Optimization of protocorm-like bodies tissue culture and clonal propagation conditions for endangered lady's slipper orchid (paphiopedilum niveum (rchb.f.) Stein)

Abstract

Paphiopedilum niveum (Rchb. f.) Stein is an endangered orchid species and has been listed in the Convention on International Trade in Endangered Species (CITES) Appendix I. Its propagation has been limited due to the fact that the explants are difficult to maintain in culture. The present study was to evaluate clonal propagation of P. niveum from two explant types; protocorm-like bodies (PLBs) and callus. For PLB proliferation, PLB clumps were cultured on modified Vacin and Went (VW) medium supplemented with coconut water (CW) (0, 10%) and Phytagel (0%, 0.1% and 0.2%). The highest increase in fresh weight (159.40±5.40 mg) was obtained on VW medium containing 10% CW and 0.2% Phytagel. For induction of PLBs-derived callus, the PLBs clumps were cultured on modified VW solid medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D) (0, 0.5 and 1.0 mg/L) in combination with thidiazuron (TDZ) (0, 0.1 and 0.5 mg/L) for 2 months. The highest increase in fresh weigh of callus (312.70±59.61 mg) was obtained on medium containing 0.5 mg/L 2, 4-D and 0.1 mg/L TDZ. Then, PLBs-derived callus were proliferated on VW solid medium supplemented with coconut water (CW) (0, 10 and 15%) and sucrose (0, 10 and 15 g/L). The highest callus growth index (0.56±0.06) was achieved from the medium containing 10% CW and 15 g/L sucrose. These proliferated PLBs-derived callus could be reinduced to form PLBs and eventually healthy plantlets.