 TREATMENT WITH SODIUM NITRATE

• Induced fusion by NaNO3 was first reported by power et al .(1970).

• Isolated protoplasts are exposed to a mixture of 5.5% carried NaNO3 in

10% sucrose solution.

• Incubation is carried out for 5 minutes at 35 ⁰C .

• Centrifugation for 5 minutes at 200x g

 HIGH pH AND HIGH SODIUM Ca ION TREATMENT

• This method was first used for the fusion of tobacco protoplasts, and is now in
use for other plants also.

• The method consists of incubating protoplasts in a solution of 0.4 M mannitol

containing 0.05 M CaCI2 at pH 10.5 (glycine-NaOH buffer) and temperature 3
7°C for 30-40 minutes.

• The protoplasts form aggregates, and fusion usually occurs within 10 minutes.

• By this method, 20-50% of the protoplasts are involved in fusion.

 POLY ETHYLENE GLYCOL (PEG) TREATMENT

• This has proved to be one of the most effective methods for protoplast
fusion .

• The cells are treated with a concentration of around 30% poly ethylene
glycol which binds to plasma membrane .

• This is treated with calcium solution which being cationic binds to PEG.

• During washing the PEG pulls out the plasma lemma leading to fusion of
protoplasts in close proximately.
 PEG ADVANTAGES
PEG treatment method is widely used protoplast fusion as it has several
advantages:

i. It results in a reproducible high-frequency of heterokaryon formation.

ii. Low toxicity to cells.

iii. Reduced formation of bi-nucleate heterokaryons.

iv. PEG-induced fusion is non-specific and therefore can be used for a wide
range of plants.
 ELECTRO FUSION TECHNIQUE

• This process involves passing low voltage electric pulses in a solution of

protoplast to be fused so that they line of for fusion .

• High voltage electric current which leads alteration of membrane the

adjacent protoplast fuse.

MECHANISM OF ELECTRO FUSION TECHNIQUE
Schematic representation of (3) successful protoplast fusion technique
 3. IDENTIFICATION AND SELECTION OF
SOMATIC HYBRID CELLS
• The protoplasts are actually involved in the fusion.

• After the fusion process, the protoplast population consists of heterogenous mixture
of unfused chloroplast, homokaryons and heterokaryons.

• It is therefore necessary to select the hybrid cells (heterokaryons).

• The commonly used methods employed for the selection of hybrid cells are as follows;
 Visual method

 Biochemical method

 Cytometric method
 VISUAL METHOD
• Visual selection of hybrid cells, although tedious is very efficient. In some of
the somatic hybridization experiments, chloroplast deficient (albino or non-
green) protoplasts of one parent are fused with green protoplasts of another
parent.

• This facilitates the visual identification of haeterokaryons under light

microscope. The heterokaryons are bigger and green in colour while the
parental protoplasts are either small or colourless.
 BIOCHEMICAL METHOD
• The biochemical methods for selection of hybrid cells are based on the use
of biochemical compounds in the medium (selection medium).
• These compounds help to sort out the hybrid and parental cells based on
their differences in the expression of characters.
• There are basically (2) type of biochemical method for selection of hybrid
cell;

 Drug sensitivity ; This method is useful for the selection hybrids of two plant species,
if one of them is sensitive to a drug i.e. actinomycin D

 Auxotrophic mutants; hybrid cell cannot grow on a minimal medium and therefore
require specific compounds to be added to the medium. i.e. Nitrogen source
 CYTOMETRIC METHOD
• Some workers use flow cytometry and fluorescent-activated cell sorting
techniques for the analysis of plant protoplasts while their viability is
maintained.

• The hybrid cells derived from such selections have proved useful for the
development of certain somatic hybrid plants.
 4. CULTURE OF THE HYBRID CELLS

Hybrid cells are cultured on suitable medium provided with the appropriate
culture conditions.

Gamborg media (B5 media)

Modified Eriksson's medium

Medium 1
 5. REGENERATION OF HYBRID PLANTS

• Plants are induced to regenerate from hybrid callus.

• 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 .

• Normally cybrids are produced when protoplast from two pythogenetically

distinct species are fused.

• Genetically are hybrids only for cytoplasmic traits.

SCHEMATIC REPRESENTATION OF CYBRIDS
SELECTED LIT OF GENETIC TRAITS TRANSFERRED THROUGH PROTOPLAST FUSION IN CROP PLANT
 TWO EXAMPLES OF CYBRIDS

• Fusion between protoplasts of potato (Solanum

tuberosum) and tomato (Lycopersicon esculentum) has
created pomato (Solanopersicon, a new genus).

• Interspecific fusion of four different species of rice

(Oryza brachyantha, O. elchngeri, O. officinalis and O.
perrieri) could be done to improve the crop.
 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.
DISADVANTAGES OF SOMATIC HYBRIDIZATION

• 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.

QUESTIONS , COMMENTS & FEEDBACK