Induction and Submerged Cultivation of Valeriana Jatamansi Adventitious Root Cultures for Production of Valerenic Acid and its Derivatives

In vitro adventitious roots were induced from Valeriana jatamansi to assess their quality as an alternative ingredient for extraction of secondary metabolites to meet the demand of phytopharmaceutical industries. A signicantly (p ≤ 0.05) high root induction (90 %) was achieved on Schenk and Hildebrandt medium fortied with 9.84 µM indole-3-butyric acid. A maximum root biomass (144.09 ± 11.36 g/L) with high relative growth rate (2.01 ± 0.04) and growth index (13.41) was achieved in submerged cultivation. The total valerenic acid derivatives (1525.14 µg/g DW) and acetoxy valerenic acid (534.91µg/g DW) were signicantly high in induced adventitious roots, with notable quantity of hydroxyl valerenic acid (919.57 µg/g DW) that otherwise not quantiable in parent plant parts. In addition, 0.059% essential oil yield consisting of 24.00% patchouli alcohol was also obtained from induced adventitious roots. The phenolic acid derivatives were also signicantly higher in adventitious roots (451.58 µg/g DW) as compared to rhizome (187.79 µg/g DW) and leaves (263.68 µg/g DW) of the parent plant. Notably, a substantial increase in phytochemicals was evident at subsequent culture stages with signicantly reduced in vitro cultivation cycle (2 months) as compared to eld grown plants (24 months). Conclusively, a comparable metabolic prole of in vitro induced V. jatamansi adventitious roots and considerably shorter growth cycle clearly determines its potential as a feasible source of phytoconstituents. 2015), Gynostemma pentaphyllum (Khai and Minh 2018), Oplopanax elatus (Han et al. 2019) and Polygonum multiorum (Ho et al. 2019) have demonstrated adventitious roots as a stable production system for array of secondary metabolites. Considering the vulnerability of V. jatamansi in Himalayas and consumer driven demand for naturals by herbal industries, the present study was carried out to assess the possibility of in vitro induced adventitious roots as an alternate source to extract bioactives and essential oil on sustainable basis. free radical scavenging activity of the samples (as described preparation of sample for determination of valerenic acid derivatives) were analyzed by 2, 2-diphenyl-1-picrylhydrazyl (DPPH, Sigma) method (Singh et al. 2016; Bhardwaj et al. 2018). 0.2 mL methanolic extracts were mixed with 3 mL of 100 µM DPPH solution and incubated at room temperature (25 ± 2°C) for 30 min under dark condition (A E ). 0.2 mL of methanol instead of above sample extracts mixed with 3 mL DPPH (100 µM) solution was taken as Blank (A B ). Finally, absorbance was measured at 517nm using UV-Visible Spectrophotometer. DPPH activity was measured as: different experiments analysed using one-way analysis of variance using statistical software (SPSS version14) for all parameters i.e. induction percent, number and length of adventitious roots, fresh-weight, dry-weight, RGR and GI. The design of experiments used in the study was completely randomized design. The experimental data for adventitious roots induction and multiplication was recorded after 4 weeks and 8 weeks, respectively. The main effects of treatment (factor) and their interactions with dependent factor were studied for the test of signicance (p ≤ 0.05) and compared using Duncan Multiple Range Test. The degree of variations was represented as mean and standard error for all the treatments. acid (AVA) and hydroxyvalerenic (HVA) are main active components of the herb V. In present study, the parent plant material (rhizome and leaves) and in vitro induced adventitious root samples were analysed for the identication and quantication of VA, AVA and HVA acid using UPLC-PDA. The yield of total valerenic acid derivatives (1525.14 ± 68.85 µg/g DW) was signicantly (p ≤ 0.05) high in adventitious roots in comparison to parent plant parts i.e. rhizome (624.78 ± 13.67 µg/g DW) and leaves (200.17 ± 4.27 µg/g DW). However, valerenic acid (506.27 µg/g DW) in rhizome found higher as compared to leaves of the parent plant (79.23 4.56 µg/g DW) as well as in vitro induced adventitious roots (70.66 0.36 µg/g DW). Whereas, AVA content was signicantly (p ≤ 0.05) high in adventitious root samples (534.91 ± 39.57 µg/g DW) than parent plant parts i.e. rhizome µg/g DW) and leaves (120.94 ± 7.48 µg/g DW). Tousi et al. (2010) higher fraction of VA (0.38 %), AVA (0.55%) and HVA (0.44%) in Valeriana ocinalis adventitious roots induced from petiole explant on MS medium with IAA. acid in different parts of planted and wild cultivated V. jatamansi plants. They reported signicantly higher content of caffeic acid (158.56 mg/100 g) and hydroxybenzoic acid (390.58 mg/100 g DW) in planted condition, whereas, wild cultivated V. jatamansi plants recorded higher content of gallic acid (8.70 mg/100 g), coumeric acid (2.89 mg/100 g), chlorogenic acid (5.52 mg/100 g) and catechin (229.59 mg/100 g). In addition, aerial portion of wild cultivated V. jatamansi plants revealled higher content of gallic acid, p-coumaric acid, chlorogenic acid and catechins. However, caffeic acid and hydroxybenzoic acid content was found higher in aerial portion of planted V. jatamansi plants. In another study, Jugran et al. (2020) observed signicant variations in different phenolic i.e. gallic acid, p-coumaric acid, chlorogenic acid, catechin, caffeic acid and hydroxy-benzoic acid in pre-owering, owering and post-owering stages of V. jatamansi plant population wrt their occurance in high, intermediate and low altitude. Gallic acid content was found to be highest (9.39 mg/100 g DW) in pre - owering stage of plant population of low altitude region. p-Coumaric acid content was maximum (24.34 mg/100 g DW) at high altitude during pre-owering stage of the plant. In case of chlorogenic acid, post-owering stage showed highest (7.45 mg/100 g DW) content at high altitude. Catechin content was maximum (6.03 mg/100 g DW) in post-owering stage of plant population at high altitude conditions. Caffeic acid and hydroxy-benzoic acid was both detected highest (2.61 & 6.24 mg/100 g DW) in pre-owering stage of plant population collected from intermediate altitudinal region. Presence of above bioactives compound in adventitious root culture showed an alternate source for their production on sustainable basis for medicinal uses. analysis antioxidant DPPH (2, adventitious shows with leaf of mL-1) free found higher in adventitious root of Platycodon


