Influence of salicylic acid, methyl jasmonate, and yeast elicitors on a shikonin level in hairy root culture of Echium khuzestanicm

Influence of salicylic acid, methyl jasmonate, and yeast elicitors on a shikonin level in hairy root culture of Echium khuzestanicm | 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 Influence of salicylic acid, methyl jasmonate, and yeast elicitors on a shikonin level in hairy root culture of Echium khuzestanicm Bahar Kazeminezhad, khosro piri, Reyhaneh Dehghan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6564667/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 Purpose Elicitors are powerful agents for enhancing the production of secondary metabolites in plants by stimulating specific cellular pathways. This study investigates Echium khuzestanicum , a native Iranian herb, with the innovative approach of utilizing a temporary immersion bioreactor in combination with biotic and abiotic elicitors to optimize growth rates and maximize shikonin production. Methods We conducted a series of treatments in a temporary immersion culture to increase the shikonin content in hairy roots. The treatments included salicylic acid, methyl jasmonate, yeast, and combinations of these compounds. Various treatment protocols were implemented using the immersion bioreactor system, which involved different cycles for immersion and drought periods. Results Our results indicate that the most effective treatment was a drying period of 45 minutes followed by 15 minutes of immersion. Notably, methyl jasmonate at a concentration of 150 µM produced the highest growth rate and shikonin yield. The combination of 150 µM methyl jasmonate with 500 µM salicylic acid further enhanced secondary metabolite synthesis. While yeast at 1000 mg/L increased shikonin content, it did not significantly influence growth rates. Conclusion These findings highlight the innovative use of temporary immersion bioreactors alongside targeted elicitors as a promising strategy to enhance secondary metabolite production in Echium khuzestanicum , thereby contributing valuable insights for future research in plant biotechnology. Echium khuzestanicum Hairy root Shikonin Temporary immersion system Elicitors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The growing global reliance on medicinal plants has sparked a surge in research aimed at exploring their therapeutic properties. This interest is largely driven by the discovery of bioactive compounds from these plants, which hold significant potential for pharmaceutical applications (Ahmadi et al. 2014). Among the innovative extraction techniques developed in recent years, the use of hairy root cultures—established through the inoculation of Agrobacterium rhizogenes —has emerged as a particularly effective method. These cultures provide a stable, hormone-free environment that enhances the yield of important secondary metabolites (Ziv 2010 ; Biswas et al. 2023 ) To scale up the production of these active pharmaceutical compounds, bioreactor technology plays a crucial role. Notably, it facilitates the efficient production of significant metabolites like shikonin and gynoids (Tripathi et al. 2003; Yazaki et al. 2017). In this context, the "temporary immersion system" represents a cost-effective approach that optimizes plant propagation by maximizing the contact between plant tissues and a fluid culture medium. This integration not only accelerates growth but also enhances metabolite production (Lyam 2012; Gianguzzi et al. 2024). A particularly noteworthy plant in this arena is Echium khuzestanicum , a native herb in Iran recognized for its shikonin production. Shikonin is a red naphthoquinone pigment celebrated for its antibacterial, anti-inflammatory, and potential anti-cancer properties (Senthilkumar 2014; Yadav et al. 2022 ). Given the growing therapeutic significance of shikonin, there is an increasing need to explore biotechnological methods to enhance its production. Recent research indicates that specific elicitors, such as salicylic acid and methyl jasmonate, can significantly boost the production of secondary metabolites. These compounds activate plant stress signaling pathways, thereby promoting higher yields (Huang et al. 2021 ). Additionally, yeast extract serves as an essential nutrient enhancer, further driving metabolite production in plant cell cultures (Tao et al. 2023 ). Furthermore, bioreactors equipped with liquid inlet and outlet systems provide precise control over critical culture parameters—such as pH, oxygenation, and gas solubility—ensuring optimal growth conditions (Paek et al. 2005 ; Tao et al. 2023 ). In light of these advancements, this study aims to assess the alone and combined effects of salicylic acid, methyl jasmonate, and yeast extract on the transgenic hairy roots of Echium khuzestanicum . By optimizing shikonin production within a temporary immersion bioreactor, we aspire to significantly enhance the efficacy and yield of this valuable compound, contributing to advancements in the biotechnological applications of medicinal plants. 2. Materials and Methods 2.1 Preparation of Hairy Roots Hairy roots of Echium khuzestanicum were generated using Agrobacterium rhizogenes strain R1000, sourced from the (No. 39299). The strain was activated by growing it in Luria-Bertani broth at 28C° for 24 hours until it reached OD 600 = 0.6, indicating suitable growth for inoculation. The transformed plant material was inoculated with the Agrobacterium culture for 48 hours. After 14 days of cocultivation, the presence of hairy roots was confirmed by PCR analysis using specific primers targeting the role C gene. Hairy roots were then cultured in Murashige and Skoog (MS) medium, subcultured every two weeks under controlled conditions (light, 16 hours/8 hours light/dark cycle, temperature of 25 C°) until they reached sufficient biomass for experimentation (Fig. 1 ). 2.2 Temporary Immersion Bioreactor Device A temporary immersion bioreactor, consisting of two interconnected enclosures, was employed to allow efficient fluid movement. The Petri dish culture with hairy roots was placed in one enclosure, while the other held the liquid medium. A timer-controlled air pump facilitated medium transfer from the bottom to the top enclosure, promoting intermittent immersion. The airflow rate was maintained at 3 L/min. To evaluate growth responses, the bioreactor was operated for four cycles with three repetitions over a total period of 72 hours (Fig. 3 ). 2.3 Investigating the Effect of Time on Growth and Secondary Metabolite Production In this experiment, the flooding duration was constant at 15 minutes across all trials, with varying pump stand-by durations of 15, 30, 45, and 60 minutes. A control setup featuring continuous immersion was also implemented for comparative analysis (Pavlov et al. 2006). Elicitors were introduced after 24 hours of culturing the hairy roots in the bioreactor to assess their impact during the exponential phase of growth. 2.4 Elicitors' Effects on Growth Rate and Secondary Metabolite Content Three elicitors were applied to enhance secondary metabolite production: Salicylic Acid : 125, 250, and 500 µM, plus control. Methyl Jasmonate : 150 µM (single concentration). Yeast Extract : 250, 500, and 1000 mg/L, with a control. The treatment applications were allocated in a completely randomized design. During treatment, the hairy roots were weighed to determine fresh weight before elicitor application, and the elicitors were carefully added to the liquid medium in a sterile environment. 2.5 Growth Assessment The growth of hairy roots was monitored by measuring fresh weight and dry weight before and after the treatment periods. These measurements were taken at the beginning, during, and at the end of the 72-hour culture period to assess changes and calculate growth rates. 