Establishment and optimization of hairy root cultures of Acanthophyllum species: a novel platform for enhanced saponin production

Acanthophyllum (Caryophyllaceae) is a valuable rangeland plant with various medicinal and industrial applications. Despite its significance, no studies have yet explored its potential for production of specialized metabolites (SMs) through hairy root culture (HRC). In this study, transgenic hairy root lines (HRLs) of three Acanthophyllum species (Acanthophyllum korshinskyi, Acanthophyllum glandulosum, and Acanthophyllum squarrosum) were successfully established for the first time via infection with wild-type strains of Rhizobium rhizogenes (LBA-9402 and C58C1) and Rhizobium tumefaciens strain C58C1(pRiA4). After 4 weeks of cultivation on solid Murashige and Skoog (MS) medium, the growth index (GI) and total saponin content of the HRLs were quantitatively assessed. Subsequently, the two HRLs exhibiting the highest total saponin content were selected for further cultivation in liquid MS medium. The growth kinetics and total saponin content of the selected HRLs were monitored at six time intervals: 10, 20, 30, 40, 50, and 60 days. The highest biomass accumulations on solid medium were recorded in HRL3 (2.24 g), HRL1 (1.72 g), and HRL4 (1.61 g), whereas HRL4 (32.15 mg/g DW) and HRL1 (26.47 mg/g DW) showed the highest total saponin contents. In liquid medium, both HRL1 and HRL4 exhibited sigmoid growth curves, with peak biomass accumulation and saponin production observed on day 40. HRL4 demonstrated a shorter doubling time (tDf) of 0.66 days and a higher specific growth rate (µf) of 1.04 day⁻¹, compared to HRL1, which had values of 0.70 days and 0.98 day⁻¹, respectively. The highest saponin content was observed on day 40 for both HRL1 (34.16 mg/g DW) and HRL4 (24.52 mg/g DW). HRL1, with its consistently higher saponin content, remains a promising candidate for future development. These findings provide a novel, scalable, and sustainable platform for the ex situ conservation and commercial-scale production of bioactive saponins in Acanthophyllum species, a genus previously unexplored in plant biotechnology.