Half gram fresh mass of calli without organogenesis was isolated
and cultivated in MS medium supplemented with 0.1 mg/l Pi
in combination with three concentrations of BAP (2, 3 and 4 mg/l)
or each phytohormone alone (0.1 mg/l of Pi or 4 mg/l of BAP). These
calli were subcultured every 4 weeks. Adventitious shoots regenerated
from these callus cultures were excised and cultivated in
Magenta vessels (Sigma–Aldrich) with 150 ml MS medium supplemented
with auxin indole-3-butyric acid (IBA) (2.5 M) and 5%
(w/v) sucrose. The plantlets were routinely subcultured every 4
weeks, and the cultures were kept in a growth chamber at 25 ◦C
under a 16/8 h light/dark photoperiod using cool-white fluorescent
light (mol m−2 s−1).