2.6 Drying and Extraction After 72 hours of treatment, the hairy roots were harvested. They were dried at laboratory temperature in the dark for 48 hours to determine dry weight (Borovaya et al. 2014 ). Shikonin was extracted according to a modified protocol by Hayashi et al. ( 1998 ). 2.7 High-Performance Liquid Chromatography (HPLC) Method The quantification of shikonin in the samples was conducted using a High-Performance Liquid Chromatography (HPLC) system (MC Company, model Bfrl, equipped with a UV detector, sy-8200). The column specifications included a length of 250 mm, a diameter of 4.6 mm, and a pore size of 5 µM. The mobile phase was acetonitrile and water in a 70:30 ratio. The HPLC analysis was performed at a flow rate of 1 mL/min with a detection wavelength of 515 nm. 2.8 Statistical Analysis Data were analyzed using one-way analysis of variance (ANOVA) to determine significant differences in growth rates and shikonin production across different treatments and times (p < 0.01). All statistical analyses were performed using SAS 9.2 software, and all experiments were conducted in triplicate. 3. Results 3.1 The Influence of Immersion Duration and Aridity on Hairy Root Development of E. khuzestanicum Analysis of variance indicated significant differences in hairy root growth related to aridity durations (F = 91.07, p < 0.01). The optimal growth was observed with a 15-minute immersion followed by 45 minutes of aridity, leading to the highest growth rate. Conversely, the lowest growth rate occurred with the treatment involving 15 minutes of immersion and 60 minutes of aridity. Notably, all treatments, aside from the 15-minute immersion with a 30-minute drought, did not exhibit significant differences when compared to the control (Table 1 ). Table 1 Growth Rates of Hairy Roots at Varying Immersion and Aridity Intervals TIME (minute) Growth rate (mg) Control (total immersion) 12.0 c 15:15 0.056 c 30:15 0.16 b 45:15 0.30 a 60:15 0.06 c Means with the same letter are not significantly different (p < 0.01). 3.2 The Impact of Elicitors on Hairy Root Growth of E. khuzestanicum 3.2.1 Effects of Salicylic Acid and Methyl Jasmonate on Hairy Root Growth Elicitors exhibited a significant influence on hairy root growth (F = 125.42, p < 0.01). The highest growth rate was achieved with 150 µM of methyl jasmonate, with a further increase in growth when combined with 500 µM salicylic acid. In contrast, the lowest growth was recorded at 125 µM salicylic acid, which showed comparability to the control sample (Table 2 ). Table 2 Effects of Salicylic Acid and Methyl Jasmonate on Hairy Root Growth Rates Methyl jasmonate and Salicylic acid (µM) Growth rate (mg) Salicylic acid (Control) 0.054 d Salicylic acid (125) 0.07 d Salicylic acid (250) 0.068 d Salicylic acid (500) 0.18 c Salicylic acid + Methyl Jasmonate 0.34 b Methyl Jasmonate (150) 0.39 a Control for Methyl Jasmonate 0.07 d Means with the same letter are not significantly different (p < 0.01). 3.2.2 Effects of Yeast Concentrations on Hairy Root Growth The analysis indicated that adding yeast did not enhance hairy root growth (F = 26047, p < 0.01). The control sample exhibited the highest growth, while lower concentrations of yeast (250 mg and 500 mg) recorded minimal growth (Table 3 ). Table 3 Effects of Yeast Concentrations on Growth Rates of Hairy Roots Yeast (mg) Growth rate (mg) Control 0.06 a 250 0.029 c 500 0.0026 c 1000 0.034 b Means with the same letter are not significantly different (p < 0.01). 3.3 Comparison of Dry Weight and Shikonin Production Under Various Elicitor Treatments and Immersion Times Significant differences were observed in both dry weight and shikonin content among various treatments with different elicitors, as summarized in Fig. 4 . The results indicate how specific elicitors and immersion times influence the growth of hairy roots of E. khuzestanicum and shikonin production. Salicylic acid exhibited varied effects on dry weight and shikonin content depending on its concentration. At 250 µM, salicylic acid resulted in a dry weight of 30.10 mg and a shikonin content of 0.14 µL, which was higher than the lower concentrations (125 µM and 500 µM) (Figs. 5 and 6 ) that yielded lower dry weights (29.30 mg and 29.1 mg respectively) and shikonin contents (0.06 µL and 0.065 µL respectively). These results suggest that a moderate concentration of salicylic acid is beneficial for enhancing both root growth and shikonin production in hairy roots. Methyl jasmonate at a concentration of 150 µM significantly enhanced both dry weight and shikonin content. The treated roots achieved a dry weight of 31.05 mg alongside a shikonin content of 0.63 µL, indicating that MeJA effectively promotes growth while concurrently stimulating shikonin biosynthesis. The combination of salicylic acid and methyl jasmonate yielded the highest results in both parameters. The treatment with salicylic acid and methyl jasmonate together resulted in a dry weight of 32.50 mg and a shikonin content of 0.67 µL, surpassing the effects of either elicitor when applied alone (Fig. 7 ). This cooperative interaction suggests a synergistic effect, wherein the combined application of these elicitors enhances growth and metabolite production more effectively than their individual applications. Yeast extract also had a notable impact on shikonin production, although it showed a more limited direct effect on growth. For instance, the 1000 mg yeast treatment resulted in a considerable shikonin content of 0.55 µL (Fig. 8 ), supporting the notion that yeast can stimulate metabolite production despite not significantly enhancing root growth compared to the other elicitors. The corresponding dry weight for this treatment was 30.9 mg, illustrating that while yeast may not be the primary driver of growth, it contributes positively to shikonin accumulation. In assessing the impact of immersion time, total immersion resulted in a higher dry weight of 32.30 mg but yielded low shikonin content (0.07 µL). In contrast, the 45-minute drying period led to a dry weight of 32.05 mg and a significantly higher shikonin content of 0.52 µL (Fig. 9 ). This indicates that while prolonged immersion may favor biomass accumulation, alternating drying periods could promote conditions more conducive to shikonin production. 4. Discussion Elicitor effects on hairy root growth Methyl jasmonate is a key player in promoting growth and shikonin biosynthesis. As a potent stress mimic, MeJA elicits a cascade of physiological responses that stimulate cellular division and differentiation (Ndiaye et al. 2022 ). Its role as an elicitor is particularly pronounced under conditions that mimic environmental stress, which invariably impacts the growth dynamics of hairy roots. Research has shown that applying MeJA in a controlled environment can lead to significant increases in root biomass, primarily attributed to the activation of growth-related pathways and the upregulation of genes involved in root development (Đurić et al. 2023 ). The effectiveness of MeJA is particularly amplified in a wet immersion system, wherein sustained exposure allows continuous signaling for stress response pathways, fundamentally driving both root development and metabolite accumulation (Kikowska et al. 2022 ). In our study, the findings indicated a marked enhancement in growth rates concurrent with increased shikonin levels, reinforcing the notion that stress-induced signaling pathways are intricately linked to secondary metabolite production. Methyl jasmonate has been shown to play a role in inducing stress responses and enhancing shikonin biosynthesis in plants like Lithospermum erythrorhizon , Arnebia euchroma , and Triticum aestivum L. (Yazaki et al. 1997 ; Hawow et al. 2014; Saher