Isolation of protoplast

1. Presented by:-
Bhoomi shah

2. Protoplast Isolation:
 The protoplast, also termed as naked plant cell refers to all the components of a plant cell
excluding the cell wall.
 The term protoplast was first introduced by Hanstein in 1880 to designate the living matter
enclosed by plant cell membrane.
 The isolated protoplast is unusual as the outer plasma membrane is totally exposed and is
the only barrier between the external environment and the interior of living cell.
 Under suitable conditions, in a number of plant species, these protoplasts have been
successfully cultured to synthesize cell walls and now genetic manipulations have also
been made.
 Protoplasts are useful for cell fusion studies. In addition, these can also take up, foreign
DNA, cell organelles, bacteria or virus particles through their naked plasma membrane.

3.  Methods of Protoplast isolation
1.mechanical methods
2.enzymatic methods.
(1) Mechanical method (Non-enzymatic method):
 Klercker (1892)was the first to isolated protoplasts from plasmolysed cell of stratiotes aloides by this method.
 Any soft parenchymatous tissue is kept in a plasmolyticum.
 The plasmolysed tissue is then finely chopped into pieces and the intact cells (plasmolysed) are released into the
medium from the cut surface.
 The suspension is then allowed for deplasmolysis and the released protoplasts attain their original size.
 The yield of protoplast in this method is very low, for large scale of protoplast yield the enzymatic method is
followed.

4. (2) Enzymatic method:
• Cocking in 1960 exhibited the possibility of enzymatic isolation of protoplasts from higher
plants.
• He used concentrated solution of cellulase to degrade the cell walls.
• Young fully expanded soft leaves, or in vitro grown callus tissue or cell suspension culture
grown cells can be used as the source material.
• The tissues or cells are incubated in plasmolyticum for 1 hr before enzymatic treatment.
• The intact tissue materials cut into smaller pieces to increase the surface area of enzymatic
activity.
• The enzymes can be used either sequentially in two step method or in a single step by mixed
enzymatic method.

5.  Two step or sequential method:
• The tissue is first treated with a macerozyme or pectinase enzyme which isolates the cells
by degrading the middle lamella.
• These free cells are then treated with cellulase which releases the protoplasts.
• In general, the cells are exposed to different enzymes for shorter periods.
 One step or simultaneous method:
• The tissue is placed to a mixture of enzymes in a one-step reaction which consists both
macerozyme and cellulase.
• One step method is commonly used because it is less laborious.
• During the enzyme treatment, the protoplasts isolated need to be stabilized because the
mechanical barrier of cell wall which served as support has been broken.
• For this reason, an osmoticum is added which prevents the protoplasts from bursting.

6.  Purification of Protoplasts:
 For purification, the protoplasts suspended in osmoticum are centrifuged using sucrose (20%)
solution.
 The viable protoplasts float on the top surface of sucrose solution forming a band.
 These protoplasts are then collected, re-suspended in osmoticum and washed several times.
 Finally the protoplasts suspended in a measured volume of protoplast culture medium after
counting the number with the help of hemocytometer.
 The viability of protoplast is checked with the help of fluorescein diacetate staining or
phenosafranine or calcofluore white.

7.  Protoplasts are purified by removing:
1. Undigested material (debris)
2. Bursts protoplasts
3. Enzymes
 Debris are removed by filtering the preparation through a nylon mesh.
 Enzymes are removed by centrifugation 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.

8.  Isolated protoplasts are cultured either in a liquid medium or semisolid agar medium in a thin
layer or as small drops of nutrient medium in petridish. The medium for protoplast culture
requires the same component as required for callus or suspension culture.
 Increasing the calcium concentration only helps to maintain the integrity of the membrane.
Generally the media require more amount of sugar. The vitamins and growth substances are
used as per requirement for cell division, callus formation and then differentiation.
 Plating density is another important measurement for protoplast culture, a density of 1 x 104 to
1 x 105 protoplast per ml is optimal, such high densities are helpful for earlier division of plant
protoplasts whereas the density is reduced subsequently during progress of culture.

9. Any questions ??
Thank you