Objective: Initiation of callus of Azadirachta indica from leaf explant.
Development of callus is an important and sometimes a prerequisite step in various tissue culture approaches. When an explant is placed on a suitable medium, which is conducive to callus development, the explant, undergoes changes and become undifferentiated in a random manner. This undifferentiated mass of cells is known as callus. Callus consists of an amorphous mass of loosely arranged thin walled cells arising from the proliferating cells of the parent tissue. Callus has no predictable Organizational pattern although localized centers of meristematic activity are present. Starting from the callus, either whole plant can be regenerated and /or suspension culture can be developed.
1. Leaves of A. indica as explant
2. Aqueous solution of 0.5% sodium hypochlorite (NaOCl) or 0.1% mercuric chloride (HgCl2)
3. Beakers, Erlenmeyer flasks
4. Petridishes, glass slides
5. Forceps, Scalpels
6. Culture Tubes
7. Sterilised distilled water (SDW)
8. Rectified spirit, 90% (v/v) alcohol, 70% (v/v) alcohol
9. Callus induction medium
A. indica - MS + IBA (8 mg/L) + BA (4 mg/L)
Surface Sterilization: As leaf explants are collected from the plants growing under natural environment, surface sterilization of explant is necessary to prevent microbial contamination.
1. Wash the leaves with 1% Savlon approximately for 10 minutes.
2. Rinse with sterile distilled water (SDW) two times.
3. Dip the explants in 70% ethanol for 30 seconds and wash with SDW two times.
4. Soak the explants in 0.1% sodium hypochlorite or HgCl2 for 3 minutes followed by several rinses of SDW.
5. Put the leaves on sterile petridish and cut into discs using cork borer.
6. Put the discs on the culture medium.
7. Incubate the explant at 25oC, 16/8 light/dark regime.
8. Observe for growth of cells at regular time intervals.