et al. 2023). However, Syclovskaya-Barank et al. (2012) and Fang et al. ( 2016 ) found that methyl jasmonate decreased shikonin production in the hairy roots of Lithp spermumcanescens and Lithospermum erythrorhizon . Salicylic acid presents an alternative pathway for promoting hairy root growth. Recognized for its role in regulating vital physiological processes, SA enhances root health by improving water retention and nutrient uptake (Zhou et al. 2022 ). Observations show that at an optimal concentration of 500 µM, SA not only contributes to vigorous growth but also synergistically interacts with other elicitors, augmenting their effects. The mechanisms via which SA promotes growth likely involve the activation of defense-related pathways that enhance overall root vitality. This further complements the stress-mimicking effects of MeJA, creating a multifaceted approach to improving root growth (Gai et al. 2019 ).In other studies adding salicylic acid to the hairy roots of Azadirachta indica resulted in increased production of active substances (Prakash et al. 2008 ; Srivastava et al. 2014; Kaur et al. 2021 ). However, Shabani et al. ( 2009 ) and Souri and Tohidloo ( 2019 ) noted a reduction in the dry weight of licorice roots treated with salicylic acid. Yeast extract, while offering limited direct stimulation to root growth, plays an essential supportive role. Rich in vital nutrients and bioactive compounds, yeast extract contributes to the overall health of the hairy roots by enhancing metabolic activity. It is hypothesized that the indirect effects of yeast extract on growth stem from its ability to create a nutrient-rich environment that promotes overall biosynthetic processes (Taha et al. 2020 ). Though its primary function may not be growth stimulation, the presence of yeast extract can thus improve the health and vigor of the roots, thereby enhancing their capacity for secondary metabolite production. Elicitor effects on shikonin production When examining the effects of these elicitors on shikonin production, a similar pattern of interaction is evident. Methyl jasmonate stands out as a robust enhancer of shikonin synthesis. The ability of MeJA to stimulate secondary metabolism is well documented across various plant species, including notable efficacy in shikonin-producing plants such as Lithospermum erythrorhizon (Ahmad et al. 2022 ) and Arnebia euchroma (Wang et al. 2014 ). In our findings, the application of MeJA at 150 µM was shown to significantly enhance not only root growth but also shikonin production, suggesting a direct correlation between stress signaling and metabolic flux towards secondary metabolite synthesis (Malik et al. 2023 ). Salicylic acid also contributes significantly to the production of shikonin, with optimal levels observed at 250 µM. The role of SA centers on its capacity to activate defense mechanisms that promote secondary metabolite biosynthesis. By triggering pathways associated with stress responses, SA can induce the upregulation of genes involved in shikonin production (Song et al. 2023 ). This observation aligns with existing literature, which highlights SA’s critical role in enhancing the levels of protective compounds produced by the plant in response to biotic and abiotic stresses (Ang et al. 2024 ). In contrast, yeast extract, despite its less pronounced effect on root growth, proves valuable in enhancing shikonin levels. The metabolic stimulation attributed to yeast extract plays a crucial role in secondary metabolite synthesis. It is suggested that the bioactive compounds and nutrients present in yeast extract can induce stress responses, potentially through the generation of reactive oxygen species, which are known to play a role in the activation of metabolic pathways leading to secondary metabolite production, including shikonin (Goncharuk et al. 2022 ). This illustrates the nuanced interactions within the culture medium that contribute to the overall yield of shikonin. In another study, yeast extract has been found to increase the production of compounds in certain plants (Hassanloo et al. 2009; El-Beltagi et al. 2022 ). Interaction with Temporary Immersion Systems (TIS) The findings from this study highlight the significant impact of Temporary Immersion Systems (TIS) on the performance of MeJA, SA, and yeast extract for optimizing hairy root growth and shikonin synthesis. TIS provides a controlled environment that enhances nutrient and oxygen availability to the roots, ultimately leading to physiological benefits critical for root development (Mirzabe et al. 2022 ). The specific immersion regimen observed in our study—a 15-minute wet immersion followed by a 45-minute aridity period—has shown promising results in maximizing root growth and secondary metabolite production. This cyclical exposure mimics natural environmental conditions, allowing for efficient nutrient uptake during immersion while promoting gas exchange and recovery during aridity. Such conditions likely stimulate auxin production and other growth-promoting factors, facilitating enhanced root vitality and reinforcing the positive effects of the elicitors (Ozyigit et al. 2023 ). The interaction between the TIS and the elicitors is multi-dimensional. In a wet immersion system, MeJA exhibits enhanced effectiveness compared to dry applications. Continuous exposure to MeJA during the immersion period allows for sustained signaling that is vital for initiating the pathways involved in secondary metabolite biosynthesis, as well as promoting growth. Conversely, dry applications may lead to suboptimal results due to limited absorption and reduced activation of stress signaling pathways (Kikowska et al. 2022 ). Salicylic acid likewise benefits from application within a wet immersion framework. The hydration afforded by this system enhances root accessibility to SA, amplifying its physiological and biochemical effects on root health and shikonin synthesis. When utilized in dry conditions, the beneficial effects of SA are likely diminished, as its accessibility to the roots may be compromised (Santos et al. 2024 ). Furthermore, yeast extract substantially benefits from the TIS. In a wet immersion context, yeast can deliver essential nutrients and bioactive compounds more uniformly, promoting optimal root health and facilitating shikonin production. The continuous nutrient flow associated with TIS enhances the bioavailability of the extract, thereby allowing for greater absorption and resulting in a more pronounced effect on shikonin yield. In contrast, dry applications may not achieve the same degree of nutrient distribution or absorption, limiting the positive impact on both root growth and metabolite production (De Carlo et al. 2021 ). Overall, the data underscore the importance of both the type of elicitor and the application method in influencing hairy root growth and shikonin production. The combined use of salicylic acid and methyl jasmonate, alongside carefully controlled immersion timing, offers a promising strategy for optimizing yields in hairy root cultures of E. khuzestanicum . Further exploration of these parameters may lead to enhanced techniques for maximizing desired secondary metabolites in plant tissue cultures. Conclusion In conclusion, the synergy of the Temporary Immersion System with methyl jasmonate, salicylic acid, and yeast extract underscores the crucial interplay between duration, mode of application, and nutrient availability in optimizing hair root cultures for enhanced growth and shikonin production. The evidence presented in this study illustrates the potential for an integrated approach to refining elicitor application strategies, thereby maximizing the yield of valuable secondary metabolites such as shikonin. Future research may explore the precise molecular mechanisms underlying these interactions and investigate the applicability of these findings across different genotypes and environmental conditions. By optimizing these elicitor combinations in conjunction with TIS, we can enhance the efficiency of plant tissue culture processes, ultimately leading to improved outcomes in secondary metabolite production and agricultural practices. