Establishment of Hairy Root Culture System in Atractylodes chinensis for Enhanced Production of Medicinal Sesquiterpenoids

Establishment of Hairy Root Culture System in Atractylodes chinensis for Enhanced Production of Medicinal Sesquiterpenoids | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Establishment of Hairy Root Culture System in Atractylodes chinensis for Enhanced Production of Medicinal Sesquiterpenoids Zhenqing Bai, Jiawen Wu, Kelu Wang, Xiuxiu Li, Beibei Shi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7670264/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Atractylodes chinensis (DC.) Koidz., a pharmacologically significant medicinal plant with various metabolites, produces sesquiterpenoids like atractylodin and β -eudesmol that dictate its medicinal quality and underpin its clinical efficacy. While hairy root systems offer industrial potential for metabolite production, no such system existed for A. chinensis . Here, we report the successful establishment of an efficient hairy root induction system for A. chinensis using Agrobacterium rhizogenes ATCC15834 with leaf and petiole explants, achieving higher induction rates than strains R1000 30 and LBA9402. Optimal growth occurred on 1/2 MS solid medium supplemented with 0.5 mg·L-1 IAA, 0.1 mg·L-1 KT, and 5% sucrose, yielding maximal biomass and metabolite accumulation over a 52-day culture period. Among the established hairy root lines, those induced from petioles (particularly line P-6) exhibited superior biomass and significantly higher levels of key metabolites, including atractylodin, β -eudesmol, total polyphenolic acids, and total flavonoids at day 52, compared to the leaf-induced line (L-1). Transcriptomic analysis confirmed the key genes involved in β -eudesmol and atractylodin biosynthesis upregulated relative to wild plants. Furthermore, treatment of the hairy roots with melatonin (MT) or its chemical homologs (5- methoxyindole, 5-MI; 5-methoxytryptamine, 5-MT) demonstrated that only 0.5 mmol·L⁻¹ 5-MI significantly enhanced β -eudesmol production. Remarkably, the β -eudesmol content achieved at 168 hours post-treatment was 1.82-fold higher than that in wild 3-year-old A. chinensis roots. This study establishes a robust platform for A. chinensis hairy root culture and highlights 5-MI as a potent inducer, providing a valuable foundation for the industrial scale production of its medicinally active compounds. Atractylodes chinensis (DC.) Koidz hairy roots atractylodin β-eudesmol 5-Methoxyindole Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Atractylodes chinensis (DC.) Koidz, a perennial herb of the Asteraceae family, is a cornerstone species in Traditional Chinese Medicine (TCM), traditionally termed "Cangzhu". Native to northern China (Inner Mongolia, Shaanxi, Jilin), it is revered for its therapeutic properties in dispelling dampness, fortifying the spleen, and alleviating rheumatic disorders (Cho et al., 2016 , Gao et al., 2023 , Wang et al., 2023 ). Modern pharmacological researches have shown that the sesquiterpenoids compounds including atractylodin and β -eudesmol, are the main ingredients of its pharmacological activities in A. chinensis in clinical practice, which possess anti-inflammatory, antibacterial, antiviral, hepatoprotective and anti-tumor effects (Kimura & Sumiyoshi, 2012 , Cheng et al., 2016 , Kim et al., 2016a , Xie et al., 2018 ). Atractylodin has a wide range of pharmacological effects and great potential for development and application. In recent years, it had been verified that atractylodin activated DRD2 to attenuate motor deficits and gait disturbances, which protected dopaminergic neurons in Parkinson's disease mice (Li et al., 2022 ), demonstrating the potential of atractylodin as a neuroprotective agent (Wang et al., 2023 ). As well as β -eudesmol, it had been shown to inhibit the growth of cancer cells (Ma et al., 2008 ) and had a regulatory effect on the gastrointestinal tract (Kimura & Sumiyoshi, 2012 ). Besides, β -eudesmol was found to significantly inhibit mast cell migration and reduce F-actin formation in a concentration-dependent manner (Nam et al., 2017 ). Due to its medicinal value, the market demand for A. chinensis is rapidly increasing, and the wild resource are insufficient, which many regions begin to cultivated A. chinensis . However, the accumulation of active metabolites such as atractylodin, β -eudesmol, atractylone, volatile oils, atractylenolide I, atractylenolide II and atractylenolide III in A. chinensis cultivated plants require 3–5 years to accumulate peak metabolite levels (Liu et al., 2024 ). So, developing standardized production of metabolites system is important. Root is commonly used tissue of Traditional Chinese Medicine, and also the main location to product secondary metabolites. Hairy roots are induced roots that synthesized various secondary metabolites, which are induced by Agrobacterium rhizogenes (Chandra & Chandra, 2011 ). Hairy roots have the effects of fast growth rate, stable genetic characteristics, and high production of secondary metabolites (Guillon et al., 2006 ). Hairy root cultures, induced by Agrobacterium rhizogenes , offer a scalable solution for metabolite biosynthesis. These systems combine rapid growth, genetic stability, and high secondary metabolite yields (Guillon et al., 2006 ), with established success in over 100 medicinal plants, including Panax ginseng , Brassica napus , Saliva miltiorrhiza , Astragalus membranaceus , Raphanus sativus , etc. (Banerjee et al., 2012 , Bai et al., 2020 , Balasubramanian et al., 2018 , Jiao et al., 2015 ). However, within the Asteraceae family, only A. lancea has reported hairy root induction (Zhang et al., 2023a ). No protocols exist for A. chinensis , hindering industrial exploitation. Various chemical inducers are employed to enhance the production of plant secondary metabolites. Among these, melatonin (MT, N-acetyl-5-methoxytryptamine), a biologically active small molecule ubiquitous in plants, functions as an effective inducer that promotes the accumulation of secondary metabolites (Kim et al., 2016b ). Jahan et al. (Jahan et al., 2020 ) found that exogenous MT treatment significantly increased the content of phenols, flavonoids, and anthocyanins in tomato seedlings. Another study had shown that the addition of exogenous MT significantly increased the total glucosinolate content of broccoli (Wei et al., 2020 ). Spraying MT resulted in the highest total isoflavone content in soybeans on the 5th day of germination (Wu et al., 2023 ). Despite its widespread presence in plants, MT's low endogenous concentrations and complex biosynthetic pathway significantly limit its practical application as an inducer in agriculture. Consequently, identifying low-cost, effective, and highly efficient functional substitutes for melatonin holds considerable value for future agricultural practices. One promising candidate is 5-methoxyindole (5-MI), a chemical homolog and synthetic intermediate of MT. 5-MI offers advantages such as low cost and high synthetic efficiency, suggesting good potential for development and application (Kong et al., 2021 ). However, the molecular mechanism underlying the induction of secondary metabolites and bioactive compounds in A. chinensis by MT and its chemical homologs like 5-MI remain unclear. Herein, we establish the hairy roots induction system for A. chinensis using A. rhizogenes ATCC15834. In addition, we optimized medium to grow A. chinensis hairy roots. We systematically evaluated petiole- and leaf-derived explants, established growth kinetics, and quantified sesquiterpenoid productions, such as atractylodin, β- eudesmol, total flavonoid, total polyphenolic acid, and transcriptional profiling of biosynthetic genes. By benchmarking metabolite yields against wild 3-year-old plants, this work bridges critical gaps in A. chinensis biotechnology, enabling sustainable production of its pharmacologically vital compounds. Additionally, melatonin (MT) and two chemical homologs of MT, 5-Methoxyindole (5-MI) and 5-Methoxytryptamine (5-MT) were used to treat A. chinensis hairy roots, and detected the expression levels of genes related to secondary metabolic pathways and the content of secondary metabolites. We explore the changes in the composition of metabolites in A. chinensis induced by MT and chemical homologs, which enrich the understanding of the important role of MT induced plant production of secondary metabolites. 2. Materials and Methods 2.1 Plant Materials The sterile seedlings of Atractylodes chinensis (DC.) Koidz were saved in our laboratory. Seeds and wild A. chinensis were collected in Chifeng City, Inner Mongolia, China (geographic coordinates: 41°17′10″N – 45°24′15″N, 116°21′07″E – 120°58′52″E) in September 2022. 2.2 Establishment of hairy roots transformation Petiole and leaf explants of A. chinensis were prepared: leaves were sectioned into 1 cm × 1 cm fragments, and petioles were trimmed to 1 cm lengths. Explants were pre-cultured on MS solid medium (pH 5.8) in darkness at 25°C for 12 h. Agrobacterium rhizogenes strains ATCC15834, R1000 and LBA9402 were cultured in YEB medium until reaching OD 600 = 0.6 at 28°C. Pre-treated explants were transferred in A. tumefaciens containing 300 µM Acetosyringone and co-cultivated at 28°C (150 rpm) for 30 min. After co-cultivation, tissues were transferred to MS solid medium supplemented with 500 mg·L − 1 cefotaxime (cef) and incubated in darkness at 25°C for 7 d. The tissues were washed with sterile water and replaced onto fresh MS solid medium every week, which the concentration of cef was gradually reduced to 0 mg·L − 1 . ` Total genomic DNA of A. chinensis hairy roots were extracted using a Plant Genomic DNA Kit (Tiangen, Beijing, WI, China). Specific primers of rolB and rolC were designed to detected the Ri plasmid of A. tumefaciens (Shkryl et al., 2023 , Djerdjouri et al., 2024 ) (Table S1). After PCR amplification, the PCR reaction mixtures were loaded directly onto 1% agarose gel for electrophoretic analysis. 2.3 Optimization of medium of hairy roots Once the hairy roots grew out, the roots were transferred on 1/2 MS solid medium and kept in the darkness at 25°C. However, the growth was slow. To increase the growth rate of A. chinensis hairy roots, different combination of hormones was added to 1/2 MS or 6,7-V solid medium, which were 1/2 MS + 0.5 mg·L − 1 IAA + 0.1 mg·L − 1 KT, 1/2 MS + 0.5 mg·L − 1 NAA + 0.1 mg·L − 1 KT, 1/2 MS + 0.5 mg·L − 1 6-BA + 0.1 mg·L − 1 KT, 6,7-V + 0.5 mg·L − 1 IAA + 0.1 mg·L − 1 KT, 6,7-V + 0.5 mg·L − 1 NAA + 0.1 mg·L − 1 KT and 6,7-V + 0.5 mg·L − 1 6-BA + 0.1 mg·L − 1 KT. Each medium was supplemented with 3% or 5% sucrose. About 0.08 g FW of hairy roots were transferred to 90 mL liquid medium in 150 mL erlenmeyer flasks, and cultured for 60 d prior to biomass assessment to determine the optimal medium for growing A. chinensis hairy roots. About 0.3 g FW of hairy roots were cultured in 90 mL 1/2 MS + 0.5 mg·L − 1 IAA + 0.1 mg·L − 1 KT + 5% sucrose medium in 150 mL erlenmeyer flasks. Fresh weight (FW) was recorded at 0, 8, 16, 24, 32, 36, 40, 44, 48 and 52 d to establish growth kinetics. 