Protoplast also known as a naked plant cell
refers to all the components of plant cell excluding the
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
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 fungus Trichoderma viride and
First achievement in protoplast fusion by Power (1970)
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. Protoplast culture and regeneration
1. Isolation of protoplast
A. Mechanical method B. Enzymatic method
A. Mechanical Method
Collection of protoplasm
Microscope Observation of cells
Cutting cell wall with knife Release of protoplasm
Experimental cells are allowed to plasmolyse by
keeping them in hypertonic solution.
In plasmolysed state, cell wall is cut with a sharp knife.
Plasmolysed cell is transferred to hypotonic solution.
This results in the release of protoplast in outer solution
through cut ends.
This method is suitable only for tissues with large cells
in which evident plasmolysis occurs.
Used for vacuolated cells like onion bulb scale,
radish and beet root tissues
Low yield of protoplast
Laborious and tedious process
Low protoplast viability
B. Enzymatic Method
Leaf sterlization, removal of
Plasmolysed cellsPlasmolysed cells
Protoplasm released Release of isolated cells
Isolated Protoplasm Protoplasm released
Surface sterilization of leaf sample
Rinsing in suitable plasmolyticum with distilled
Peeling of off the lower epidermis towards margin
with sharp forceps below the junction of a lateral
vein and midrib.
Purification of isolated protoplasts.
Enzymatic protoplast isolation steps
Cell walls are dissolved by enzymes.
Such enzymes are extracted from fungi, bacteria
Macerozyme, a pectinase enzyme, from Rhizopus fungus,
Driselase a mixture of cellulase and pectinase, from
Pectinase breaks the tissues into cells by dissolving
calcium pectate of middle lamella.
Hemicellulase and cellulase break down the cell wall.
Commercially available enzymes are "Pectolyase Y-
23", Onozuka R-1O.
Protoplast using enzymes may be isolated by sequential
method or mixed enzyme method.
In the first process two enzymes-pectinase and cellulase
are used sequentially, while in the second process two
enzymes are used simultaneously.
The enzyme mixture macerates the cells and
simultaneously destroys their walls.
Sequential method is useful in isolating protoplasts
from palisade layer. While mixture enzyme method is
useful in isolating protoplasts from spongy parenchyma
and upper epidermis.
Used for variety of tissues and organs including
leaves, petioles, fruits, roots, coleoptiles, hypocotyls,
stem, shoot apices, embryo microspores
Mesophyll tissue - most suitable source
High yield of protoplast
Easy to perform
More protoplast viability
Enzyme solutions are filtered with nylon mesh to remove
Filtrate is centrifuged for 5 minutes at 700 rpm.
The protoplast forms pellet and goes at the bottom of
Supernatant is removed with Pasteur pipett.
The pellet at the base is suspended in 10 ml of MS medium
plus mannitol and the process is repeated thrice.
The resultant protoplast is pure.
2. Protoplast Fusion
A. Spontaneous fusion B. Induced fusion
Intraspecific Intergeneric Electrofusion
A. Spontaneous fusion
Protoplast fuse spontaneously during isolation
process mainly due to physical contact
Intraspecific protoplast fusion
Intraspecific protoplast fusion is the cross between the
This technique offers the only way of carrying out
crosses and genetic analysis.
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.
B. Induced fusion
fusion induced by chemicals
3. Ca 2+ ions
4. Polyvinyl alcohal
Physical fusion of protoplasts
under microscope by using micromanipulator and
Fusion induced by electrical stimulation
Fusion of protoplasts of pearl chain is induced by the
application of high strength electric field (100kv m-1)
for few microsec.
Fig. 1: A schematic representation of the three most
successful protoplast fusion strategies
Fig. 2: Two tobacco plant protoplast are fused to produce a
cell that acquires some of the characteristics of both parents
3. Identification and Selection
Hybrid identification- Based on difference between the
parental cells and hybrid cell with respect to
ii. Cytoplasmic markers
Fluorochromes like FITC (fluoroscein
isothiocyanate) and RITC (Rhodamine
isothiocyanate) are used for labelling of hybrid
iii. Presence of chloroplast
iv. Nuclear staining
Heterokaryon is stained by carbol-fuschin,
aceto-carmine or aceto-orcein stain
v. Several markers are used
Specific amino acid
Auxotrophic and metabolic mutants
4. Protoplast culture and regeneration
Plants are induced to regenerate from hybrid calli.
Hybrid cells are cultured on sterile and cooled down
nutrient medium in petri dishes.
The plates are incubated at 25°C in a dim white light.
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.
These hybrid plants must be at least partially fertile, in
addition to having some useful property, to be of any use in
Advantages of somatic hybridization
Production of novel interspecific and intergenic
hybrid e.g. 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
Production of heterozygous lines in the single species
Studies on the fate of plasma genes
Production of unique hybrids of nucleus and
Limitations 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
No confirmation of expression of
particular trait in somatic hybrids
Application of Somatic hybridization
Protoplast fusion to create somatic hybrids
"wide crosses" where embryo culture won't work
i. Citopsis gilletiana (wild) x Citrus sinensis
ii. citrus sexually incompatible spp.
iii. wild relative has disease/nematode resistance
iv. somatic hybrid used as a rootstock
Solanum somatic hybrids
i. 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