protoplast fusion

1. Protoplast fusion
SURYA PRAKASH GAUTAM Ph.D
Asstt.Professor
HIPER, Nadaun

2. Contents:
Introduction
Isolation of Protoplast
 Fusion products - the hybrids and cybrids
 techniques of protoplast fusion
Procedure for successful somatic hybridization
 Applications

3. Protoplast Fusion
PROTOPLAST FUSION:
•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.
•The protoplast an excellent tool for the synthesis of novel combination of genes and are essential
part of the overall process required for the genetic manipulation of plants.

4. Protoplast Fusion
ISOLATION OF PROTOPLAST:
Protoplast can be isolated from almost all plant parts i.e.
roots,leaves,fruits,tubers, root nodules,endosperm,pollen mother cell, callus and
suspension culture.
Protoplasts are isolated from cells by two methods-
MECHANICAL METHOD
MECHANICAL METHOD:
ENZYMATIC METHOD
The cells are kept in a suitable plasmolyticum & cut with a fine knife, so that
protoplast are released from cells cut through the cell wall, when the tissue is again
deplasmolysed.
This method is suitable for isolation of protoplasts from higher plant tissue such as
leaf, bulb scale, fruit epidermis,raddish roots

5. Protoplast Fusion
DISADVANTAGE-
It yields a very small number of protoplasts after a rather tedious procedure.
It is not suitable for isolating protoplasts from meristmatic & less vacuolated cells.
A. Tissue is cut along the dotted line B. Release of Protoplasts from Damaged cells

6. Protoplast Fusion
Enzymes for the preparation of protoplasts
•Cell walls are made of a variety of polysaccharides. Protoplasts can be made by
degrading cell walls with a mixture of the appropriate polysaccharide-degrading
enzymes:
•During and subsequent to digestion of the cell wall, the protoplast becomes very
sensitive to osmotic stress. This means cell wall digestion and protoplast storage must
be done in an isotonic solution to prevent rupture of the plasma membrane.
Type of cell Enzyme
Plant cells Cellulase, pectinase, xylanase
Gram-positive bacteria Lysozyme (+EDTA)
Fungal cells Chitinase

7. Method of isolation of protoplast
Fig. Method of isolation of protoplast

8. A. Shoot Culture B. Remove Leaves C. Cut Leaves
G.
Protoplasts
F. Centrifuge 500g 1 min
Ready for E. Isolated
Protoplasts
D. Incubate in
Purification Enzyme
8

9. Purification, culture and regeneration of protoplasts
Fig: Purification, culture and regeneration of protoplasts

10. PURIFICATION:
Only two commonly used methods:-
1.Sedimentation & washing
2.Flotation
1. 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. Protoplasts being lighter (low density) then other cell debris, gradients may be used,
which will allow the protoplasts to float and the cell debris to sediment.
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 can be pipetted off from the top of the tube after
centrifugation.

11. FUSION PRODUCTS - THE HYBRIDS AND CYBRIDS
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 .

13. TECHNIQUES OF PROTOPLAST FUSION
1. Spontaneous fusion
2. Mechanical fusion
3. Induced fusion
1. During isolation of protoplasts for culture, when enzymatic degradation of cell
walls is affected, some of the protoplasts, lying in close proximity, may undergo
fusion to produce homokaryons or homokaryocytes, each with 2-40 nuclei.
The occurrence of multinucleate fusion bodies is more frequent, when protoplasts are
prepared from actively dividing cells.

14. Somatic hybridization is generally used for fusion of protoplasts either from two
different species (interspecific fusion) or from two diverse sources belonging to the
same species.
To achieve this objective, spontaneous fusion may be of no value, and induced fusion
requiring a suitable agent (fusogen) is necessary. In animals, inactivated Sendai virus is
needed to induce fusion.
NaNO3 treatment
This method was successfully utilized fro fusion of protoplasts from root tips of oat and
maize seedlings but is not preferred due to low frequency of fusion, particularly when
highly vacuolated mesophyll protoplasts are used

15. Treatment with calcium ions (Ca++) at high pH.
This method involves spinning (centrifugation the protoplasts in a fusion inducing
solution (0.05M CaCl2 2H2O in 0.4M mannitol at pH 10.5) 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. The details of the protocol are
described by Bhojwani and Razdan (1983). The method was found to be superior to
other methods in some cases, but high pH was toxic in other cases.

16. Polyethylene glycol (PEG) treatment
Since 1974, protoplast fusion has been successfully achieved in several crops, using
polyethylene glycol (PEG) as a fusogen. The technique gives high frequency of
fusion with reproducible results and involves low cytotoxicity.
The technique can be used for fusion of protoplasts from unrelated plant taxa (e.g.
soybean – tobacco, soybean – maize, and soybean – barley), from unrelated animal
taxa and also between those from animal and plant cells.

17. Electrical fusion
If Protoplasts are placed into a small culture vessel containing electrodes and a
potential difference is applied, then the protoplasts will line up between the
electrodes.
If now an extremely short, electric shock is applied, protoplasts can be induced to
fuse.

18. Protoplast Fusion Induced by the Application of Electric Shock

19. Fig. Fusion of protoplasts of potato and tomato, and production of hybrid plant (pomato).

20. 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. PEG induces fusion of plant
protoplasts and animal cells and produces
(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.

21. 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.