Elucidating the role of different culture medium on in vitro plant regeneration of Valeriana jatamansi Jones ex Roxb. a high value medicinal plant of the temperate Himalayas

Elucidating the role of different culture medium on in vitro plant regeneration of Valeriana jatamansi Jones ex Roxb. a high value medicinal plant of the temperate Himalayas | 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 Elucidating the role of different culture medium on in vitro plant regeneration of Valeriana jatamansi Jones ex Roxb. a high value medicinal plant of the temperate Himalayas Geken Riba, Babita Patni, Rajeev Ranjan Kumar, Devesh Jangpangi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9539107/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Valeriana jatamansi (Tagar) is a threatened Himalayan medicinal plant facing population decline due to habitat degradation and overexploitation . This study aimed to develop an efficient micropropagation protocol and evaluate biochemical attributes under in vitro and field conditions. A completely randomized design (CRD) was used to compare Murashige and Skoog (MS) and Gamborg’s B5 media, with data analyzed using two-way ANOVA and DMRT, revealing significant effects (p ≤ 0.01) of basal media and plant growth regulators (PGRs). Gamborg’s B5 medium showed superior performance, with maximum shoot proliferation (3 ± 0.81 shoots per explant) and leaf number (11.33 ± 4.71) at 2.0 mg L⁻¹ BAP + 0.5 mg L⁻¹ IAA. Root induction was optimal on MS medium with IBA, producing the highest root number (35.66 ± 9.8) at 4.0 mg L⁻¹ and maximum root length (4.33 ± 0.94 cm) at 3.0 mg L⁻¹. Field-grown plants exhibited higher carotenoids, soluble sugars, starch, and antioxidant activity, whereas in vitro cultures showed higher amino acid content (5.44 ± 0.08 mg g⁻¹ FW). These findings indicate that B5 medium is superior for in vitro regeneration, providing a reliable protocol for large-scale propagation and conservation of V. jatamansi . Valeriana jatamansi Direct organogenesis Gamborg’s medium Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message Optimized micropropagation of threatened Himalayan via Gamborg’s B5 medium achieves superior shoot proliferation; biochemical profiling reveals distinct vs. field metabolite profiles for effective conservation. Introduction Valeriana is a genus with about 200 distributed worldwide and these plants are used to treat various ailments on the place where the plant is endemic. Valeriana species have been used for generations. These species are often utilized in traditional medicine systems, particularly in areas where they are naturally found. The use of Valeriana species dates back centuries, with historical records documenting their medicinal applications in multiple cultural contexts. The most important commercial species are V. offincinalis, V. wallichii , V. fauriei and V. edulis. ( Houghton, 1997 ). Indian valerian Valeriana jatamansi Jones ex Roxb. also known as Valeriana wallichii DC. belongs to the Valerianaceae family and is an important medicinal and aromatic plant in the temperate Himalayan area. Indian valerian is commonly referred to as Tagar or Sugandhbala in Sanskrit, and Mushkbala in Hindi. It thrives in subtropical to temperate regions, growing naturally at altitudes ranging from 1500 to 3000 meters. Natural populations of the species are found mostly as undercover on moist slopes along the streams ( Gautam et al., 2021 ). The plant can also be found in the eastern Himalayan region ( Raina & Negi, 2015 ). V. jatamansi roots are used as one of the ingredients in the preparation of snake bites and it is also used for liver, kidney and spleen disorder. V. jatamansi is also used in various industries such as cosmetics and perfumery ( Charmakar et al., 2021 ). Important constituents from rhizomes and roots of V. jatamansi used by different industries are ( Wills et al., 2000 ) bakkenoloids type sesquiterpenoids, essential oils and phenolics ( Gautam et al., 2021 ). Overexploitation of the plant for industrial and pharmaceutical purposes has led to a decline in its wild populations. If the current rate of exploitation continues unchecked, the plant faces the risk of becoming extinct in the near future ( Nawchoo et al., 2012 ). V. jatamansi has been assessed as a Vulnerable (VU) species under the IUCN criteria due to overharvesting, habitat degradation, and restricted distribution in the Himalayan region ( Gowthami et al., 2021 ). Sustainable alternatives to wild collection are urgently needed to ensure the continued availability of this valuable species for industrial and pharmacological applications. In recent years, plant tissue culture has gained substantial importance in the propagation and preservation of medicinal and aromatic plants (MAPs), many of which are facing population decline due to anthropogenic pressures such as habitat fragmentation, overharvesting, climate change, and unregulated trade. The sustainable cultivation of these plants through plant tissue culture significantly reduces the dependency on wild populations, thereby alleviating pressure on natural ecosystems and contributing to biodiversity conservation. ( Cuce & Inceer 2024 ), ( Cuce et al., 2022 ), ( Inceer et al., 2022 ), ( Cüce & Karaismailoğlu, 2023 ), ( Cuce et al., 2025 ). In addition to propagation, plant tissue culture also serves as a platform for secondary metabolite enhancement through advanced biotechnological interventions such as elicitation, precursor feeding, and genetic transformation. These strategies have been successfully employed to boost the production of bioactive compounds in several commercially important plant species ( Murthy et al., 2014 ). Such approaches are essential for developing plant lines with improved compatibility and productivity for industrial applications, particularly in terms of biomass yield and the accumulation of pharmacologically active constituents. ( Fazili et al., 2022 ), However, the success of such in vitro applications is strongly influenced by the composition of the culture medium, which governs cell differentiation, organogenesis, and metabolite biosynthesis (Neumann et al. , 2005). Among the various commonly used basal media, Murashige and Skoog (MS) medium, developed in 1962, is widely used for general-purpose in vitro culture due to its high nutrient and salt content ( Murashige & Skoog 1962 ). Nevertheless, plant species exhibit varied responses to the same culture medium, and the optimization of medium composition including macro and micronutrients, vitamins, carbon sources, and growth regulators is crucial for enhancing growth and productivity in vitro ( Pierik 1997 ) ( George et al., 2007 ). Gamborg’s B5 medium, formulated in 1968 for the culture of soybean suspension cells, has been shown to be effective for certain plant species, especially for those sensitive to the high nitrogen content of MS medium. Comparative evaluations have highlighted Gamborg's B5 medium’s superior performance in promoting organogenesis and root induction in selected medicinal plants, suggesting its broader applicability across different plant species. ( Gamborg et al., 1968 ) ( Hussain et al., 2012 ) ( Verma et al., 2015 ), (Jacob et al. , 2005) . In the current investigation, V. jatamansi an important high-altitude medicinal herb endemic to the Himalayan region and valued for its essential oil and valepotriates was cultured under in vitro conditions using Murashige and Skoog (MS) medium and Gamborg’s B5 medium supplemented with varying concentrations and combinations of auxins and cytokinins. The objective was to determine the optimal medium for achieving high-frequency shoot proliferation, leaf development, and rooting. The experimental results revealed that Gamborg’s B5 medium demonstrated significantly higher efficacy compared to MS medium in promoting all observed growth parameters. This study is the first documented report highlighting the superior response of V. jatamansi to B5 medium, which not only contributes to the understanding of species-specific media requirements but also opens new research areas for more effective tissue culture protocols for this medicinally important species. The successful micropropagation of V. jatamansi using Gamborg’s B5 medium offers a reliable way to produce plants in large numbers, which can help both pharmaceutical industries and conservation efforts. Finally, the study strengthens the case for further exploration of alternative and species-specific media formulations for other endangered or commercially valuable medicinal plants. Materials and methods Plant material and explant inoculation Three- to four-year-old plants of V. jatamansi were procured from the Pothibasa field station (2200 m above sea level) of the High-Altitude Plant Physiology Research Centre, Hemvati Nandan Bahuguna Garhwal University, Srinagar, Uttarakhand. The plants were maintained in the glasshouse of the HAPPRC main research complex (550 m above sea level) at Srinagar, Uttarakhand. Healthy nodal segments of V. jatamansi were selected as explants (Fig. 5 B). Nodal segments were excised using sterilized scissors under aseptic conditions. The explants were initially washed under running tap water using fine mesh nets to prevent material loss and to remove surface debris. They were then trimmed to uniform sizes and rinsed thoroughly 3 to4 times with tap water, followed by washing with double-distilled water. Surface sterilization was done by treating with surfactant Tween-20 for 2 to 4 minutes. For antifungal treatment, the explants were cleaned with 0.1–0.5% Bavistin solution using a soft brush and subsequently soaked for 30 to 40 min. Following fungicide treatment, explants were thoroughly rinsed 5 to 6 times with double-distilled water to remove any residual chemicals. Nodal segments of V. jatamansi were treated with 0.5% (w/v) mercuric chloride (HgCl₂) for 5 minutes to establish an optimal surface sterilization protocol for in vitro culture initiation. Culture conditions Two basal media were used in this study: Murashige and Skoog (MS) medium ( Murashige & Skoog, 1962 ) and Gamborg’s B5 medium ( Gamborg et al., 1968 ), both supplemented with 3% (w/v) sucrose. Plant growth regulators (PGRs) were incorporated in different concentrations and combinations, namely T0: control (no PGR), T1: BAP 0.5 mg L − 1 , T2: BAP 1.0 mg L − 1 , T3: BAP 1.5 mg L − 1 , T4: BAP 2.0 mg L − 1 , T1A1: BAP 0.5 mg L − 1 + IAA 0.125 mg L − 1 , T2A2: BAP 1.0 mg L − 1 + IAA 0.25 mg L − 1 , T3A3: BAP 1.5 mg L − 1 + IAA 0.375 mg L − 1 , and T4A4: BAP 2.0 mg L − 1 + IAA 0.5 mg L − 1 . For rooting, regenerated shoots were transferred to MS medium supplemented with auxins: T0: control, T1: NAA 0.2 mg L − 1 , T2: IBA 0.2 mg L − 1 , T3: IAA 0.2 mg L − 1 , T4: IBA 2.0 mg L − 1 , T5: IBA 3.0 mg L − 1 , and T6: IBA 4.0 mg L − 1 . The pH of the media was adjusted to 5.8 and sterilized by autoclaving at 121°C and 105 kPa for 15 min and solidified with 0.7% (w/v) plant tissue culture grade agar. Cultures were maintained in a growth chamber at 24 ± 2°C, 50–60% relative humidity, under a 16/8 h light/dark photoperiod with a photosynthetic photon flux density of 60 µmol m⁻² s⁻¹ provided by cool daylight fluorescent lamps. The PGRs were added to the growth medium after adjusting the pH and before adding the gelling agent. Cultures were maintained in 250 mL Erlenmeyer flasks sealed with sterile cotton plugs wrapped in absorbent fabric. Rooted plantlets were removed from the culture vessels, washed, and transferred to pots containing sand, soil, perlite, cocopeat, and vermiculite in a 1:1:1 (v/v) ratio for hardening under greenhouse conditions (27°C, 90% humidity). All media components, PGRs, and chemicals were procured from HiMedia Pvt. Ltd., India. Chlorophyll and Carotenoid Estimation For pigment extraction, 50 mg of fresh leaf tissue from both in vitro –grown and field-grown plants of V. jatamansi was homogenized in 5 mL of absolute acetone and incubated in the dark to prevent pigment degradation. The homogenate was centrifuged at 3,000 rpm for 15 min, and the resulting supernatant was collected and diluted with an additional 10 mL of acetone. The absorbance of the extract was recorded at 480, 645, and 663 nm using a UV–VIS spectrophotometer. Chlorophyll a, chlorophyll b, and total carotenoid contents were quantified following the method of Arnon ( 1949 ), using the following equations: Chlorophyll a (mg L − 1 ) = 12.7 (A663) – 2.69 (A645) Chlorophyll b (mg L − 1 ) = 22.9 (A645) – 4.68 (A663) Total chlorophyll (mg L − 1 ) = 20.2 (A645) + 8.02 (A663) Total Carbohydrate Estimation The anthrone method was employed to estimate the total soluble sugar and starch contents in leaf tissues of V. jatamansi . Freshly harvested leaves (200 mg) from in vitro grown and field grown plants were extracted with 5 mL of 80% boiling ethanol and centrifuged at 3,000 rpm for 10 minutes. The resulting supernatant was used for the estimation of soluble sugars, while the pellet was reserved for starch quantification. Sucrose was used as the standard reference for both estimations. For total soluble sugar estimation, appropriate volumes of the ethanol supernatant were diluted to 1 mL with distilled water. To this, 4 mL of cold anthrone reagent (prepared by dissolving 200 mg of anthrone in 100 mL of 95% sulfuric acid) was added. The mixture was cyclomixed for 2–3 minutes and the absorbance was measured at 620 nm using a UV-VIS double beam spectrophotometer. All estimations were carried out in triplicates For starch estimation, the ethanol-insoluble pellet was resuspended in 5 mL of 52% perchloric acid and centrifuged at 3,000 rpm for 12 minutes. The supernatant was then diluted to 1 mL with distilled water, and 4 mL of anthrone reagent was added. After thorough mixing, absorbance was recorded at 620 nm. A standard curve was prepared using known concentrations of sucrose for quantification. Estimation of Reducing Power Assay 2.5 ml of methanol plant extract (1mg/ml in methanol) was mixed with 2.5 ml of sodium phosphate buffer (0.2M; pH 6.6) and 2.5 ml of potassium ferricyanide (1%) in a test tube. The test tubes were then covered by silver foil and placed in water bath at 50°C for 20 mins. The test tubes were then removed from the water bath and allowed to cool down in room temperature and then 2.5 ml of trichloro-acetic acid (TCA 10%) was added. The mixture was then centrifuged at 3000 rpm for 10 minutes. Different concentrations of 25µl to 125 µl supernatant, 1.25 ml distilled water and 250 µl ferric chloride (0.1%) was added to the test tubes. The test tubes mixture was mixed well and absorbance was taken at 700 nm using spectrophotometer. Estimation of total free amino acids Total amino acid (TAA) content was estimated using the ninhydrin method (Moore et al. , 1954) . An acetate buffer (pH 5.0) was prepared by dissolving 1.64 g of sodium acetate in 100 mL of distilled water. Ninhydrin reagent was prepared by dissolving 750 mg of ninhydrin in 26.25 mL of 80% ethanol and 11.25 mL of acetate buffer. Ethanolic extract (50 mg) was dissolved in 5 mL of ethanol, centrifuged