PROTOPLAST FUSIONPresented by- Neetu NandPh.D 1st Semester

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

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.)

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.

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

Method of isolation of protoplast

PURIFICATION OF PROTOPLASTo 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.

Purification, culture and regeneration of protoplasts

Fig: Purification, culture and regeneration of protoplasts

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.

A. MECHANICAL FUSION-

In this the isolated protoplast are brought into intimate physical contact mechanically. Under microscope and using micromanipulator or perfusion micropipette.

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

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.

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 .

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.

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

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.

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.

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.

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.

Thank you