Introduction
Valeriana jatamansi (Synonyms Valeriana wallichii) is an important medicinal and aromatic plant of Valerianaceae family grows between 1000-3000m elevation in Himalayan region. It is known by various vernacular names such as Indian Valerian, Muskbala, Sugandhbala (Hindi) and Tagar (Sanskrit) in India (Ved et al. 2003). V. jatamansi is an vulnerable plant species used in number of herbal medicine as a substitute for European V. o cinalis . As a consequence, Indian government included V. jatamansi as a prioritized plant species under cultivation programme of National Medicinal Plants Board (NMPB). The rhizome extract is known for its tranquilizer activities, besides antimicrobial, anti-in ammatory, insecticidal, antiviral, anti-leishmanial and antioxidant properties (Jugran et al. 2019). It is the most commonly used herbal supplement in combination with other herbs and ranked among top 10 therapeutics sold in United States (Lanje et al. 2020). In 2020, Valerian is ranked among top ten herbal medicines used in phytopharmaceutical industry around the globe (Link 1) as compared to 19th position during 2018 (Link 2). There are number of bioactive compounds reported from V. jatamansi including sesquiterpenes that consists of valerenic acid, acetoxy valerenic acid and hydroxyl valerenic acid (Bos et al. 1996;Singh et al. 2006). Valerenic acid is known to have spasmolytic and muscle relaxant properties, while isovalerenic acid contribute for its aroma (Murti et al. 2011). The hydroxy valerenic acid acts on GABA receptor of the central nervous system and is a major inhibitory neurotransmitter that helps in muscle relaxation Neuhaus et al. 2008; Felgentreff et al. 2012). In addition, the essential oil extracted from V. jatamansi roots, which varies from 0.05% -1.66% (Singh et al. 2010). The yield of essential oil in uenced by method and duration of distillation, species and growing habitat (Verma et al. 2013). The major component of essential oil are monoterpenes and sesquiterpenes (Jugran et al. 2019). An array of valerian products (root extracts, dried roots, powdered roots, essential oils and tinctures) alone or in combination with other plant ingredients are commercially available in market such as 'VitaGreen' (Brain tonic), 'Mushkbala Valerian root oil' (Perfume, Spa, Beauty), 'Calm Bliss' (Good sleep) etc.
In India, the estimated annual trade of dried V. jatamansi roots was about 1000-2000 MT in 2019, 3 which increased by more than 10 fold from 111.5 tonnes in 2001-2002 (Purohit et al. 2015). This huge jump in demand of raw ingredient by local healer, trader and exporter led to overexploitation of V. jatamansi from natural habitats and thus, adversely affecting the demand-supply chain of quality raw ingredient on sustainable basis. In addition, the low supply-high demand also led to adulteration in raw ingredients, thus affecting the human health at large (Dhiman et al. 2020).
In this regard, a number of biotechnological strategies are available and can be explored to produce bioactive ingredients. Among different plant cell and organ cultures, in vitro adventitious roots revealed to be an e cient system for the production of phytoconstituents at industrial scale (Murthy et al. 2016; Rahmat and Kang 2019). Generally, the adventitious roots formed by above ground plant parts such as leaf, stem, hypocotyl and non-pericycle tissues of the primary roots under adverse environmental condition (Verstraeten et al. 2014). There formation is an intricate process consists of three interdependent phases (induction, initiation and expression) and known to be in uenced by plant hormones, especially auxin (Kevers et al. 1997). Primarily, the particular cells of an explant or wounded tissue undergo fate transition, divide and form root primordia that enlarge through cortex to get expressed as adventitious roots (Lischweski et al. 2015). Under in vitro conditions, the establishment of adventitious root cultures in uenced by number of factors such as substrate, light, temperature, humidity and phytohormones (Murthy et al. 2008). These roots are easy to grow and have fast proliferation rate with capability of synthesizing speci c bioactive compounds ( (Ho et al. 2019) have demonstrated adventitious roots as a stable production system for array of secondary metabolites. Considering the vulnerability of V. jatamansi in Himalayas and consumer driven demand for naturals by herbal industries, the present study was carried out to assess the possibility of in vitro induced adventitious roots as an alternate source to extract bioactives and essential oil on sustainable basis.

