Shoots (1.570.3 cm long) obtained from micropropagated
plants of D. Second Love (D. Peace x
D. Awayuki), which were grown on Vacin and Went
(VW) (1949) basal medium modified by substituting
Fe2(C4H4O6)3 for FeEDTA and supplemented with
the Murashige and Skoog (1962) micronutrients,
0.22 mM TDZ, 2% sucrose, 0.4 mg/L thiamine,
100 mg/L myo-inositol and 0.2% Phytagel (maintenance
medium), were used as explants in this
experiment. The pH of the medium was adjusted to
5.8570.01 before autoclaving it at 120 1C for
15 min.
At first, the explants were kept in TDZ-free
maintenance medium (flowering non-inductive
medium) for 4 weeks and then transferred to VW
basal medium supplemented with 1.8 mM TDZ, 2%
sucrose, 0.4 mg/L thiamine, 100 mg/L myo-inositol
and 0.2% Phytagel (flowering inductive medium).
The control treatment consisted of transferring the
explants to fresh flowering non-inductive medium.
Explants were grown in 125 mL Erlenmeyer flasks
closed with perforated rubber stoppers. The perforations
were filled with cotton on which two
drops of a saturated potassium permanganate
solution were placed after explant inoculation.
Cultures were kept in a growth room at 2671 1C
with a 16-h photoperiod provided by cool-white
fluorescent lamps at 35–45 mmolm2 s1.