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contributions All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by [Bahar Kazemzadeh], [Khosro Piri] and [Reyhane Dehghan]. The first draft of the manuscript was written by [Bahar Kazemzadeh], and all authors commented on previous versions. All authors read and approved the final manuscript. Data availability All data generated or analyzed during this study are included in this published article. 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Yadav S, Sharma A, Nayik GA, Cooper R, Bhardwaj G, Sohal HS, Mutreja V, Kaur R, Areche FO, AlOudat M, Shaikh AM (2022) Review of shikonin and derivatives: isolation, chemistry, biosynthesis, pharmacology and toxicology. Front Pharmacol 13:905755. Yazaki K, Takeda K, Tabata M (1997) Effects of methyl jasmonate on shikonin and dihydroechinofuran production in Lithospermum cell cultures. J Plant Cell Physiol 38:776–782. https://doi.org/10.1093/oxfordjournals.pcp.a029235. Yazaki K (2017) Lithospermum erythrorhizon cell cultures: present and future aspects. J Plant Biotechnol 34(3):131-42. Ziv M (2010) Bioreactor technology for plant micropropagation. J Hortic Rev 24: 1-30. Zhou H, Ge H, Chen J, Li X, Yang L, Zhang H, Wang Y (2022) Salicylic acid regulates root gravitropic growth via clathrin-independent endocytic trafficking of PIN2 auxin transporter in Arabidopsis thaliana. Int J Mol Sci 23(16): 9379. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6564667","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470032375,"identity":"b506d351-f79b-4046-9f32-1df3326f88b1","order_by":0,"name":"Bahar Kazeminezhad","email":"","orcid":"","institution":"Bu Ali Sina University","correspondingAuthor":false,"prefix":"","firstName":"Bahar","middleName":"","lastName":"Kazeminezhad","suffix":""},{"id":470032376,"identity":"5387f8aa-93dc-4a1e-854b-eb3e87ab3e4b","order_by":1,"name":"khosro piri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYFACNjYwxc/AA+UTrUWygWQtBgd4iHSWOQNb2oMPf+7JGZ8/e0yCocaOgU/6AH4tlg1sxw1nthUbm93IS5NgOJbMwMaXgF+LwQH2NmnehoTEbTd4zCQY2A4wsBFyIFgLz5+ExM39Z4Ba/hGlhe2YNA9bQuIGhhwzCcY2IrQA/ZImObMtwVjiRl6yRWJfMg9BLcAQM5P48CdBjr//7MEbH77Zycn3EHKY/AMkXgIDA+HYMSCoYhSMglEwCkYBABGVNWXupNQ0AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9435-2378","institution":"Shahid Beheshti University","correspondingAuthor":true,"prefix":"","firstName":"khosro","middleName":"","lastName":"piri","suffix":""},{"id":470032377,"identity":"2a2b0b59-56bd-4e2f-b663-1c734c15a0de","order_by":2,"name":"Reyhaneh Dehghan","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"prefix":"","firstName":"Reyhaneh","middleName":"","lastName":"Dehghan","suffix":""}],"badges":[],"createdAt":"2025-04-30 11:47:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6564667/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6564667/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84678977,"identity":"8df3f6a8-8c31-4f68-abec-568dba03163a","added_by":"auto","created_at":"2025-06-16 08:04:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":408491,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHairy root samples under subculture.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/1a51a8064714f8cc0d0af413.png"},{"id":84680141,"identity":"97d454a2-b35e-4dde-aea5-4a29b1c24e48","added_by":"auto","created_at":"2025-06-16 08:12:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 3: Temporary Immersion Bioreactor Cycle.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/8fc790ce604fd813ec749a2c.png"},{"id":84678985,"identity":"74be4484-9bf2-414c-bb9e-e96e110ed999","added_by":"auto","created_at":"2025-06-16 08:04:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 4: Effect of time and different stimuli on shikonin production in hairy roots of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ekhuzestanicum\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/70303de837d3540dc38db147.png"},{"id":84680143,"identity":"3aaf243a-a6ea-4f56-95ed-b387cbd38d36","added_by":"auto","created_at":"2025-06-16 08:12:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 5: Standard shikonin peak curve in HPLC.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/2a0333fa51e52d29295d4ce0.png"},{"id":84680145,"identity":"baf571fb-a1b2-4bf2-ad5a-d23699f1f226","added_by":"auto","created_at":"2025-06-16 08:12:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 6: Peak diagram from the concentration of 250 μM of salicylic acid in the HPLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/e75288db255084adfd89eca2.png"},{"id":84680844,"identity":"108bbace-e2d4-4194-8ed9-f9e58f08a993","added_by":"auto","created_at":"2025-06-16 08:20:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 7: Peak diagram from the interaction of salicylic acid and methyl jasmonate in the HPLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/3374603e6df9b0b007d72a7c.png"},{"id":84679004,"identity":"18a91b67-5e65-4c76-9fa9-5ff79c74b016","added_by":"auto","created_at":"2025-06-16 08:04:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 8: Peak diagram from yeast at a concentration of 1000 mg in the HPLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/9ca75a65158ef53d47511488.png"},{"id":84678982,"identity":"b6c6b7aa-5b9a-4807-8403-ef1902b9a6ff","added_by":"auto","created_at":"2025-06-16 08:04:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":54675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 9: Peak diagram from 45 minutes of dry time and 15 minutes of immersion in the HPLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/60a3fce8adc91c820ebee0a6.png"},{"id":93788361,"identity":"057015c3-e4f6-4699-9a3e-d444039004c8","added_by":"auto","created_at":"2025-10-17 14:33:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2124428,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/1b969979-3c34-4a9d-b040-6b2588483e1d.pdf"},{"id":84678980,"identity":"90428f64-80d6-4f60-a0d5-bcea1e6aeb60","added_by":"auto","created_at":"2025-06-16 08:04:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":629268,"visible":true,"origin":"","legend":"","description":"","filename":"suply.docx","url":"https://assets-eu.researchsquare.com/files/rs-6564667/v1/e9a66e247cdc5fc2e0426393.docx"}],"financialInterests":"","formattedTitle":"Influence of salicylic acid, methyl jasmonate, and yeast elicitors on a shikonin level in hairy root culture of Echium khuzestanicm","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe growing global reliance on medicinal plants has sparked a surge in research aimed at exploring their therapeutic properties. This interest is largely driven by the discovery of bioactive compounds from these plants, which hold significant potential for pharmaceutical applications (Ahmadi et al. 2014). Among the innovative extraction techniques developed in recent years, the use of hairy root cultures\u0026mdash;established through the inoculation of \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e\u0026mdash;has emerged as a particularly effective method. These cultures provide a stable, hormone-free environment that enhances the yield of important secondary metabolites (Ziv \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Biswas et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTo scale up the production of these active pharmaceutical compounds, bioreactor technology plays a crucial role. Notably, it facilitates the efficient production of significant metabolites like shikonin and gynoids (Tripathi et al. 2003; Yazaki et al. 2017). In this context, the \"temporary immersion system\" represents a cost-effective approach that optimizes plant propagation by maximizing the contact between plant tissues and a fluid culture medium. This integration not only accelerates growth but also enhances metabolite production (Lyam 2012; Gianguzzi et al. 2024).