Anoectochilus roxburghii (Wall.) Lindl has been used in Chinese herbal medicine for treating various ailments. However, its wild resources are endangered, and artificial cultivation of the plant is limited by the low regeneration rate of conventional propagation methods. The lack of A. roxburghii resources is detrimental to the commercial production of the plant and kinsenoside, which is unique to Anoectochilus species. To develop highly efficient methods for A. roxburghii micropropagation and find alternative resources for kinsenoside production, we created an induction, proliferation, and regeneration of PLBs (IPR-PLB) protocol for A. roxburghii. We also analyzed the kinsenoside and flavonoid contents during the induction and proliferation of PLBs. The best media of IPR-PLB for PLB induction and proliferation (secondary PLB induction and proliferation), shoot formation, and rooting medium were Murashige and Skoog (MS) + 3 mg/L 6-benzylaminopurine (6-BA) + 0.5 mg/L naphthaleneacetic acid (NAA) + 0.8 mg/L zeatin (ZT) + 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2, 4-D), MS + 3 mg/L 6-BA + 0.5 mg/L NAA, and MS + 0.5 mg/L NAA, respectively. On these optimized media, the PLB induction rate was 89 ± 2.08%, secondary PLB induction rate was 120 ± 5%, secondary PLB proliferation rate was 400 ± 10% and 350 ± 10 % in terms of the quantity and biomass at approximately 1 month, shoot induction rate was 10.5 shoots/PLB mass, and root induction rate was 98%. All plantlets survived after acclimation. Darkness or weak light were essential for PLB proliferation, and light was crucial for PLB differentiation on these optimized media. The kinsenoside contents of PLBs and secondary PLBs were 10.38 ± 0.08 and 12.30 ± 0.08 mg/g fresh weight (FW), respectively. Moreover, the peak kinsenoside content during the proliferation of secondary PLBs was 34.27 ± 0.79 mg/g FW, which was slightly lower than that of the whole plant (38.68 ± 3.12 mg/g FW). Two flavonoids exhibited tissue- or temporal-specific accumulation patterns, and astragalin accumulated exclusively during the first 2 weeks of cultivation. The IPR-PLB protocol for A. roxburghii may facilitate the efficient micropropagation of A. roxburghii plants. Furthermore, the PLBs are a good alternative resource for kinsenoside production.

The flavonoids in Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) have high medicinal value. However, because of slow growth and harsh ecological environments, T. hemsleyanum is currently an endangered species. In light of this, we present a detailed hairy root induction procedure as a promising alternative to true roots with medicinal value. The percentage of explants induced by Agrobacterium rhizogenes (A. rhizogenes) to produce hairy roots out of the total number of explants infected (induction rate 1) was 95.83 ± 7.22%, and the proportion of hairy roots that contained Rol B fragments among all the hairy roots with or without Rol B fragments (positive rate) was 96.57 ± 1.72%. The transformation was further confirmed by the expression of the GUS protein. A high-productive hairy root line was screened for the comparative profiling of six flavonoids with true roots using high-performance liquid chromatography (HPLC). The contents of (+)-catechin, (−)-epicatechin, neochlorogenic acid, luteolin-6-C-glucoside, and orientin were 692.63 ± 127.24, 163.34 ± 31.86, 45.95 ± 3.46, 209.68 ± 6.03, and 56.82 ± 4.75 μg/g dry weight (DW) of 30-day-old hairy roots, respectively, which were higher than those of 3-year-old true roots. Hairy roots have stronger antioxidant activity than true roots. Overall, the hairy roots of T. hemsleyanum could serve as promising alternative sources for the production of flavonoids with medicinal uses.