Plant Materials
V. jatamansi (approximately 2 years old) plants were collected from Chamba District, Himachal Pradesh of north-west Himalayan region during May, 2018 and maintained under poly-house conditions at CSIR-IHBT. The leaves of V. jatamansi was used as explant for in vitro induction of adventitious root.

Explants preparation and surface sterilization
Explants were washed with autoclaved distilled water having 2-3 drops of Tween-20. Thereafter, the leaf explants were treated with Bavistin (0.1 % w/v) and Finally, surface sterilized with mercuric chloride (0.1 % w/v) for 2 min. under laminar ow cabinet and washed thoroughly with distilled water for three-four times. Under aseptic condition, explants were placed on autoclaved blotting paper to remove free water before inoculation.

Media preparation and culture conditions
For optimization of adventitious root initiation process, sterilized leaf explants were inoculated on hormone free Murashige

Determination of adventitious root biomass and growth parameters
The adventitious roots removed from semi-solid medium with the help of forceps after 8 weeks of cultivation and fresh weight was measured. In case of liquid cultures, roots were drained on sieve (1.0 mm) under aseptic condition and then rinsing once with autoclaved distilled water. Adventitious roots were dried on sterile lter paper to remove free surface water, after that fresh weight was measured. Finally, roots were dried in hot air oven at 40º C, until constant dry weight reached. The relative growth rate (RGR) and growth index (GI) was measured as per method reported by Ho et al. (2017) on fresh weight basis using following equations: Where, ln: natural log, W1: initial weight, W2: nal weight and CP: culture period.