\u003c/p\u003e \u003cp\u003eA particularly noteworthy plant in this arena is \u003cem\u003eEchium khuzestanicum\u003c/em\u003e, a native herb in Iran recognized for its shikonin production. Shikonin is a red naphthoquinone pigment celebrated for its antibacterial, anti-inflammatory, and potential anti-cancer properties (Senthilkumar 2014; Yadav et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given the growing therapeutic significance of shikonin, there is an increasing need to explore biotechnological methods to enhance its production.\u003c/p\u003e \u003cp\u003eRecent research indicates that specific elicitors, such as salicylic acid and methyl jasmonate, can significantly boost the production of secondary metabolites. These compounds activate plant stress signaling pathways, thereby promoting higher yields (Huang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, yeast extract serves as an essential nutrient enhancer, further driving metabolite production in plant cell cultures (Tao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, bioreactors equipped with liquid inlet and outlet systems provide precise control over critical culture parameters\u0026mdash;such as pH, oxygenation, and gas solubility\u0026mdash;ensuring optimal growth conditions (Paek et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn light of these advancements, this study aims to assess the alone and combined effects of salicylic acid, methyl jasmonate, and yeast extract on the transgenic hairy roots of \u003cem\u003eEchium khuzestanicum\u003c/em\u003e. By optimizing shikonin production within a temporary immersion bioreactor, we aspire to significantly enhance the efficacy and yield of this valuable compound, contributing to advancements in the biotechnological applications of medicinal plants.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of Hairy Roots\u003c/h2\u003e \u003cp\u003eHairy roots of \u003cem\u003eEchium khuzestanicum\u003c/em\u003e were generated using \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e strain R1000, sourced from the (No. 39299). The strain was activated by growing it in Luria-Bertani broth at 28C\u0026deg; for 24 hours until it reached OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6, indicating suitable growth for inoculation. The transformed plant material was inoculated with the Agrobacterium culture for 48 hours. After 14 days of cocultivation, the presence of hairy roots was confirmed by PCR analysis using specific primers targeting the role C gene. Hairy roots were then cultured in Murashige and Skoog (MS) medium, subcultured every two weeks under controlled conditions (light, 16 hours/8 hours light/dark cycle, temperature of 25 C\u0026deg;) until they reached sufficient biomass for experimentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Temporary Immersion Bioreactor Device\u003c/h2\u003e \u003cp\u003eA temporary immersion bioreactor, consisting of two interconnected enclosures, was employed to allow efficient fluid movement. The Petri dish culture with hairy roots was placed in one enclosure, while the other held the liquid medium. A timer-controlled air pump facilitated medium transfer from the bottom to the top enclosure, promoting intermittent immersion. The airflow rate was maintained at 3 L/min. To evaluate growth responses, the bioreactor was operated for four cycles with three repetitions over a total period of 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Investigating the Effect of Time on Growth and Secondary Metabolite Production\u003c/h2\u003e \u003cp\u003eIn this experiment, the flooding duration was constant at 15 minutes across all trials, with varying pump stand-by durations of 15, 30, 45, and 60 minutes. A control setup featuring continuous immersion was also implemented for comparative analysis (Pavlov et al. 2006). Elicitors were introduced after 24 hours of culturing the hairy roots in the bioreactor to assess their impact during the exponential phase of growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Elicitors' Effects on Growth Rate and Secondary Metabolite Content\u003c/h2\u003e \u003cp\u003eThree elicitors were applied to enhance secondary metabolite production:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSalicylic Acid\u003c/b\u003e: 125, 250, and 500 \u0026micro;M, plus control.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eMethyl Jasmonate\u003c/b\u003e: 150 \u0026micro;M (single concentration).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eYeast Extract\u003c/b\u003e: 250, 500, and 1000 mg/L, with a control.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe treatment applications were allocated in a completely randomized design. During treatment, the hairy roots were weighed to determine fresh weight before elicitor application, and the elicitors were carefully added to the liquid medium in a sterile environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Growth Assessment\u003c/h2\u003e \u003cp\u003eThe growth of hairy roots was monitored by measuring fresh weight and dry weight before and after the treatment periods. These measurements were taken at the beginning, during, and at the end of the 72-hour culture period to assess changes and calculate growth rates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Drying and Extraction\u003c/h2\u003e \u003cp\u003eAfter 72 hours of treatment, the hairy roots were harvested. They were dried at laboratory temperature in the dark for 48 hours to determine dry weight (Borovaya et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Shikonin was extracted according to a modified protocol by Hayashi et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 High-Performance Liquid Chromatography (HPLC) Method\u003c/h2\u003e \u003cp\u003eThe quantification of shikonin in the samples was conducted using a High-Performance Liquid Chromatography (HPLC) system (MC Company, model Bfrl, equipped with a UV detector, sy-8200). The column specifications included a length of 250 mm, a diameter of 4.6 mm, and a pore size of 5 \u0026micro;M. The mobile phase was acetonitrile and water in a 70:30 ratio. The HPLC analysis was performed at a flow rate of 1 mL/min with a detection wavelength of 515 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using one-way analysis of variance (ANOVA) to determine significant differences in growth rates and shikonin production across different treatments and times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). All statistical analyses were performed using SAS 9.2 software, and all experiments were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The Influence of Immersion Duration and Aridity on Hairy Root Development of \u003cem\u003eE. khuzestanicum\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAnalysis of variance indicated significant differences in hairy root growth related to aridity durations (F\u0026thinsp;=\u0026thinsp;91.07, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The optimal growth was observed with a 15-minute immersion followed by 45 minutes of aridity, leading to the highest growth rate. Conversely, the lowest growth rate occurred with the treatment involving 15 minutes of immersion and 60 minutes of aridity. Notably, all treatments, aside from the 15-minute immersion with a 30-minute drought, did not exhibit significant differences when