2. INTRODUCTION
•A protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially
removed using either mechanical or enzymatic means.
Protoplasts: Have their cell wall entirely removed
Spheroplasts: Have their cell wall only partially removed
• More generally protoplast refers to that unit of biology which is composed of a cell's nucleus
and the surrounding protoplasmic materials.
Protoplast are naked spherical cells obtained from plants by removing of cell wall and it is
cultivated in liquid as well as on solid media.
Protoplast are the cells of which cell walls are removed and cytoplasmic membrane is the
outermost layer in such cells
3. HISTORY
Term protoplast introduced in 1880 by Hanstein
First isolation of protoplast was achieved by Klercker in 1892 by
using mechanical method
Real beginning in protoplast research was made by Cocking in 1960
who used enzymatic method for cell wall removal.
Rakabe and his associates (1971) were successful to achieve the
regeneration of whole tobacco plant from protoplasts.
(Source- Satyanarayana, U. 2005. Biotechnology, 1st edn. Uppala
Publisher, Vijayawada (A.P.)
4. ISOLATION OF PROTOPLAST
Protoplast can be isolated from almost all plant parts i.e. roots, leaves, fruits, tubers, root
nodules, endosperm, pollen mother cell etc.
Protoplasts are isolated from cells by two methods-
MECHANICAL METHOD
ENZYMATIC METHOD
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.
5. MECHANICAL METHOD
Small piece of epidermis from a plant is selected
The cells are subjected to plasmolysis this results in shrinking of protoplast away
from cell walls
The tissue is dissected to release the protoplast
DISADVANTAGE-
It yields a very small number of protoplasts
It is not suitable for isolating protoplasts from meristmatic & less vacuolated cells
This method is laborious and tedious
7. PURIFICATION OF PROTOPLAST
o Two commonly used methods:-
1. Sedimentation & washing:- In this method, the crude protoplasts suspension is
centrifuged at low speed (50-100g for 5 min). The intact protoplasts form a pellet
and supernatant containing cell debris can be pipetted off. The pellet is gently
resuspended in fresh culture media plus mannitol and rewashed. This process is
repeated two or three times to get relatively clean protoplast preparation.
2. Flotation:- A concentrated solution of mannitol, Sorbitol and sucrose (0.3-0.6M)
can be used as a gradient and crude protoplasts suspension may be centrifuged in
this gradient at an appropriate speed. Protoplasts being lighter (low density) then
other cell debris allow the protoplasts to float and the cell debris to sediment.
Protoplasts can be pipetted off from the top of the tube after centrifugation.
8. Purification, culture and regeneration of protoplasts
Fig: Purification, culture and regeneration of protoplasts
9. METHODS OF PROTOPLAST FUSION
Protoplast fusion can be broadly classified into two categories-
1. Spontaneous fusion (fuse through their plasmodesmata)
2. Induced fusion (needs a fusion inducing chemicals):-
a) Mechanical fusion
b) Chemo fusion
c) Electro fusion
1. Spontaneous fusion- During enzymatic degradation of cell walls some of
the adjoining protoplasts may fuse to form homokaryocytes
(homokaryons). These fused cells sometimes contains high number of
nuclei (2-40) because of expansion & subsequent coalescence of
plasmodermal connections between cells.
10. A. MECHANICAL FUSION-
In this the isolated protoplast are
brought into intimate physical contact
mechanically. Under microscope and
using micromanipulator or perfusion
micropipette.
11. B. CHEMOFUSION
Several chemicals has been used to induced protoplast fusion such as NaNo3,
polyethylene glycol and Calcium ions-
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. - The method consists of
incubating protoplasts in a solution of 0.4 M mannitol containing 0.05 M CaCl2 at
pH 10.5 (glycine-NaOH buffer) at 370C for 30-40 mins. Fusion occurs within 10
mins
12. Polyethylene glycol (PEG) treatment- Isolated protoplast 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.
C. Electro fusion- In this method an electric field of low strength (10Kv/m)
gives rise to dielectrophoretic dipole generation within the protoplast suspension or
a high strength of electric field (100Kv/m) for some micro seconds are applied this
lead to fusion.
13.
14. FUSION PRODUCT
FUSION PRODUCTS - THE HYBRIDS AND CYBRIDS
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.
The binucleate cells are known as heterokaryon or heterocyte .
When nuclei are fused the cells are known as hybrid or synkaryocyte .
Only cytoplasms fuse and genetic information from one of the two nuclei is lost
is known as cybrid i.e. cytoplasmic hybrid or heteroplast .
15.
16. Procedure for successful somatic hybridization is as below:
(i) Isolation of protoplasts from suitable plants.
(ii) Mixing of protoplasts in centrifuge tube containing fugigenic chemicals i.e.
chemicals promoting protoplast fusion, such as polyethylene glycol (PEG)
(20%, W/V), sodium nitrate (NaNO3), maintenance of high pH 10.5 and
temperature 37°C (as a result of fusion of protoplasts viable heterokaryons are
produced.
(iii) Wall regeneration by heterokaryotic cells.
(iv) Fusion of nuclei of heterokaryon to produce hybrid cells.
(v) Plating and production of colonies of hybrid cells.
(vi) Selection of hybrid, subculture and induction of organogenesis in the
hybrid colonies.
(vii) Transfer of mature plants from the regenerated callus.
17. Fig. Fusion of protoplasts of potato and tomato, and production of hybrid plant
(pomato).
18. APPLICATION OF SOMATIC
HYBRIDIZATION AND CYBRIDIZATION
1. Somatic cell fusion appears to be the only means through which two different
parental genomes can be recombined among plants that cannot reproduce sexually
(asexual or sterile).
2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused
to produce fertile diploids and polyploids.
3. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases
somatic hybrids between two incompatible plants have also found application in
industry or agriculture.
4. Somatic cell fusion is useful in the study of cytoplasmic genes and their activities
and this information can be applied in plant-breeding experiments.
19. CONTD……
5. Many disease resistance genes (eg- TMV, potato virus X, club rot disease) could be
successfully transferred from one species to other.
6. Genes responsible for tolerance to cold, frost and salt could be successfully
introduced through somatic hybridization. Eg, Introduction of cold tolerance gene in
tomato.
7. Somatic hybrids with high nicotine content and low erucic acid have been
developed.
8. Cybridization has made it possible to transfer cytoplasmic male sterility.
20. LIMITATIONS
It does not always produce plants that give fertile and visible seeds
Regenerated plants obtained from somatic hybridization are often variable due to
somaclonal variations, chromosomal eliminations etc.
Protoplast culture is associated with genetic instability
Production of viable somatic hybrids is not possible in all instances
There is no certainity as regards the expression of any specific character
There are limitations in the selection methods of hybrids, as many of them are not
efficient.
Somatic hybridization between two diploids results in formation of an amphidiploid
which is not favourable that’s why haploid protoplasts are recommended.
21. REFRENCES
Chawala, H. S. 2002. Introduction to Plant Biotehnology, 2nd
edn. Oxford & IBH Publising C./ Pvt. Ltd. New Delhi India.
Gupta, P.K. 2013. Elements of Biotechnology, 2nd edn. Rastogi
Publications, Meerut, New Delhi India.
Satyanarayana, U. 2005. Biotechnology, 1st edn. Uppala
Publisher, Vijayawada (A.P.) India.