Preparation of sample and determination of valerenic acid derivatives
The parent plant rhizome, leaves and in vitro induced adventitious roots from different culture stages were dried and powdered for phytochemical analysis. From each sample, 200 mg dried powder was extracted with 3.0 mL of methanol (HPLC grade) using sonication method for 60 min. and thereafter centrifuged at 2000 rpm for 10 min. The left-over residual of each sample was again extracted with 2 mL of solvent and supernatant pooled together in 5.0 mL sample collecting vials. Finally, respective supernatants were ltered with Puradisc syringe lter (0.2 µm) and stored at 4 ºC. Final concentration of extract in respective samples was made to 40.0 mg/mL. The standards of valerenic acid (VA), acetoxyvalerenic acid (AVA) and hydroxyvalerenic acid (HVA) were procured from Sigma India and stock solutions (1 mg/mL) made in HPLC grade methanol to prepare calibration curve for comparative evaluation. The valerenic acid derivatives were quanti ed using Acquity Ultra Performance Liquid Chromatography (UPLC) -e photodiode array detector (Waters, India).
System have two mobile phases: (A) phosphoric acid (0.1 %) in water and (B) acetonitrile. All the samples were injected at 5 µL concentration. The absorbance was measured at 280 nm and quanti cation performed using standard curve of VA, AVA and HVA. Each sample was analysed in triplicate.
Distillation and phytochemical analysis of essential oil Freshly harvested V. jatamansi plant parts (rhizome and leaves) and in vitro grown adventitious roots were distilled in Clevenger apparatus. Chopped samples were washed with distilled water and air-dried on sterile lter paper. Thereafter, 1 Kg (FW) of each sample was added to 5.0 L round bottom ask and hydrodistilled (400 mL water) for 4 hours. The extracted essential oil ltered and stored under dark in sealed vials at 4°C.
Gas Chromatography-Mass Spectrometry (GC-MS) analysis of essential oil extracted from all the samples was performed on Shimadzu QP2010 series tted with AOC-500 auto-sampler and SH-RXI-55ILMS capillary column (30 m x 0.25 mm i.d., lm thickness 0.25 µm). Helium (99.99 % pure) was used as carrier gas with 1.05 mL/min ow rate, linear velocity 37.60 cm/s, pressure 65.30 kPa, split ratio 1: 10, mass scan 40-800 amu at a sampling rate of 1.0 scan/s, scan speed: 1666 u/s and interval: 0.5 s. The oven temperature was programmed as mentioned for GC analysis. Electron impact ionization at 70 eV with 0.9 kV detector voltage was used. 10 µL oil samples were mixed with 2 mL DCM (GC grade) and 2 µL of this solution was injected. Ion source temperature was 200°C, interface temperature was 240°C and injector temperature was maintained at 250°C. The constituents were identi ed with the help of relative retention indices and by comparison with known mass spectral data, National Institute of Standards and Technology (NIST) libraries. A mixture of n-alkanes (C 8 -C 24 ) was used as reference for the calculation of relative retention indices (RRI) in temperature-programmed run. Moreover, decane was also used as an internal standard. The analysis of every sample was performed in triplicate.

Phenolic acids derivatives characterization
Plant part (rhizome and leaves) and adventitious root samples (100 mg each) were analysed for determination of phenolic acids derivatives i.e. gallic acid, pcoumaric acid, rutin, ferulic acid, cinnamic acid and kaempferol (Sigma) using Ultra Performance Liquid Chromatography -e photodiode array detector (UPLC-PDA, Waters, India) system. All the samples were dried, powdered and extracted in 70 % Methanol using sonication methods for 10 min. followed by centrifugation at 8000 rpm for 10 min. This process was repeated three times upto 5 ml of solvent. The samples were ltered through 0.22 µm syringe lter and transferred into vials for further analysis. In the UPLC system, the analytical column used was Waters HSS-T3 C18 column (2.1mm 100 mm, 5 mm, 1.8 µm). Detection wavelength was set at 270 nm. The gradient elution system was used, mobile phase A contain 0.1% formic acid in water, mobile phase B was 0.1 % formic acid in acetonitrile (ACN). The gradient started from 0 min. at 10% B; then from 0-1 min, 10% B; 1-8 min, linear gradient from 10% B to 45% B; 8-9 min, 95% B; 9-10 min, 95% B, then again mobile phase ran on initial conditions, 10-11 min, 10% B, 11-13 min, 10% B. Elution was performed at a solvent ow rate of 0.25 mL/min. The entire sample was analysed in triplicate.
Analysis of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) activity The free radical scavenging activity of the samples (as described preparation of sample for determination of valerenic acid derivatives) were analyzed by 2, 2- Design of experiment and data analysis The resultant data obtained from different experiments were analysed using one-way analysis of variance using statistical software (SPSS version14) for all parameters i.e. induction percent, number and length of adventitious roots, fresh-weight, dry-weight, RGR and GI. The design of experiments used in the study was completely randomized design. The experimental data for adventitious roots induction and multiplication was recorded after 4 weeks and 8 weeks, respectively. The main effects of treatment (factor) and their interactions with dependent factor were studied for the test of signi cance (p ≤ 0.05) and compared using Duncan Multiple Range Test. The degree of variations was represented as mean and standard error for all the treatments.