at 4°C, 3000 rpm for 10 minutes, and the supernatant diluted to prepare 1 mL test solutions. To each, 2 mL of ninhydrin reagent was added, vortexed, and incubated at 100°C for 20–30 minutes. After cooling, absorbance was measured at 570 nm. TAA was calculated as mg/g fresh weight using a glycine standard curve. Statistical analysis The experiment was executed following a completely randomized design (CRD), to evaluate the effects of different plant growth regulators (PGRs) on the direct organogenesis of Valeriana jatamansi using two different culture media (MS and B5). For shoot proliferation study three nodal explants were transferred in one 250ml erlenmeyer flask and three flasks were used for each treatment. a factorial two-way analysis of variance (ANOVA) was used to evaluate the effects of two independent factors, namely types of growth medium and concentrations of PGRs on the in vitro regeneration capacity. Results were evaluated as mean ± SD. Significant differences for multiple comparisons were determined using Duncan’s multiple range test by SPSS 26.0, Correlation and box plot analysis was also done using R software version 2025.09.2. Results and Discussions Direct Organogenesis and Shoot Proliferation Shoot regeneration in Valeriana jatamansi was significantly influenced by both basal medium composition and plant growth regulator (PGR) treatments (Fig. 3 ). Two-way ANOVA confirmed that the main effects of medium and PGRs were highly significant for shoot number ( p ≤ 0.01) and shoot length ( p ≤ 0.001 for medium), while their interaction effects varied by parameter (Table 1). Box plot simulation showed higher growth potential across all shoot growth parameters (Fig. 1 ). Table 1. Interaction effects of basal medium composition and plant growth regulator (PGR) concentrations on the in vitro morphogenic response of Valeriana jatamansi . Experimental parameters Variance source of two-way ANOVA Interaction P × M Growth medium (M) PGR treatments (P) F P-value F P-value F P-value No. of shoots 11.560** 0.002 3.19** 0.008 1.21 NS 0.321 NS Shoot length 43.416*** 0.000 4.37** 0.001 2.33** 0.039 NS No. of leaves 9.58** 0.004 1.92 NS 0.086 1.16 NS 0.346 NS NS non-significant * P <0.05; ** P <0.01; *** P <0.001 Shoot Number The interaction between growth medium and PGR treatments for shoot number was non-significant ( F = 1.21; p = 0.321), indicating that the influence of the basal medium remained consistent across different hormonal regimes (Table 1). Consequently, the main effects were analyzed independently. When data were pooled across all PGR levels, Gamborg’s B5 medium produced a significantly higher mean shoot number (1.96 ± 0.97 per explant) compared to MS medium (1.33 ± 0.55 per explant) (Table 2 ). Among the PGR treatments, the highest shoot proliferation (2.33 ± 1.03 shoots per explant) was achieved using either 1.5 mg L − 1 BAP alone or the combination of 2.0 mg L − 1 BAP with 0.5 mg L − 1 IAA (Table 2 ) (Fig. 5 C). Mean separation using Duncan’s multiple range test confirmed that these treatments were statistically superior to the control (1.00 ± 0.00) (Fig. 5 E). Table 2 Main effects of basal media and plant growth regulators on the quantitative growth characteristics of Valeriana jatamansi under in vitro conditions. Factor Shoot number/explant Shoot length/explant Leaf number/explant Growth Medium MS 1.33 ± 0.55 1.69 ± 0.61 4.48 ± 2.81 Gamborg’s B5 1.96 ± 0.97 2.89 ± 1.07 7.11 ± 3.84 PGR Treatments (mg L − 1 ) Control BAP IAA 0.0 1.00 ± 0.00 a 1.00 ± 0.00 a 2.00 ± 0.00 a 0.5 0.0 1.16 ± 0.40 a 2.4 ± 0.87 b 5.16 ± 3.12 ab 1.0 0.0 1.5 ± 0.54 ab 3.11 ± 1.75 b 6.66 ± 4.13 b 1.5 0.0 2.33 ± 1.03 b 2.41 ± 0.80 b 5.5 ± 3.33 ab 2.0 0.0 1.66 ± 0.81 ab 2.33 ± 1.40 b 5.5 ± 4.18 ab 0.5 0.125 1.16 ± 0.40 a 2.08 ± 0.82 ab 6 ± 2.75 ab 1.0 0.5 2 ± 1.09 ab 2.63 ± 0.47 b 7.16 ± 3.81 b 1.5 2.0 1.66 ± 0.81 ab 2.16 ± 0.51 ab 5.66 ± 2.33 ab 2.0 0.5 2.33 ± 1.03 b 2.5 ± 1.04 b 8.5 ± 4.88 b Values represent mean ± SD. Means followed by distinct letters within a column indicate significant differences (DMRT, p ≤ 0.05). Data were recorded 5 weeks after the culture and represent a total of three replicates of 10 plants per treatment BAP 6-Benzylaminopurine, Indole-3-Acetic Acid. Shoot Length In contrast to shoot number, shoot elongation exhibited a significant interaction between the basal medium and PGR treatments ( F = 2.33; p = 0.039), indicating that the response to hormonal supplementation depended on the specific nutrient composition of the basal medium (Table 1). The longest shoots (4.33 ± 1.25 cm) were recorded on B5 medium supplemented with 1.0 mg L − 1 BAP, followed by B5 medium containing 2.0 mg L − 1 BAP (3.50 ± 0.00 cm). Across all PGR concentrations, shoots regenerated on B5 medium (2.89 ± 1.07 cm) were consistently longer than those on MS medium (1.69 ± 0.61 cm) (Table 2 ). Correlation analysis further supported these findings, revealing a strong positive association between BAP concentration and shoot proliferation in Gamborg’s B5 medium ( r = 0.86), (Fig. 2 A), whereas a weaker correlation was observed in MS medium ( r = 0.41), (Fig. 2 B). Leaf Number The interaction effect for leaf number was non-significant ( F = 1.16; p = 0.346), (Table 1). When pooled across PGR treatments, Gamborg’s B5 medium supported significantly higher leaf production (7.11 ± 3.84 leaves per explant) compared to MS medium (4.48 ± 2.81 leaves per explant) (Table 2 ). Among the hormonal treatments, the combination of 2.0 mg L − 1 BAP and 0.5 mg L − 1 IAA yielded the maximum leaf count (8.5 ± 4.88), (Fig. 5 C) which was significantly different from the control according to the DMRT (Table 2 ). A strong positive correlation was observed between leaf number and shoot length in both MS ( r = 0.81) and Gamborg’s B5 ( r = 0.79) media, (Fig. 2 ), indicating that enhanced shoot elongation is closely linked to increased leaf development under in vitro conditions. The superior performance of Gamborg’s B5 medium suggests that Valeriana jatamansi exhibits a preferential response to the nutrient composition of Gamborg’s formulation. Gamborg’s B5 medium contains relatively higher levels of potassium nitrate and additional nitrogen sources, including ammonium sulfate, which are known to enhance nitrogen assimilation, protein synthesis, and overall morphogenic competence in plant tissue cultures ( Aziz et al., 2010 ; Yin et al., 2017 ; Bari et al., 2022 ) . Furthermore, the elevated thiamine content of Gamborg’s B5 medium may support enzymatic activity and metabolic processes essential for active cell division and tissue differentiation (Goyer et al ., 2010; Fitzpatrick et al ., 2020; Vollmer et al., 2023 ). Comparable medium-specific responses have been reported in Bergenia ciliata , underscoring the importance of species-specific optimization of basal salt composition for efficient in vitro regeneration of threatened Himalayan medicinal plants (Kurmi et al ., 2020) Rooting and acclimatization. In direct organogenesis, rooting was induced in Gamborg’s B5 medium with treatments of BAP (0.5–2 mg L − 1 ) and BAP combined with IAA (0.5 mg L − 1 + IAA 0.125 mg L − 1 , 1 mg L − 1 + IAA 0.25 mg L − 1 ), (Fig. 5 F) while MS medium failed to induce rooting. These findings align with earlier studies demonstrating the role of nitrogen sources in improving root architecture in various species. For instance, ammonium sulfate has been linked to enhanced lateral root growth, while potassium nitrate supports nutrient uptake and antioxidant defences, improving plant resilience in challenging environments ( Rafi et al., 2018 ; Wang et al., 2016 ). This suggests that Gamborg’s B5 medium's formulation is well-suited to promoting effective rooting in tissue culture systems. Shoot-induced plantlets without roots were sub-cultured in MS medium supplemented with auxins (NAA, IBA, IAA) for root induction. No rooting occurred in the control or treatments with NAA (0.2 mg L − 1 ), IBA (0.2 mg L − 1 ), or IAA (0.2 mg L − 1 ). The maximum number of roots (35.66 ± 9.8 roots per explant) was obtained with IBA at a concentration of 4 mg L − 1 , followed by progressively lower root numbers at 3 mg L − 1 and 2 mg L − 1 concentrations of IBA, respectively. Maximum root length (3.66 ± 0.94 cm) and the least root induction time (21.66 ± 2.36 days) were achieved with IBA (3 mg L − 1 and 4 mg L − 1 , respectively) (Fig. 6 ) (Table 3 ). These results suggest that IBA is more effective than NAA and IAA for adventitious root induction in Valeriana jatamansi , which may be attributed to its conversion into active auxin (IAA) and its role in promoting root initiation processes ( Fattorini et al., 2017 ; Frick & Strader, 2018 ). Plantlets with well-developed roots were transferred to potting mixture containing cocopeat + perlite (1:1) in small clay pots under controlled condition. (27°c and 90% relative humidity) (Fig. 6 E). The plants achieved a height of 3–4 cm after four weeks of transplantation. After raising under controlled conditions plants were transferred to large pots containing soil, sand and farmyard manure (1:1:1) and were maintained in green house. Table 3 Influence of exogenous auxin supplementation on the in vitro rhizogenic response and root architecture of Valeriana jatamansi. Treatment No. of days for root emergence Rooting (%) No of roots (Per explant) Root length (cm) Control 0 ± 0 a 0% 0 ± 0 a 0 ± 0 a MS + NAA 0.2 L − 1 0 ± 0 a 0% 0 ± 0 a 0 ± 0 a MS + IBA 0.2 L − 1 0 ± 0 a 0% 0 ± 0 a 0 ± 0 a MS + IAA 0.2 L − 1 0 ± 0 a 0% 0 ± 0 a 0 ± 0 a MS + IBA 2 L − 1 24 ± 4.32 b 100% 19.33 ± 7.02 b 3.83 ± 1.17 b MS + IBA 3 L − 1 25 ± 4.08 b 100% 32.33 ± 5.73 c 4.33 ± 0.94 b MS + IBA 4 L − 1 21.66 ± 2.36 b 100% 35.66 ± 9.8 c 3.66 ± 0.94 b Values represent mean ± SD. Means followed by distinct letters within a column indicate significant differences (DMRT, p ≤ 0.05). Data were recorded 5 weeks after the culture and represent a total of three replicates of 10 plants per treatment BAP 6-Benzylaminopurine, Indole-3-Acetic Acid. Phytochemical analysis Pigment Analysis Pigment analysis revealed that field-grown plants contained significantly higher levels of chlorophyll a (2.80 ± 0.01 mg/g FW), chlorophyll b (4.41 ± 0.009 mg/g FW), (Fig. 4 A), and total carotenoids (336 ± 22.86 mg/g FW), (Fig. 4 B), compared to in vitro -grown plants. In contrast, in vitro -grown plants exhibited lower pigment concentrations, with chlorophyll a at 1.773 ± 0.004 mg/g FW, chlorophyll b at 2.932 ± 0.01 mg/g FW, and carotenoids at 167.2 ± 1.30 mg/g FW, (Table 4 ). Similar results could be found in studies conducted on various phytochemical comparison of field grown and in vitro plants. ( Talreja, 2011 ; Khan et al., 2021 ; Malik et al., 2020 ). This suggests that field conditions favor the synthesis of both chlorophyll a, chlorophyll b and carotenoids, possibly due to enhanced light exposure and nutrient availability. Table 4 Comparative evaluation of photosynthetic pigments and primary metabolite profiles in in vitro propagated and field-grown Valeriana jatamansi Jones. Chlorophyll a Field grown plants In vitro plants 2.80 ± 0.01 mg/g FW 1.773 ± 0.004 mg/g FW Chlorophyll b 4.41 ± 0.009 mg/g FW 2.932 ± 0.01 mg/g FW Carotenoids 336 ± 22.86 mg/g FW 167.2 ± 1.30 mg/g FW Total soluble sugar 31.25 ± 1.8 mg/g FW 14.5 ± 0.7 mg/g FW Total soluble starch 28.125 ± 1.6 mg/g FW 13.05 ± 0.6 mg/g FW Total amino acids 5.17 ± 0.18 mg/g FW 5.44 ± 0.08 mg/g FW FW: Fresh weight Total Carbohydrate estimation Total soluble sugar was found in higher amount in field grown plants (31.25 ± 1.8 mg/g FW) as compared to in vitro plants (14.5 ± 0.7 mg/g FW), (Fig. 4 C), (Table 4 ). Total soluble starch was found in higher quantity in the field grown plants (28.125 ± 1.6 mg/g FW) in comparison with in vitro grown plants (13.05 ± 0.6 mg/g FW), (Fig. 4 D), (Table 4 ). Similar results could be found in studies conducted on various phytochemical comparison of field grown and in vitro plants. ( Talreja, 2011 ; Khan et al., 2021 ; Malik et al., 2020 ). This suggests that field conditions favour the synthesis of these compounds, possibly due to enhanced light exposure and nutrient availability in field condition. Total amino acid The total amino acids were present in higher amount in the plants grown in vitro (5.44 ± 0.08 mg/g FW), as compared to plants in cultivation (5.17 ± 0.18 mg/g FW), (Fig. 4 E), (Table 4 ). The higher accumulation of amino acids in in vitro -grown plants compared to field-grown plants can be attributed to the controlled nutrient conditions in the in vitro environment. In contrast to field conditions, which present a complex and variable nutrient landscape, in vitro conditions provide a consistent supply of nutrients that can enhance amino acid uptake and accumulation. Moreover, the controlled environment of in vitro culture may induce specific stress responses that lead to increased amino acid levels, similar to how certain amino acids are involved in plant adaptation to stress. These findings align with the concept that controlled conditions can amplify the effects of biostimulants, leading to higher concentrations of bioactive compounds in plant tissues. ( Lardos et al., 2024 ). Total reducing power assay When compared with standard of ascorbic acid with methanolic extracts of in vitro grown plants and field grown plants, field grown plants showed highest reducing power 0.334 as compared to in vitro grown plants that is 0.111 (Fig. 4 F), (Table 4 ). A higher absorbance of the reaction mixture corresponds to an increased reducing power. Several similar studies have shown that field-grown plants typically demonstrate a higher reducing power compared to in vitro cultured plants. ( Hakkim et al., 2007 ; Khorasani et al. , 2015; Kutty et al., 2014 ). Field-grown plants are subjected to various environmental stressors such as extreme temperatures, drought, and pathogen exposure, which can induce the production of higher levels of antioxidants as a defence mechanism ( Bansal et al., 2024 ). These natural conditions stimulate the synthesis of phenolic compounds and other antioxidant metabolites, contributing to a greater reducing power compared to plants grown in controlled, sterile in vitro environments (Thiruvengadam et al. , 2015). Conclusion This study successfully established a comprehensive and reproducible micropropagation protocol for V. jatamansi , a high-value medicinal plant endemic to the Himalayas, whose natural populations are critically threatened by overexploitation and a lack of organized cultivation. The study aimed to address this urgent need for conservation and sustainable propagation strategies by comparing the efficacy of two widely used culture media, Murashige and Skoog (MS) and Gamborg’s B5, for direct organogenesis. The findings unequivocally demonstrate that the Gamborg’s B5 medium significantly outperforms the MS medium in key growth parameters, offering a more effective solution for the in vitro propagation of this species. The primary focus of this investigation was to determine the optimal medium for achieving high-frequency shoot proliferation, leaf development, and rooting. The