2.4 Determination of metabolites of A. chinensis hairy roots The A. chinensis hairy roots were selected at 0, 8, 16, 24, 32, 36, 40, 44, 48 and 52 d. Hairy roots were washed and dried at 37°C. For determinating of atractylodin and β -eudesmol, 0.2 g hairy roots were added 4 mL 70% methanol for 12 h. The extraction was centrifuged for 8 min at 10,000 × g to collect the supernatant. After filtering through a 0.22 mm PTFE membrane, the samples were detected by High Performance Liquid Chromatography (HPLC). Atractylodin and β- eudesmol chemical molecular standard samples (Sigma) were prepared according to the concentration gradient. Atractylodin and β- eudesmol were quantified based on standard curves (Fig. S1 and S2). After grinding, 0.05 g hairy roots were added 1 mL 70% ethanol for ultrasonic extraction for 2 h at 40 ° C. The Folin-C assay was used to detect total polyphenolic acid (Kupina et al., 2018 ). The total polyphenolic acid was quantified based on gallic acid standard curves (Fig. S3). The total flavonoid was measured by Aluminum chloride method (Chang et al., 2002 ), and was quantified based on rutin standard curves (Fig. S4). 2.5 Real time-quantitative PCR (RT-qPCR) analysis and statistical analysis According to the growth curve of A. chinensis hairy roots, hairy roots at 8, 24 and 52 d were collected, and the wild 1-year-old roots of A. chinensis were as the control. The key genes involved in the biosynthesis of β -eudesmol ( AcOGF30G6 ), and genes involved in the biosynthesis of atractylodin ( AcZFP706 , AcCTA12 , AcPSPTA25520 , AcGTF25 ) were used for RT-qPCR, and the Acactin was as the control gene (Zhang et al. 2024) (Table S1). Total RNA was extracted according to the MiniBEST Universal RNA Extraction Kit (TaKaRa, Beijing, China). First-strand cDNA was performed according to the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Beijing, China). The RT-qPCR reaction system and procedures were referred to the TB Green Premix Ex TaqⅡ kit (TaKaRa, Beijing, China). The 2 −ΔΔt method was used to calculate the relative expression of genes. Each sample has three independent biological replicates. SPSS 23.0 was used to determine the statistical significance ( P < 0.05 ). 2.6 Compounds treatments N-acetyl-5-Methoxytryptamine (melatonin, MT), 5-Methoxytryptamine (5-MT) and 5-Methoxyindole (5-MI) (Solarbio, Beijing, China) were used in this study. The A. chinensis hairy roots P-6 were treated with these three compounds. After 52 d of A. chinensis hairy roots cultivation, different compounds (MT, 5-MI and 5-MT) with different concentrations (0, 0.01, 0.05, 0.1, 0.2, and 0.5 mmol · L − 1 ) were added to medium to continue cultivating hairy roots. Samples were taken at 168 h to detect the biomass accumulation of hairy roots and contents of β- eudesmol, total polyphenolic acid and total flavonoid. Samples were taken at 0, 12, 72 and 168 h for RT-qPCR. Each sample has three independent biological replicates. 3. Results 3.1 The induction and identification of Atractylodes chinensis hairy roots The sterile seedlings of A. chinensis preserving in the laboratory were used. The explants were infected by the Agrobacterium tumefaciens strains ATCC15834, R1000 and LBA9402, respectively. The induction rates were first calculated, which A. tumefaciens strain LBA9402 did not induce A. chinensis hairy roots. And induction rate of R1000 was low, being 5%, however, the hairy roots were death quick. Therefore, we focused on the hairy roots induced by ATCC15834, with an induction rate of 85–91% (Table 1 ). After infection by ATCC15834, hairy roots were observed to grow at the petioles wound site at 20 d in 1/2 MS solid medium used A. chinensis petioles. At 80 d, the hairy roots extended and the number of roots increased. And the biomass of A. chinensis hairy roots increased extremely at 170 d. On the other side, hairy roots were observed at the leaf wound site at 50 d used A. chinensis leaves. At 130 d, the length of hairy roots increased, and branches were observed. And the branches of A. chinensis hairy roots increased about 2–3 cm at 170 d (Fig. 1 A). To determine the growing hairy roots induced by A. tumefaciens , we designed specific primers ( rolB and rolC ) and used hairy roots DNA as templates for PCR amplification. The results showed that rolB and rolC genes were detected in A. chinensis hairy roots (Fig. 1 B), illustrating that the A. tumefaciens Ri plasmid was successfully transferred into A. chinensis to induce hairy roots. Table 1 Different Agrobacterium tumefaciens strains induced hairy roots rates of petiole and leaf explants. A. tumefaciens strains Petiole Leaf Number of infections Number of hairy roots Induction rate (%) Number of infections Number of hairy roots Induction rate (%) ATCC15834 34 31 91.2 42 36 85.7 R1000 38 2 5.3 39 2 5.1 LBA9402 35 0 0 40 0 0 3.2 The determination of the optimal medium for growing A. chinensis hairy roots Because the hairy roots grew slowly in 1/2 MS medium, six hairy roots amplification medium with different concentrations of sucrose (3%, 5%) were mixed to screen the optimal medium for growing A. chinensis hairy roots. After adding hormones KT with IAA, or NAA, or 6-BA to the 1/2 MS solid medium and culturing with 3% sucrose for 60 d, there were no significant changes. On the contrary, A. chinensis hairy roots grew well after adding 5% sucrose for 60 d, with the effect of adding IAA, NAA, and 6-BA to the same concentration of KT on the accumulation of hairy root biomass being IAA > NAA > 6-BA. On the other side, based on the 6,7-V solid medium, adding NAA and 6-BA to the same concentration of KT resulted in higher biomass accumulation in 3% sucrose than in 5% sucrose, and adding 5% sucrose to KT and IAA resulted in higher biomass accumulation than in 3% sucrose (Fig. 2 A). According to the above results, our conclusion is that the order of the effect of adding IAA, NAA, and 6-BA to the same concentration of KT at 5% sucrose on the biomass accumulation of A. chinensis hairy roots was: IAA > NAA > 6-BA. Additionally, the biomass of A. chinensis hairy roots were also measured to select the optimal medium for growing hairy roots intuitively. The accumulation of biomass in 5% sucrose is generally higher than in 3% sucrose except B2 and B3. And the optimal hormone combination was 0.5 mg·L − 1 IAA + 0.1 mg·L − 1 KT. Finally, A. chinensis hairy roots accumulated more biomass in 1/2 MS than in 6,7-V solid medium (Fig. 2 B). Our results showed that 1/2 MS solid medium supplemented with 0.5 mg·L − 1 IAA, 0.1 mg·L − 1 KT, and 5% sucrose was the most effective medium combination in increasing the value of hairy roots of A. chinensis . Due to the induction of hairy roots by leaves (L-1) and petioles (P-6 and P-14) of A. chinensis . We measured growth curve of these three lines for 52 d, which weighed FW of hairy roots. With the passage of time, the biomass of hairy roots in all three lines increased remarkedly at 52 d (Fig. S5). However, the growth rate of different lines varies. The growth of L-1 was rapid from 8 to 44 d, and tended to stabilize after 44 d. P-6 had two rapid growth phases, including 16–24 d and 48–52 d. And the growth of P-14 was rapid from 0 to 48 d. Overall, among the three lines of hairy roots, the P-6 had the highest biomass accumulation at 52 d, reaching 3.7 g FW. The biomass accumulation of L-1 was the lowest at 52 d, reaching 2.7 g FW. And P-14 was 3.2 g at 52 d. In addition, changes in sucrose concentration in the medium were measured (Fig. S6). There was a significant difference before 24 d of sucrose concentration. After decreasing, there was no significant difference changes from 24 to 36 d. The sucrose concentration decreased from 36 to 40 d, and there was no significant difference in sucrose concentration changes from 40 to 48 d. 3.3 Analysis of metabolites of A. chinensis hairy roots We measured atractylodin, β- eudesmol, total polyphenolic acids and total flavonoid of three induced lines to further determine the line with the highest efficiency in inducing metabolites. Firstly, the main metabolites of atractylodin and β -eudesmol in A. chinensis hairy roots of different lines at different time. Due that the hairy roots on the 0 d were separated from the later stage of the growth cycle, therefore, the hairy roots of 0 d were not included in the comparison of material contents with other time. There were no significant differences of atractylodin contents of L-1 and P-14. The contents of atractylodin in P-6 at 52 d were significantly higher than other time, reached 1.76 mg·g − 1 DW (Table 2 ). In addition, we also compared three lines contents of atractylodin at 52 d with the wild 3-year-old A. chinensis (Fig. 3 A). The results showed that the content of atractylodin in P-6 was significantly higher than the other two lines, which was no significant difference compared to wild 3-year-old plants. Table 2 The determination of atractylodin contents of A. chinensis hairy roots at different time. Lines Atractylodin contents (mg·g − 1 DW) 0 d 8 d 16 d 24 d 32 d 36 d 40 d 44 d 48 d 52 d L-1 0.54 ± 0.15 a 0.54 ± 0.16 a 0.53 ± 0.21 a 0.37 ± 0.01 a 0.38 ± 0.00 a 0.37 ± 0.01 a 0.38 ± 0.01 a 0.37 ± 0.01 a 0.39 ± 0.02 a 0.37 ± 0.02 a P-6 1.78 ± 0.08 a 0.40 ± 0.00 c 0.36 ± 0.08 c 0.37 ± 0.01 c 0.95 ± 0.28 b 0.74 ± 0.02 b 0.75 ± 0.02 b 0.78 ± 0.04 b 0.74 ± 0.05 b 1.76 ± 0.08 a P-14 0.78 ± 0.01 a 0.76 ± 0.02 a 0.75 ± 0.01 a 0.74 ± 0.03 a 0.73 ± 0.04 a 0.73 ± 0.01 a 0.76 ± 0.02 a 0.75 ± 0.01 a 0.53 ± 0.32 a 0.77 ± 0.03 a Notes: Each value represents the mean ± standard deviation (n = 3). The lower-case letters indicate significant differences among different times via ANOVA followed by Duncan's post-test ( P < 0.05). Similarly, we also determined β -eudesmol contents. The β -eudesmol contents showed a trend of first decreasing and then increasing at 8–52 d, which L-1 and P-6 reached the lowest contents at 32 d, and P-14 reached the lowest contents at 16 d. The contents of β -eudesmol in L-1 increased sharply from 32 to 36 d, which reached the maximum at 36 d, and was no significant change at 36–52 d. The contents of β -eudesmol in P-6 increased significantly from 36 to 40 d, and was no significant change at 40–52 d. The accumulation of β -eudesmol in P-14 was significantly higher at 52 d than other time (Table 3 ). Therefore, the β -eudesmol contents of three lines at 52 d were compared with the wild 3-year-old A. chinensis roots (Fig. 3 B). Although the β- eudesmol contents of hairy roots was significantly lower than control, the P-6 induced by petioles was significantly higher than L-1 induced by leaves. Table 3 The determination of β -eudesmol contents of A. chinensis hairy roots at different time. Lines β -eudesmol contents (mg·g − 1 DW) 0 d 8 d 16 d 24 d 32 d 36 d 40 d 44 d 48 d 52 d L-1 5.45 ± 0.88 ab 4.69 ± 0.35 ab 5.28 ± 0.57 ab 3.94 ± 1.24 b 3.71 ± 1.02 b 6.09 ± 1.20 a 5.14 ± 0.38 ab 5.02 ± 0.33 ab 4.89 ± 1.01 ab 5.55 ± 0.52 ab P-6 6.91 ± 0.27 ab 4.89 ± 0.04 cd 5.06 ± 0.14 cd 4.11 ± 0.33 e 3.45 ± 1.01 e 4.04 ± 1.19 e 7.72 ± 0.23 a 7.84 ± 0.23 a 7.12 ± 0.27 ab 7.24 ± 0.26 a P-14 6.41 ± 0.68 ab 4.96 ± 0.59 bc 3.10 ± 0.36 d 3.71 ± 0.35 cd 4.72 ± 1.03 cd 4.32 ± 0.95 cd 4.71 ± 0.55 bc 5.00 ± 0.06 bc 5.70 ± 0.99 bc 7.10 ± 0.34 a Notes: Each value represents the mean ± standard deviation (n = 3). The lower-case letters indicate significant differences among different times via ANOVA followed by Duncan's post-test ( P < 0.05). Total phenolic acids and total flavonoids were determined and analyzed. The results showed that total phenolic acids of A. chinensis hairy roots had no significant change at different time expect L-1 at 8 d and P-14 at 8 and 16 d (Fig. S7 A). And the total flavonoids in P-14 was significantly higher than the other two lines and wild 3-year-old plants (Fig. S7 B). On the other side, the total flavonoids of three A. chinensis hairy roots were no significant at different time (Fig. 3 C). And the total flavonoids of L-1 were significantly lower than control and other two lines, while P-6 and P-14 at 52 d were no significant difference compared to wild 3-year-old plants (Fig. 3 D). 