Plant Protoplast: Isolation, Purification and Culturing with Viability test of protoplast cells

1. PLANT PROTOPLAST:
Isolation, Purification & Culturing
Dr. Divya Sharma
Assistant Professor
A Biodiction (A Unit of Dr. Divya Sharma)

2. Plant’s Protoplast

3. Protoplast
❑ The word “Protoplast” coined by Hanstein in 1880, for the living matter surrounded
by the cell membrane.
❑ Protoplast is a plant, bacterial or fungal cell that had its cell wall completely or
partially removed using either mechanical or enzymatic methods
Protoplast = Cell – Cell wall (without cell wall a cell)
❑ Protoplasts are plant cell with the plasma membrane but without the cell wall.
Protoplast allows the fusion of similar or different species and the fused product
can generate into the whole plant.
Protoplast = Completely removal of cell wall
Spheroplast = Partially removal of cell wall

4. ❑ First report on protoplast fusion derived somatic hybrid plants of Nicotiana glauca
(wild tobacco)
❑ Protoplast isolation and their culture provide millions of single cells for a variety
of studies
❑ Less energy required for spherical shape and are able to fuse with each other for
hybrid production
Production of Protoplast by EnzymeTreatment

5. History Isolation of Protoplast
❑ In 1880, Hanstein – Introduced Protoplast term.
❑ In 1892, Klercker – Isolate first time protoplast using plasmolysed cell and
subsequently slicing the tissue.
❑ In 1960, Cocking – Demonstrate the enzymatic method for isolation of a large
number of protoplast from cells of higher plants (using concentrated cellulose
enzyme solution)
❑ In 1968, Tkebe et al. – employed firstly the commercial preparation of the enzyme
for protoplast isolation (using macerozyme and cellulose).
❑ In 1920, Power – Performed firstly Protoplast fusion experiment
❑ In 1971, Rakabe and his team – Regeneration of whole tobacco plant from
protoplast.

6. Protoplast
❑ Protoplast are plant cell with the plasma membrane but without the cell wall.
Protoplast allow the fusion of similar or different species and the fused product can
generate into the whole plant.
❑ Protoplast can be isolated from almost all plant parts viz namely roots, leaves,
tubers, root nodules, endosperms, pollen mother cells, pollen, pollen tetras,
embryogenic or non-embryogenic suspension culture.
❑ More recently viable protoplast have been isolated from male and female gametes.

7. Use of Protoplast:
▪ Used for DNA transformation (for making) genetically modified organisms
▪ Membrane biology (including the uptake of macromolecules and virus)
▪ Plant breeding

8. Importance of Protoplast
 Regenerate into whole plant
 Hybrids can be developed from protoplast
 Easy to perform single cell cloning with protoplast
 Genetic transformation can be achieved through genetic engineering of protoplast
DNA
 Isolation of cells organelles and chromosome is easy from protoplast
 Protoplast are used for membrane studies
 Isolation of mutant from protoplast is easy

9. Source of Protoplast
❑ Leaves (most convenient)
❑ Callus culture (mostly used)
❑ Cell suspension culture (provide excellent source material)

10. Material Required for Protoplast
Isolation
❑ Plant leaves
❑ 13% Mannitol
❑ CPW salt solution (Cell protoplast washing media)
❑ Enzyme solutions: Macerase and cellulose
❑ sorbitol
❑ Petriplate/ glass tubes

11. Methods of Protoplast Isolation
Mechanical method
Enzymatic
method

12. Plant Protoplast Isolation:An overview

13. Plant tissue
Cells plasmolysis
Microscope observation of cells
Cutting cell wall with knife
Release of protoplasm
Collection of protoplasm
Mechanical Method
❑ Tissue is immersed in 1.0 M sucrose until protoplasm shrunk away from their
enclosing the cell wall (plasmolysis)
❑ Plasmolysed tissue is cut with a sharp knife at such a thickness that only cell walls
are cut plasmolysed cell

14. Limitations of Mechanical Method
❑ Used for vacuolated cells like onion bulb scale, radish and beat root tissues
❑ Low yield of protoplast
❑ Laborious and tedious process
❑ Low protoplast viability

15. Enzymatic Method
❑ Used for variety of tissues and organ including leaves, petioles, fruit, roots,
coleoptiles, hypocotyls, stem, shoot apices, embryo microspores
❑ Mesophyll tissues – most suitable source
❑ High yield of protoplast
❑ Easy to perform
❑ More protoplast viability

16. Incubation of leaf segment overnight in enzymatic solutions
[0.5% maceroenzyme + 2% cellulose in 13% sorbitol or mannitol at pH 5.4) at 25 ֯C]
Mixture is filtered and remove the leaf debris, then centrifuged at 100g for 10 min
Protoplast forms pellets and supernatant removed
Solution replaced with 20% sucrose solution and again centrifuged for 10 min
Clean protoplast float
Direct Enzymatic Method: One step
Procedure only
Procedure repeat 3 times and protoplast washed with 13% sorbital solution
Pipetted out protoplast

