Efficient adventitious shoot regeneration from shoot tip culture of Rhynchostylis gigantea (Lindl.) Ridl. (amethyst-purple), a rare thai orchid species

Shoot tip culture was applied to Rhynchostylis gigantea (Lindl.) Ridl. (amethyst-purple) to increase the number of orchids that have a low population in natural habitats. The effects of sucrose concentrations, plant growth regulators (PGRs), and activated charcoal (AC) in different culture media on protocorm-like body (PLB) induction, multiple shoots, root formation, and ploidy level stability were investigated. The effects of various concentrations of sucrose (0 - 20 g L^-1) incorporated in 5 liquid media; Vacin and Went, 1949 medium (VW), VW medium supplemented with 150 ml L^-1 coconut water (modified VW), Schenk & Hildebrandt, 1976 medium (SH), Murashige and Skoog, 1962 medium (MS), and half strength MS medium (1/2 MS) were studied. The highest PLBs (7.9 pieces) and survival (92.6%) were observed in modified VW liquid medium supplemented with 10 g L^-1 sucrose without plant growth regulators after 8 weeks of culture. Shoot and root induction were observed in SH agar medium supplemented with PGRs. The supplementation of 0.1 mg L^-1 TDZ on SH agar medium induced the highest shoot formation at 73.3 shoots per explant, while in SH agar medium supplemented with NAA induced root formation, the highest root formation was at 4.2 mm. After 12 weeks of culture, SH agar medium showed the release of phenolic compounds. AC was used to inhibit phenolic exudates and to induce root formation in SH agar medium. The combination of 1 g L^-1 AC and 1.0 g L^-1 NAA showed an optimum root formation at 51% and rooting from a shoot at 2.3 roots per shoot. A similar pattern of ploidy level was observed by flow cytometry from the leaves of both stock plantlets and regenerated plantlets. There was no difference in the morphology of stock plantlets and regenerated plantlets.