3.4 The expression levels of genes related to metabolites synthesis of A. chinensis hairy roots In order to analyze the ability of A. chinensis hairy roots to synthesize metabolites at the molecular level, the key genes involved in the biosynthesis of β -eudesmol ( AcOGF30G6 ), and genes involved in the biosynthesis of atractylodin ( AcZFP706 , AcCTA12 , AcPSPTA25520 , AcGTF25 ) were used for RT-qPCR. The expression of AcOGF30G6 were significantly up-regulated of three lines at 52 d compared to wild 1-year plants (Fig. 4 ). The trends of AcZFP706 , AcPSPTA25520 and AcGTF25 increased with the growth of hairy roots in three lines, which AcPSPTA25520 increased significantly of three lines at 52 d compared to wild 1-year plants. AcZFP706 in line L-1 and AcGTF25 in line P-14 increased significantly of three lines at 52 d compared to control. The expression level of AcCTA12 in line P-14 at 52 d increased significantly compared to control. 3.5 Different concentrations of MT, 5-MI and 5-MT treated to the A. chinensis hairy roots Compared to hairy roots induced from leaves, those induced from petioles exhibited greater biomass accumulation and higher metabolic productivity, particularly for atractylodin and total flavonoids. Among the petiole-derived lines, the P-6 line displayed significantly higher atractylodin content than P-14 and was therefore selected for compound treatments. To investigate the effects of MT, 5-MT, and 5-MI on sesquiterpenoid accumulation in A. chinensis hairy roots, the P-6 line was treated with varying concentrations (0, 0.01, 0.05, 0.1, 0.2, and 0.5 mmol·L⁻¹) of each compound. Growth (biomass) and the contents of β -eudesmol, total phenolic acids, and total flavonoids were measured. At 168 hours post-treatment (hpt), the biomass of the treated hairy roots showed no significant differences compared to the control (Fig. S8). Analysis of the main sesquiterpenoid metabolite, β -eudesmol, revealed that its levels in the hairy roots were significantly lower than in wild 3-year-old A. chinensis roots (Fig. 3 B). Treatment with 0.5 mmol·L⁻¹ 5-MI significantly increased the β -eudesmol content to 17.37 mg·g⁻¹ DW (Fig. 5 A). In contrast, treatments with MT and 5-MT across all concentrations induced no significant changes in β -eudesmol levels at 168 hpt. Conversely, treatment with 0.5 mmol·L⁻¹ 5-MI significantly decreased the levels of total phenolic acids and total flavonoids (Fig. 5 B and C). Treatments with MT and 5-MT at all concentrations resulted in no significant changes in these metabolites at 168 hpt. The increase induced by 5-MI compensated for the initially lower β-eudesmol content in the hairy roots. Specifically, the β-eudesmol level at 168 hpt after 0.5 mmol·L⁻¹ 5-MI treatment was significantly higher than that in wild 3-year-old A. chinensis roots (Fig. 6 ). Furthermore, this treatment significantly upregulated the expression of AcOGF30G6 , a key gene involved in β-eudesmol biosynthesis (Fig. S9). 4. Discussion The conservations of Traditional Chinese Medicine germplasm resources currently mainly include methods such as seed propagation, artificial cultivation, plant tissue cultivation, and hairy roots cultivation. As an important Traditional Chinese Medicine, Atractylodes chinensis (DC.) Koidz. has not been reported to be induced hairy roots, which is unfavorable for its researches and reproduction. Medicinal plants produce a plenty of phytocompounds such as cyclopeptides, quinones, alkaloids, flavonoids and terpenes, which are mainly used for pharmaceutical and flavor industries (Roy, 2021 , Miao et al., 2021 ). Commercial source of these metabolites is field-grown plants, which are generally influenced by seasonal changes and regional differences. Hairy roots culture is considered an alternative method of high-value secondary metabolites for industrial production, which is mass reproduction in a short period of time (Shao et al., 2021 ). In this study, we established the hairy roots cultivation system of A. chinensis efficiently, including determination of the medium, selection of explants and definition of cultivation time. The establishment of the hairy root cultivation system requires Agrobacterium rhizogenes , which contains a special plasmid that induces rooting (Chandra & Chandra, 2011 ). The efficiency of inducing hairy roots varies among different strains of A. rhizogenes . The strains ATCC15834, R1000, A4, and C58 of A. rhizogenes were used to induce hairy roots of Trigonella foenumgraecum , which A4 was more conducive to the accumulation of diosgenin in hairy roots than other strains (Zolfaghari et al., 2020 ). Similarly, A4 strain showed a better transformation frequency compared with ATCC15384, LBA9402, and ACCC10060 to induce Rubia yunnanensis hairy roots (Miao et al. 2021 ). ATCC15834 was found the most efficient strain for Trachyspermum ammi L. hairy roots induction (Vamenani et al. 2020). The C58C1 was the best strain for inducing Atractylodes lancea hairy roots than A4, K599 and LBA9402 (Zhang et al., 2023a ). Other researches indicated that different plants infected by the same strain of A. rhizogenes , the induction of hairy roots varies. The induction rates of hairy roots between the Scutellaria orientalis and S. araxensis infected with ATCC15834, which were 93.3% and 56.6%, respectively (Gharari et al. 2020). In this study, A. chinensis hairy roots were successfully induced by A. rhizogenes ATCC15834, which the induction rate was highest compared with A. rhizogenes R1000 and LBA9402, reaching 85–91%. However, the growth of hairy roots was slow in MS solid medium, which the biomass increased extremely at 170 d. Therefore, it is urgently to optimize the hairy roots culture medium for faster growth. Previous studies have shown that changing medium composition (Yosephine et al., 2015 ) or adding exogenous hormones (Putalun et al., 2004 ) increased the contents of secondary metabolites in hairy roots. Different combinations of exogenous hormones were added to induce different hairy roots. Medium supplemented with 1 mg·L − 1 NAA and 1 mg·L − 1 BA induced Physalis minima L. hairy roots efficiently (Putalun et al., 2004 ). Hairy roots of A. lancea , also belonging to the Asteraceae crops, were grown on MS0 solid medium supplemented with 3% sucrose, 1.0 mg·L − 1 6-BA, and 0.1 mg·L − 1 NAA for two months (Zhang et al., 2023a ). Therefore, adding exogenous hormones and sucrose were shorten the growth cycle of hairy roots effectively. Sucrose had commonly been used as the carbohydrate source for hairy root growth, and proclaimed superior to other carbohydrate sources such as glucose, mannitol and sorbitol (Banerjee et al., 2012 ). In this study, we designed 6 combinations of medium with 3% or 5% sucrose to determine the optimal medium for growing A. chinensis hairy roots. Combining morphological and biomass growth, 1/2 MS solid medium supplemented with 0.5 mg·L − 1 IAA, 0.1 mg·L − 1 KT, and 5% sucrose was the most effective medium combination. Hairy roots cultivation has been developed as an innovative approach for the large-scale production of secondary metabolites and plant chemicals, which was cultivated large number of secondary metabolites in a short period of time and continuously supplied high-value products (Korde et al., 2016 ). And the yield of effective components of secondary metabolites produced by plants takes a long time, which were harvested after 3–5 years of growth to accumulate secondary metabolites a relatively high level (Liu et al., 2019 , Ran et al., 2022 , Liu et al., 2024 ). The main active ingredients of A. chinensis are volatile oils, eucalyptol and guaiacol sesquiterpenes, and polyacetylenes, which are considered characteristic phytochemicals (Meng et al., 2010 ). Among them, atractylone, β- eudesmol, atractylodinol and atractylodin are main active ingredients (Wang et al., 2023 ). In this study, we focused on atractylodin and β -eudesmol, which possess anti-inflammatory, antibacterial, antiviral and anti-tumor effects (Kimura & Sumiyoshi, 2012 , Cheng et al., 2016 ). And the contents of these two sesquiterpenoids compounds were similar with Zhang et al. (Zhang et al., 2023b ), which the atractylodin contents in hairy roots was significantly higher than normal roots, and β -eudesmol contents in hairy roots was lower than normal roots. Moreover, we found that the two lines induced by petioles, especially P-6, produced more metabolites than hairy roots induced by leaves (L-1), including atractylodin, β -eudesmol, total polyphenolic acids and total flavonoid. And we were surprised to find that there was no significant difference in the content of atractylodin produced by P-6 compared to the wild 3-year-old plants, reached 1.76 mg/g. Although the β -eudesmol of P-6 hairy roots (7.24 mg/g) was significantly lower than control plants (9.53 mg/g) in Inner Mongolia, it was higher than 5-year-old plants (1.61 mg/g) collected in Good Agricultural Practice database, Liuhe City, Changchun, China Changchun City (Liu et al., 2024 ), which were caused by regional differences. Moreover, the expression levels of genes related to β -eudesmol and atractylodin synthesis of A. chinensis hairy roots were significantly up-regulated at 52 d compared to wild 1-year plants. These results demonstrated that the contents of secondary metabolites produced by hairy roots that induced by petioles were consistent with wild 3-year-old A. chinensis . We estimated profits based on effective content, and the profit of hairy roots was much higher than field planting. To specifically enhance β- eudesmol content in A. chinensis hairy roots, this study explored the application of chemical inducers. Melatonin (MT), a well-established inducer, is known to promote the accumulation of various plant secondary metabolites (Wei et al., 2020 ). For instance, treatment of cabbage with 100 µmol/L MT upregulated anthocyanin biosynthesis genes and stimulated anthocyanin accumulation (Luo et al., 2018 ). Furthermore, MT-mediated nitric oxide (NO) production has been shown to enhance isoflavone accumulation by boosting phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) activity and upregulating key biosynthetic genes (Yin et al., 2023 ). However, in our experiments, treatment with varying concentrations of MT failed to significantly induce β -eudesmol accumulation in A. chinensis hairy roots. We therefore evaluated two chemical homologs of MT, 5-methoxyindole (5-MI) and 5-methoxytryptamine (5-MT). Notably, only treatment with 0.5 mmol·L⁻¹ 5-MI resulted in a significant increase in β -eudesmol content. This induced level reached 17.37 mg·g⁻¹ DW, representing a 1.82-fold increase compared to levels found in wild 3-year-old A. chinensis roots. These findings demonstrate that 5-MI acts as an effective inducer for significantly improving β -eudesmol content, a key quality marker, in A. chinensis hairy roots. This enhancement holds substantial promise for increasing the commercial value and profitability of A. chinensis cultivation utilizing hairy root technology. Declarations CRediT authorship contribution statement Z.B. and B.S. conceived the research. J.W., K.W., and X.L. conducted the research. X.L., B.S., and Z.B. analyzed the data. Z.B. and B.S. wrote the manuscript. Funding This research was supported by the Inner Mongolia Autonomous Region Science and Technology Plan Project (Program No. 2022YFDZ0015), the Shaanxi Provincial Department of Education Research Program (Program No. 23JK0722), and the Doctoral Scientific Research Start-up Program of Yan’an University (Program No. YAU202303846). