• After a period of time it becomes necessary to subculture callus into a fresh medium, due to depletion of essential nutrients and drying out of the gel.
• Metabolites secreted by the callus may also accumulate to toxic levels in the medium.
• The subcultured callus must be of a sufficient size to ensure renewed growth on the fresh medium.
• Subcultures are usually performed every 3 to 6 weeks.

Typical callus growth curve. X indicates time for subculture