compared to the control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eGrowth Rates of Hairy Roots at Varying Immersion and Aridity Intervals\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTIME (minute)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrowth rate (mg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (total immersion)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15:15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.056\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30:15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45:15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60:15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.06\u003csup\u003ec\u003c/sup\u003e\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 \u003cb\u003eMeans with the same letter are not significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The Impact of Elicitors on Hairy Root Growth of \u003cem\u003eE. khuzestanicum\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Effects of Salicylic Acid and Methyl Jasmonate on Hairy Root Growth\u003c/h2\u003e \u003cp\u003eElicitors exhibited a significant influence on hairy root growth (F\u0026thinsp;=\u0026thinsp;125.42, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The highest growth rate was achieved with 150 \u0026micro;M of methyl jasmonate, with a further increase in growth when combined with 500 \u0026micro;M salicylic acid. In contrast, the lowest growth was recorded at 125 \u0026micro;M salicylic acid, which showed comparability to the control sample (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\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\u003eEffects of Salicylic Acid and Methyl Jasmonate on Hairy Root Growth Rates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl jasmonate and Salicylic acid (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrowth rate (mg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid (Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.054\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid (125)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.07\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid (250)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.068\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid (500)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylic acid\u0026thinsp;+\u0026thinsp;Methyl Jasmonate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.34 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl Jasmonate (150)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl for Methyl Jasmonate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.07 \u003csup\u003ed\u003c/sup\u003e\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 \u003cb\u003eMeans with the same letter are not significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Effects of Yeast Concentrations on Hairy Root Growth\u003c/h2\u003e \u003cp\u003eThe analysis indicated that adding yeast did not enhance hairy root growth (F\u0026thinsp;=\u0026thinsp;26047, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The control sample exhibited the highest growth, while lower concentrations of yeast (250 mg and 500 mg) recorded minimal growth (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\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\u003eEffects of Yeast Concentrations on Growth Rates of Hairy Roots\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYeast (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGrowth rate (mg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.029\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0026\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.034\u003csup\u003eb\u003c/sup\u003e\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 \u003cb\u003eMeans with the same letter are not significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Comparison of Dry Weight and Shikonin Production Under Various Elicitor Treatments and Immersion Times\u003c/h2\u003e \u003cp\u003eSignificant differences were observed in both dry weight and shikonin content among various treatments with different elicitors, as summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The results indicate how specific elicitors and immersion times influence the growth of hairy roots of \u003cem\u003eE. khuzestanicum\u003c/em\u003e and shikonin production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSalicylic acid exhibited varied effects on dry weight and shikonin content depending on its concentration. At 250 \u0026micro;M, salicylic acid resulted in a dry weight of 30.10 mg and a shikonin content of 0.14 \u0026micro;L, which was higher than the lower concentrations (125 \u0026micro;M and 500 \u0026micro;M) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e) that yielded lower dry weights (29.30 mg and 29.1 mg respectively) and shikonin contents (0.06 \u0026micro;L and 0.065 \u0026micro;L respectively). These results suggest that a moderate concentration of salicylic acid is beneficial for enhancing both root growth and shikonin production in hairy roots.\u003c/p\u003e \u003cp\u003eMethyl jasmonate at a concentration of 150 \u0026micro;M significantly enhanced both dry weight and shikonin content. The treated roots achieved a dry weight of 31.05 mg alongside a shikonin content of 0.63 \u0026micro;L, indicating that MeJA effectively promotes growth while concurrently stimulating shikonin biosynthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe combination of salicylic acid and methyl jasmonate yielded the highest results in both parameters. The treatment with salicylic acid and methyl jasmonate together resulted in a dry weight of 32.50 mg and a shikonin content of 0.67 \u0026micro;L, surpassing the effects of either elicitor when applied alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This cooperative interaction suggests a synergistic effect, wherein the combined application of these elicitors enhances growth and metabolite production more effectively than their individual applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eYeast extract also had a notable impact on shikonin production, although it showed a more limited direct effect on growth. For instance, the 1000 mg yeast treatment resulted in a considerable shikonin content of 0.55 \u0026micro;L (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e), supporting the notion that yeast can stimulate metabolite production despite not significantly enhancing root growth compared to the other elicitors. The corresponding dry weight for this treatment was 30.9 mg, illustrating that while yeast may not be the primary driver of growth, it contributes positively to shikonin accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn assessing the impact of immersion time, total immersion resulted in a higher dry weight of 32.30 mg but yielded low shikonin content (0.07 \u0026micro;L). In contrast, the 45-minute drying period led to a dry weight of 32.05 mg and a significantly higher shikonin content of 0.52 \u0026micro;L (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This indicates that while prolonged immersion may favor biomass accumulation, alternating drying periods could promote conditions more conducive to shikonin production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cb\u003eElicitor effects on hairy root growth\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMethyl jasmonate is a key player in promoting growth and shikonin biosynthesis. As a potent stress mimic, MeJA elicits a cascade of physiological responses that stimulate cellular division and differentiation (Ndiaye et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Its role as an elicitor is particularly pronounced under conditions that mimic environmental stress, which invariably impacts the growth dynamics of hairy roots. Research has shown that applying MeJA in a controlled environment can lead to significant increases in root biomass, primarily attributed to the activation of growth-related pathways and the upregulation of genes involved in root development (Đurić et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effectiveness of MeJA is particularly amplified in a wet immersion system, wherein sustained exposure allows continuous signaling for stress response pathways, fundamentally driving both root development and metabolite accumulation (Kikowska et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In our study, the findings indicated a marked enhancement in growth rates concurrent with increased shikonin levels, reinforcing the notion that stress-induced signaling pathways are intricately linked to secondary metabolite production.