17. Two enzyme mixture (mixture A and mixture B) are used one after the other
Leaf segment with Enzyme mixture A (0.5% maceroenzyme + 0.3% potassium dextran sulphate in
13% mannitol at pH 5.8) are vacuumed filtered for 5 min., transferred to a water bath at 25 ֯C and
subjected to slow shaking
Enzyme mixture is then replaced by fresh enzyme mixture A and leaf segment are incubated for
another hour
Mixture is filtered using nylon mesh and centrifuged for 1 min
Sequential Enzymatic Method: Two step
procedure
Then, washed three times with 13% mannitol, to isolate pure cells
Cells are then incubated with Enzyme mixture B (2% cellulose in 13% mannitol solution at pH 5.4)
for above 90 min at 30 ֯C
The mixture is centrifuged for 1 min, so that protoplast form a pellet and clean 3 times with sorbital
Protoplast float to the surface of sucrose solution

18. Plasmolyzed cells
Release of isolated cells
Protoplast released
Plasmolyzed cells
Protoplast released
Pectinase + Cellulose Cellulose
Pectinase

19. Advantages of Enzymatic Methods
 Large scale and reproducible isolation of protoplast from various tissues
 Osmotic shrinkage is minimum and the deleterious effects of excessive
plasmolysis are minimized
 Cells remain intact and are not injured as is the case of mechanical methods of
isolation

20. Purification of Protoplast

21. Sedimentation and Washing
 Crude protoplast suspension id centrifuged at low speed (50-100g for 5 min).
 The intact protoplast 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.
 Repeat this procedure 2-3 times to get relatively clean protoplast preparation.

22. Floating
 Protoplast being lighter (low density) than other cell debris, gradients maybe
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 protoplast suspension may be centrifuged in this gradient
at an appropriate speed
 Protoplasts can be pipetted off from the top of the tube after centrifugation

23. Protoplast are separated after digestion period from enzyme and cellular debris,
transfer to the suitable method
Concentrated solution of Mannitol, sorbitol and sucrose (0.3 – 0.6 M) can be
used as a gradient
This is done by centrifugation at low speed followed by filtration through nylon
mesh (60-70 µm)
Again, followed by centrifugation or by density gradient
Centrifugation step
Pellets dissolved and washed three times and finally suspended in culture
medium

24. Culturing of Protoplast:
Culture techniquesCulturetechniques
Liquid culture
Nutrient culture

25. Culture of Protoplast
 Isolated protoplast on their hybrid cells are cultured either in a liquid or agar
medium
1. Liquid culture medium:
Includes either incubating protoplasts in a thin layer or as small drops of nutrient
medium inside a petri-dish which, in turn, is covered by another petri-dish, and
finally sealed with paraffin.The culture dish is maintained at 25-28 ֯C.
2. Nutrient medium:
About 2 mL aliquots of isolated protoplasts of suitable density (10^-3 – 10^-5
cells/mL) are mixed with an equal volume of agar medium, the temperature of
which should not exceed 45 ֯C.
On solidification of the agar, the culture plates are sealed and maintained in an
inverted position at 25-28 ֯C.

26. Purification, Culturing and Regeneration of Protoplast

27. Importance of Protoplast culture
 Study of osmotic behavior
 Study of IAA action
 Study of plasmalemma
 Study of cell wall formation
 Study of isolation
 Study of morphogenesis
 Virus uptake and replication
 Study of photosynthesis
 Gene transfer

28. Viability test
Phase Contrast Microscopy
Tetrazolium Chloride (TTC)Test
Fluorescein Diacetate (FDA)Test
Evan’s Blue

29. Viability Test
 Isolated protoplast should be healthy and viable in order to undergo proper
division and regeneration.
 Its can be performed by microscopic observation of untreated cells or after
staining the cells with suitable chemicals to indicate active metabolism in the
protoplasts

30. Phase Contrast Microscopy
 Cytoplasmic streaming movement
(cyclosis) and the presence of clear,
healthy nucleus indicate that the cells
are in viable state
 Observation of cyclosis (unstained
cells) under bright field, as an
indication of active metabolism in
Phase Contrast Microscopy
Microscopy image of Protoplast of plant cells

31. Tetrazolium Chloride (TTC) Test
 Respiratory efficiency of cells is
measured by the reduction of 2,3,5-
triphenyl tetrazolium chloride (TTC)
to the Red Dye Formazon
 Formed formazon can be extracted
and measured by
spectrophotometrically
TTCTest: Deep red stain of the endosperm
and viable embryo; Non-red stain developed
seeds, that are dead cells

32. Fluorescein Diacetate (FDA) Test
 Inside the living cell FDA is cleaved by
enzyme Esterase to release Fluorescein
(Green) in color, that is impermeable
across the plasma membrane
 0.5% FDA in acetone is prepared and
stored at 0ºC. This solution mixed
with 0.01% final concentration to
protoplasts suspension with osmotic
stabilizer
 After 5 min incubation, cells are
observed under microscope with
suitable filter FDATest under blue light: Micrograph of
protoplast and dividing cells in culture

33. Evan’s Blue Staining
 0.025% Evan’s Blue stain solution
used as a coloring agent
 The stain gives color to the dead
protoplasts by becoming permeable to
dead ones. While viable protoplasts
remains colorless due to
impermeability of plasma membrane
to the stain
Evan’s Blue test: Cell death evidenced by
Evans blue staining: Untreated viable cells;
Dead cells indicates in blue color

34. Thank you
A Biodiction (A Unit of Dr. Divya Sharma)