A. Formation of cell wall :
The process of cell wall formation may be completed in two to several days although protoplasts in culture generally start regenerating a cell wall within a few hours after isolation.
Protoplasts lose their characteristic spherical shape once the wall formation is complete.
The freshly formed cell wall is composed of loosely arranged microfibrils, the process requiring 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.
There is a direct relationship between wall formation and cell divisions. Protoplasts with a poorly developed wall often show budding and those which are not able to generated a proper wall fail to undergo normal mitosis.
B. Development of callus/Whole Plant:
After the formation of a wall around the protoplasts, the reconstituted cells show considerable increase in size and first divisions generally occur within a week.
Subsequent divisions give rise to small cell colonies. After 2-3 weeks, macroscopic colonies are formed which can be transferred to an osmotic free medium to develop a callus.
The callus may be induced to undergo organogenic differentiation or whole plant regeneration.
C. Regeneration via Embryogenesis
Plant regeneration of protoplasts via embryogenesis mostly occurs in species belonging to Gramineae, Umbelliferae, Rutaceae, and leguminosae among angiosperms and Pinaceae of gymnosperms.
Plant species in which complete plants have been regenerated from protoplasts are as follows:
Apium graveolens (Celery)
Daucus carota (carrot)
Helianthus annus (Sunflower)
Cichorium endivae (endive)
Brassica rapa (turnip)
B. napus (rapeseed)
B. oleracea (cabbage)
Medicago sativa (alfalfa)
Trifolium pratense (red clover)
Triticum aestivum (Wheat)
Zea mays (maize)
Saccharum officinarum (sugarcane)
Pennisetum americanum (Pearl millet)
Lycopersicon esculentum (Tromato)
Nicotiana tabaccum (Tobacco)
Solanum melongena (Brinjal)
S tuberosum (Potato)