results showed that B5 medium fortified with BAP (2 mg L -1 ) and IAA (0.5 mg L -1 ) was the most effective for shoot regeneration, producing a maximum of 3 ± 0.816 shoots per plant, which was a notably superior response compared to the best MS medium treatment (BAP 1.5 mg L -1 ), which yielded 2.33 ± 0.471 shoots per plant. This finding aligns with the observation that Gamborg’s B5 medium’s distinct nutrient profile, including higher levels of potassium nitrate and ammonium sulfate, enhances nutrient uptake and protein synthesis, which are critical for tissue development. Furthermore, the maximum shoot length (4.33 ± 1.247 cm) and the highest number of leaves (11.33 ± 4.714 leaves per plant) were achieved on B5 medium, confirming its overall superior performance for the aerial parts of the plant. Beyond shoot induction, this study also provided a detailed analysis of rooting and acclimatization. The results indicate that rooting was observed exclusively in B5 medium during the direct organogenesis phase. However, root development was also observed in MS medium when shoot induced plantlets were subcultured in MS medium supplemented with auxins. The maximum number of roots (35.66 ± 9.8) and the longest root length (4.33 ± 0.94 cm) were recorded in MS medium supplemented with IBA at 4 mg L -1 and 3 mg L -1 , respectively. This two-step protocol initial shoot proliferation on B5 medium followed by rooting on MS medium provides a comprehensive and highly efficient strategy for mass multiplication. The study further evaluated the biochemical attributes of in vitro -grown plants in comparison to field-grown counterparts. Phytochemical analysis confirmed that field conditions promote the synthesis of a number of key compounds. Field-grown plants had significantly higher levels of chlorophyll a (2.80 ± 0.01 mg/g FW), chlorophyll b (4.41 ± 0.009 mg/g FW), total carotenoids (336 ± 22.86 mg/g FW), total soluble sugars (31.25 ± 1.8 mg/g FW), and total starch (28.125 ± 1.6 mg/g FW). Moreover, field-grown plants showed superior antioxidant potential, with a higher reducing power (absorbance of 0.334) compared to in vitro -grown plants (absorbance of 0.111). Conversely, in vitro -grown plants exhibited a higher total amino acid content (5.44 ± 0.08 mg/g FW) compared to field-grown plants (5.17 ± 0.18 mg/g FW). These differences are likely due to environmental stressors in the field that stimulate the production of secondary metabolites as a defence mechanism, while the controlled in vitro environment may enhance the accumulation of primary metabolites like amino acids. In conclusion, this research provides an efficient species-specific tissue culture protocol that offers a sustainable and scalable method for the mass propagation of Valeriana jatamansi . By enabling the production of genetically uniform, high-quality plantlets, this approach can alleviate the pressure on wild populations and support the establishment of commercial cultivation, thereby ensuring the long-term availability of this valuable plant for pharmaceutical, cosmetic, and perfumery industries. This study establishes the importance of optimizing media formulations for specific plant species and highlights the potential of plant tissue culture as a critical tool for both conservation and the sustainable production of threatened medicinal plants. Abbreviations ANOVA Analysis of Variance B5 Gamborg’s Medium BAP Benzyl acetyl pyruvate Cm Centimetre ºC Degree Celsius DMRT Duncan's Multiple Range Test FW Fresh weight h Hour(s) HgCl2 Mercuric Chloride IAA Indole-3-acetic acid IBA Indole-3-butyric acid kPa kilopascal mg Milli gram Mg/g milligrams per gram ml Millilitre MS Murashige and Skoog’s (1962) medium NAA Naphthalene Acetic Acid nm nanometer pH Potential of hydrogen PGRs Plant growth regulators rpm Revolution per minute SD Standard deviation UV Ultra violet UV-VIS Ultraviolet-Visible µl microlitres µmol micromole v/v Volume by volume w/v Weight by volume Declarations Acknowledgement The authors sincerely acknowledge the Department of High-Altitude Plant Physiology Research Centre, H.N.B. Garhwal University, Srinagar, for providing essential facilities and continuous guidance throughout the course of this study. Conflict of interest The authors declare no conflict of interest. Data availability statement The data that support the findings of this study are available from the corresponding author, [Geken Riba], upon reasonable request. Funding Declaration The authors declare that no specific funding was received for this research from any funding agency in the public, commercial, or not-for-profit sectors. Ethics Declaration Not applicable. Author Contribution G.R conceptualized and designed the study, performed the plant collection and laboratory experiments, analyzed the data, and drafted the manuscript. B.P provided institutional supervision and critical insights throughout the research process. R.R.K contributed to the experiment conceptualization phase and provided technical assistance during the experimental work. D.J assisted in writing and was responsible for formatting the manuscript. All authors have read and approved the final version of the manuscript. References Arnon D (1949) Estimation of total chlorophyll. Plant Physiol 24(1):1–15 Aziz EE, El-Danasoury MM, Craker LE (2010) Impact of sulfur and ammonium sulfate on dragonhead plants grown in newly reclaimed soil. J herbs spices Med plants 16(2):126–135 Aziz EE, El-Danasoury MM, Craker LE (2010) Impact of sulfur and ammonium sulfate on dragonhead plants grown in newly reclaimed soil. J herbs spices Med plants 16(2):126–135 Bansal Y et al (2024) Synthesis and accumulation of phytocompounds in field-, tissue-culture grown (stress) root tissues and simultaneous defense response activity in Glycyrrhiza glabra L. Sustainability 16(4):1613 Bari M, Latiful, and Sabina Yeasmin (2022). Microbes Cult methods : 77–98 Bisht S, Singh, Verma S (2019) Standardization of in vitro Multiplication Protocol for Picrorhiza kurroa (Royle Ex. Benth). Int. J Curr Microbiol App Sci 8(8):2043–2049 Chapman K, Chomchalow N (2003), February Production of medicinal plants in Asia. In III WOCMAP Congress on Medicinal and Aromatic Plants-Volume 5: Quality, Efficacy, Safety, Processing and Trade in Medicinal 679 (pp. 45–59) Charmakar S, Kunwar RM, Sharma HP, Rimal B, Baral S, Joshi N, Oli BN (2021) Production, distribution, use and trade of Valeriana jatamansi Jones in Nepal. Global Ecol Conserv, 30, e01792 Chen R, Zhang M, Lü J, Zhang X, da Silva JAT, Ma G (2014) Shoot organogenesis and somatic embryogenesis from leaf explants of Valeriana jatamansi Jones. Sci Hort 165:392–397 Cuce M, Inceer H (2024) Micropropagation and reintroduction of the endemic Tripleurospermum ziganaense (Asteraceae) to its natural habitat. Vitro Cell Dev Biology-Plant 60(5):646–658 Cüce M, Karaismailoğlu MC (2023) An improved micropropagation protocol for the ex situ conservation of Thlaspi cariense A. Carlström (Brassicaceae): an endangered Turkish endemic plant species. Vitro Cell Dev Biology-Plant 59(5):574–583 Cuce M, Inceer H, Bagdatli E, Ertürk Ö, Çil E, Sagan R (2025) In vitro propagation, antioxidant and antimicrobial activities of Tripleurospermum baytopianum (Asteraceae): a threatened endemic species in Türkiye. South Afr J Bot 184:40–49 Cuce M, Inceer H, Imamoglu KV, Ergin T, Ucler AO (2022) Towards ex situ conservation of globally rare Turkish endemic Tripleurospermum fissurale (Asteraceae). In Vitro Cellular & Developmental Biology-Plant, 58(6), 1002–1011 Das J, Mao AA, Handique PJ (2013) Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones). Acta Physiol Plant 35:55–63 Dwivedi T, Kanta C, Singh LR, Prakash I (2019) A list of some important medicinal plants with their medicinal uses from Himalayan State Uttarakhand, India. J Med Plants 7(2):106–116 Fattorini L, Veloccia A, Della Rovere F, D’angeli S, Falasca G, Altamura MM (2017) Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity. BMC Plant Biol 17(1):121 Fazili MA, Bashir I, Ahmad M, Yaqoob U, Geelani SN (2022) In vitro strategies for the enhancement of secondary metabolite production in plants: a review. Bull Natl Res Centre 46(1):35 Fitzpatrick TB, Lottie M (2020) Chapman. The importance of thiamine (vitamin B1) in plant health: From crop yield to biofortification. J Biol Chem 295(34):12002–12013 Frick EM, Strader LC (2018) Roles for IBA-derived auxin in plant development. J Exp Bot 69(2):169–177 Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):148–151 Gautam RD, Kumar A, Kumar R, Chauhan R, Singh S, Kumar M, Singh S (2021) Clonal propagation of Valeriana jatamansi retains the essential oil profile of mother plants: An approach toward generating homogenous grade of essential oil for industrial use. Front Plant Sci 12:738247 Gautam RD, Kumar A, Kumar R, Chauhan R, Singh S, Kumar M, Singh S (2021) Clonal propagation of Valeriana jatamansi retains the essential oil profile of mother plants: An approach toward generating homogenous grade of essential oil for industrial use. Front Plant Sci 12:738247 George EF, Hall MA, De Klerk GJ (eds) (2007) Plant propagation by tissue culture: volume 1. the background, vol 1. Springer Science & Business Media Gowthami R, Sharma N, Pandey R, Agrawal A (2021) Status and consolidated list of threatened medicinal plants of India. Genet Resour Crop Evol 68(6):2235–2263 Goyer A (2010) Thiamine in plants: aspects of its metabolism and functions. Phytochemistry 71.14-15 : 1615–1624 Hakkim FL, Shankar CG, Girija S (2007) Chemical composition and antioxidant property of holy basil (Ocimum sanctum L.) leaves, stems, and inflorescence and their in vitro callus cultures. J Agric Food Chem 55(22):9109–9117 Hamid R, Kamili AN, Zaffar M, da Silva JAT, Mujib A, Parray JA (2010) Callus Mediated Shoot Organogenesis from Shoot Tips of Cichorium intybus. J Med Aromatic Plant Sci Biotechnol 4 Special Issue 1:84–86 hiruvengadam M, Chung I-M (2015) Phenolic compound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L). Electron J Biotechnol 18(4):295–301 Houghton P (ed) (1997) Valerian: the genus Valeriana. Crc Hussain A, Qarshi IA, Nazir H, Ullah I (2012) Plant tissue culture: current status and opportunities. Recent advances in plant in vitro culture. IntechOpen Inceer H, Cuce M, Imamoglu KV, Ergin T, Ucler AO (2022) In vitro propagation and cytogenetic stability of Tripleurospermum insularum (Asteraceae)-a critically endangered insular endemic species from Turkey. Plant Biosystems-An Int J Dealing all Aspects Plant Biology 156(5):1213–1221 Jacob A, Malpathak N (2005) Manipulation of MS and B5 components for enhancement of growth and solasodine production in hairy root cultures of Solanum khasianum Clarke. Planr Cell Tissue Organ Cult 80(3):247–257 Khan A, Shah AH, Ali N (2021) In-vitro propagation and phytochemical profiling of a highly medicinal and endemic plant species of the Himalayan region (Saussurea costus). Scientific reports 11.1 : 23575 Khorasani Esmaeili A et al (2015) Antioxidant activity and total phenolic and flavonoid content of various solvent extracts from in vivo and in vitro grown Trifolium pratense L. (Red Clover). BioMed Research International, 2015, 643285 Kiani Z, Mashayekhi K, Mousavizadeh SJ, Nezhad KZ (2022) Changes in carbohydrate and phytochemical content in the bulbs and leaves of ten onion accessions between two developmental stages. Italus Hortus 29:47–55 Kumar A, Sathyakumar S, Goraya GS, Gupta AK, Adhikari BS, Rawat GS (2021) Assessment of medicinal and aromatic plant species on their collection, usage, demand, markets, price trends and life cycle in Lahaul and Pangi landscape, Himachal Pradesh. A report submitted to Himachal Pradesh Forest Department and United Nations Development Programme, p 141 Kurmi K, Marcia C (2020) Haigis. Nitrogen metabolism in cancer and immunity. Trends Cell Biol 30:408–424 Kutty NN et al (2014) Evaluation of antioxidant capacity and acetylcholinesterase inhibitory activity of field-grown plants and in vitro plantlets of Enicostemma littorale. Free Radicals Antioxid 4(2):21–26 Kutty NN et al (2014) Evaluation of antioxidant capacity and acetylcholinesterase inhibitory activity of field-grown plants and in vitro plantlets of Enicostemma littorale. Free Radicals Antioxid 4(2):21–26 Lardos M et al (2024) Discovery of the biostimulant effect of asparagine and glutamine on plant growth in Arabidopsis thaliana. Front Plant Sci 14:1281495 Leghari S, Jahan et al (2016) Role of nitrogen for plant growth and development: A review. Adv Environ Biology 10(9):209–219 Malik A et al (2020) A comparative biochemical evaluation of in vivo and in vitro propagated Alhagi maurorum: an important medicinal plant. Legume Research-An Int J 43(5):627–633 Mathur J, Ahuja PS, Mathur A, Kukreja AK, Shah NC (1988) In vitro propagation of Valeriana wallichii. Planta Med 54(01):82–83 Mishra Y, Rawat R, Nema B, Shirin F (2013) Effect of Seed Orientation and Medium Strength on in vitro Germination of Pterocarpus marsupium Roxb. Notulae Scientia Biologicae 5:476–479 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant, 15(3) Murthy HN, Lee EJ, Paek KY (2014) Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Cult (PCTOC) 118:1–16 Nawchoo AI, Rather MA, Ganie HA, Jan RT (2012) Need for unprecedented impetus for monitoring and conservation of Valeriana jatamansi, a valuable medicinal plant of Kashmir Himalaya. Agricultural Sci Res Journals 2(7):369–373 Neumann KH, Kumar A, Imani J (2009) Plant cell and tissue culture: a tool in biotechnology, vol 12. Springer, Berlin Phillips GC, Garda M (2019) Plant tissue culture media and practices: An overview. In Vitro Cellular & Developmental Biology - Plant, 55(3), 242–257 Phillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. Vitro Cell Dev Biology-Plant 55:242–257 Pierik RLM (1997) vitro culture of higher plants. Springer science & business media Rafi S, Wani SH, Wani ZA, Parveen K, Bhat MY (2018) In vitro culture and biochemical attributes of Bergenia ciliata (Haw.) Sternb. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 88(2), 609–619 Rafi S et al (2018) In vitro Culture and Biochemical Attributes of Bergenia ciliata (Haw.) Sternb. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences 88 : 609–619 Raina AP, Negi KS (2015) Essential oil composition of Valeriana jatamansi Jones from Himalayan regions of India. Indian J Pharm Sci 77(2):218–221 Ruiz Herrera LF, Shane MW, López-Bucio J (2015) Nutritional regulation of root development. Wiley Interdisciplinary Reviews: Dev Biology 4(4):431–443 Talreja T (2011) Biochemical estimation of three primary metabolites from medicinally important plant Moringa oleifera. Int J Pharm Sci Rev Res 7(2):186–188 Thiruvengadam M, Chung I-M (2015) Phenolic compound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L). Electron J Biotechnol 18(4):295–301 Tomar NS, Sharma M, Agarwal RM (2015) Phytochemical analysis of Jatropha curcas L. during different seasons and developmental stages and seedling growth of wheat (Triticum aestivum L) as affected by extracts/leachates of Jatropha curcas L. Physiol Mol Biology Plants 21(1):83–92 Verma PC, Singh H, Negi AS, Saxena G, Rahman LU, Banerjee S (2015) Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth. Plant Signal Behav, 10(5), e1023976 Vollmer R, Espirilla J, Sánchez JC, Arroyo L, Acosta M, Flores G, Azevedo V (2023) Thiamine improves in vitro propagation of sweetpotato [Ipomoea batatas (L.) Lam.]