Enhancement of medium components and the effects of cytokinins and auxins on in vitro propagation and successful reintroduction of Thai endangered orchid Dendrobium proteranthum Seidenf

An efficient in vitro propagation and reintroduction protocol was developed for Dendrobium proteranthum Seidenf., an endemic and rare orchid species of Thailand. Six-month-old seedlings were cultured on full-, half-, and quarter-strength MS media with varying sucrose levels. All treatments showed high survival (86.7–100%). Full-strength MS with 30 g L−1 sucrose was optimal, yielding the highest shoot (3.0), leaf (7.0), and root (7.2) numbers per plantlet. Seedlings were cultured on MS medium with 0–4.0 mg L−1 BA, kinetin, or TDZ for 12 weeks. The highest shoot induction (3.4 shoots and 8.3 leaves/plantlet) occurred with 1.0 mg L−1 BA, while root formation peaked in the control (5.9 roots/plantlet). The combined effects of BA and NAA (0–2.0 mg L−1) were tested. The combination of 0.5 mg L−1 BA + 1.0 mg L−1 NAA achieved 100% shoot formation, the highest shoot number (4.8 shoots/plantlet), and tallest plantlets (8.4 mm). Meanwhile, 0.5 mg L−1 BA + 0.5 mg L−1 NAA yielded 100% leaf and root formation with the most roots (14.4 roots/plantlet). The greatest leaf number (14.7 leaves/plantlet) occurred with 1.0 mg L−1 BA + 1.0 mg L−1 NAA. For ex situ conservation, plantlets acclimatized on sphagnum moss at Ban Romklao Botanical Garden showed 88.5% survival. After reintroduction to Phu Luang Wildlife Sanctuary, 78.7% survived after 40 weeks and completed both vegetative and reproductive stages. This study establishes an effective, reproducible protocol for mass propagation and in situ restoration of D. proteranthum.