\u003c/p\u003e \u003cp\u003eMethyl jasmonate has been shown to play a role in inducing stress responses and enhancing shikonin biosynthesis in plants like \u003cem\u003eLithospermum erythrorhizon\u003c/em\u003e, \u003cem\u003eArnebia euchroma\u003c/em\u003e, and \u003cem\u003eTriticum aestivum\u003c/em\u003e L. (Yazaki et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Hawow et al. 2014; Saher et al. 2023). However, Syclovskaya-Barank et al. (2012) and Fang et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that methyl jasmonate decreased shikonin production in the hairy roots of \u003cem\u003eLithp spermumcanescens\u003c/em\u003e and \u003cem\u003eLithospermum erythrorhizon\u003c/em\u003e .\u003c/p\u003e \u003cp\u003eSalicylic acid presents an alternative pathway for promoting hairy root growth. Recognized for its role in regulating vital physiological processes, SA enhances root health by improving water retention and nutrient uptake (Zhou et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Observations show that at an optimal concentration of 500 \u0026micro;M, SA not only contributes to vigorous growth but also synergistically interacts with other elicitors, augmenting their effects. The mechanisms via which SA promotes growth likely involve the activation of defense-related pathways that enhance overall root vitality. This further complements the stress-mimicking effects of MeJA, creating a multifaceted approach to improving root growth (Gai et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).In other studies adding salicylic acid to the hairy roots of \u003cem\u003eAzadirachta indica\u003c/em\u003e resulted in increased production of active substances (Prakash et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Srivastava et al. 2014; Kaur et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, Shabani et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Souri and Tohidloo (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) noted a reduction in the dry weight of licorice roots treated with salicylic acid.\u003c/p\u003e \u003cp\u003eYeast extract, while offering limited direct stimulation to root growth, plays an essential supportive role. Rich in vital nutrients and bioactive compounds, yeast extract contributes to the overall health of the hairy roots by enhancing metabolic activity. It is hypothesized that the indirect effects of yeast extract on growth stem from its ability to create a nutrient-rich environment that promotes overall biosynthetic processes (Taha et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Though its primary function may not be growth stimulation, the presence of yeast extract can thus improve the health and vigor of the roots, thereby enhancing their capacity for secondary metabolite production.\u003c/p\u003e \u003cp\u003e \u003cb\u003eElicitor effects on shikonin production\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhen examining the effects of these elicitors on shikonin production, a similar pattern of interaction is evident. Methyl jasmonate stands out as a robust enhancer of shikonin synthesis. The ability of MeJA to stimulate secondary metabolism is well documented across various plant species, including notable efficacy in shikonin-producing plants such as \u003cem\u003eLithospermum erythrorhizon\u003c/em\u003e (Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and \u003cem\u003eArnebia euchroma\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In our findings, the application of MeJA at 150 \u0026micro;M was shown to significantly enhance not only root growth but also shikonin production, suggesting a direct correlation between stress signaling and metabolic flux towards secondary metabolite synthesis (Malik et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSalicylic acid also contributes significantly to the production of shikonin, with optimal levels observed at 250 \u0026micro;M. The role of SA centers on its capacity to activate defense mechanisms that promote secondary metabolite biosynthesis. By triggering pathways associated with stress responses, SA can induce the upregulation of genes involved in shikonin production (Song et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This observation aligns with existing literature, which highlights SA\u0026rsquo;s critical role in enhancing the levels of protective compounds produced by the plant in response to biotic and abiotic stresses (Ang et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, yeast extract, despite its less pronounced effect on root growth, proves valuable in enhancing shikonin levels. The metabolic stimulation attributed to yeast extract plays a crucial role in secondary metabolite synthesis. It is suggested that the bioactive compounds and nutrients present in yeast extract can induce stress responses, potentially through the generation of reactive oxygen species, which are known to play a role in the activation of metabolic pathways leading to secondary metabolite production, including shikonin (Goncharuk et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This illustrates the nuanced interactions within the culture medium that contribute to the overall yield of shikonin. In another study, yeast extract has been found to increase the production of compounds in certain plants (Hassanloo et al. 2009; El-Beltagi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInteraction with Temporary Immersion Systems (TIS)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe findings from this study highlight the significant impact of Temporary Immersion Systems (TIS) on the performance of MeJA, SA, and yeast extract for optimizing hairy root growth and shikonin synthesis. TIS provides a controlled environment that enhances nutrient and oxygen availability to the roots, ultimately leading to physiological benefits critical for root development (Mirzabe et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe specific immersion regimen observed in our study\u0026mdash;a 15-minute wet immersion followed by a 45-minute aridity period\u0026mdash;has shown promising results in maximizing root growth and secondary metabolite production. This cyclical exposure mimics natural environmental conditions, allowing for efficient nutrient uptake during immersion while promoting gas exchange and recovery during aridity. Such conditions likely stimulate auxin production and other growth-promoting factors, facilitating enhanced root vitality and reinforcing the positive effects of the elicitors (Ozyigit et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe interaction between the TIS and the elicitors is multi-dimensional. In a wet immersion system, MeJA exhibits enhanced effectiveness compared to dry applications. Continuous exposure to MeJA during the immersion period allows for sustained signaling that is vital for initiating