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Protoplast
and
its isolation
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
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.
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.
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
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
1. Mechanical method
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.
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.
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
Protoplast culture
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.
Protoplast culture
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.
• 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.
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
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.
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.
Application
THANK YOU

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Protoplast culture

  • 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
  • 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.