The newly synthesized cell wall can be observed under a fluorescence microscope using calcofluor white. The freshly formed cell wall is composed of loosely arranged microfibrils; this requires an exogenous supply of a readily metabolized carbon source (sucrose) in the nutrient medium. Ionic osmotic stabilizers in the medium are reported to suppress the development of a proper wall.

Soon after the formation of a wall around the protoplast the reconstituted cells show considerable increase in size and first divisions generally occur between 2 to 7 days. Subsequent divisions give rise to small cell colonies. A direct relationship exists between wall formation and cell divisions.

Successive cell divisions resulting in the formation of a protoplast derived callus depend on the kind, concentration, and ratio of growth regulators such as auxins and cytokinins used in the medium and its composition, genotype of the donor tissue, and environmental culture conditions.
In contrast to regeneration of protoplasts into a callus mass, more often multinucleate protoplasts are observed in a protoplast culture. They form as a result of incomplete cytokinesis during first division which, results in spontaneous fusion.
Such multinucleate protoplasts in most cases will not undergo continued growth and will die; some, however, are capable of continued growth and differentiation into polyploid plants.Regeneration of protoplast derived callus into whole plants occurs either through embryogenesis or through organogenesis.