the pathways involved in secondary metabolite biosynthesis, as well as promoting growth. Conversely, dry applications may lead to suboptimal results due to limited absorption and reduced activation of stress signaling pathways (Kikowska et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSalicylic acid likewise benefits from application within a wet immersion framework. The hydration afforded by this system enhances root accessibility to SA, amplifying its physiological and biochemical effects on root health and shikonin synthesis. When utilized in dry conditions, the beneficial effects of SA are likely diminished, as its accessibility to the roots may be compromised (Santos et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, yeast extract substantially benefits from the TIS. In a wet immersion context, yeast can deliver essential nutrients and bioactive compounds more uniformly, promoting optimal root health and facilitating shikonin production. The continuous nutrient flow associated with TIS enhances the bioavailability of the extract, thereby allowing for greater absorption and resulting in a more pronounced effect on shikonin yield. In contrast, dry applications may not achieve the same degree of nutrient distribution or absorption, limiting the positive impact on both root growth and metabolite production (De Carlo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverall, the data underscore the importance of both the type of elicitor and the application method in influencing hairy root growth and shikonin production. The combined use of salicylic acid and methyl jasmonate, alongside carefully controlled immersion timing, offers a promising strategy for optimizing yields in hairy root cultures of \u003cem\u003eE. khuzestanicum\u003c/em\u003e. Further exploration of these parameters may lead to enhanced techniques for maximizing desired secondary metabolites in plant tissue cultures.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, the synergy of the Temporary Immersion System with methyl jasmonate, salicylic acid, and yeast extract underscores the crucial interplay between duration, mode of application, and nutrient availability in optimizing hair root cultures for enhanced growth and shikonin production. The evidence presented in this study illustrates the potential for an integrated approach to refining elicitor application strategies, thereby maximizing the yield of valuable secondary metabolites such as shikonin. Future research may explore the precise molecular mechanisms underlying these interactions and investigate the applicability of these findings across different genotypes and environmental conditions. By optimizing these elicitor combinations in conjunction with TIS, we can enhance the efficiency of plant tissue culture processes, ultimately leading to improved outcomes in secondary metabolite production and agricultural practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting Interests\u003c/strong\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by [Bahar Kazemzadeh], [Khosro Piri] and [Reyhane Dehghan]. The first draft of the manuscript was written by [Bahar Kazemzadeh], and all authors commented on previous versions. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad M, Varela Alonso A, Koletti AE, Rodić N, Reichelt M, R\u0026ouml;del P, Assimopoulou AN, Paun O, Declerck S, Schneider C, Molin EM (2022) Dynamics of alkannin/shikonin biosynthesis in response to jasmonate and salicylic acid in \u003cem\u003eLithospermum officinale\u003c/em\u003e. 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Res 2(2):243-253. https://10.4314/tjpr.v2i2.14607.\u003c/li\u003e\n\u003cli\u003eWang S, Guo LP, Xie T, Yang J, Tang JF, Li X, Wang X, Huang LQ (2014) Different secondary metabolic responses to MeJA treatment in shikonin-proficient and shikonin-deficient cell lines from \u003cem\u003eArnebia euchroma (Royle) Johnst\u003c/em\u003e. Plant Cell Tissue Organ Cult (PCTOC) 119:587-98.\u003c/li\u003e\n\u003cli\u003eYadav S, Sharma A, Nayik GA, Cooper R, Bhardwaj G, Sohal HS, Mutreja V, Kaur R, Areche FO, AlOudat M, Shaikh AM (2022) Review of shikonin and derivatives: isolation, chemistry, biosynthesis, pharmacology and toxicology. Front Pharmacol 13:905755.\u003c/li\u003e\n\u003cli\u003eYazaki K, Takeda K, Tabata M (1997) Effects of methyl jasmonate on shikonin and dihydroechinofuran production in Lithospermum cell cultures. J Plant Cell Physiol 38:776\u0026ndash;782. https://doi.org/10.1093/oxfordjournals.pcp.a029235.\u003c/li\u003e\n\u003cli\u003eYazaki K (2017) \u003cem\u003eLithospermum erythrorhizon\u003c/em\u003e cell cultures: present and future aspects. J Plant Biotechnol 34(3):131-42. \u003c/li\u003e\n\u003cli\u003eZiv M (2010) Bioreactor technology for plant micropropagation. J Hortic Rev 24: 1-30.\u003c/li\u003e\n\u003cli\u003eZhou H, Ge H, Chen J, Li X, Yang L, Zhang H, Wang Y (2022) Salicylic acid regulates root gravitropic growth via clathrin-independent endocytic trafficking of PIN2 auxin transporter in Arabidopsis thaliana. Int J Mol Sci 23(16): 9379.\u003c/li\u003e\n\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":"Echium khuzestanicum, Hairy root, Shikonin, Temporary immersion system, Elicitors","lastPublishedDoi":"10.21203/rs.3.rs-6564667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6564667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eElicitors are powerful agents for enhancing the production of secondary metabolites in plants by stimulating specific cellular pathways. This study investigates \u003cem\u003eEchium khuzestanicum\u003c/em\u003e, a native Iranian herb, with the innovative approach of utilizing a temporary immersion bioreactor in combination with biotic and abiotic elicitors to optimize growth rates and maximize shikonin production.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe conducted a series of treatments in a temporary immersion culture to increase the shikonin content in hairy roots. The treatments included salicylic acid, methyl jasmonate, yeast, and combinations of these compounds. Various treatment protocols were implemented using the immersion bioreactor system, which involved different cycles for immersion and drought periods.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur results indicate that the most effective treatment was a drying period of 45 minutes followed by 15 minutes of immersion. Notably, methyl jasmonate at a concentration of 150 \u0026micro;M produced the highest growth rate and shikonin yield. The combination of 150 \u0026micro;M methyl jasmonate with 500 \u0026micro;M salicylic acid further enhanced secondary metabolite synthesis. While yeast at 1000 mg/L increased shikonin content, it did not significantly influence growth rates.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings highlight the innovative use of temporary immersion bioreactors alongside targeted elicitors as a promising strategy to enhance secondary metabolite production in \u003cem\u003eEchium khuzestanicum\u003c/em\u003e, thereby contributing valuable insights for future research in plant biotechnology.\u003c/p\u003e","manuscriptTitle":"Influence of salicylic acid, methyl jasmonate, and yeast elicitors on a shikonin level in hairy root culture of Echium khuzestanicm","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 08:04:20","doi":"10.21203/rs.3.rs-6564667/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":"7c9d6086-f447-4f35-984c-f07b61326039","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-17T14:25:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 08:04:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6564667","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6564667","identity":"rs-6564667","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}