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability The original contributions presented in the study are included in the article/supplementary material. Ethics declaration : not applicable. References Bai Z, Wu J, Huang W et al (2020) The ethylene response factor SmERF8 regulates the expression of SmKSL1 and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots. 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Industrial Crops Prod 145:112075 Supplementary Files SF.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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09:16:34","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151009,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/87cc823b07317f02f6ce03b4.png"},{"id":93574538,"identity":"22bd6cad-4e45-4b8b-92e6-af52eafe1b9a","added_by":"auto","created_at":"2025-10-15 09:16:34","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":442247,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/ff113b87992081e801d85391.png"},{"id":93574545,"identity":"db64254f-3e7d-481f-964e-ed41886cacde","added_by":"auto","created_at":"2025-10-15 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09:16:34","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126873,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25006890structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/04f946e4e5a225753f71709b.xml"},{"id":93575866,"identity":"a362acc1-9212-462e-ae72-8cf7d4328a10","added_by":"auto","created_at":"2025-10-15 09:24:34","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137397,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/63880111153672c5e28510c6.html"},{"id":93574523,"identity":"36caa917-f885-4bb8-96eb-e4741d6fd511","added_by":"auto","created_at":"2025-10-15 09:16:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":912256,"visible":true,"origin":"","legend":"\u003cp\u003eThe induction of \u003cem\u003eAtractylodes\u003c/em\u003e \u003cem\u003echinensis\u003c/em\u003e hairy root. \u003cstrong\u003eA\u003c/strong\u003e: The hairy roots induced by \u003cem\u003eA. chinensis\u003c/em\u003e petioles at different time in 1/2 MS medium. \u003cstrong\u003eB\u003c/strong\u003e: The hairy roots induced by \u003cem\u003eA. chinensis\u003c/em\u003e leave at different time in 1/2 MS medium. \u003cstrong\u003eC\u003c/strong\u003e: Identification of \u003cem\u003erolB\u003c/em\u003e and \u003cem\u003erolC\u003c/em\u003e genes in \u003cem\u003eA. chinensis\u003c/em\u003ehairy roots.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/9cec7df863abc0584bdf973e.png"},{"id":93575856,"identity":"cc3e2b8d-0c50-4f63-9154-203b495fc430","added_by":"auto","created_at":"2025-10-15 09:24:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1377353,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots in different medium. \u003cstrong\u003eA\u003c/strong\u003e: The phenotypes of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots in different medium. \u003cstrong\u003eB\u003c/strong\u003e: Biomass accumulation of \u003cem\u003eA. chinensis\u003c/em\u003ehairy roots in different medium. Each value represents the mean ± standard deviation (n = 3).\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/0c116de7cdef88e88a8e6be7.png"},{"id":93574524,"identity":"794fd984-8287-4536-b032-865d6fa024aa","added_by":"auto","created_at":"2025-10-15 09:16:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":479377,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of metabolites in wild 3-year-old rootsand three hairy roots lines of \u003cem\u003eA. chinensis\u003c/em\u003e.\u003cstrong\u003e A\u003c/strong\u003e: Atractylodin contents. \u003cstrong\u003eB\u003c/strong\u003e: \u003cem\u003eβ\u003c/em\u003e-eudesmol contents.\u003cstrong\u003e C\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eTotal phenolic acids contents. \u003cstrong\u003eD\u003c/strong\u003e: Total flavonoids contents. Three \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots lines were collected at 52 d. Each value represents the mean ± standard deviation (n = 3). The lower-case letters indicate significant differences among different lines via ANOVA followed by Duncan's post-test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/253f162dd53148dca68c89ba.png"},{"id":93574530,"identity":"2c96c3df-be95-4f85-a195-6cb06bddf86c","added_by":"auto","created_at":"2025-10-15 09:16:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":874922,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of genes related to metabolites synthesis of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots. \u003cstrong\u003eA\u003c/strong\u003e: Lines L-1. \u003cstrong\u003eB\u003c/strong\u003e: Lines P-6. \u003cstrong\u003eC\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eLines P-14. The wild 1-year old roots of \u003cem\u003eA. chinensis\u003c/em\u003e were as the control. The\u003cem\u003e AcOGF30G6 \u003c/em\u003egene was involved in the biosynthesis of \u003cem\u003eβ\u003c/em\u003e-eudesmol, and \u003cem\u003eAcZFP706\u003c/em\u003e, \u003cem\u003eAcCTA12\u003c/em\u003e, \u003cem\u003eAcPSPTA25520\u003c/em\u003e, \u003cem\u003eAcGTF25\u003c/em\u003e genes were involved in the biosynthesis of atractylodin. The lower-case letters indicate significant differences among different lines via ANOVA followed by Duncan's post-test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/750dd55f862237d6d1795655.png"},{"id":93575857,"identity":"f19eb445-077f-44a8-987b-982b7d0648f9","added_by":"auto","created_at":"2025-10-15 09:24:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":885665,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of metabolites in\u003cem\u003e A. chinensis\u003c/em\u003e hairy roots after different concentrations of melatonin and its chemical homologs treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: \u003cem\u003eβ\u003c/em\u003e-eudesmol contents. \u003cstrong\u003eB\u003c/strong\u003e: Total phenolic acids contents.\u003cstrong\u003e C\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eTotal flavonoids contents. \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots were collected at 168 hpt. Each value represents the mean ± standard deviation (n = 3). The lower-case letters indicate significant differences among different lines via ANOVA followed by Duncan's post-test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/f3078227e34102b5ad6874b3.png"},{"id":93574526,"identity":"2212a5d4-36d2-44d0-8362-5c2b53e1e744","added_by":"auto","created_at":"2025-10-15 09:16:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":162253,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of \u003cem\u003eβ\u003c/em\u003e-eudesmol contents in wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e roots and 0.5 mmol·L\u003csup\u003e-1\u003c/sup\u003e 5-MI treatment \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots at 168 hpt.\u003c/p\u003e\n\u003cp\u003eEach value represents the mean ± standard deviation (n = 3). The *** indicate significant differences among different lines via ANOVA followed by Duncan's post-test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/8caa534c4749836e660a4045.png"},{"id":94107842,"identity":"136ff8a1-b21c-47d9-857d-275407a4a8c3","added_by":"auto","created_at":"2025-10-22 12:43:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6407086,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/608d9b7d-18ad-49b9-be87-0e9a61465eec.pdf"},{"id":93574531,"identity":"b9e69583-cea8-4b52-8b34-4dd455c998ff","added_by":"auto","created_at":"2025-10-15 09:16:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":274232,"visible":true,"origin":"","legend":"","description":"","filename":"SF.docx","url":"https://assets-eu.researchsquare.com/files/rs-7670264/v1/88839792e4c3f0754a1f292e.docx"}],"financialInterests":"","formattedTitle":"Establishment of Hairy Root Culture System in Atractylodes chinensis for Enhanced Production of Medicinal Sesquiterpenoids","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eAtractylodes chinensis\u003c/em\u003e (DC.) Koidz, a perennial herb of the Asteraceae family, is a cornerstone species in Traditional Chinese Medicine (TCM), traditionally termed \"Cangzhu\". Native to northern China (Inner Mongolia, Shaanxi, Jilin), it is revered for its therapeutic properties in dispelling dampness, fortifying the spleen, and alleviating rheumatic disorders (Cho et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Gao et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Modern pharmacological researches have shown that the sesquiterpenoids compounds including atractylodin and \u003cem\u003eβ\u003c/em\u003e-eudesmol, are the main ingredients of its pharmacological activities in \u003cem\u003eA. chinensis\u003c/em\u003e in clinical practice, which possess anti-inflammatory, antibacterial, antiviral, hepatoprotective and anti-tumor effects (Kimura \u0026amp; Sumiyoshi, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Cheng et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Kim et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, Xie et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Atractylodin has a wide range of pharmacological effects and great potential for development and application. In recent years, it had been verified that atractylodin activated DRD2 to attenuate motor deficits and gait disturbances, which protected dopaminergic neurons in Parkinson's disease mice (Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), demonstrating the potential of atractylodin as a neuroprotective agent (Wang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As well as \u003cem\u003eβ\u003c/em\u003e-eudesmol, it had been shown to inhibit the growth of cancer cells (Ma et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and had a regulatory effect on the gastrointestinal tract (Kimura \u0026amp; Sumiyoshi, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Besides, \u003cem\u003eβ\u003c/em\u003e-eudesmol was found to significantly inhibit mast cell migration and reduce F-actin formation in a concentration-dependent manner (Nam