-confirmed with a wide range of genotypes. Plant Cell Tissue Organ Cult (PCTOC) 152(2):253–266 Wang J et al (2016) Different responses of alpine plants to nitrogen addition: effects on plant-plant interactions. Sci Rep 6(1):38320 Wills RB, Bone K, Morgan M (2000) Herbal products: active constituents, modes of action and quality control. Nutr Res Rev 13(1):47–77 Yıldıztekin M (2021) Ameliorative effects of exogenous ascorbic acid and potassium nitrate on antioxidant defense system and mineral nutrient uptake in tomatoes under salt stress Yin T-F et al (2017) Species-specific phenological responses to long-term nitrogen fertilization in an alpine meadow. J Plant Ecol 10(2):301–309 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviewers agreed at journal 13 May, 2026 Reviews received at journal 07 May, 2026 Reviewers agreed at journal 05 May, 2026 Reviewers agreed at journal 04 May, 2026 Reviewers invited by journal 04 May, 2026 Editor assigned by journal 03 May, 2026 Submission checks completed at journal 03 May, 2026 First submitted to journal 27 Apr, 2026 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. 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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-9539107","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":636023889,"identity":"56d1d563-c46f-44f0-b860-aeb8665ed020","order_by":0,"name":"Geken 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09:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9539107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9539107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109067610,"identity":"4d094cab-4a12-4fc7-a905-99b6a685ed40","added_by":"auto","created_at":"2026-05-12 09:57:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1004921,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot representation of shoot growth parameters (Number of leaves, number of shoots, and shoot length) of \u003cem\u003eValeriana jatamansi\u003c/em\u003e cultured on Murashige and Skoog (MS) and Gamborg B5 media under \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9539107/v1/a4e05268e2332b2462fd9e19.png"},{"id":109005494,"identity":"96a2da8a-2c58-4519-91c0-764c86bb5b7f","added_by":"auto","created_at":"2026-05-11 15:37:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":736257,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation heatmap illustrating the relationships between shoot growth parameters of \u003cem\u003eValeriana jatamansi\u003c/em\u003e and plant growth regulators under \u003cem\u003ein vitro\u003c/em\u003e conditions on (A) Murashige and Skoog (MS) medium and (B) Gamborg B5 medium.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9539107/v1/62f6573eeb126fa0cfb4b690.png"},{"id":109005496,"identity":"e1a62cd7-2e76-44b1-a6c3-69d90fe23076","added_by":"auto","created_at":"2026-05-11 15:37:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76227,"visible":true,"origin":"","legend":"\u003cp\u003eSynergistic effects of growth media and plant growth regulators on shoot growth parameters of \u003cem\u003eValeriana jatamansi\u003c/em\u003eunder \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9539107/v1/ee2fe147dc78d985ea9df9f7.png"},{"id":109005498,"identity":"1259c3c2-1e60-4500-8397-d0fb17599284","added_by":"auto","created_at":"2026-05-11 15:37:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39257,"visible":true,"origin":"","legend":"\u003cp\u003ePhytochemical analysis of field grown and \u003cem\u003ein vitro\u003c/em\u003e grown \u003cem\u003eValeriana jatamansi. \u0026nbsp;\u003c/em\u003e(A) Total chlorophyll content, (B) Total Caretonoids content, (C) Total sugar content Mg/g FW, (D) Total starch mg/g FW, (E) Total amino acid content mg/g FW, (F) Comparison of total reducing power in \u003cem\u003ein vitro\u003c/em\u003e grown and field grown \u003cem\u003eValeriana jatamansi.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9539107/v1/bcac2db3a141c7722190ca1f.png"},{"id":109005495,"identity":"52b295e4-b319-43e4-84c6-0c153e0d9b9e","added_by":"auto","created_at":"2026-05-11 15:37:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":153612,"visible":true,"origin":"","legend":"\u003cp\u003eDirect organogenesis of\u003cem\u003e Valeriana jatamansi\u003c/em\u003ein MS and B5 medium. Inoculated nodal segments under growth chamber conditions; (B) Nodal segments cultured on Murashige and Skoog (MS) medium; (C) Direct organogenesis on Gamborg B5 medium supplemented with BAP (2 mg L⁻¹) and IAA (0.5 mg L⁻¹) after 5 weeks of inoculation; (D) Direct organogenesis on MS medium supplemented with BAP (1.5 mg L⁻¹) after 5 weeks of inoculation; (E) Control (MS medium without plant growth regulators); (F) Root induction on Gamborg B5 medium supplemented with BAP (2 mg L⁻¹) and IAA (0.5 mg L⁻¹) after 5 weeks of inoculation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9539107/v1/a3a720dd9e5edc7d475c11c3.png"},{"id":109068142,"identity":"fa1e0a25-0d77-40d8-a296-43eeb729125c","added_by":"auto","created_at":"2026-05-12 10:03:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90396,"visible":true,"origin":"","legend":"\u003cp\u003eRoot induction and acclimatization of \u003cem\u003eValeriana jatamansi\u003c/em\u003e. (A) Root induction on Murashige and Skoog (MS) medium supplemented with IBA (3 mg L⁻¹) after 20 days of subculture; (B) Rooted plantlets on MS medium supplemented with IBA (4 mg L⁻¹) ready for acclimatization; (C) Well-developed rooted plantlet on MS medium supplemented with IBA (4 mg L⁻¹); (D) Transfer of plantlets to clay pots; (E) Pots covered with polythene sheets to maintain high humidity; (F) Plants after 5 weeks of acclimatization.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9539107/v1/c14e143e4b1236a12ae21072.png"},{"id":109206609,"identity":"d1d53ade-715b-438c-b621-9905eeb2d6a1","added_by":"auto","created_at":"2026-05-13 15:13:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2894138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9539107/v1/4e5af92d-6e10-44a7-9e70-419e284d235a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elucidating the role of different culture medium on in vitro plant regeneration of Valeriana jatamansi Jones ex Roxb. a high value medicinal plant of the temperate Himalayas","fulltext":[{"header":"Key message","content":"\u003cp\u003eOptimized micropropagation of threatened Himalayan via Gamborg\u0026rsquo;s B5 medium achieves superior shoot proliferation; biochemical profiling reveals distinct vs. field metabolite profiles for effective conservation.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eValeriana\u003c/em\u003e is a genus with about 200 distributed worldwide and these plants are used to treat various ailments on the place where the plant is endemic. \u003cem\u003eValeriana\u003c/em\u003e species have been used for generations. These species are often utilized in traditional medicine systems, particularly in areas where they are naturally found. The use of \u003cem\u003eValeriana\u003c/em\u003e species dates back centuries, with historical records documenting their medicinal applications in multiple cultural contexts. The most important commercial species are \u003cem\u003eV. offincinalis, V. wallichii\u003c/em\u003e, \u003cem\u003eV. fauriei\u003c/em\u003e and \u003cem\u003eV. edulis.\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eHoughton, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Indian valerian \u003cem\u003eValeriana jatamansi\u003c/em\u003e Jones ex Roxb. also known as \u003cem\u003eValeriana wallichii\u003c/em\u003e DC. belongs to the Valerianaceae family and is an important medicinal and aromatic plant in the temperate Himalayan area. Indian valerian is commonly referred to as Tagar or Sugandhbala in Sanskrit, and Mushkbala in Hindi. It thrives in subtropical to temperate regions, growing naturally at altitudes ranging from 1500 to 3000 meters. Natural populations of the species are found mostly as undercover on moist slopes along the streams \u003cb\u003e(\u003c/b\u003eGautam et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The plant can also be found in the eastern Himalayan region \u003cb\u003e(\u003c/b\u003eRaina \u0026amp; Negi, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e \u003cem\u003eV. jatamansi\u003c/em\u003e roots are used as one of the ingredients in the preparation of snake bites and it is also used for liver, kidney and spleen disorder. \u003cem\u003eV. jatamansi\u003c/em\u003e is also used in various industries such as cosmetics and perfumery \u003cb\u003e(\u003c/b\u003eCharmakar et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Important constituents from rhizomes and roots of \u003cem\u003eV. jatamansi\u003c/em\u003e used by different industries are \u003cb\u003e(\u003c/b\u003eWills et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) bakkenoloids type sesquiterpenoids, essential oils and phenolics \u003cb\u003e(\u003c/b\u003eGautam et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overexploitation of the plant for industrial and pharmaceutical purposes has led to a decline in its wild populations. If the current rate of exploitation continues unchecked, the plant faces the risk of becoming extinct in the near future \u003cb\u003e(\u003c/b\u003eNawchoo et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eV. jatamansi\u003c/em\u003e has been assessed as a Vulnerable (VU) species under the IUCN criteria due to overharvesting, habitat degradation, and restricted distribution in the Himalayan region \u003cb\u003e(\u003c/b\u003eGowthami et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Sustainable alternatives to wild collection are urgently needed to ensure the continued availability of this valuable species for industrial and pharmacological applications.\u003c/p\u003e \u003cp\u003eIn recent years, plant tissue culture has gained substantial importance in the propagation and preservation of medicinal and aromatic plants (MAPs), many of which are facing population decline due to anthropogenic pressures such as habitat fragmentation, overharvesting, climate change, and unregulated trade. The sustainable cultivation of these plants through plant tissue culture significantly reduces the dependency on wild populations, thereby alleviating pressure on natural ecosystems and contributing to biodiversity conservation. \u003cb\u003e(\u003c/b\u003eCuce \u0026amp; Inceer \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e\u003cb\u003e), (\u003c/b\u003eCuce et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), \u003cb\u003e(\u003c/b\u003eInceer et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), \u003cb\u003e(\u003c/b\u003eC\u0026uuml;ce \u0026amp; Karaismailoğlu, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003cb\u003e), (\u003c/b\u003eCuce et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition to propagation, plant tissue culture also serves as a platform for secondary metabolite enhancement through advanced biotechnological interventions such as elicitation, precursor feeding, and genetic transformation. These strategies have been successfully employed to boost the production of bioactive compounds in several commercially important plant species \u003cb\u003e(\u003c/b\u003eMurthy et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Such approaches are essential for developing plant lines with improved compatibility and productivity for industrial applications, particularly in terms of biomass yield and the accumulation of pharmacologically active constituents. \u003cb\u003e(\u003c/b\u003eFazili et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), However, the success of such \u003cem\u003ein vitro\u003c/em\u003e applications is strongly influenced by the composition of the culture medium, which governs cell differentiation, organogenesis, and metabolite biosynthesis \u003cb\u003e(Neumann\u003c/b\u003e \u003cb\u003eet al.\u003c/b\u003e, \u003cb\u003e2005).\u003c/b\u003e Among the various commonly used basal media, Murashige and Skoog (MS) medium, developed in 1962, is widely used for general-purpose \u003cem\u003ein vitro\u003c/em\u003e culture due to its high nutrient and salt content \u003cb\u003e(\u003c/b\u003eMurashige \u0026amp; Skoog \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1962\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Nevertheless, plant species exhibit varied responses to the same culture medium, and the optimization of medium composition including macro and micronutrients, vitamins, carbon sources, and growth regulators is crucial for enhancing growth and productivity \u003cem\u003ein vitro\u003c/em\u003e \u003cb\u003e(\u003c/b\u003ePierik \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1997\u003c/span\u003e\u003cb\u003e) (\u003c/b\u003eGeorge et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Gamborg\u0026rsquo;s B5 medium, formulated in 1968 for the culture of soybean suspension cells, has been shown to be effective for certain plant species, especially for those sensitive to the high nitrogen content of MS medium. Comparative evaluations have highlighted Gamborg's B5 medium\u0026rsquo;s superior performance in promoting organogenesis and root induction in selected medicinal plants, suggesting its broader applicability across different plant species. \u003cb\u003e(\u003c/b\u003eGamborg et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1968\u003c/span\u003e) \u003cb\u003e(\u003c/b\u003eHussain et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) \u003cb\u003e(\u003c/b\u003eVerma et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), \u003cb\u003e(Jacob\u003c/b\u003e \u003cb\u003eet al.