2.3. Callus induction
The shoot tip (3–8 mm) and basal end of immature leaf (5–8 mm) explants from three to four-month-old plantlets were cultured on sterile MS medium (0.8% Gelrite and 30 g l−1 sucrose) containing 6-benzyladenine (BA; 0.2–3.0 mg l−1) and 2,4-dichlorophenoxyacetic acid (2,4-D; 0.1–2.0 mg l−1) or
alpha-naphthalene acetic acid (NAA; 0.1–1 mg l−1) for callus induction.
All cultures were maintained in glass jars (60 mm × 90 mm) containing30 ml medium.
Four explants were cultured per jar,
and each treatment contained twenty explants.
Every experiment was repeated three times.
During the first few weeks,
the cultures were incubated in the dark until callus was induced.
The calli induced from the explants were then sub-cultured onto the same medium every 4 weeks.
Each callus was divided into 4–6 mm diameter pieces during the transfer and subcultured up to two to three times.