Within 2-4 days, protoplasts start developing cell walls, which can be detected by staining with 0.1% calcofluor white (CPW) fluorescent stain.While the presence of a proper wall is essential for a regular division, it is not always a prerequisite for nuclear division; not all protoplasts with cell walls formed, embark upon division.

The protoplasts, which are capable of dividing, undergo first division within 2-7 days and form multicellular colonies after 2- 3 weeks. After another two weeks, these colonies can be treated as standard tissue cultures. From these colonies or tissues in culture, plants can be regenerated, although success in this effort has been achieved only in a few species.

Different steps from isolated protoplasts to regeneration of plants are depicted. However, the variation in regenerated plants derived from protoplasts is much greater than that observed in plants regenerated from a meristem, a stem segment or a leaf disc.