In vitro propagation through transverse thin cell layer (tTCL) culture system of lady’s slipper orchid: Paphiopedilum callosum var. sublaeve

Paphiopedilum callosum var. sublaeve, a lady’s slipper orchid native to southern Thailand, is protected under CITES (appendix I) due to habitat destruction and over-collection. The novel micropropagation technique for this endemic species was investigated. A 2-week preculture of seeds in distilled water before transfer to 1/2MS solid medium was the optimal conditions to promote seed germination in vitro. Shoot tip-derived transverse thin cell layer (tTCL) explants cultured on MVW solid medium containing 1.0 mg/L TDZ for 8 weeks provided the highest percentage of regenerated protocorm-like bodies (46.67±6.67), shoot formation (40.00±5.16), root formation (30.00±12.38) and survival rate (70.00±4.47). The acclimatized plantlets potted in sphagnum moss in the greenhouse grew well at 80% survival rate. No genetic variation was detected between the regenerated plantlets and their own mother plants based on RAPD marker