Large scale in vitro propagation of Anoectochilus roxburghii for commercial application: Pharmaceutically important and ornamental plant

• An improved and efficient propagation system of Anoectochilus roxburghii was investigated.
• Half-strength MS medium containing 1.5 mg l−1 BA was proved optimum for shoot bud induction.
• Media and plant growth regulators for shoot growth and proliferation were compared.
• The addition of banana homogenate to media is appropriate for in vitro rooting.

Anoectochilus roxburghii (Orchidaceae family) is an important medicinal and ornamental plant which is in extinct condition because of the over-collected and the loss of its suitable habitats. Several protocols for A. roxburghii micropropagation have been tested but none has provided an effective method for large-scale micropropagation. The study investigates an improved and efficient propagation method including shoots generation and formation, shoot proliferation, inducing root and acclimatization. The nodal segments cut from field-grown plants were adopted as explants. Half-strength Murashige and Skoog (MS) medium supplemented with 1.5 mg l−1 6-benzyladenine (BA) was proved to be the optimal medium for shoots generation and induction, on which, the shoot formation rate was 91.67%. Shoots were inoculated on such four media as half-strength MS, MS, Vacin and Went (VW), and Gamborg (B5), containing various concentrations and combinations of the cytokinins BA, 0.5–4.0 mg l−1, kinetin (Kn), 0.1–2.0 mg l−1, and the auxin 1-α-naphthaleneacetic acid (NAA), 0–0.5 mg l−1, to select the optimal conditions for shoot growth and proliferation. Among those, the highest proliferation rate was 4.33, which was obtained on half-strength MS medium fortified with 3.0 mg l−1 BA, 1.0 mg l−1 Kn, 0.5 mg l−1 NAA and additives. The half-strength MS medium supplemented with 0.6 mg l−1 NAA, 0.3 mg l−1 indole-3-butyric acid (IBA) and 100 mg l−1 banana mashes turned out to be best medium for in vitro rooting, which has a inducing root rate of 93.33%. The rooted plantlets were successfully transferred into plastic cups containing a sterile sand and peat soil mixture in a ratio of 1:2, and the surface was covered with live moss. The survival rate for A. roxburghii was 90.2%. The described protocol can be efficiently used for the large-scale propagation and conservation of the germplasm of this endangered medicinal and ornamental plant.