Plant material, culture media and culture conditions
Flowers of hybrid orchid Dendrobium Madame Thong-In
were self-pollinated and the seedpods (3-month-old) obtained
were surfaced sterilized by cleaning with 100%
Cloroxr (active ingredient 5.25% sodium hypochlorite),
and then dipped into 95% (v/v) ethanol and burnt off. The
seedpods were then dissected lengthwise and seeds were
cultured in 9 cm petri dishes with 25 ml of modified KC
medium (Knudson 1946) supplemented with 2% (w/v) sucrose,
15% (v/v) coconut water (CW, obtained from local
green coconuts), with or without 0.03% (w/v) activated charcoal
(AC, Sigma) and 0.3% (w/v) Gelrite (Kelco, San Diego,
USA). Media were adjusted to pH 5.3 before autoclaving
at 1 kg cm−2 (121◦C) for 20 min. KC medium was used
throughout the experiments as the basal medium.
Two-month-old green protocorms (2–3 mm height with
two to three leaflets) were transferred to 40 ml basal liquid
medium supplemented with 4.4 μM BA (K1) in 100 ml Erlenmeyer
flasks on rotary shakers at 120 rpm for proliferation
and multiplication. All cultures were incubated at 26±2◦C
under a 16-h photoperiod of 35 μmol m−2 s−1 from daylight
fluorescent lamps.