1. Selection of explant and pretreatment: Selection of explant depends on objective and experimental requirements. For callus culture, and healthy, part of the plant can be used as explant. The explant is washed under running water with the help of chemical Tween-20. This treatment cleans dust and microbes present on the surface.
2. Surface Sterilization of explant: The selected explants are surface sterilized by appropriate disinfectant chemical under laminar airflow. This results in to complete sterilization of explant. Depending upon the nature of explant various types of disinfectants such as Mercuric chloride (HgCl2), Ethanol, Silver nitrate (AgNO3), Bromine or Chlorine water can be used.
3. Initiation of Culture: The surface-sterilized explant is transferred on a suitable nutrient medium under aseptic conditions and transferred to the culture room. In a culture chamber, callus formation is initiated in due course of time. The explant and callus may be transferred to fresh medium for multiplication.