\u003c/b\u003e, \u003cb\u003e2005)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn the current investigation, \u003cem\u003eV. jatamansi\u003c/em\u003e an important high-altitude medicinal herb endemic to the Himalayan region and valued for its essential oil and valepotriates was cultured under \u003cem\u003ein vitro\u003c/em\u003e conditions using Murashige and Skoog (MS) medium and Gamborg\u0026rsquo;s B5 medium supplemented with varying concentrations and combinations of auxins and cytokinins. The objective was to determine the optimal medium for achieving high-frequency shoot proliferation, leaf development, and rooting. The experimental results revealed that Gamborg\u0026rsquo;s B5 medium demonstrated significantly higher efficacy compared to MS medium in promoting all observed growth parameters. This study is the first documented report highlighting the superior response of \u003cem\u003eV. jatamansi\u003c/em\u003e to B5 medium, which not only contributes to the understanding of species-specific media requirements but also opens new research areas for more effective tissue culture protocols for this medicinally important species. The successful micropropagation of \u003cem\u003eV. jatamansi\u003c/em\u003e using Gamborg\u0026rsquo;s B5 medium offers a reliable way to produce plants in large numbers, which can help both pharmaceutical industries and conservation efforts. Finally, the study strengthens the case for further exploration of alternative and species-specific media formulations for other endangered or commercially valuable medicinal plants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and explant inoculation\u003c/h2\u003e \u003cp\u003eThree- to four-year-old plants of \u003cem\u003eV. jatamansi\u003c/em\u003e were procured from the Pothibasa field station (2200 m above sea level) of the High-Altitude Plant Physiology Research Centre, Hemvati Nandan Bahuguna Garhwal University, Srinagar, Uttarakhand. The plants were maintained in the glasshouse of the HAPPRC main research complex (550 m above sea level) at Srinagar, Uttarakhand. Healthy nodal segments of \u003cem\u003eV. jatamansi\u003c/em\u003e were selected as explants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Nodal segments were excised using sterilized scissors under aseptic conditions. The explants were initially washed under running tap water using fine mesh nets to prevent material loss and to remove surface debris. They were then trimmed to uniform sizes and rinsed thoroughly 3 to4 times with tap water, followed by washing with double-distilled water. Surface sterilization was done by treating with surfactant Tween-20 for 2 to 4 minutes. For antifungal treatment, the explants were cleaned with 0.1\u0026ndash;0.5% Bavistin solution using a soft brush and subsequently soaked for 30 to 40 min. Following fungicide treatment, explants were thoroughly rinsed 5 to 6 times with double-distilled water to remove any residual chemicals. Nodal segments of \u003cem\u003eV. jatamansi\u003c/em\u003e were treated with 0.5% (w/v) mercuric chloride (HgCl₂) for 5 minutes to establish an optimal surface sterilization protocol for \u003cem\u003ein vitro\u003c/em\u003e culture initiation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCulture conditions\u003c/h3\u003e\n\u003cp\u003eTwo basal media were used in this study: Murashige and Skoog (MS) medium \u003cb\u003e(\u003c/b\u003eMurashige \u0026amp; Skoog, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1962\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e and Gamborg\u0026rsquo;s B5 medium \u003cb\u003e(\u003c/b\u003eGamborg et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1968\u003c/span\u003e), both supplemented with 3% (w/v) sucrose. Plant growth regulators (PGRs) were incorporated in different concentrations and combinations, namely T0: control (no PGR), T1: BAP 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T2: BAP 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T3: BAP 1.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T4: BAP 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T1A1: BAP 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e + IAA 0.125 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T2A2: BAP 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e + IAA 0.25 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T3A3: BAP 1.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e + IAA 0.375 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and T4A4: BAP 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e + IAA 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. For rooting, regenerated shoots were transferred to MS medium supplemented with auxins: T0: control, T1: NAA 0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T2: IBA 0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T3: IAA 0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T4: IBA 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, T5: IBA 3.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and T6: IBA 4.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The pH of the media was adjusted to 5.8 and sterilized by autoclaving at 121\u0026deg;C and 105 kPa for 15 min and solidified with 0.7% (w/v) plant tissue culture grade agar. Cultures were maintained in a growth chamber at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026ndash;60% relative humidity, under a 16/8 h light/dark photoperiod with a photosynthetic photon flux density of 60 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; provided by cool daylight fluorescent lamps. The PGRs were added to the growth medium after adjusting the pH and before adding the gelling agent. Cultures were maintained in 250 mL Erlenmeyer flasks sealed with sterile cotton plugs wrapped in absorbent fabric. Rooted plantlets were removed from the culture vessels, washed, and transferred to pots containing sand, soil, perlite, cocopeat, and vermiculite in a 1:1:1 (v/v) ratio for hardening under greenhouse conditions (27\u0026deg;C, 90% humidity). All media components, PGRs, and chemicals were procured from HiMedia Pvt. Ltd., India.\u003c/p\u003e\n\u003ch3\u003eChlorophyll and Carotenoid Estimation\u003c/h3\u003e\n\u003cp\u003eFor pigment extraction, 50 mg of fresh leaf tissue from both \u003cem\u003ein vitro\u003c/em\u003e\u0026ndash;grown and field-grown plants of \u003cem\u003eV. jatamansi\u003c/em\u003e was homogenized in 5 mL of absolute acetone and incubated in the dark to prevent pigment degradation. The homogenate was centrifuged at 3,000 rpm for 15 min, and the resulting supernatant was collected and diluted with an additional 10 mL of acetone. The absorbance of the extract was recorded at 480, 645, and 663 nm using a UV\u0026ndash;VIS spectrophotometer. Chlorophyll a, chlorophyll b, and total carotenoid contents were quantified following the method of Arnon (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1949\u003c/span\u003e), using the following equations:\u003c/p\u003e \u003cp\u003eChlorophyll a (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;12.7 (A663) \u0026ndash; 2.69 (A645)\u003c/p\u003e \u003cp\u003eChlorophyll b (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;22.9 (A645) \u0026ndash; 4.68 (A663)\u003c/p\u003e \u003cp\u003eTotal chlorophyll (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;20.2 (A645)\u0026thinsp;+\u0026thinsp;8.02 (A663)\u003c/p\u003e\n\u003ch3\u003eTotal Carbohydrate Estimation\u003c/h3\u003e\n\u003cp\u003eThe anthrone method was employed to estimate the total soluble sugar and starch contents in leaf tissues of \u003cem\u003eV. jatamansi\u003c/em\u003e. Freshly harvested leaves (200 mg) from \u003cem\u003ein vitro\u003c/em\u003e grown and field grown plants were extracted with 5 mL of 80% boiling ethanol and centrifuged at 3,000 rpm for 10 minutes. The resulting supernatant was used for the estimation of soluble sugars, while the pellet was reserved for starch quantification. Sucrose was used as the standard reference for both estimations.\u003c/p\u003e \u003cp\u003eFor total soluble sugar estimation, appropriate volumes of the ethanol supernatant were diluted to 1 mL with distilled water. To this, 4 mL of cold anthrone reagent (prepared by dissolving 200 mg of anthrone in 100 mL of 95% sulfuric acid) was added. The mixture was cyclomixed for 2\u0026ndash;3 minutes and the absorbance was measured at 620 nm using a UV-VIS double beam spectrophotometer. All estimations were carried out in triplicates\u003c/p\u003e \u003cp\u003eFor starch estimation, the ethanol-insoluble pellet was resuspended in 5 mL of 52% perchloric acid and centrifuged at 3,000 rpm for 12 minutes. The supernatant was then diluted to 1 mL with distilled water, and 4 mL of anthrone reagent was added. After thorough mixing, absorbance was recorded at 620 nm. A standard curve was prepared using known concentrations of sucrose for quantification.\u003c/p\u003e\n\u003ch3\u003eEstimation of Reducing Power Assay\u003c/h3\u003e\n\u003cp\u003e2.5 ml of methanol plant extract (1mg/ml in methanol) was mixed with 2.5 ml of sodium phosphate buffer (0.2M; pH 6.6) and 2.5 ml of potassium ferricyanide (1%) in a test tube. The test tubes were then covered by silver foil and placed in water bath at 50\u0026deg;C for 20 mins. The test tubes were then removed from the water bath and allowed to cool down in room temperature and then 2.5 ml of trichloro-acetic acid (TCA 10%) was added. The mixture was then centrifuged at 3000 rpm for 10 minutes. Different concentrations of 25\u0026micro;l to 125 \u0026micro;l supernatant, 1.25 ml distilled water and 250 \u0026micro;l ferric chloride (0.1%) was added to the test tubes. The test tubes mixture was mixed well and absorbance was taken at 700 nm using spectrophotometer.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of total free amino acids\u003c/h2\u003e \u003cp\u003eTotal amino acid (TAA) content was estimated using the ninhydrin method \u003cb\u003e(Moore\u003c/b\u003e \u003cb\u003eet al.\u003c/b\u003e, \u003cb\u003e1954)\u003c/b\u003e. An acetate buffer (pH 5.0) was prepared by dissolving 1.64 g of sodium acetate in 100 mL of distilled water. Ninhydrin reagent was prepared by dissolving 750 mg of ninhydrin in 26.25 mL of 80% ethanol and 11.25 mL of acetate buffer. Ethanolic extract (50 mg) was dissolved in 5 mL of ethanol, centrifuged at 4\u0026deg;C, 3000 rpm for 10 minutes, and the supernatant diluted to prepare 1 mL test solutions. To each, 2 mL of ninhydrin reagent was added, vortexed, and incubated at 100\u0026deg;C for 20\u0026ndash;30 minutes. After cooling, absorbance was measured at 570 nm. TAA was calculated as mg/g fresh weight using a glycine standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experiment was executed following a completely randomized design (CRD), to evaluate the effects of different plant growth regulators (PGRs) on the direct organogenesis of \u003cem\u003eValeriana jatamansi\u003c/em\u003e using two different culture media (MS and B5). For shoot proliferation study three nodal explants were transferred in one 250ml erlenmeyer flask and three flasks were used for each treatment. a factorial two-way analysis of variance (ANOVA) was used to evaluate the effects of two independent factors, namely types of growth medium and concentrations of PGRs on the \u003cem\u003ein vitro\u003c/em\u003e regeneration capacity. Results were evaluated as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Significant differences for multiple comparisons were determined using Duncan\u0026rsquo;s multiple range test by SPSS 26.0, Correlation and box plot analysis was also done using R software version 2025.09.2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussions","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDirect Organogenesis and Shoot Proliferation\u003c/h2\u003e \u003cp\u003eShoot regeneration in \u003cem\u003eValeriana jatamansi\u003c/em\u003e was significantly influenced by both basal medium composition and plant growth regulator (PGR) treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Two-way ANOVA confirmed that the main effects of medium and PGRs were highly significant for shoot number (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01) and shoot length (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001 for medium), while their interaction effects varied by parameter (Table\u0026nbsp;1). Box plot simulation showed higher growth potential across all shoot growth parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cp\u003eTable 1. Interaction effects of basal medium composition and plant growth regulator (PGR) concentrations on the \u003cem\u003ein vitro\u003c/em\u003e morphogenic response of \u003cem\u003eValeriana jatamansi\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"619\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eExperimental parameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 342px;\"\u003e\n \u003cp\u003eVariance source of two-way ANOVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003eInteraction P \u0026times; M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003eGrowth medium (M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 169px;\"\u003e\n \u003cp\u003ePGR treatments (P)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNo. of shoots\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003e11.560**\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.002\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cem\u003e3.19**\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.008\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cem\u003e1.21 NS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.321 NS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eShoot length\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003e43.416***\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.000\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cem\u003e4.37**\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.001\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cem\u003e2.33**\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.039 NS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNo. of leaves\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003e9.58**\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.004\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cem\u003e1.92 NS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.086\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cem\u003e1.16 NS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cem\u003e0.346 NS\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 619px;\"\u003e\n \u003cp\u003e\u003cem\u003eNS\u003c/em\u003e non-significant\u003c/p\u003e\n \u003cp\u003e*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003c/br\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eShoot Number\u003c/h2\u003e \u003cp\u003eThe interaction between growth medium and PGR treatments for shoot number was non-significant (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.21; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.321), indicating that the influence of the basal medium remained consistent across different hormonal regimes (Table\u0026nbsp;1). Consequently, the main effects were analyzed independently. When data were pooled across all PGR levels, Gamborg\u0026rsquo;s B5 medium produced a significantly higher mean shoot number (1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97 per explant) compared to MS medium (1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 per explant) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among the PGR treatments, the highest shoot proliferation (2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 shoots per explant) was achieved using either 1.