Rhizome induction and proliferation in Anoectochilus lylei for biomass and bioactive compounds accumulation

Anoectochilus lyei is overexploited in the wild, especially for medicinal and ornamental purposes. Uncontrolled harvesting, in particular, has led to a significant decline in wild populations. This study aimed to induce and optimize culture conditions for rhizome biomass cultivation in A. lylei , providing a sustainable method for biomass and bioactive compound production. The highest rhizome induction rate (98.9%) with fresh weight (FW) of 12.64 g and dry weight (DW) of 1.14 g was achieved using Murashige and Skoog (MS) medium supplemented with 2 g/L hyponex I, 2 g/L hyponex II, 2 g/L peptone, and 1.0 g/L activated charcoal. To optimize the culture condition, different salt strengths (MS; Schenk and Hildebrandt, SH; Gamborg, B5) were tested, and ¾ ×MS medium with 2 mg/L BA (6-benzylaminopurin), 0.5 mg/L NAA (α-naphthalene acetic acid), 0.2 mg/L Kin (kinetin), and 35 g/L sucrose was most effective for rhizome proliferation (127.33 g/L FW and 20.40 g/L DW). The highest total phenolic content (4.9 mg/g DW) was observed in ½ × MS medium, while the highest flavonoid (1.05 mg/g DW) and polysaccharide content (150.66 mg/g DW) was recorded in control. Optimal bioactive productivity was observed in ¾×MS medium. In sucrose concentrations treatments, 35 g/L sucrose yielded the highest rhizome biomass (157.17 g/L FW; 20.67 g/L DW). The highest phenolic (4.44 mg/g DW), and flavonoid (1.15 mg/g DW) were recorded at 10 g/L sucrose, while polysaccharide (115.87 mg/g DW) content was observed at 35 g/Lsucrose concentrations. Additionally, rhizome cultures exhibited higher kinsenoside (2.94 mg/g DW) and polysaccharide content than both ex vitro and in vitro plants. Furthermore, the rhizome extract suppressesed the growth of Staphylococcus aureus and Escherichia coli , demonstrating potential for antibacterial applications. These results highlight the potential for large-scale bioreactor cultivation of A. lylei rhizomes for enhanced biomass and bioactive compound production. Graphical Abstract