This study presented the rapid multiplication system of Anoectochilus roxburghii (Wall.) Lindl. in vitro and the liquid shake culture technique for biomass production capable of being used as a source of pharmaceutical materials.
Stem segments (1.5-2 cm in length with a single node) were dipped in 70% (v/v) ethanol for 1 min and then rinsed in 0.1% (v/v) mercury chloride for 5 min, washed thoroughly in sterile distilled water under the aseptic condition in the laminar airflow hood. The aseptic culture was initiated by culturing stem nodal segments on hormone-free MS medium (Murashige & Skoog, 1962) for axillary bud formation in 60 days. For rapid shoot proliferation stage and elongation, axillary buds were cultured on MS medium supplemented with 1.5 mg L-1 6-benzyl adenine (BAP) and 0.2 mg.L-1 alpha-naphthaleneacetic acid (NAA) and shoot elongation on half-strength MS medium (MS½) in 90 days. Rooting induction on MS½ medium with 0.5 mg.L-1 NAA in 60 days of culture.
Rapid and efficient multiplication of plant biomass production was successful established by culturing shoots (approximately 3.5 to 4 cm in height with 4 leaves) semi-submerged on 250mL conical flask contain hormone-free MS½ liquid medium (30g.L-1 sucrose, 10% coconut water) supplemented with separate polyamine sources (1.5g.L-1 casein or 1g.L-1 yeast extract) in a refrigerated incubator shaker (New Brunswick Innova 4230) at 80 rpm
