Protoplast isolation

1. PROTOPAST ISOATION,
PROTOPAST FUSION AND
SOMATIC HYBRIDISATION

2. Protoplast
The entire pant cell without its cellulosic cell wall and they are nacked plant cell including all the
components of a cell excluding the cell wall are called protoplast.
Protoplasts are function individual cell with plasma membrane as outer layer.
Hanstein (1880) proposed the term protoplast.
The isolation of protoplast was first reported by klercker (1892).

3. What is protoplast culture
In vitro culture of protoplast is known as protoplast culture.
SOURCES OF PROTOPLAST
1. Mesophyll cells of leaves
2. Culture suspension cells
3. Callus culture.
4. Preconditioned plant material

4. Isolation of protoplast
Procedure for protoplast isolation-
Young cell suspension are particularly ideal for isolation of
protoplasts in large quantities.
However, the most commonly used organs are leaves which can be employed for
isolation of protoplasts using the following steps:
1. Sterilization of leaves ,
2. Peeling off the epidermis,
3. Enzymatic treatment and,
4. Preconditioned plant material

5. Fully expanded leaves are obtained from about 10 weeks old plants (in tobacco) and are sterilized
by dipping them into 70% ethyl alcohol for about a minute and then treating them with 2 % solution
of sodium hyperchlorite fur 20-30 minutes.
The leaves are then rinsed three times with sterile distilled water and subsequent operation are
carried out under aseptic condition (under “laminar air flow chamber’).
The lower epidermis of the sterilized leaves is carefully peeled off and the stripped leaves are cut
into small pieces.
In cereals, where it is difficult to peel off the epidermis, leaves are cut in long strips and used with
enzyme mixture.
Digestion is usually carried out after incubation in osmoticum (a solution of higher concentration
than the cell contents which causes the cells to plasmolyse).

6. This makes the cell walls easier to digest. Debrise is filtered and/or centrifused out of the
suspension and the protoplasts are than centrifused to form a pallet.
On resuspension the protoplast can be cultured on media which induce cell division and
differentiation.
A large number number of plants can be regenerated from a single experiment – (a gram
of potato leaf tissue can produce more than a million protoplasts)
1. Mechanical method (non enzymatic)
It is done by cutting plasmolysed cells with a sharp edged micro scalpel or knife after
keeping the material under microscope.
The protoplasts are released and the cells are deplamolysed. This method is useful for
isolation of protoplasts from vacuolated cells.
Example- Onion bulbs, scales, radish roots.
This method gives a poor yield of protoplasts and is not suitable for isolating protoplasts
from meristematic and less vacuolated cells.

7. Enzyamtic method ( cocking, 1960)
Enzymatic
Sequential method Mixed method
(Two step method) (Single step method)
Commercially available enzymes used for protoplast culture –
The role of enymes is to dissolve middle lamella and dissolving cell wall.
A combination of these enzyme in a concentration of 0.5 to 20 % is used.
In many cases the macroenzyme and cellulase are sufficient to obtain protoplast is significant number.

8. The enzyme solution is prepared in 10-15% sorbitol or mannitol containing small amount of CaCl2
(7mM) for membrane stability.
The enzyme method is done in two ways i-e., one is by sequential method and another one is by
mixed enzymatic method.
Enyme Source
Cellulases
Cellulases Onozuka R-10 Trichoderma viride
Cellulases Onozuka RS T. viride
Cellulases YC T. viride
Cellulases YC T. viride
Cellulases CEL T. viride
Cellulysin T. viride

9. Driselase Trpex lacteus
Meicelase T. viride
Hemicellulases
Helicase Helixpomatia
Henicellulases Aspergillus niger
Henicelellulases H-
2125
Rhizopus sp.
Rhozyme HP 150 Aspergillus niger
Pectinases
Macerase Rhizopus arrhizus
Macerozyme R-10 R. arrhizus
PATE Bacillus polymyra
Pectind Aspergillus sp.
Pectolyase Aspergillus japonicus
Zymolase Arthrobacter luteus

10. A. Sequential Method
This involves initial incubation of macerated plant tissue with pectinase
macroenzyme .
Which inturn are then converted into protoplasts by cellulase treatement.

11. B. Mixed Enzymatic
Plant tissue are plasmolysed in the presence of a mixture of pectinase and
cellulase thus inducing simultaneous separation of cells and degradation of cell
wall to release protoplast directly.
After enzyme treatment protoplast suspensions are collected by centrifugation
(60-100rpm) for 2-5 minutes.
Then washed in medium without enzyme.
Cell debrise are removed and the protoplasts are placed in a medium with
appropriate concentration of sucrose or mannitol.