Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes-mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rolB and rolC genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g⁻¹ DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g⁻¹ DW for shoots and roots respectively) and calli (1.082 μg g⁻¹ DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g⁻¹ DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g⁻¹ DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g⁻¹ DW). Hairy root culture of R. yunnanensis, with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.

云南茜草是一种重要的药用植物，具有多种生物活性次生代谢产物。为了减少该物种对野生种群的依赖，我们旨在建立可以生产茜草科环肽（RA）和醌类的体外培养系统。使用四种发根土壤杆菌菌株研究了发根农杆菌介导的离体生长的云南松植物茎段的转化并优化了转化条件。当未经预培养的茎段（带叶）浸入发根土壤中时，毛状根出现的频率最高（68.89%）A4株菌悬液30分钟，在Murashige和Skoog（MS）固体培养基上避光共培养3天，然后避光孵育。rol B 和rol C 基因的PCR 分析证实了六个毛状根克隆的转化性质。与体外生长的植物和愈伤组织相比，毛状根生长迅速，特别是在 MS 液体培养基中显示出最高的生物量积累。毛状根的组织学观察揭示了血管圆柱体的解剖学差异，其中细胞表现出以旺盛生长为特征的高有丝分裂活性。UPLC-MS/MS 分析表明，在 ½MS 液体培养基（4.611 μg g ^-1 DW）中生长的毛状根中 RA 的数量高于体外培养的生长植物（芽和根分别为0.331 和 4.096 μg g ^-1 DW）和愈伤组织（1.082 μg g ^-1 DW），但仍远低于种子传播植物的根（80.296 μg g ^-1 DW） . 然而，毛状根积累了高水平的醌（MS 和 ½MS 液体培养基分别为2320.923 和 5067.801 μg g ^-1 DW），与种子植物的根（7409.973 μg g ^-1 DW）处于同一数量级. R. yunnanensis 的毛状根培养具有高生物量积累和醌类生产，可为 RA 和醌类的生产提供有吸引力的前景，可进一步优化药物用途。