In vitro propagation of Paphiopedilum hangianum Perner & Gruss

Paphiopedilum hangianum Perner & Gruss is a tropical lithophytic orchid that is threatened with extinction due to over-collection and loss of suitable habitats. Asymbiotic germination and tissue culture can provide useful means for its conservation and commercial propagation. Seeds collected 180 days after pollination (DAP) were optimum for culture in vitro. Seed germination (72.67%) was possible on H026 medium containing 0.5 mg l−1 α-naphthaleneacetic acid (NAA), 10% coconut water (CW), and 1.0 g l−1 activated charcoal (AC). Half-strength Murashige and Skoog (MS) medium with 5.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 thidiazuron (TDZ) was most suitable for callus formation and proliferation from protocorms 60 days after germination. Half-strength MS supplemented with 5.0 mg l−1 kinetin (Kn) and 2.0 or 1.0 mg l−1 N6-benzyladenine (BA) was most suitable for the induction of protocorm-like bodies (PLBs) from protocorms 60 days after germination or for PLB proliferation. Half-strength MS supplemented with 5.0 mg l−1 Kn and 0.5 mg l−1 NAA was most suitable for callus differentiation. H026 medium containing 1.0 g l−1 peptone, 30 g l−1 sucrose, 1.0 g l−1 AC, 1.0–2.0 mg l−1 NAA and 100 g l−1 banana homogenate (BH) was suitable for PLB differentiation and plantlet growth, half-strength MS medium supplemented with 1.0 mg l−1 BA and 2.0 mg l−1 NAA was suitable for shoot proliferation while Hyponex N016 medium containing 1.0 mg l−1 NAA and 100 g l−1 BH was suitable for rooting. About 5000 plantlets were successfully produced within 3 years from plant regeneration, showing no obvious phenotypic variation. This protocol is an efficient means for the large-scale propagation of P. hangianum.

► Degree of seed maturity affects Paphiopedilum hangianum seed germination in vitro.
► Plant growth regulators affect callus formation, proliferation and differentiation from protocorms.
► Plant growth regulators affect protocorm-like bodies formation, proliferation and differentiation from protocorms.
► Plant growth regulators affect shoot formation and proliferation from seedlings in vitro.