et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDue to its medicinal value, the market demand for \u003cem\u003eA. chinensis\u003c/em\u003e is rapidly increasing, and the wild resource are insufficient, which many regions begin to cultivated \u003cem\u003eA. chinensis\u003c/em\u003e. However, the accumulation of active metabolites such as atractylodin, \u003cem\u003eβ\u003c/em\u003e-eudesmol, atractylone, volatile oils, atractylenolide I, atractylenolide II and atractylenolide III in \u003cem\u003eA. chinensis\u003c/em\u003e cultivated plants require 3\u0026ndash;5 years to accumulate peak metabolite levels (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). So, developing standardized production of metabolites system is important. Root is commonly used tissue of Traditional Chinese Medicine, and also the main location to product secondary metabolites. Hairy roots are induced roots that synthesized various secondary metabolites, which are induced by \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e (Chandra \u0026amp; Chandra, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Hairy roots have the effects of fast growth rate, stable genetic characteristics, and high production of secondary metabolites (Guillon et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Hairy root cultures, induced by \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e, offer a scalable solution for metabolite biosynthesis. These systems combine rapid growth, genetic stability, and high secondary metabolite yields (Guillon et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), with established success in over 100 medicinal plants, including \u003cem\u003ePanax ginseng\u003c/em\u003e, \u003cem\u003eBrassica napus\u003c/em\u003e, \u003cem\u003eSaliva miltiorrhiza\u003c/em\u003e, \u003cem\u003eAstragalus membranaceus\u003c/em\u003e, \u003cem\u003eRaphanus sativus\u003c/em\u003e, etc. (Banerjee et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Bai et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Balasubramanian et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Jiao et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, within the Asteraceae family, only \u003cem\u003eA. lancea\u003c/em\u003e has reported hairy root induction (Zhang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). No protocols exist for \u003cem\u003eA. chinensis\u003c/em\u003e, hindering industrial exploitation.\u003c/p\u003e\u003cp\u003eVarious chemical inducers are employed to enhance the production of plant secondary metabolites. Among these, melatonin (MT, N-acetyl-5-methoxytryptamine), a biologically active small molecule ubiquitous in plants, functions as an effective inducer that promotes the accumulation of secondary metabolites (Kim et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). Jahan et al. (Jahan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that exogenous MT treatment significantly increased the content of phenols, flavonoids, and anthocyanins in tomato seedlings. Another study had shown that the addition of exogenous MT significantly increased the total glucosinolate content of broccoli (Wei et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Spraying MT resulted in the highest total isoflavone content in soybeans on the 5th day of germination (Wu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite its widespread presence in plants, MT's low endogenous concentrations and complex biosynthetic pathway significantly limit its practical application as an inducer in agriculture. Consequently, identifying low-cost, effective, and highly efficient functional substitutes for melatonin holds considerable value for future agricultural practices. One promising candidate is 5-methoxyindole (5-MI), a chemical homolog and synthetic intermediate of MT. 5-MI offers advantages such as low cost and high synthetic efficiency, suggesting good potential for development and application (Kong et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the molecular mechanism underlying the induction of secondary metabolites and bioactive compounds in \u003cem\u003eA. chinensis\u003c/em\u003e by MT and its chemical homologs like 5-MI remain unclear.\u003c/p\u003e\u003cp\u003eHerein, we establish the hairy roots induction system for \u003cem\u003eA. chinensis\u003c/em\u003e using \u003cem\u003eA. rhizogenes\u003c/em\u003e ATCC15834. In addition, we optimized medium to grow \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots. We systematically evaluated petiole- and leaf-derived explants, established growth kinetics, and quantified sesquiterpenoid productions, such as atractylodin, \u003cem\u003eβ-\u003c/em\u003eeudesmol, total flavonoid, total polyphenolic acid, and transcriptional profiling of biosynthetic genes. By benchmarking metabolite yields against wild 3-year-old plants, this work bridges critical gaps in \u003cem\u003eA. chinensis\u003c/em\u003e biotechnology, enabling sustainable production of its pharmacologically vital compounds. Additionally, melatonin (MT) and two chemical homologs of MT, 5-Methoxyindole (5-MI) and 5-Methoxytryptamine (5-MT) were used to treat \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots, and detected the expression levels of genes related to secondary metabolic pathways and the content of secondary metabolites. We explore the changes in the composition of metabolites in \u003cem\u003eA. chinensis\u003c/em\u003e induced by MT and chemical homologs, which enrich the understanding of the important role of MT induced plant production of secondary metabolites.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plant Materials\u003c/h2\u003e\u003cp\u003eThe sterile seedlings of \u003cem\u003eAtractylodes chinensis\u003c/em\u003e (DC.) Koidz were saved in our laboratory. Seeds and wild \u003cem\u003eA. chinensis\u003c/em\u003e were collected in Chifeng City, Inner Mongolia, China (geographic coordinates: 41\u0026deg;17\u0026prime;10\u0026Prime;N \u0026ndash; 45\u0026deg;24\u0026prime;15\u0026Prime;N, 116\u0026deg;21\u0026prime;07\u0026Prime;E \u0026ndash; 120\u0026deg;58\u0026prime;52\u0026Prime;E) in September 2022.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Establishment of hairy roots transformation\u003c/h2\u003e\u003cp\u003ePetiole and leaf explants of \u003cem\u003eA. chinensis\u003c/em\u003e were prepared: leaves were sectioned into 1 cm \u0026times; 1 cm fragments, and petioles were trimmed to 1 cm lengths. Explants were pre-cultured on MS solid medium (pH 5.8) in darkness at 25\u0026deg;C for 12 h. \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e strains ATCC15834, R1000 and LBA9402 were cultured in YEB medium until reaching OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6 at 28\u0026deg;C. Pre-treated explants were transferred in \u003cem\u003eA. tumefaciens\u003c/em\u003e containing 300 \u0026micro;M Acetosyringone and co-cultivated at 28\u0026deg;C (150 rpm) for 30 min. After co-cultivation, tissues were transferred to MS solid medium supplemented with 500 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cefotaxime (cef) and incubated in darkness at 25\u0026deg;C for 7 d. The tissues were washed with sterile water and replaced onto fresh MS solid medium every week, which the concentration of cef was gradually reduced to 0 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e`\u003c/p\u003e\u003cp\u003eTotal genomic DNA of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots were extracted using a Plant Genomic DNA Kit (Tiangen, Beijing, WI, China). Specific primers of \u003cem\u003erolB\u003c/em\u003e and \u003cem\u003erolC\u003c/em\u003e were designed to detected the Ri plasmid of \u003cem\u003eA. tumefaciens\u003c/em\u003e (Shkryl et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Djerdjouri et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (Table S1). After PCR amplification, the PCR reaction mixtures were loaded directly onto 1% agarose gel for electrophoretic analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Optimization of medium of hairy roots\u003c/h2\u003e\u003cp\u003eOnce the hairy roots grew out, the roots were transferred on 1/2 MS solid medium and kept in the darkness at 25\u0026deg;C. However, the growth was slow. To increase the growth rate of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots, different combination of hormones was added to 1/2 MS or 6,7-V solid medium, which were 1/2 MS\u0026thinsp;+\u0026thinsp;0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT, 1/2 MS\u0026thinsp;+\u0026thinsp;0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT, 1/2 MS\u0026thinsp;+\u0026thinsp;0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 6-BA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT, 6,7-V\u0026thinsp;+\u0026thinsp;0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT, 6,7-V\u0026thinsp;+\u0026thinsp;0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT and 6,7-V\u0026thinsp;+\u0026thinsp;0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 6-BA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT. Each medium was supplemented with 3% or 5% sucrose. About 0.08 g FW of hairy roots were transferred to 90 mL liquid medium in 150 mL erlenmeyer flasks, and cultured for 60 d prior to biomass assessment to determine the optimal medium for growing \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots.\u003c/p\u003e\u003cp\u003eAbout 0.3 g FW of hairy roots were cultured in 90 mL 1/2 MS\u0026thinsp;+\u0026thinsp;0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT\u0026thinsp;+\u0026thinsp;5% sucrose medium in 150 mL erlenmeyer flasks. Fresh weight (FW) was recorded at 0, 8, 16, 24, 32, 36, 40, 44, 48 and 52 d to establish growth kinetics.