This document summarizes the process of isolating plant protoplast cells. It defines a protoplast as a plant cell without its cell wall. There are two main methods for isolating protoplasts - mechanical and enzymatic. The enzymatic method uses enzymes like pectinase and cellulase to gently dissolve the cell wall and is preferred. The steps of the enzymatic isolation process are described, including incubating tissue in the enzyme solution, filtering, washing, and testing viability. Finally, some applications of isolated protoplasts in research are mentioned like transformation and somatic hybridization.

1. Protoplast
and
its isolation

2. Introduction
• Is a naked cell surrounded by a plasma
membrane. It can regenerate cell wall, grow and
divide.
• Is fragile but can be cultured and grow into a
whole plant.
• Is used by biotechnologists, microbiologists,
pathologists.
• In 1960, E.C Cocking contributed to the
enzymatic isolation and culture of protoplast

3. A typical Plant cell wall
 The Plant cell wall is
multilayered structure
1. The middle lamella- Pectin
compounds and proteins.
2. The primary wall- Cellulose
and hemicelluloses.
3. The secondary wall-
Cellulose, hemicelluloses and
lignin.

4. Definition
Protoplast:
• Unit of biology which is composed of a cell's
nucleus and the surrounding protoplasmic
materials. Hanstein (1880).
OR
• It is a plant cell that has its cell wall completely
removed using either mechanical or enzymatic
means.

5. Why do we need to remove cell wall?
• Large molecules can not pass through
the cell wall.
• Somatic hybridization and genetic
Manipulation can not be done.
So we have to remove the cell wall. After
removing it, we will get protoplasts

6. ISOLATION OF PROTOPLAST
• Important to isolate as gently and as quickly
as possible viable and uninjured protoplast.
• There are two methods for the isolation of
protoplast from plant tissues.
1. Mechanical method
2. Enzymatic method

7. 1. Mechanical method

8. Mechanical method
• In this method, the cells are kept in suitable
plasmolyticum (for example CPW salts
containing 13% w/v mannitol or immersed in 1.0
M sucrose).
• Once the plasmolysis is complete, while
remaining in the osmoticum, the leaf lamina
would be cut with a sharp-edged knife
• Now, cells are cut only through the cell wall,
releasing intact protoplasts.
• The released protoplasts then have to be
separated from damaged ones and cell debris.

9. Drawbacks
• Used for vacuolated cells like onion
bulb scale, radish and beet root tissues
only.
• Tissue damage is caused by mechanical
method
• maximum osmotic shrinkage
• Low yield.
• Laborious and tedious process.
• Low viability.

10. 2. Enzymatic method: One step method
(direct method)
• Refers to the use of enzymes to dissolve the cell
wall for releasing protoplasts.
• Cocking 1960.
Advantages:
Used for variety of tissues and organs such as
fruits, roots, petioles, leaves, shoots, etc.
Osmotic shrinkage is minimum
Cells remain intact and not injured
Protoplast readily obtained

12. Protocol for direct method
• Incubation of leaf segments over night in
enzyme solution (pectinase and cellulase)
• Treated with enzymes to liberate protoplasts,
Mixture is filtered and Centrifugation at
600rpm for 5mins.
• Protoplast forms pellet
• These are washed with sorbitol 3X,
recentrifuge
• Cleaned protoplast float
• Pipettes out.

14. Protocal: Sequential method
(indirect method)
• Two enzyme mixtures (mixture A and 'mixture
B) are used one after the other.
• Leaf segments with mixture A (macerozyme in
manifold at pH 5.8) are vacuumed infiltrated
for 5 min. (WASHING)
• After 15 min. the enzyme mixture is replaced
by fresh 'enzyme mixture A' and leaf segments
are incubated for another hour.

15. • The mixture is filtered using nylon mesh,
centrifuged at 600rpm for 1 min.
• Washed three times with 13% mannitol to
get a pure sample of isolated cells.
• Cells are then, incubated with 'enzyme
mixture B' (cellulase in a solution of mannitol
at pH 5.4) for above 90 min, at 30° C.
• After incubation, the mixture is centrifuged
for 1 min, so that protoplasts form a pellet,
which are cleaned three times as in 'one step
method.

16. Purification of protoplasts
Protoplasts are purified by removing:
 Undigested material (debris)
 Bursts protoplasts(damaged protoplasts)
 Enzymes(pectinase+cellulase)
● Debris are removed by filtering the preparation through a
nylon mesh of 100µ pore size.
● Enzymes are removed by centrifugation at 600rpm for 5
mins whereby the protoplasts settle to the bottom of the
tube and the supernatant removed with the help of a
pipette
● Intact protoplasts are separated from broken protoplasts
through centrifugation and removed by a pipette as they
are collected at the top of tube

17. Protoplast viability and density
To check the viability of isolated protoplasts,
1. Fluorescein diacetate test: It accumulates only inside
the plasma lemma of viable protoplasts, can be
detected with fluorescence/UV microscopy.
2 . Evans blue test: Intact viable protoplasts, exclude
the Evans blue stain. Impermeability of the cell to
Evans blue indicates a living cell.
3. Cyclosis test: Protoplasmic streaming can be a
measure of viability.
4. Density test: The density of the protoplasts in the
suspension (number/unit volume) is determined by
counting with a modified haemocytometer such as
Neubauer with a field depth of 0.2mm.

18. Application of protoplast
Protoplasts can be used to study;
• Metabolic studies including photosynthesis.
• For DNA transformation.
• For somatic hybridization.
• Ingesting "foreign" material into the
cytoplasm.
• Single cell systems.
• Cell Wall synthesis.

19. Application

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