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP alone or the combination of 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP with 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Mean separation using Duncan\u0026rsquo;s multiple range test confirmed that these treatments were statistically superior to the control (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\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 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMain effects of basal media and plant growth regulators on the quantitative growth characteristics of \u003cem\u003eValeriana jatamansi\u003c/em\u003e under \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShoot number/explant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShoot length/explant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLeaf number/explant\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eGrowth Medium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eGamborg\u0026rsquo;s B5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003ePGR Treatments (mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;3.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.18\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.16\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eValues represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Means followed by distinct letters within a column indicate significant differences (DMRT, p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data were recorded 5 weeks after the culture and represent a total of three replicates of 10 plants per treatment BAP 6-Benzylaminopurine, Indole-3-Acetic Acid.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eShoot Length\u003c/h2\u003e \u003cp\u003eIn contrast to shoot number, shoot elongation exhibited a significant interaction between the basal medium and PGR treatments (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.33; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039), indicating that the response to hormonal supplementation depended on the specific nutrient composition of the basal medium (Table\u0026nbsp;1). The longest shoots (4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 cm) were recorded on B5 medium supplemented with 1.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP, followed by B5 medium containing 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP (3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 cm). Across all PGR concentrations, shoots regenerated on B5 medium (2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 cm) were consistently longer than those on MS medium (1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 cm) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Correlation analysis further supported these findings, revealing a strong positive association between BAP concentration and shoot proliferation in Gamborg\u0026rsquo;s B5 medium (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.86), (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whereas a weaker correlation was observed in MS medium (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.41), (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLeaf Number\u003c/h2\u003e \u003cp\u003eThe interaction effect for leaf number was non-significant (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.16; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.346), (Table\u0026nbsp;1). When pooled across PGR treatments, Gamborg\u0026rsquo;s B5 medium supported significantly higher leaf production (7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84 leaves per explant) compared to MS medium (4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81 leaves per explant) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among the hormonal treatments, the combination of 2.0 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BAP and 0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e IAA yielded the maximum leaf count (8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.88), (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) which was significantly different from the control according to the DMRT (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A strong positive correlation was observed between leaf number and shoot length in both MS (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.81) and Gamborg\u0026rsquo;s B5 (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.79) media, (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that enhanced shoot elongation is closely linked to increased leaf development under \u003cem\u003ein vitro\u003c/em\u003e conditions.\u003c/p\u003e \u003cp\u003eThe superior performance of Gamborg\u0026rsquo;s B5 medium suggests that \u003cem\u003eValeriana jatamansi\u003c/em\u003e exhibits a preferential response to the nutrient composition of Gamborg\u0026rsquo;s formulation. Gamborg\u0026rsquo;s B5 medium contains relatively higher levels of potassium nitrate and additional nitrogen sources, including ammonium sulfate, which are known to enhance nitrogen assimilation, protein synthesis, and overall morphogenic competence in plant tissue cultures \u003cb\u003e(\u003c/b\u003eAziz et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bari et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Furthermore, the elevated thiamine content of Gamborg\u0026rsquo;s B5 medium may support enzymatic activity and metabolic processes essential for active cell division and tissue differentiation \u003cb\u003e(Goyer\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e., \u003cb\u003e2010; Fitzpatrick\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e., \u003cb\u003e2020;\u003c/b\u003e Vollmer et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Comparable medium-specific responses have been reported in \u003cem\u003eBergenia ciliata\u003c/em\u003e, underscoring the importance of species-specific optimization of basal salt composition for efficient \u003cem\u003ein vitro\u003c/em\u003e regeneration of threatened Himalayan medicinal plants \u003cb\u003e(Kurmi\u003c/b\u003e \u003cb\u003eet al\u003c/b\u003e., \u003cb\u003e2020)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eRooting and acclimatization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn direct organogenesis, rooting was induced in Gamborg\u0026rsquo;s B5 medium with treatments of BAP (0.5\u0026ndash;2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and BAP combined with IAA (0.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e + IAA 0.125 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e + IAA 0.25 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) while MS medium failed to induce rooting. These findings align with earlier studies demonstrating the role of nitrogen sources in improving root architecture in various species. For instance, ammonium sulfate has been linked to enhanced lateral root growth, while potassium nitrate supports nutrient uptake and antioxidant defences, improving plant resilience in challenging environments \u003cb\u003e(\u003c/b\u003eRafi et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e This suggests that Gamborg\u0026rsquo;s B5 medium's formulation is well-suited to promoting effective rooting in tissue culture systems.\u003c/p\u003e \u003cp\u003eShoot-induced plantlets without roots were sub-cultured in MS medium supplemented with auxins (NAA, IBA, IAA) for root induction. No rooting occurred in the control or treatments with NAA (0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), IBA (0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), or IAA (0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The maximum number of roots (35.66\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8 roots per explant) was obtained with IBA at a concentration of 4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, followed by progressively lower root numbers at 3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentrations of IBA, respectively. Maximum root length (3.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 cm) and the least root induction time (21.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36 days) were achieved with IBA (3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 4 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that IBA is more effective than NAA and IAA for adventitious root induction in \u003cem\u003eValeriana jatamansi\u003c/em\u003e, which may be attributed to its conversion into active auxin (IAA) and its role in promoting root initiation processes \u003cb\u003e(\u003c/b\u003eFattorini et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Frick \u0026amp; Strader, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Plantlets with well-developed roots were transferred to potting mixture containing cocopeat\u0026thinsp;+\u0026thinsp;perlite (1:1) in small clay pots under controlled condition. (27\u0026deg;c and 90% relative humidity) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The plants achieved a height of 3\u0026ndash;4 cm after four weeks of transplantation. After raising under controlled conditions plants were transferred to large pots containing soil, sand and farmyard manure (1:1:1) and were maintained in green house.\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 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInfluence of exogenous auxin supplementation on the in vitro rhizogenic response and root architecture of \u003cem\u003eValeriana jatamansi.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of days for root emergence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRooting (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo of roots (Per explant)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRoot length (cm)\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\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;NAA 0.2 L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;IBA 0.2 L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;IAA 0.2 L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;IBA 2 L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u0026thinsp;\u0026plusmn;\u0026thinsp;4.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;IBA 3 L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;4.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.73\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u0026thinsp;+\u0026thinsp;IBA 4 L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.66\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Means followed by distinct letters within a column indicate significant differences (DMRT, p\u0026thinsp;\u0026le;\u0026thinsp;0.05). Data were recorded 5 weeks after the culture and represent a total of three replicates of 10 plants per treatment BAP 6-Benzylaminopurine, Indole-3-Acetic Acid.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhytochemical analysis\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003ePigment Analysis\u003c/h2\u003e \u003cp\u003ePigment analysis revealed that field-grown plants contained significantly higher levels of chlorophyll a (2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g FW), chlorophyll b (4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 mg/g FW), (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and total carotenoids (336\u0026thinsp;\u0026plusmn;\u0026thinsp;22.86 mg/g FW), (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), compared to \u003cem\u003ein vitro\u003c/em\u003e-grown plants. In contrast, \u003cem\u003ein vitro\u003c/em\u003e-grown plants exhibited lower pigment concentrations, with chlorophyll a at 1.773\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 mg/g FW, chlorophyll b at 2.932\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g FW, and carotenoids at 167.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30 mg/g FW, (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similar results could be found in studies conducted on various phytochemical comparison of field grown and \u003cem\u003ein vitro\u003c/em\u003e plants. \u003cb\u003e(\u003c/b\u003eTalreja, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Malik et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e This suggests that field conditions favor the synthesis of both chlorophyll a, chlorophyll b and carotenoids, possibly due to enhanced light exposure and nutrient availability.\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 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative evaluation of photosynthetic pigments and primary metabolite profiles in \u003cem\u003ein vitro\u003c/em\u003e propagated and field-grown \u003cem\u003eValeriana jatamansi\u003c/em\u003e Jones.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eChlorophyll a\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eField grown plants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e plants\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g FW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.773\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004 mg/g FW\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eChlorophyll b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.932\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCarotenoids\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e336\u0026thinsp;\u0026plusmn;\u0026thinsp;22.86 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e167.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal soluble sugar\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal soluble starch\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.125\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal amino acids\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/g FW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eFW: Fresh weight\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTotal Carbohydrate estimation\u003c/h2\u003e \u003cp\u003eTotal soluble sugar was found in higher amount in field grown plants (31.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mg/g FW) as compared to \u003cem\u003ein vitro\u003c/em\u003e plants (14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mg/g FW), (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Total soluble starch was found in higher quantity in the field grown plants (28.125\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mg/g FW) in comparison with \u003cem\u003ein vitro\u003c/em\u003e grown plants (13.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mg/g FW), (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similar results could be found in studies conducted on various phytochemical comparison of field grown and \u003cem\u003ein vitro\u003c/em\u003e plants. \u003cb\u003e(\u003c/b\u003eTalreja, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Malik et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e This suggests that field conditions favour the synthesis of these compounds, possibly due to enhanced light exposure and nutrient availability in field condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTotal amino acid\u003c/h2\u003e \u003cp\u003eThe total amino acids were present in higher amount in the plants grown \u003cem\u003ein vitro\u003c/em\u003e (5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/g FW), as compared to plants in cultivation (5.