Generally, isolation of protoplast is categorized into mechanical and enzymatic. Also learn about the techniques involved in the isolation of leaf mesophyll protoplast.
Mechanical Method:
This method is crude and oldest method for protoplast isolation. It is only an historical perspective. It involves chopping and plasmolysis of tissues. Time consuming and low yield of protoplast are the main constraints in this method.
Enzymatic Method:
The unprecedented success in protoplast culture was accomplished by employing several enzymes released commercially. The enzymes for protoplast isolation are available in different commercial names. These enzymes are pectinase, cellulase, macerozyme and hemi-cellulase.
Pectinase dissolves middle lamella embedded with pectin component. The cellulase and hemi-cellulase degrade the cellulose component of the cell wall, and consequently releases protoplast. The most widely used commercial enzymes are onozuka R-10, and macerozyme R-10 pectolyase Y-23.
Isolation Techniques of Leaf Mesophyll Protoplast:
The techniques involved in the isolation of leaf mesophyll protoplast are as follows:
(a) Fully expanded leaves obtained from 3 to 10 week old plants are subjected to sterilization by dipping in 70% alcohol for 30-60 s followed by treating them for 20-30 min in 2% sodium hypochlorite or calcium hypochlorite. The leaves are then rinsed in sterile water in the final step to remove all traces of sterilants.
(b) The lower epidermis of leaves are carefully peeled off and then cut into small pieces.
(c) The lower epidermis of the peeled segments is placed in a filter sterilized enzyme mixture containing 0.5% macerozyme, 2% cellulase in 13% sorbitol or mannitol. The pH is set to 5.4. The enzyme mixture is incubated for 15-18 h at 26°C.
(d) The enzyme mixture is then filtered through nylon mesh (50-100 µ) to remove leaf debris and transferred to screw capped tubes and centrifuged at (100 x g) for 1 min and the process is repeated three times.
(e) Protoplast is placed in 20% sucrose for final cleaning before washed with sorbitol solution (0.1M) at (200 × g) for 1 min. The floating protoplasts are then harvested by gently pipetting out with Pasteur pipette.
Alternatively, a two-step method is also followed for the isolation of protoplast as follows:
(a) The peeled segments are placed in enzyme mixture containing macerozyme (0.5%), potassium dextran sulfate (0.3%) in 13% mannitol. After adjusting pH to 5.8, incubation is carried out in water bath at 25°C. Enzyme mixture is then replaced with fresh enzyme after 15 min of incubation. These are then filtered through a nylon mesh, centrifuged at 100 × g for 1 min. Pelleted cells are then washed three times with mannitol (13%) to procure isolated cells.
(b) The cells obtained are mixed with second set of enzyme mixture containing 2% cellulase in 13% mannitol at pH 5.4. The incubation is carried out for 90 min at 30°C. Centrifuge the suspension at 100 g for 1 min. Pelleted protoplasts are subjected to final cleaning by placing them in 2% sucrose and harvested gently by Pasture pipette.
Protoplast can be isolated from cells and cell suspension, pollen grains and pollen mother cells. The filtered cell suspension is directly treated with 2-4% onozuka cellulase in 0.7M mannitol for 5-6 h at 30-32°C in water bath. Isolation of protoplast from mature pollen grain is comparatively a difficult task due to the nature of pollen wall, which comprises mainly a durable sporopollenin.
This polymer can be dissolved by treatment with KOH and certain organic bases. The pollen grain is squeezed out from the anther and subjected to enzyme treatment. Additional enzymes are required for protoplast isolation. The enzyme mixture contains cellulase 2%, macerozyme 1%, and helicase 0.5%, to release protoplast.