Results And Discussion
The ever-rising demand for natural products and continuously diminishing plant population in natural habitat necessitates to develop alternative method to generate herbal ingredients of industrial importance. Hence, in vitro adventitious roots of V. jatamansi are worked out in this study as a substitute to obtain valerenic acid derivatives, essential oil and other derivatives.
Induction of adventitious root cultures Murashige and Skoog (1962) Schenk and Hildebrandt (1972) and Gamborg (1968) medium were tried to induce in vitro adventitious roots from leaf explant of V. jatamansi. For this purpose, leaves were surface sterilized and cut into 4-5 mm sections and inoculated on different hormone free media under aseptic conditions.
The selection of explant was based on earlier reports that showed leaf explant as a best source for initiation of adventitious roots cultures in P. vietnamensis For instance, SH media have low ammonium: nitrate ratio (1:9) as compared to MS medium (1:2), which probably be helping in organogenesis. SH media also have 10 times higher myo-inositol (1000 mg/L) concertation than B5 and MS medium. It is pertinent to mention that myo-inositol known to accelerate cell division rather than increasing the cell size (Staudt 1984), which may be one of the reason for induction of adventitious root in SH medium. Similar observation was reported for adventitious root growth in ve species of Scutellaria genus in SH medium forti ed with IBA (1.0 mg/L) as compared to B5 and MS medium (Barska et al. 2011).
In general, adventitious root formation is a complex process and tightly regulated by various phytohormones. However, auxin is a key hormone for induction and regulation of adventitious root development. There are two types of auxins i.e. synthetic (NAA [1-naphthaleneacetic acid]) and natural analogue (IAA . The induction of adventitious roots was evident as hair-like outgrowth from cut ends of the leaves within 8 days of inoculation, as compared to 28 days in hormone free medium. Furthermore, induction of roots was also observed from the margins of the leaf sections at several points during extended period up to four weeks of incubation. Experimental data releveled a signi cantly (p ≤ 0.05) high percentage of adventitious root induction (90%) as well as their number (5.72 ± 0.18) and length (1.73 ± 0.06 cm) in medium forti ed with 9.84 µM IBA (Table 1). However, the rooting potential found to be decreased beyond this concentration of IBA. It was noticed that above this IBA concertation i.e. 9.84 µM IBA, leaves showed more callusing than adventitious root formation. It can be deduced from the results that SH medium with 9.84 µM IBA forti cation is e cient for inducing adventitious roots from V. jatamansi leaf explant at reasonably shorter time as compared to other combinations. In accordance to present study, IBA has been demonstrated to be an e cient hormone for induction and multiplication of adventitious roots in number of plants ( The effect of auxin on adventitious root induction was further con rmed by forti cation of basal SH, MS and B5 media with optimized concentration of IBA (9.84 µM). Surprisingly, adventitious roots were induced from inoculated leaf explants in all the media. However, signi cantly high (p ≤ 0.05) rhizogenic potentials in terms of induction (90.0%) as well as number (5.44 ± 0.32) and length (1.47 ± 0.09 cm) of adventitious roots was obtained in SH medium as compared to MS and B5 (Fig. 1). Thus, the earlier results hold true that the SH medium with 9.84 µM IBA concentration was optimum for induction of adventitious roots. These roots were repeatedly sub-cultured on the above optimized medium for further multiplication and maintenance of mother stock. Consistent with observation of present investigation, Kim