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mg/g FW), (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The higher accumulation of amino acids in \u003cem\u003ein vitro\u003c/em\u003e-grown plants compared to field-grown plants can be attributed to the controlled nutrient conditions in the \u003cem\u003ein vitro\u003c/em\u003e environment. In contrast to field conditions, which present a complex and variable nutrient landscape, \u003cem\u003ein vitro\u003c/em\u003e conditions provide a consistent supply of nutrients that can enhance amino acid uptake and accumulation. Moreover, the controlled environment of \u003cem\u003ein vitro\u003c/em\u003e culture may induce specific stress responses that lead to increased amino acid levels, similar to how certain amino acids are involved in plant adaptation to stress. These findings align with the concept that controlled conditions can amplify the effects of biostimulants, leading to higher concentrations of bioactive compounds in plant tissues. \u003cb\u003e(\u003c/b\u003eLardos et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTotal reducing power assay\u003c/h2\u003e \u003cp\u003eWhen compared with standard of ascorbic acid with methanolic extracts of \u003cem\u003ein vitro\u003c/em\u003e grown plants and field grown plants, field grown plants showed highest reducing power 0.334 as compared to \u003cem\u003ein vitro\u003c/em\u003e grown plants that is 0.111 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A higher absorbance of the reaction mixture corresponds to an increased reducing power. Several similar studies have shown that field-grown plants typically demonstrate a higher reducing power compared to \u003cem\u003ein vitro\u003c/em\u003e cultured plants. \u003cb\u003e(\u003c/b\u003eHakkim et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; \u003cb\u003eKhorasani\u003c/b\u003e \u003cb\u003eet al.\u003c/b\u003e, \u003cb\u003e2015;\u003c/b\u003e Kutty et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Field-grown plants are subjected to various environmental stressors such as extreme temperatures, drought, and pathogen exposure, which can induce the production of higher levels of antioxidants as a defence mechanism \u003cb\u003e(\u003c/b\u003eBansal et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e These natural conditions stimulate the synthesis of phenolic compounds and other antioxidant metabolites, contributing to a greater reducing power compared to plants grown in controlled, sterile \u003cem\u003ein vitro\u003c/em\u003e environments \u003cb\u003e(Thiruvengadam\u003c/b\u003e \u003cb\u003eet al.\u003c/b\u003e, \u003cb\u003e2015).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study successfully established a comprehensive and reproducible micropropagation protocol for \u003cem\u003eV. jatamansi\u003c/em\u003e, a high-value medicinal plant endemic to the Himalayas, whose natural populations are critically threatened by overexploitation and a lack of organized cultivation. The study aimed to address this urgent need for conservation and sustainable propagation strategies by comparing the efficacy of two widely used culture media, Murashige and Skoog (MS) and Gamborg\u0026rsquo;s B5, for direct organogenesis. The findings unequivocally demonstrate that the Gamborg\u0026rsquo;s B5 medium significantly outperforms the MS medium in key growth parameters, offering a more effective solution for the \u003cem\u003ein vitro\u003c/em\u003e propagation of this species. The primary focus of this investigation was to determine the optimal medium for achieving high-frequency shoot proliferation, leaf development, and rooting. The results showed that B5 medium fortified with BAP (2 mg L\u003csup\u003e-1\u003c/sup\u003e) and IAA (0.5 mg L\u003csup\u003e-1\u003c/sup\u003e) was the most effective for shoot regeneration, producing a maximum of 3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.816 shoots per plant, which was a notably superior response compared to the best MS medium treatment (BAP 1.5 mg L\u003csup\u003e-1\u003c/sup\u003e), which yielded 2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.471 shoots per plant. This finding aligns with the observation that Gamborg\u0026rsquo;s B5 medium\u0026rsquo;s distinct nutrient profile, including higher levels of potassium nitrate and ammonium sulfate, enhances nutrient uptake and protein synthesis, which are critical for tissue development. Furthermore, the maximum shoot length (4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.247 cm) and the highest number of leaves (11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.714 leaves per plant) were achieved on B5 medium, confirming its overall superior performance for the aerial parts of the plant. Beyond shoot induction, this study also provided a detailed analysis of rooting and acclimatization. The results indicate that rooting was observed exclusively in B5 medium during the direct organogenesis phase. However, root development was also observed in MS medium when shoot induced plantlets were subcultured in MS medium supplemented with auxins. The maximum number of roots (35.66\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8) and the longest root length (4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 cm) were recorded in MS medium supplemented with IBA at 4 mg L\u003csup\u003e-1\u003c/sup\u003e and 3 mg L\u003csup\u003e-1\u003c/sup\u003e, respectively. This two-step protocol initial shoot proliferation on B5 medium followed by rooting on MS medium provides a comprehensive and highly efficient strategy for mass multiplication.\u003c/p\u003e \u003cp\u003eThe study further evaluated the biochemical attributes of \u003cem\u003ein vitro\u003c/em\u003e-grown plants in comparison to field-grown counterparts. Phytochemical analysis confirmed that field conditions promote the synthesis of a number of key compounds. Field-grown plants had significantly higher levels of chlorophyll a (2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg/g FW), chlorophyll b (4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 mg/g FW), total carotenoids (336\u0026thinsp;\u0026plusmn;\u0026thinsp;22.86 mg/g FW), total soluble sugars (31.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mg/g FW), and total starch (28.125\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mg/g FW). Moreover, field-grown plants showed superior antioxidant potential, with a higher reducing power (absorbance of 0.334) compared to \u003cem\u003ein vitro\u003c/em\u003e-grown plants (absorbance of 0.111). Conversely, \u003cem\u003ein vitro\u003c/em\u003e-grown plants exhibited a higher total amino acid content (5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/g FW) compared to field-grown plants (5.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mg/g FW). These differences are likely due to environmental stressors in the field that stimulate the production of secondary metabolites as a defence mechanism, while the controlled \u003cem\u003ein vitro\u003c/em\u003e environment may enhance the accumulation of primary metabolites like amino acids.\u003c/p\u003e \u003cp\u003eIn conclusion, this research provides an efficient species-specific tissue culture protocol that offers a sustainable and scalable method for the mass propagation of \u003cem\u003eValeriana jatamansi\u003c/em\u003e. By enabling the production of genetically uniform, high-quality plantlets, this approach can alleviate the pressure on wild populations and support the establishment of commercial cultivation, thereby ensuring the long-term availability of this valuable plant for pharmaceutical, cosmetic, and perfumery industries. This study establishes the importance of optimizing media formulations for specific plant species and highlights the potential of plant tissue culture as a critical tool for both conservation and the sustainable production of threatened medicinal plants.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnalysis of Variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eB5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGamborg\u0026rsquo;s Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBenzyl acetyl pyruvate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCentimetre\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026ordm;C\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDegree Celsius\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDuncan's Multiple Range Test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFresh weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eh\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHour(s)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHgCl2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMercuric Chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIndole-3-acetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIBA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIndole-3-butyric acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ekPa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekilopascal\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emg\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMilli gram\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMg/g\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emilligrams per gram\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eml\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMillilitre\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMurashige and Skoog\u0026rsquo;s (1962) medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNaphthalene Acetic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003enm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enanometer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePotential of hydrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlant growth regulators\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erpm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRevolution per minute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltra violet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUV-VIS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltraviolet-Visible\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;l\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicrolitres\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026micro;mol\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicromole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ev/v\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVolume by volume\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ew/v\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWeight by volume\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eThe authors sincerely acknowledge the Department of High-Altitude Plant Physiology Research Centre, H.N.B. Garhwal University, Srinagar, for providing essential facilities and continuous guidance throughout the course of this study.\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, [Geken Riba], upon reasonable request.\u003c/p\u003e\n\u003cp\u003eFunding Declaration\u003c/p\u003e\n\u003cp\u003eThe authors declare that no specific funding was received for this research from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003eEthics Declaration\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eG.R conceptualized and designed the study, performed the plant collection and laboratory experiments, analyzed the data, and drafted the manuscript. B.P provided institutional supervision and critical insights throughout the research process. R.R.K contributed to the experiment conceptualization phase and provided technical assistance during the experimental work. D.J assisted in writing and was responsible for formatting the manuscript. All authors have read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArnon D (1949) Estimation of total chlorophyll. Plant Physiol 24(1):1\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAziz EE, El-Danasoury MM, Craker LE (2010) Impact of sulfur and ammonium sulfate on dragonhead plants grown in newly reclaimed soil. J herbs spices Med plants 16(2):126\u0026ndash;135\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAziz EE, El-Danasoury MM, Craker LE (2010) Impact of sulfur and ammonium sulfate on dragonhead plants grown in newly reclaimed soil. J herbs spices Med plants 16(2):126\u0026ndash;135\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBansal Y et al (2024) Synthesis and accumulation of phytocompounds in field-, tissue-culture grown (stress) root tissues and simultaneous defense response activity in Glycyrrhiza glabra L. Sustainability 16(4):1613\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBari M, Latiful, and Sabina Yeasmin (2022). Microbes Cult methods : 77\u0026ndash;98\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBisht S, Singh, Verma S (2019) Standardization of in vitro Multiplication Protocol for Picrorhiza kurroa (Royle Ex. Benth). Int. J Curr Microbiol App Sci 8(8):2043\u0026ndash;2049\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChapman K, Chomchalow N (2003), February Production of medicinal plants in Asia. In III WOCMAP Congress on Medicinal and Aromatic Plants-Volume 5: Quality, Efficacy, Safety, Processing and Trade in Medicinal 679 (pp. 45\u0026ndash;59)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCharmakar S, Kunwar RM, Sharma HP, Rimal B, Baral S, Joshi N, Oli BN (2021) Production, distribution, use and trade of Valeriana jatamansi Jones in Nepal. Global Ecol Conserv, 30, e01792\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen R, Zhang M, L\u0026uuml; J, Zhang X, da Silva JAT, Ma G (2014) Shoot organogenesis and somatic embryogenesis from leaf explants of Valeriana jatamansi Jones. Sci Hort 165:392\u0026ndash;397\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuce M, Inceer H (2024) Micropropagation and reintroduction of the endemic Tripleurospermum ziganaense (Asteraceae) to its natural habitat. Vitro Cell Dev Biology-Plant 60(5):646\u0026ndash;658\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC\u0026uuml;ce M, Karaismailoğlu MC (2023) An improved micropropagation protocol for the ex situ conservation of Thlaspi cariense A. Carlstr\u0026ouml;m (Brassicaceae): an endangered Turkish endemic plant species. Vitro Cell Dev Biology-Plant 59(5):574\u0026ndash;583\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuce M, Inceer H, Bagdatli E, Ert\u0026uuml;rk \u0026Ouml;, \u0026Ccedil;il E, Sagan R (2025) In vitro propagation, antioxidant and antimicrobial activities of Tripleurospermum baytopianum (Asteraceae): a threatened endemic species in T\u0026uuml;rkiye. South Afr J Bot 184:40\u0026ndash;49\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuce M, Inceer H, Imamoglu KV, Ergin T, Ucler AO (2022) Towards ex situ conservation of globally rare Turkish endemic Tripleurospermum fissurale (Asteraceae). In Vitro Cellular \u0026amp; Developmental Biology-Plant, 58(6), 1002\u0026ndash;1011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas J, Mao AA, Handique PJ (2013) Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones). Acta Physiol Plant 35:55\u0026ndash;63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDwivedi T, Kanta C, Singh LR, Prakash I (2019) A list of some important medicinal plants with their medicinal uses from Himalayan State Uttarakhand, India. J Med Plants 7(2):106\u0026ndash;116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFattorini L, Veloccia A, Della Rovere F, D\u0026rsquo;angeli S, Falasca G, Altamura MM (2017) Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity. BMC Plant Biol 17(1):121\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFazili MA, Bashir I, Ahmad M, Yaqoob U, Geelani SN (2022) In vitro strategies for the enhancement of secondary metabolite production in plants: a review. Bull Natl Res Centre 46(1):35\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitzpatrick TB, Lottie M (2020) Chapman. The importance of thiamine (vitamin B1) in plant health: From crop yield to biofortification. J Biol Chem 295(34):12002\u0026ndash;12013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrick EM, Strader LC (2018) Roles for IBA-derived auxin in plant development. J Exp Bot 69(2):169\u0026ndash;177\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):148\u0026ndash;151\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGautam RD, Kumar A, Kumar R, Chauhan R, Singh S, Kumar M, Singh S (2021) Clonal propagation of Valeriana jatamansi retains the essential oil profile of mother plants: An approach toward generating homogenous grade of essential oil for industrial use. Front Plant Sci 12:738247\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGautam RD, Kumar A, Kumar R, Chauhan R, Singh S, Kumar M, Singh S (2021) Clonal propagation of Valeriana jatamansi retains the essential oil profile of mother plants: An approach toward generating homogenous grade of essential oil for industrial use. Front Plant Sci 12:738247\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeorge EF, Hall MA, De Klerk GJ (eds) (2007) Plant propagation by tissue culture: volume 1. the background, vol 1. Springer Science \u0026amp; Business Media\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGowthami R, Sharma N, Pandey R, Agrawal A (2021) Status and consolidated list of threatened medicinal plants of India. Genet Resour Crop Evol 68(6):2235\u0026ndash;2263\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoyer A (2010) Thiamine in plants: aspects of its metabolism and functions. Phytochemistry 71.14-15 : 1615\u0026ndash;1624\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHakkim FL, Shankar CG, Girija S (2007) Chemical composition and antioxidant property of holy basil (Ocimum sanctum L.) leaves, stems, and inflorescence and their in vitro callus cultures. J Agric Food Chem 55(22):9109\u0026ndash;9117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamid R, Kamili AN, Zaffar M, da Silva JAT, Mujib A, Parray JA (2010) Callus Mediated Shoot Organogenesis from Shoot Tips of Cichorium intybus. J Med Aromatic Plant Sci Biotechnol 4 Special Issue 1:84\u0026ndash;86\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ehiruvengadam M, Chung I-M (2015) Phenolic compound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L). Electron J Biotechnol 18(4):295\u0026ndash;301\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoughton P (ed) (1997) Valerian: the genus Valeriana. Crc\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain A, Qarshi IA, Nazir H, Ullah I (2012) Plant tissue culture: current status and opportunities. Recent advances in plant in vitro culture. IntechOpen\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInceer H, Cuce M, Imamoglu KV, Ergin T, Ucler AO (2022) In vitro propagation and cytogenetic stability of Tripleurospermum insularum (Asteraceae)-a critically endangered insular endemic species from Turkey. Plant Biosystems-An Int J Dealing all Aspects Plant Biology 156(5):1213\u0026ndash;1221\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacob A, Malpathak N (2005) Manipulation of MS and B5 components for enhancement of growth and solasodine production in hairy root cultures of Solanum khasianum Clarke. Planr Cell Tissue Organ Cult 80(3):247\u0026ndash;257\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan A, Shah AH, Ali N (2021) In-vitro propagation and phytochemical profiling of a highly medicinal and endemic plant species of the Himalayan region (Saussurea costus). Scientific reports 11.1 : 23575\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhorasani Esmaeili A et al (2015) Antioxidant activity and total phenolic and flavonoid content of various solvent extracts from in vivo and in vitro grown Trifolium pratense L. (Red Clover). BioMed Research International, 2015, 643285\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiani Z, Mashayekhi K, Mousavizadeh SJ, Nezhad KZ (2022) Changes in carbohydrate and phytochemical content in the bulbs and leaves of ten onion accessions between two developmental stages. Italus Hortus 29:47\u0026ndash;55\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar A, Sathyakumar S, Goraya GS, Gupta AK, Adhikari BS, Rawat GS (2021) Assessment of medicinal and aromatic plant species on their collection, usage, demand, markets, price trends and life cycle in Lahaul and Pangi landscape, Himachal Pradesh. A report submitted to Himachal Pradesh Forest Department and United Nations Development Programme, p 141\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurmi K, Marcia C (2020) Haigis. Nitrogen metabolism in cancer and immunity. Trends Cell Biol 30:408\u0026ndash;424\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKutty NN et al (2014) Evaluation of antioxidant capacity and acetylcholinesterase inhibitory activity of field-grown plants and in vitro plantlets of Enicostemma littorale. Free Radicals Antioxid 4(2):21\u0026ndash;26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKutty NN et al (2014) Evaluation of antioxidant capacity and acetylcholinesterase inhibitory activity of field-grown plants and in vitro plantlets of Enicostemma littorale. Free Radicals Antioxid 4(2):21\u0026ndash;26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLardos M et al (2024) Discovery of the biostimulant effect of asparagine and glutamine on plant growth in Arabidopsis thaliana. Front Plant Sci 14:1281495\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeghari S, Jahan et al (2016) Role of nitrogen for plant growth and development: A review. Adv Environ Biology 10(9):209\u0026ndash;219\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik A et al (2020) A comparative biochemical evaluation of in vivo and in vitro propagated Alhagi maurorum: an important medicinal plant. Legume Research-An Int J 43(5):627\u0026ndash;633\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMathur J, Ahuja PS, Mathur A, Kukreja AK, Shah NC (1988) In vitro propagation of Valeriana wallichii. Planta Med 54(01):82\u0026ndash;83\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra Y, Rawat R, Nema B, Shirin F (2013) Effect of Seed Orientation and Medium Strength on in vitro Germination of Pterocarpus marsupium Roxb. Notulae Scientia Biologicae 5:476\u0026ndash;479\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant, 15(3)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurthy HN, Lee EJ, Paek KY (2014) Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Cult (PCTOC) 118:1\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNawchoo AI, Rather MA, Ganie HA, Jan RT (2012) Need for unprecedented impetus for monitoring and conservation of Valeriana jatamansi, a valuable medicinal plant of Kashmir Himalaya. Agricultural Sci Res Journals 2(7):369\u0026ndash;373\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeumann KH, Kumar A, Imani J (2009) Plant cell and tissue culture: a tool in biotechnology, vol 12. Springer, Berlin\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillips GC, Garda M (2019) Plant tissue culture media and practices: An overview. In Vitro Cellular \u0026amp; Developmental Biology - Plant, 55(3), 242\u0026ndash;257\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. Vitro Cell Dev Biology-Plant 55:242\u0026ndash;257\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePierik RLM (1997) vitro culture of higher plants. Springer science \u0026amp; business media\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRafi S, Wani SH, Wani ZA, Parveen K, Bhat MY (2018) In vitro culture and biochemical attributes of Bergenia ciliata (Haw.) Sternb. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 88(2), 609\u0026ndash;619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRafi S et al (2018) In vitro Culture and Biochemical Attributes of Bergenia ciliata (Haw.) Sternb. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences 88 : 609\u0026ndash;619\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaina AP, Negi KS (2015) Essential oil composition of Valeriana jatamansi Jones from Himalayan regions of India. Indian J Pharm Sci 77(2):218\u0026ndash;221\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuiz Herrera LF, Shane MW, L\u0026oacute;pez-Bucio J (2015) Nutritional regulation of root development. Wiley Interdisciplinary Reviews: Dev Biology 4(4):431\u0026ndash;443\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalreja T (2011) Biochemical estimation of three primary metabolites from medicinally important plant Moringa oleifera. Int J Pharm Sci Rev Res 7(2):186\u0026ndash;188\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThiruvengadam M, Chung I-M (2015) Phenolic compound production and biological activities from in vitro regenerated plants of gherkin (Cucumis anguria L). Electron J Biotechnol 18(4):295\u0026ndash;301\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomar NS, Sharma M, Agarwal RM (2015) Phytochemical analysis of Jatropha curcas L. during different seasons and developmental stages and seedling growth of wheat (Triticum aestivum L) as affected by extracts/leachates of Jatropha curcas L. Physiol Mol Biology Plants 21(1):83\u0026ndash;92\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma PC, Singh H, Negi AS, Saxena G, Rahman LU, Banerjee S (2015) Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth. Plant Signal Behav, 10(5), e1023976\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVollmer R, Espirilla J, S\u0026aacute;nchez JC, Arroyo L, Acosta M, Flores G, Azevedo V (2023) Thiamine improves in vitro propagation of sweetpotato [Ipomoea batatas (L.) Lam.]-confirmed with a wide range of genotypes. Plant Cell Tissue Organ Cult (PCTOC) 152(2):253\u0026ndash;266\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J et al (2016) Different responses of alpine plants to nitrogen addition: effects on plant-plant interactions. Sci Rep 6(1):38320\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWills RB, Bone K, Morgan M (2000) Herbal products: active constituents, modes of action and quality control. Nutr Res Rev 13(1):47\u0026ndash;77\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYıldıztekin M (2021) Ameliorative effects of exogenous ascorbic acid and potassium nitrate on antioxidant defense system and mineral nutrient uptake in tomatoes under salt stress\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin T-F et al (2017) Species-specific phenological responses to long-term nitrogen fertilization in an alpine meadow. J Plant Ecol 10(2):301\u0026ndash;309\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Valeriana jatamansi, Direct organogenesis, Gamborg’s medium","lastPublishedDoi":"10.21203/rs.3.rs-9539107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9539107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eValeriana jatamansi (Tagar) is a threatened Himalayan medicinal plant facing population decline due to habitat degradation and overexploitation\u003c/em\u003e. This study aimed to develop an efficient micropropagation protocol and evaluate biochemical attributes under \u003cem\u003ein vitro\u003c/em\u003eand field conditions. A completely randomized design (CRD) was used to compare Murashige and Skoog (MS) and Gamborg’s B5 media, with data analyzed using two-way ANOVA and DMRT, revealing significant effects (p ≤ 0.01) of basal media and plant growth regulators (PGRs). Gamborg’s B5 medium showed superior performance, with maximum shoot proliferation (3 ± 0.81 shoots per explant) and leaf number (11.33 ± 4.71) at 2.0 mg L⁻¹ BAP + 0.5 mg L⁻¹ IAA. Root induction was optimal on MS medium with IBA, producing the highest root number (35.66 ± 9.8) at 4.0 mg L⁻¹ and maximum root length (4.33 ± 0.94 cm) at 3.0 mg L⁻¹. Field-grown plants exhibited higher carotenoids, soluble sugars, starch, and antioxidant activity, whereas in vitro cultures showed higher amino acid content (5.44 ± 0.08 mg g⁻¹ FW). These findings indicate that B5 medium is superior for \u003cem\u003ein vitro\u003c/em\u003e regeneration, providing a reliable protocol for large-scale propagation and conservation of \u003cem\u003eV. jatamansi\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Elucidating the role of different culture medium on in vitro plant regeneration of Valeriana jatamansi Jones ex Roxb. a high value medicinal plant of the temperate Himalayas","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 15:37:08","doi":"10.21203/rs.3.rs-9539107/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T12:15:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274005318512594594079934464378299497799","date":"2026-05-13T08:24:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T08:22:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171854786267736265698754736288576792821","date":"2026-05-05T06:27:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154885502438099184882496020456598217414","date":"2026-05-04T08:56:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-04T07:40:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-03T13:52:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-03T13:52:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2026-04-27T08:56:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ad7bf4f3-0f95-4dce-b97e-50eced506b69","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T12:15:03+00:00","index":18,"fulltext":""},{"type":"reviewerAgreed","content":"274005318512594594079934464378299497799","date":"2026-05-13T08:24:17+00:00","index":17,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T08:22:46+00:00","index":16,"fulltext":""},{"type":"reviewerAgreed","content":"171854786267736265698754736288576792821","date":"2026-05-05T06:27:54+00:00","index":15,"fulltext":""},{"type":"reviewerAgreed","content":"154885502438099184882496020456598217414","date":"2026-05-04T08:56:50+00:00","index":12,"fulltext":""},{"type":"reviewersInvited","content":"5","date":"2026-05-04T07:40:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-03T13:52:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-03T13:52:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T15:37:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 15:37:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9539107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9539107","identity":"rs-9539107","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}