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Determination of metabolites of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots were selected at 0, 8, 16, 24, 32, 36, 40, 44, 48 and 52 d. Hairy roots were washed and dried at 37\u0026deg;C. For determinating of atractylodin and \u003cem\u003eβ\u003c/em\u003e-eudesmol, 0.2 g hairy roots were added 4 mL 70% methanol for 12 h. The extraction was centrifuged for 8 min at 10,000 \u0026times; \u003cem\u003eg\u003c/em\u003e to collect the supernatant. After filtering through a 0.22 mm PTFE membrane, the samples were detected by High Performance Liquid Chromatography (HPLC). Atractylodin and \u003cem\u003eβ-\u003c/em\u003eeudesmol chemical molecular standard samples (Sigma) were prepared according to the concentration gradient. Atractylodin and \u003cem\u003eβ-\u003c/em\u003eeudesmol were quantified based on standard curves (Fig. S1 and S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter grinding, 0.05 g hairy roots were added 1 mL 70% ethanol for ultrasonic extraction for 2 h at 40 \u0026deg; C. The Folin-C assay was used to detect total polyphenolic acid (Kupina et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The total polyphenolic acid was quantified based on gallic acid standard curves (Fig. S3). The total flavonoid was measured by Aluminum chloride method (Chang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and was quantified based on rutin standard curves (Fig. S4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Real time-quantitative PCR (RT-qPCR) analysis and statistical analysis\u003c/h2\u003e\u003cp\u003eAccording to the growth curve of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots, hairy roots at 8, 24 and 52 d were collected, and the wild 1-year-old roots of \u003cem\u003eA. chinensis\u003c/em\u003e were as the control. The key genes involved in the biosynthesis of \u003cem\u003eβ\u003c/em\u003e-eudesmol (\u003cem\u003eAcOGF30G6\u003c/em\u003e), and genes involved in the biosynthesis of atractylodin (\u003cem\u003eAcZFP706\u003c/em\u003e, \u003cem\u003eAcCTA12\u003c/em\u003e, \u003cem\u003eAcPSPTA25520\u003c/em\u003e, \u003cem\u003eAcGTF25\u003c/em\u003e) were used for RT-qPCR, and the \u003cem\u003eAcactin\u003c/em\u003e was as the control gene (Zhang et al. 2024) (Table S1). Total RNA was extracted according to the MiniBEST Universal RNA Extraction Kit (TaKaRa, Beijing, China). First-strand cDNA was performed according to the PrimeScript\u0026trade; RT Reagent Kit with gDNA Eraser (TaKaRa, Beijing, China). The RT-qPCR reaction system and procedures were referred to the TB Green Premix Ex TaqⅡ kit (TaKaRa, Beijing, China). The 2\u003csup\u003e\u0026minus;ΔΔt\u003c/sup\u003e method was used to calculate the relative expression of genes. Each sample has three independent biological replicates. SPSS 23.0 was used to determine the statistical significance (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Compounds treatments\u003c/h2\u003e\u003cp\u003eN-acetyl-5-Methoxytryptamine (melatonin, MT), 5-Methoxytryptamine (5-MT) and 5-Methoxyindole (5-MI) (Solarbio, Beijing, China) were used in this study. The \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots P-6 were treated with these three compounds. After 52 d of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots cultivation, different compounds (MT, 5-MI and 5-MT) with different concentrations (0, 0.01, 0.05, 0.1, 0.2, and 0.5 mmol \u0026middot; L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were added to medium to continue cultivating hairy roots. Samples were taken at 168 h to detect the biomass accumulation of hairy roots and contents of \u003cem\u003eβ-\u003c/em\u003eeudesmol, total polyphenolic acid and total flavonoid. Samples were taken at 0, 12, 72 and 168 h for RT-qPCR. Each sample has three independent biological replicates.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 The induction and identification of \u003cem\u003eAtractylodes chinensis\u003c/em\u003e hairy roots\u003c/h2\u003e\u003cp\u003eThe sterile seedlings of \u003cem\u003eA. chinensis\u003c/em\u003e preserving in the laboratory were used. The explants were infected by the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strains ATCC15834, R1000 and LBA9402, respectively. The induction rates were first calculated, which \u003cem\u003eA. tumefaciens\u003c/em\u003e strain LBA9402 did not induce \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots. And induction rate of R1000 was low, being 5%, however, the hairy roots were death quick. Therefore, we focused on the hairy roots induced by ATCC15834, with an induction rate of 85\u0026ndash;91% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After infection by ATCC15834, hairy roots were observed to grow at the petioles wound site at 20 d in 1/2 MS solid medium used \u003cem\u003eA. chinensis\u003c/em\u003e petioles. At 80 d, the hairy roots extended and the number of roots increased. And the biomass of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots increased extremely at 170 d. On the other side, hairy roots were observed at the leaf wound site at 50 d used \u003cem\u003eA. chinensis\u003c/em\u003e leaves. At 130 d, the length of hairy roots increased, and branches were observed. And the branches of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots increased about 2\u0026ndash;3 cm at 170 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To determine the growing hairy roots induced by \u003cem\u003eA. tumefaciens\u003c/em\u003e, we designed specific primers (\u003cem\u003erolB\u003c/em\u003e and \u003cem\u003erolC\u003c/em\u003e) and used hairy roots DNA as templates for PCR amplification. The results showed that \u003cem\u003erolB\u003c/em\u003e and \u003cem\u003erolC\u003c/em\u003e genes were detected in \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), illustrating that the \u003cem\u003eA. tumefaciens\u003c/em\u003e Ri plasmid was successfully transferred into \u003cem\u003eA. chinensis\u003c/em\u003e to induce hairy roots.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferent \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strains induced hairy roots rates of petiole and leaf explants.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eA. tumefaciens\u003c/em\u003e strains\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003ePetiole\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eLeaf\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber of infections\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNumber of hairy roots\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInduction rate (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNumber of infections\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNumber of hairy roots\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInduction rate (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eATCC15834\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e91.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e85.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eR1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLBA9402\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 The determination of the optimal medium for growing \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots\u003c/h2\u003e\u003cp\u003eBecause the hairy roots grew slowly in 1/2 MS medium, six hairy roots amplification medium with different concentrations of sucrose (3%, 5%) were mixed to screen the optimal medium for growing \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots. After adding hormones KT with IAA, or NAA, or 6-BA to the 1/2 MS solid medium and culturing with 3% sucrose for 60 d, there were no significant changes. On the contrary, \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots grew well after adding 5% sucrose for 60 d, with the effect of adding IAA, NAA, and 6-BA to the same concentration of KT on the accumulation of hairy root biomass being IAA\u0026thinsp;\u0026gt;\u0026thinsp;NAA\u0026thinsp;\u0026gt;\u0026thinsp;6-BA. On the other side, based on the 6,7-V solid medium, adding NAA and 6-BA to the same concentration of KT resulted in higher biomass accumulation in 3% sucrose than in 5% sucrose, and adding 5% sucrose to KT and IAA resulted in higher biomass accumulation than in 3% sucrose (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). According to the above results, our conclusion is that the order of the effect of adding IAA, NAA, and 6-BA to the same concentration of KT at 5% sucrose on the biomass accumulation of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots was: IAA\u0026thinsp;\u0026gt;\u0026thinsp;NAA\u0026thinsp;\u0026gt;\u0026thinsp;6-BA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, the biomass of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots were also measured to select the optimal medium for growing hairy roots intuitively. The accumulation of biomass in 5% sucrose is generally higher than in 3% sucrose except B2 and B3. And the optimal hormone combination was 0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA\u0026thinsp;+\u0026thinsp;0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT. Finally, \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots accumulated more biomass in 1/2 MS than in 6,7-V solid medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Our results showed that 1/2 MS solid medium supplemented with 0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA, 0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT, and 5% sucrose was the most effective medium combination in increasing the value of hairy roots of \u003cem\u003eA. chinensis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eDue to the induction of hairy roots by leaves (L-1) and petioles (P-6 and P-14) of \u003cem\u003eA. chinensis\u003c/em\u003e. We measured growth curve of these three lines for 52 d, which weighed FW of hairy roots. With the passage of time, the biomass of hairy roots in all three lines increased remarkedly at 52 d (Fig. S5). However, the growth rate of different lines varies. The growth of L-1 was rapid from 8 to 44 d, and tended to stabilize after 44 d. P-6 had two rapid growth phases, including 16\u0026ndash;24 d and 48\u0026ndash;52 d. And the growth of P-14 was rapid from 0 to 48 d. Overall, among the three lines of hairy roots, the P-6 had the highest biomass accumulation at 52 d, reaching 3.7 g FW. The biomass accumulation of L-1 was the lowest at 52 d, reaching 2.7 g FW. And P-14 was 3.2 g at 52 d. In addition, changes in sucrose concentration in the medium were measured (Fig. S6). There was a significant difference before 24 d of sucrose concentration. After decreasing, there was no significant difference changes from 24 to 36 d. The sucrose concentration decreased from 36 to 40 d, and there was no significant difference in sucrose concentration changes from 40 to 48 d.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Analysis of metabolites of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots\u003c/h2\u003e\u003cp\u003eWe measured atractylodin, \u003cem\u003eβ-\u003c/em\u003eeudesmol, total polyphenolic acids and total flavonoid of three induced lines to further determine the line with the highest efficiency in inducing metabolites. Firstly, the main metabolites of atractylodin and \u003cem\u003eβ\u003c/em\u003e-eudesmol in \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots of different lines at different time. Due that the hairy roots on the 0 d were separated from the later stage of the growth cycle, therefore, the hairy roots of 0 d were not included in the comparison of material contents with other time. There were no significant differences of atractylodin contents of L-1 and P-14. The contents of atractylodin in P-6 at 52 d were significantly higher than other time, reached 1.76 mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, we also compared three lines contents of atractylodin at 52 d with the wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results showed that the content of atractylodin in P-6 was significantly higher than the other two lines, which was no significant difference compared to wild 3-year-old plants.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe determination of atractylodin contents of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots at different time.