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1. Plant cells treated with cellulase & pectinase to remove the cell wall which is composed of cellulose, pectin and small amount of hemicellulose. These enzymes only break down the cell wall leaving the plasma membrane intact
2. The cells are then incubated with a mineral salt solution containing mannitol for several hours. This sugar exerts osmotic pressure causing PLASMOLYSIS leading to easier digestion 3. In order for the protoplasts to grow they must be put in a suitable medium to encourage cell wall growth