Usually, 50-­100 m mol 1-1 CaCl₂ is added to the osmoticum as it improves plasma membrane stability. The cells and tissues are incubated in the enzyme mixture for few to several (generally, 16-18) hours; naked protoplasts devoid of cell wall are gradually released in the enzyme mixture.

Protoplasts have been isolated from virtually all plant parts, but leaf mesophyll is the most preferred tissue, for this purpose. In general, fully expanded leaves are surface sterilized, their lower epidermis is peeled off with a pair of forceps and the peeled areas are cut into small (Ca. I cm2) pieces with a scalpel and suspended in the enzyme mixture. When epidermis can not be peeled, leaf may be cut into Ca. 1 mm2 pieces and treated with the enzyme mixture; vacuum infiltration may be used to facilitate the entry of enzymes into the tissues.

Generally, MS and B5 media, and their modifications are used for protoplast culture. The media are supplemented with a suitable osmoticum and, almost always, with an auxin and a cytokinin, their types and concentrations depending mainly on the plant species. After 7-10 days of culture, protoplasts regenerate cell wall and the osmolarity of medium is gradually reduced to that of normal medium.

The macroscopic colonies are transferred onto normal tissue culture media. Protoplasts are very sensitive to light; therefore, they are cultured in diffuse light or dark for the first 4-7 days.