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLines\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e\u003cp\u003eAtractylodin contents (mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e32 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e40 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e44 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e48 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e52 d\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003eNotes: Each value represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). The lower-case letters indicate significant differences among different times via ANOVA followed by Duncan's post-test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, we also determined \u003cem\u003eβ\u003c/em\u003e-eudesmol contents. The \u003cem\u003eβ\u003c/em\u003e-eudesmol contents showed a trend of first decreasing and then increasing at 8\u0026ndash;52 d, which L-1 and P-6 reached the lowest contents at 32 d, and P-14 reached the lowest contents at 16 d. The contents of \u003cem\u003eβ\u003c/em\u003e-eudesmol in L-1 increased sharply from 32 to 36 d, which reached the maximum at 36 d, and was no significant change at 36\u0026ndash;52 d. The contents of \u003cem\u003eβ\u003c/em\u003e-eudesmol in P-6 increased significantly from 36 to 40 d, and was no significant change at 40\u0026ndash;52 d. The accumulation of \u003cem\u003eβ\u003c/em\u003e-eudesmol in P-14 was significantly higher at 52 d than other time (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, the \u003cem\u003eβ\u003c/em\u003e-eudesmol contents of three lines at 52 d were compared with the wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Although the \u003cem\u003eβ-\u003c/em\u003eeudesmol contents of hairy roots was significantly lower than control, the P-6 induced by petioles was significantly higher than L-1 induced by leaves.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe determination of \u003cem\u003eβ\u003c/em\u003e-eudesmol contents of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots at different time.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLines\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e\u003cp\u003e\u003cem\u003eβ\u003c/em\u003e-eudesmol contents (mg\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e32 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e40 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e44 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e48 d\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e52 d\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e7.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e7.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e7.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 \u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 \u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e7.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003eNotes: Each value represents the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). The lower-case letters indicate significant differences among different times via ANOVA followed by Duncan's post-test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTotal phenolic acids and total flavonoids were determined and analyzed. The results showed that total phenolic acids of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots had no significant change at different time expect L-1 at 8 d and P-14 at 8 and 16 d (Fig.\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS7\u003c/span\u003e A). And the total flavonoids in P-14 was significantly higher than the other two lines and wild 3-year-old plants (Fig.\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS7\u003c/span\u003e B). On the other side, the total flavonoids of three \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots were no significant at different time (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). And the total flavonoids of L-1 were significantly lower than control and other two lines, while P-6 and P-14 at 52 d were no significant difference compared to wild 3-year-old plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 The expression levels of genes related to metabolites synthesis of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots\u003c/h2\u003e\u003cp\u003eIn order to analyze the ability of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots to synthesize metabolites at the molecular level, the key genes involved in the biosynthesis of \u003cem\u003eβ\u003c/em\u003e-eudesmol (\u003cem\u003eAcOGF30G6\u003c/em\u003e), and genes involved in the biosynthesis of atractylodin (\u003cem\u003eAcZFP706\u003c/em\u003e, \u003cem\u003eAcCTA12\u003c/em\u003e, \u003cem\u003eAcPSPTA25520\u003c/em\u003e, \u003cem\u003eAcGTF25\u003c/em\u003e) were used for RT-qPCR. The expression of \u003cem\u003eAcOGF30G6\u003c/em\u003e were significantly up-regulated of three lines at 52 d compared to wild 1-year plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The trends of \u003cem\u003eAcZFP706\u003c/em\u003e, \u003cem\u003eAcPSPTA25520\u003c/em\u003e and \u003cem\u003eAcGTF25\u003c/em\u003e increased with the growth of hairy roots in three lines, which \u003cem\u003eAcPSPTA25520\u003c/em\u003e increased significantly of three lines at 52 d compared to wild 1-year plants. \u003cem\u003eAcZFP706\u003c/em\u003e in line L-1 and \u003cem\u003eAcGTF25\u003c/em\u003e in line P-14 increased significantly of three lines at 52 d compared to control. The expression level of \u003cem\u003eAcCTA12\u003c/em\u003e in line P-14 at 52 d increased significantly compared to control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Different concentrations of MT, 5-MI and 5-MT treated to the \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots\u003c/h2\u003e\u003cp\u003eCompared to hairy roots induced from leaves, those induced from petioles exhibited greater biomass accumulation and higher metabolic productivity, particularly for atractylodin and total flavonoids. Among the petiole-derived lines, the P-6 line displayed significantly higher atractylodin content than P-14 and was therefore selected for compound treatments. To investigate the effects of MT, 5-MT, and 5-MI on sesquiterpenoid accumulation in \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots, the P-6 line was treated with varying concentrations (0, 0.01, 0.05, 0.1, 0.2, and 0.5 mmol\u0026middot;L⁻\u0026sup1;) of each compound. Growth (biomass) and the contents of \u003cem\u003eβ\u003c/em\u003e-eudesmol, total phenolic acids, and total flavonoids were measured. At 168 hours post-treatment (hpt), the biomass of the treated hairy roots showed no significant differences compared to the control (Fig. S8). Analysis of the main sesquiterpenoid metabolite, \u003cem\u003eβ\u003c/em\u003e-eudesmol, revealed that its levels in the hairy roots were significantly lower than in wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Treatment with 0.5 mmol\u0026middot;L⁻\u0026sup1; 5-MI significantly increased the \u003cem\u003eβ\u003c/em\u003e-eudesmol content to 17.37 mg\u0026middot;g⁻\u0026sup1; DW (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, treatments with MT and 5-MT across all concentrations induced no significant changes in \u003cem\u003eβ\u003c/em\u003e-eudesmol levels at 168 hpt. Conversely, treatment with 0.5 mmol\u0026middot;L⁻\u0026sup1; 5-MI significantly decreased the levels of total phenolic acids and total flavonoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). Treatments with MT and 5-MT at all concentrations resulted in no significant changes in these metabolites at 168 hpt. The increase induced by 5-MI compensated for the initially lower β-eudesmol content in the hairy roots. Specifically, the β-eudesmol level at 168 hpt after 0.5 mmol\u0026middot;L⁻\u0026sup1; 5-MI treatment was significantly higher than that in wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Furthermore, this treatment significantly upregulated the expression of \u003cem\u003eAcOGF30G6\u003c/em\u003e, a key gene involved in β-eudesmol biosynthesis (Fig. S9).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe conservations of Traditional Chinese Medicine germplasm resources currently mainly include methods such as seed propagation, artificial cultivation, plant tissue cultivation, and hairy roots cultivation. As an important Traditional Chinese Medicine, \u003cem\u003eAtractylodes chinensis\u003c/em\u003e (DC.) Koidz. has not been reported to be induced hairy roots, which is unfavorable for its researches and reproduction. Medicinal plants produce a plenty of phytocompounds such as cyclopeptides, quinones, alkaloids, flavonoids and terpenes, which are mainly used for pharmaceutical and flavor industries (Roy, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Miao et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Commercial source of these metabolites is field-grown plants, which are generally influenced by seasonal changes and regional differences. Hairy roots culture is considered an alternative method of high-value secondary metabolites for industrial production, which is mass reproduction in a short period of time (Shao et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, we established the hairy roots cultivation system of \u003cem\u003eA. chinensis\u003c/em\u003e efficiently, including determination of the medium, selection of explants and definition of cultivation time.\u003c/p\u003e\u003cp\u003eThe establishment of the hairy root cultivation system requires \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e, which contains a special plasmid that induces rooting (Chandra \u0026amp; Chandra, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The efficiency of inducing hairy roots varies among different strains of \u003cem\u003eA. rhizogenes\u003c/em\u003e. The strains ATCC15834, R1000, A4, and C58 of \u003cem\u003eA. rhizogenes\u003c/em\u003e were used to induce hairy roots of \u003cem\u003eTrigonella foenumgraecum\u003c/em\u003e, which A4 was more conducive to the accumulation of diosgenin in hairy roots than other strains (Zolfaghari et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, A4 strain showed a better transformation frequency compared with ATCC15384, LBA9402, and ACCC10060 to induce \u003cem\u003eRubia yunnanensis\u003c/em\u003e hairy roots (Miao et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). ATCC15834 was found the most efficient strain for \u003cem\u003eTrachyspermum ammi\u003c/em\u003e L. hairy roots induction (Vamenani et al. 2020). The C58C1 was the best strain for inducing \u003cem\u003eAtractylodes lancea\u003c/em\u003e hairy roots than A4, K599 and LBA9402 (Zhang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Other researches indicated that different plants infected by the same strain of \u003cem\u003eA. rhizogenes\u003c/em\u003e, the induction of hairy roots varies. The induction rates of hairy roots between the \u003cem\u003eScutellaria orientalis\u003c/em\u003e and \u003cem\u003eS. araxensis\u003c/em\u003e infected with ATCC15834, which were 93.3% and 56.6%, respectively (Gharari et al. 2020). In this study, \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots were successfully induced by \u003cem\u003eA. rhizogenes\u003c/em\u003e ATCC15834, which the induction rate was highest compared with \u003cem\u003eA. rhizogenes\u003c/em\u003e R1000 and LBA9402, reaching 85\u0026ndash;91%. However, the growth of hairy roots was slow in MS solid medium, which the biomass increased extremely at 170 d. Therefore, it is urgently to optimize the hairy roots culture medium for faster growth.