Sequential Two Step Method - In two step method, leaf segments with mixture A (0.5% macerozyme + 0.3% potassium dextran sulphate in 13% mannitol at pH 5.8) are vacuum infiltrated for 5 min., transferred to a water bath at 25°C and subjected to slow shaking.

After 15 min. the enzyme mixture is replaced by fresh 'enzyme mixture A' and leaf segments incubated for another hour.The mixture is filtered using nylon mesh, centrifuged (100g) for 1 min. and washed three times with 13% mannitol to get a pure sample of isolated cells.