Effect of IBA on multiplication of adventitious roots in submerged cultivation
In order to evaluate the capability of adventitious roots for large-scale cultivation, in vitro induced roots were inoculated (1.0 % inoculum density) in SH liquid medium augmented with various IBA concentrations. The growth of adventitious roots was determined based on biomass yield and relative growth rate (RGR) after 8 weeks of cultivation. Experimental results showed a signi cant (p ≤ 0.05) effect of IBA concentration on multiplication of adventitious roots in liquid medium. A signi cantly high root biomass yield (123.39 ± 7.11 g/L FW) and relative growth rate (RGR) (1.95 ± 0.03) was recorded in SH liquid medium at 4.92 µM IBA concentration after 8 weeks of cultivation (Table 2). In addition, the growth index (11.34 ± 0.71) (GI) was also found highest in same medium (Fig. 2a).
However, the IBA concertation beyond 4.92 µM was not able to support further growth of adventitious roots. Furthermore, these results suggest that the multiplication of adventitious root in submerged condition was obtained in just half IBA concentration to that of induction medium (9.48 µM).

Effect of medium strength on multiplication of adventitious roots in submerged cultivation
In general, medium type and their elemental composition found to in uence growth as well as overall productivity of in vitro plant cultures.  (Table 3) as compared to other medium strength tested in the study. GI was also highest in half strength (1/2X) SH medium (Fig. 2b). SH medium having 1/4x concentration exhibited slowest growth as evident from the root biomass yield. From the above results, it can be deduced that optimal concentration of macro and micronutrients in media seems to be the determining factor for in vitro induced adventitious root growth.  In uence of various concentrations of sucrose on multiplication of adventitious roots in submerged cultivation In plant cell and tissue cultures, sucrose is the principal carbohydrate used as energy source for growth and development. It is catabolized into glucose and fructose and it's absorption rate varies with type of cultures (George et al. 2008). In the present work, diverse sucrose (1 to 5 % w/v) concentration in optimized media i.e. ½ strength SH media having 4.92 µM IBA were tried in submerged cultivation to improve further V. jatamansi adventitious roots multiplication.
Among different concentrations of sucrose tested, a signi cantly high (p ≤ 0.05) root biomass (144.09 ± 11.36 g/L FW) and relative growth rate (2.01 ± 0.04) was obtained in medium augmented with 2% (w/v) sucrose after cultivation of 8 weeks (Table 4). Also, same media showed highest growth index (13.41 ± 1.14) as compared to other concentration (Fig. 2c). However, the root biomass yield on dry weight basis depicted dissimilar pattern with subsequent increase in sucrose concentration (4-5%). It may be due to formation of more extracellular polysaccharides with respect to the higher concentration of sucrose in the medium (Saiman et al. 2012).  Interestingly, in vitro adventitious roots showed presence of HVA at signi cantly high amount (919.57 ± 28.85 µg/g DW), which otherwise not quanti able in leaves as well as rhizome parts of the parent plants (Table 5 and Fig. 3). It is also pertinent to mention here that the comparative evaluation performed was between two-month (eight weeks) old in vitro adventitious roots versus rhizomes and leaves of approximately 2-year old plants grown under conventional agricultural cultivation condition. Thus, results of present study clearly suggesting the potential of V. jatamansi adventitious roots as a good alternative source of valerenic acid and its derivatives. In addition, in vitro induced adventitious roots could be a novel source of hydroxyvalerenic acid that was unquanti able in parent plant parts.  cultivation followed by nursery grown (3.18 mg/g) and aeroponic cultivation (1.78 mg/g). AVA content was found higher (2.38 mg/g) in roots grown under aeroponic condition.

