Direct One Step Method - In one step method, the leaf segments are incubated overnight (15-18h) with enzyme mixture at 25°C and teased gently to liberate the protoplasts. The mixture is filtered through fine wire gauze to remove leaf debris, transferred to 13 X 1000 mm screw capped tubes and centrifuged at 100g for 1 min. The protoplasts form a pellet and supernatant removed.

The process is repeated three times and protoplasts washed with 13% sorbitol solution, which is later replaced by 20% sucrose solution and centrifuged at a speed of 200g for 1 min. The cleaned protoplasts, which will now float (debris settles do), can be pipetted out and bulked.