Principles of Callus Culture
For successful initiation of callus, three important criteria should be accomplished.
i) Aseptic preparation of plant material.
ii) Selection of suitable nutrient medium supplemented with appropriate ration of auxin and cytokinins or only appropriate auxin, and
iii) Incubation of culture under controlled physical condition.
Different plant parts carry a number of surface borne micro-organism like bacteria, fungus, etc. The excised plant parts called explants are at first washed with liquid detergent. Then explants are surface sterilized by the most commonly used chemical such as 0.1% w/v mercuric chloride (Hgcl2) or sodium hypochloride (0.8% to 1.6% available chloride) for a limited time ( generally 10-15 minutes). After sterilization, the explants are repeatedly rinsed with autoclaved distilled water.
The surface sterilized plant material is cut aseptically into small segments (a few millimetres in size) and are transferred aseptically on a suitable nutrient medium solidified with agar.
Agar solidified or semi-solid nutrient medium after its preparation and sterilization by autoclave at 15 lbs, pressure for 15 minutes is used for induction of callus tissue. For most successful callus culture and for healthy callus growth usually both an auxin and cytokinins are required.
The suitable temperature for in vitro callus initiation and growth is usually 25+- 2 0C. In some plant materials initiation and growth of callus take place in totally dark condition. However, in some cases a particular photoperiod (16 hrs light and 8hrs dark) is necessary for initiation and growth of callus tissues. Approximately, 2000 to 3000 lux artificial light intensity is needed. Generally, 55 to 60 % relative humidity is maintained in culture room.
Once the growth of the callus tissue is well established, portions of the callus tissue can be removed and transferred directly on to fresh nutrient medium to continue growth. In this manner, callus cultures can be continuously maintained by serial subcultures.