Determination of essential oil, patchouli alcohol and other derivatives using Gas Chromatographic -Mass Spectroscopic (GC -MS) analysis
Valeriana jatamansi plant parts (rhizome & leaves) and adventitious root (1000g each, FW) were used for the extraction of essential oil using hydro-distillation method in a Clevenger apparatus. The plant rhizome yielded relatively higher essential oil content (0.4% v/w) as compared to in vitro induced adventitious root (0.059% v/w). However, trace amount of essential oil was obtained from the leaves of parent plants. In present study, adventitious root induced from leaves yielded 0.059% v/w essential oil, which is reasonably low as compared to rhizomes harvested from eld grown parent plants. However, it is worth mentioning that in vitro cultivation period of adventitious root is signi cantly low (2 months) as compared to eld conditions (2 years).
Furthermore, the essential oil extracted from different samples was analysed by GC-MS to characterize its individual constituents. In this regard, a total of thirty-one phytochemical constituents were characterized and identi ed (Table 6, Fig. 4). These constituents represent 98.15%, 79.11% and 96.56% of essential oil obtained from rhizome, leaves and adventitious root samples, respectively. Overall, the GC-MS analysis exhibited presence of nine common constituents i.e.
Effect of culture stages on production of valerenic acid derivatives In general, in vitro plant cell and tissue culture shows variability in growth as well as metabolite production at various culture stages. Therefore, production of valerenic acid derivatives at different culture stages i.e. a) induced from leaf explants on semi -solid SH media + 9.84 µM IBA (P 0 ), b) multiplied on semi -solid SH media + 4.92 µM IBA (P 1 ) and c) submerged cultivation in liquid SH media + 4.92 µM IBA (P 2 ) of V. jatamansi adventitious root development was also studied in present investigation. The results showed presence of VA, AVA and HVA in all the stages of adventitious root formation and multiplication (Fig. 7).
Total valerenic acid derivatives yield were signi cantly (p ≤ 0.05) increased (302.28-1625.98 µg/g DW) from culture stage 1 to stage 3. In case of individual metabolite, there was signi cant (p ≤ 0.05) enhancement in AVA (92.51-620.72 µg/g DW) and HVA (140.42-933.95 µg/g DW) content with respect to different culture stages (Fig. 7). From the trend, it can be deduced that in culture stage 1 the priority of the tissue is speci cally towards its growth and development, whereas during stage 2 and 3 the high metabolic yield indicates the shift or activation of secondary metabolism. Also, there might be a possibility that submerged cultivation create more stressing environment, thus enhancing biosynthesis of valerenic acid derivatives, especially in stage 3 cultures.
Similar information on secondary metabolite production in relation to in vitro plant tissue culture stages/passage is very limited. Hagimori

Overall process of induction, multiplication and submerged cultivation
The complete process of V. jatamansi adventitious root culture cultivation can be divided into three stages; namely, adventitious root induction, multiplication and submerged cultivation for scale up production (Fig. 8). The detailed bioprocess can be summarized under following heads: Adventitious root induction: In this stage, adventitious roots were induced from leaf explants on optimized SH media forti ed with 9.84 µM IBA within eight days of inoculation under aseptic condition.
Multiplication of adventitious roots: After four weeks of induction, induced roots were further ampli ed on semi-solid ½ strength SH media having 4.92 µM IBA and 2.0 % sucrose.
Submerged cultivation of adventitious roots: Considering the development of alternative route for production of valerenic acid derivatives, large-scale multiplication of adventitious roots was done through submerged cultivation in optimized ½ strength liquid SH media enriched with 4.92 µM IBA and 2.0 % sucrose for two months (eight weeks) under in vitro condition.
Considering the developed in vitro protocol, the complete process took two months (eight weeks) after induction of adventitious roots from leaf explants of V. jatamansi. Whereas, the generation of commercially valued rhizomes or root biomass through conventional means generally takes 2 years after transplanting in eld condition (Singh et al. 2010). In addition, the conventional cultivation gets jeopardised by slow germination, poor viability and long dormancy of seeds as well as limited planting material through vegetative propagation (Rana et al. 2004). These issues not only affecting the availability of quality raw material to herbal industries on sustainable basis, but also affecting the plant population in its natural habitat. The feasibility of submerged cultivation bioprocess as compared to conventional cultivation can be assessed on the basis that in vitro adventitious roots can yield over two times higher essential oil (Table 8). Table 8 Comparative analysis of essential oil and total valerenic acid derivative yield in conventional and submerged cultivation. However, a signi cantly shorter in vitro cultivation cycle (2 month) than conventional means (2 years) could be a crucial factor to support the feasibility of developed process at industrial scale. Conclusively, the results of present study are clearly exhibiting the potential of in vitro induced adventitious roots as an sustainable source to meet industrial demand around the year.     Growth cycle of in vitro induced adventitious root culture of V. jatamansi.