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that changing medium composition (Yosephine et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) or adding exogenous hormones (Putalun et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) increased the contents of secondary metabolites in hairy roots. Different combinations of exogenous hormones were added to induce different hairy roots. Medium supplemented with 1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA and 1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BA induced \u003cem\u003ePhysalis minima\u003c/em\u003e L. hairy roots efficiently (Putalun et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Hairy roots of \u003cem\u003eA. lancea\u003c/em\u003e, also belonging to the \u003cem\u003eAsteraceae\u003c/em\u003e crops, were grown on MS0 solid medium supplemented with 3% sucrose, 1.0 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 6-BA, and 0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAA for two months (Zhang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Therefore, adding exogenous hormones and sucrose were shorten the growth cycle of hairy roots effectively. Sucrose had commonly been used as the carbohydrate source for hairy root growth, and proclaimed superior to other carbohydrate sources such as glucose, mannitol and sorbitol (Banerjee et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this study, we designed 6 combinations of medium with 3% or 5% sucrose to determine the optimal medium for growing \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots. Combining morphological and biomass growth, 1/2 MS solid medium supplemented with 0.5 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA, 0.1 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KT, and 5% sucrose was the most effective medium combination.\u003c/p\u003e\u003cp\u003eHairy roots cultivation has been developed as an innovative approach for the large-scale production of secondary metabolites and plant chemicals, which was cultivated large number of secondary metabolites in a short period of time and continuously supplied high-value products (Korde et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). And the yield of effective components of secondary metabolites produced by plants takes a long time, which were harvested after 3\u0026ndash;5 years of growth to accumulate secondary metabolites a relatively high level (Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Ran et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The main active ingredients of \u003cem\u003eA. chinensis\u003c/em\u003e are volatile oils, eucalyptol and guaiacol sesquiterpenes, and polyacetylenes, which are considered characteristic phytochemicals (Meng et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Among them, atractylone, \u003cem\u003eβ-\u003c/em\u003eeudesmol, atractylodinol and atractylodin are main active ingredients (Wang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, we focused on atractylodin and \u003cem\u003eβ\u003c/em\u003e-eudesmol, which possess anti-inflammatory, antibacterial, antiviral and anti-tumor effects (Kimura \u0026amp; Sumiyoshi, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Cheng et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). And the contents of these two sesquiterpenoids compounds were similar with Zhang et al. (Zhang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), which the atractylodin contents in hairy roots was significantly higher than normal roots, and \u003cem\u003eβ\u003c/em\u003e-eudesmol contents in hairy roots was lower than normal roots. Moreover, we found that the two lines induced by petioles, especially P-6, produced more metabolites than hairy roots induced by leaves (L-1), including atractylodin, \u003cem\u003eβ\u003c/em\u003e-eudesmol, total polyphenolic acids and total flavonoid. And we were surprised to find that there was no significant difference in the content of atractylodin produced by P-6 compared to the wild 3-year-old plants, reached 1.76 mg/g. Although the \u003cem\u003eβ\u003c/em\u003e-eudesmol of P-6 hairy roots (7.24 mg/g) was significantly lower than control plants (9.53 mg/g) in Inner Mongolia, it was higher than 5-year-old plants (1.61 mg/g) collected in Good Agricultural Practice database, Liuhe City, Changchun, China Changchun City (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which were caused by regional differences. Moreover, the expression levels of genes related to \u003cem\u003eβ\u003c/em\u003e-eudesmol and atractylodin synthesis of \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots were significantly up-regulated at 52 d compared to wild 1-year plants. These results demonstrated that the contents of secondary metabolites produced by hairy roots that induced by petioles were consistent with wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e. We estimated profits based on effective content, and the profit of hairy roots was much higher than field planting.\u003c/p\u003e\u003cp\u003eTo specifically enhance \u003cem\u003eβ-\u003c/em\u003eeudesmol content in \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots, this study explored the application of chemical inducers. Melatonin (MT), a well-established inducer, is known to promote the accumulation of various plant secondary metabolites (Wei et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For instance, treatment of cabbage with 100 \u0026micro;mol/L MT upregulated anthocyanin biosynthesis genes and stimulated anthocyanin accumulation (Luo et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, MT-mediated nitric oxide (NO) production has been shown to enhance isoflavone accumulation by boosting phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) activity and upregulating key biosynthetic genes (Yin et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, in our experiments, treatment with varying concentrations of MT failed to significantly induce \u003cem\u003eβ\u003c/em\u003e-eudesmol accumulation in \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots. We therefore evaluated two chemical homologs of MT, 5-methoxyindole (5-MI) and 5-methoxytryptamine (5-MT). Notably, only treatment with 0.5 mmol\u0026middot;L⁻\u0026sup1; 5-MI resulted in a significant increase in \u003cem\u003eβ\u003c/em\u003e-eudesmol content. This induced level reached 17.37 mg\u0026middot;g⁻\u0026sup1; DW, representing a 1.82-fold increase compared to levels found in wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e roots. These findings demonstrate that 5-MI acts as an effective inducer for significantly improving \u003cem\u003eβ\u003c/em\u003e-eudesmol content, a key quality marker, in \u003cem\u003eA. chinensis\u003c/em\u003e hairy roots. This enhancement holds substantial promise for increasing the commercial value and profitability of \u003cem\u003eA. chinensis\u003c/em\u003e cultivation utilizing hairy root technology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.B. and B.S. conceived the research. J.W., K.W., and X.L. conducted the research. X.L., B.S., and Z.B. analyzed the data. Z.B. and B.S. wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Inner Mongolia Autonomous Region Science and Technology Plan Project (Program No. 2022YFDZ0015), the Shaanxi Provincial Department of Education Research Program (Program No. 23JK0722), and the Doctoral Scientific Research Start-up Program of Yan\u0026rsquo;an University (Program No. YAU202303846).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBai Z, Wu J, Huang W et al (2020) The ethylene response factor SmERF8 regulates the expression of SmKSL1 and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots. 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Front Plant Sci 14:1237800\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZolfaghari F, Rashidi-Monfared S, Moieni A, Abedini D, Ebrahimi A (2020) Improving diosgenin production and its biosynthesis in Trigonella foenum-graecum L. hairy root cultures. Industrial Crops Prod 145:112075\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Atractylodes chinensis (DC.) Koidz, hairy roots, atractylodin, β-eudesmol, 5-Methoxyindole","lastPublishedDoi":"10.21203/rs.3.rs-7670264/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7670264/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAtractylodes chinensis\u003c/em\u003e (DC.) Koidz., a pharmacologically significant medicinal plant with various metabolites, produces sesquiterpenoids like atractylodin and \u003cem\u003eβ\u003c/em\u003e-eudesmol that dictate its medicinal quality and underpin its clinical efficacy. While hairy root systems offer industrial potential for metabolite production, no such system existed for \u003cem\u003eA. chinensis\u003c/em\u003e. Here, we report the successful establishment of an efficient hairy root induction system for \u003cem\u003eA. chinensis\u003c/em\u003e using \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e ATCC15834 with leaf and petiole explants, achieving higher induction rates than strains R1000 30 and LBA9402. Optimal growth occurred on 1/2 MS solid medium supplemented with 0.5 mg\u0026middot;L-1 IAA, 0.1 mg\u0026middot;L-1 KT, and 5% sucrose, yielding maximal biomass and metabolite accumulation over a 52-day culture period. Among the established hairy root lines, those induced from petioles (particularly line P-6) exhibited superior biomass and significantly higher levels of key metabolites, including atractylodin, \u003cem\u003eβ\u003c/em\u003e-eudesmol, total polyphenolic acids, and total flavonoids at day 52, compared to the leaf-induced line (L-1). Transcriptomic analysis confirmed the key genes involved in \u003cem\u003eβ\u003c/em\u003e-eudesmol and atractylodin biosynthesis upregulated relative to wild plants. Furthermore, treatment of the hairy roots with melatonin (MT) or its chemical homologs (5- methoxyindole, 5-MI; 5-methoxytryptamine, 5-MT) demonstrated that only 0.5 mmol\u0026middot;L⁻\u0026sup1; 5-MI significantly enhanced \u003cem\u003eβ\u003c/em\u003e-eudesmol production. Remarkably, the \u003cem\u003eβ\u003c/em\u003e-eudesmol content achieved at 168 hours post-treatment was 1.82-fold higher than that in wild 3-year-old \u003cem\u003eA. chinensis\u003c/em\u003e roots. This study establishes a robust platform for \u003cem\u003eA. chinensis\u003c/em\u003e hairy root culture and highlights 5-MI as a potent inducer, providing a valuable foundation for the industrial scale production of its medicinally active compounds.\u003c/p\u003e","manuscriptTitle":"Establishment of Hairy Root Culture System in Atractylodes chinensis for Enhanced Production of Medicinal Sesquiterpenoids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 09:16:29","doi":"10.21203/rs.3.rs-7670264/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fcd4a473-1350-4948-90f5-8dc61af87320","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-22T12:35:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 09:16:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7670264","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7670264","identity":"rs-7670264","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}