Abstract

Anoectochilus formosanus Hayata is a valuable medicinal plant with well-documented health benefits. However, it has become increasingly rare due to habitat loss and overharvesting. To reduce harvesting pressure on wild populations, several studies have reported in vitro and ex vitro cultivation of A. formosanus for biomass production. Nevertheless, information on in vitro culture using media supplemented with natural organic substances remains limited. In this study, natural organic sources, including fresh coconut water (Cocos nucifera L.) (CW), finely homogenized ripe banana pulp (Musa acuminata Colla), finely homogenized potato tuber (Solanum tuberosum L.), and finely homogenized soybean seeds (Glycine max (L.) Merr.), were evaluated for their effects on the in vitro culture of A. formosanus, with the aim of producing high-quality medicinal biomass and assessing kinsenoside accumulation. The results showed that, when applied individually, Murashige and Skoog (MS) medium supplemented with 10% or 15% (v/v) CW, 70 g L-¹ banana, 50 g L-¹ potato, or 50 g L-¹ soybean supported optimal in vitro growth. Under combined supplementation, MS medium containing 10% coconut water with 15–20 g L-¹ of each organic additive produced the best growth performance, achieving 100% regeneration, maximum plant height (14.65 cm), root number (8.10 roots per plant), and fresh biomass (2.79 g per plant). Plant growth increased progressively over 1, 3, 5 and 7 months of in vitro culture. The accumulated kinsenoside content reached 10.01%, 10.88%, 11.47%, and 11.56% after 1, 3, 5 and 7 months, respectively. The findings indicate a strong association between plant developmental stage, biomass accumulation, and secondary metabolite production. Overall, the results demonstrated that defined combinations of natural organic additives effectively support in vitro propagation and promote kinsenoside accumulation in A. formosanus. These findings provide a robust experimental basis for the development of a sustainable tissue culture system for large-scale production of high-quality medicinal biomass, with potential applications in conservation and commercial exploitation of this species.