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One of the most significant developments in the field of plant tissue culture, Witnessed during the last few decades,is the isolation, culture and fusion of protoplasts.

Since in plant cells, the plasma membrane is bound by a rigid cellulose wall, it has been relatively difficult to handle plant cells. Only in 1960, it was demonstrated by E.C. Cocking at the University of, Nottingham (U.K.), that nakedcells called protoplasts can be obtained through enzymatic degradation of cell walls. Cultured protoplasts can beused not only for somatic cell fusions, but also for taking up foreign DNA, cell organelles, bacteria and virus

particles. Protoplasts can be isolated by mechanical or enzymatic methods. In mechanical method, cells arekept in a suitable plasmolyticum (in plasmolysed cells, protoplasts shrink away from cell wall)and cut with a fine knife, so that protoplasts are released from cells cut through the cell wall,when the tissue is again deplasmolysed. This method is suitable for isolation of protoplasts fromvacuolated cells (e.g. onion bulbs, scales, radish roots). The enzymatic method is almostinvariably used now for the isolation of protoplasts, since it gives large quantities of protoplasts,where cells are not broken and osmotic shrinkage is minimum. However, sometimes mechanicaland enzymatic methods are combined, where cells are first separated mechanically and later usedfor isolation of protoplasts through enzymatic treatment.

The protoplasts can be isolated from a variety of tissues including leaves, roots, in vitro shoot cultures,callus, cell suspension and pollen. However, the most commonly used organs are leaves which can be

employed for isolation of protoplasts. The many advantages of the enzymatic method of isolationenables large scale reproducible isolation of protoplasts with minimum osmotic shrinkage andminimization of deleterious effects of plasmolysis.

Materials Required for Protoplast Isolationy 7 0% Ethanoly 0.1% Mercuric Chloridey 0.5M Mannitol

y Enzyme solution :y 1% Cellulase 0.25% Macerozyme R-10y 27 .2 mg/l KH2PO4y 101 mg/l KNO3y 1480 mg/l CaCl2.2H2Oy 246 mg/l MgSO4. 7 H2Oy 0.5M Mannitol (pH 5.6)

y Cell Protoplast Washing Medium :y 0.5M Mannitol (pH 5.6)y 27 .2 mg/l KH2PO4y 101 mg/l KNO3

8/6/2019 One of the Most Significant Developments in the Field of Plant Tissue Culture(01)

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y 1480 mg/l CaCl2.2H2Oy 246 mg/l MgSO4. 7 H2O

Protocol for the isolation of Protoplasts

y Take fresh leaves from 4-5 week old aseptically growing plants.y Surface sterilize in 7 0% Ethanol followed by treating with 0.1% Mercuric chloride for 1

minute.y Wash the leaves thoroughly with sterile distilled water.y Peel the lower epidermis of the leaf tissue and place it in 0.5M Mannitol solution for one

hour in a petriplate.y Remove the Mannitol solution and replace it with filter sterilized enzyme solution.y Incubate the plates in dark for 16-20 hours at 25oC.y Separate the protoplast layer by filtration using 0.45M nylon membrane.y Centrifuge the filtrate at 1000g for 10 minutes at 4 oC.y Remove the supernatant and resuspend the pellet in cell protoplast washing medium.y Centrifuge at 1000g for 10 minutes.y Repeat step 9 twice.y F inally suspend the protoplast pellet at a density of 1*105 ml/l in the modified protoplast

culture medium.y Plate the protoplasts as fine thin layer in petriplates.y Incubate the plates at 25 oC in dark.