Optimization of micropropagation protocol and elicitation of luteolin and rutin biomolecules using phenylalanine and chitosan in root callus of Rumex hastatus D. Don

Rumex hastatus D. Don, a traditional medicinal plant is known for its antitumorous, antiangiogenic and antibacterial potential. HPLC analysis of different plant parts showed that root had the maximum quantity of luteolin and rutin and thus was selected for the establishment of aseptic cultures. Murashige and Skoog’s medium supplemented with 5 µM TDZ proved best for developing greenish-brown and compact calli in 98% root cultures within 30 days. Upon subculturing such callus pieces differentiated an average of 10.50 ± 1.29 shoots per culture. The excised shoots were transferred on MS + 0.1 µM IBA medium where they developed an average of 12.03 ± 2.49 roots per shoot. These plantlets were successfully acclimatized under greenhouse conditions and their genetic fidelity was evaluated employing SRAP and SCoT markers which revealed their high similarity with mother plant proving their true-to-type nature. Interestingly, the in vitro regenerants showed elevated levels of phenols, flavonoids, luteolin and rutin contents and higher antioxidant activities over the mother plant as revealed by phosphomolybdenum and DPPH assays. For elicitation, 100 mg/L phenylalanine was best for enhancing luteolin content (407.18 ± 10.58 μg/g d.w.) up to 17.42-fold over control (22.10 ± 0.57 μg/g d.w.) whereas, 25 mg/L chitosan proved most effective for accumulation of rutin content (1169.07 ± 46.49 μg/g d.w.) up to 2.74-fold over control (312.41 ± 12.42 μg/g d.w.). To the best of our knowledge, this is the first report on micropropagation and elicitation of luteolin and rutin in R. hastatus through root culture. The present investigation focused on selection of an elite plant part, regeneration through in vitro root callus, genetic fidelity analysis, evaluation of secondary metabolites, antioxidant activity and enhancement of luteolin and rutin using elicitors in Rumex hastatus.


Introduction
Rumex hastatus D. Don (Arrowleaf dock), a traditional medicinal plant belonging to the family polygonaceae is a perennial, small bushy shrub with many ascending branches, arrow shaped leaves, having terminal inflorescence. A native to Afghanistan, China and north west India, it grows at an altitude of 700-2500 m and is traditionally used to cure rheumatism, skin diseases, bilious complaints, piles, bleeding of lungs etc. (Ahmad et al. 2016). Plant is also used as flavouring agent, carminative, purgative and diuretic (Ullah et al. 2014). Phytochemical investigations have revealed that roots are the major source of several important bioactive compounds such as resveratrol, epicatechin, epicatechin gallate, rumexneposide A and B, hastatusides A and B, rutin, nepodin, orientaloside, emodin, physcion (Liang et al. 2010), luteolin, luteolin glucoside, kaempherol (Sahreen et al. 2011), phytol, dihydrojasmone, nonivamide, silane,  eicosanol, aristolone, sitostrenone (Ahmad et al. 2016). Due to the occurrence of these bioactive compounds, this plant has immense antioxidant, antitumor, anti-inflammatory, hepatoprotective, antiangiogenic, anthelminthic, anticholinesterase and antibacterial properties (Dahiya et al. 2012;Sahreen et al. 2015Sahreen et al. , 2017Ahmad et al. 2015Ahmad et al. , 2016. However, due to extensive over-exploitation by the pharmaceutical and nutraceutical industries, the plant is losing its natural habitat and valuable germplasm. It is thus imperative to develop an efficient micropropagation protocol for its conservation, sustainability and production. Tissue culture has been widely used for the large-scale regeneration of valuable medicinal plants including the production of pharmaceutically active secondary metabolites through callus, cell suspension, hairy root cultures, etc. (Shinde et al. 2009a(Shinde et al. , 2009bSharma and Agrawal 2018;Singh et al. 2020aSingh et al. , 2020b. This technology has been beneficial in improving the yield of various bioactive molecules such as phenols, flavonoids, alkaloids, etc. under in vitro conditions contributing to the production of elite genotypes or chemotypes (Chavan et al. 2014;Bhattacharyya et al. 2014;Sharma and Agrawal 2018). Therefore, phytochemical evaluation and antioxidant activities in in vitro grown plants are of immense importance. To the best of our knowledge, in vitro micropropagation studies using root explants in R. hastatus have been presented for the first time here. Since, the in vitro raised plants particularly derived through callus-mediated pathway are liable to show somaclonal variations (Sharma and Agrawal 2018;Devarumath et al. 2002;Singh et al. 2013), there remains a need to assess the genetic fidelity of such plants through various strategies e.g., detection of chromosome numbers, isozyme profiling and PCR based DNA markers (Bose et al. 2016;Sharma and Agrawal 2018). PCR based markers are proven to be sensitive, reliable, rapid, and cost-effective and not influenced by environmental factors for evaluation of genetic stability (Bose et al. 2016). Besides, several other DNA molecular markers such as start codon targeted (SCoT) polymorphism, sequence related amplified polymorphism (SRAP), intersimple sequence repeats (ISSR), random amplification of polymorphic DNA (RAPD), or intron splice junction (ISJ) have also been employed to assess the genetic stability of regenerants in different plant systems e.g., Tetrastigma hemsleyanum (Peng et al. 2015), Nardostachys jatamansi (Bose et al. 2016;Dhiman et al. 2021), Rumex nepalensis (Bhattacharyya et al. 2017), Plumbago zeylanica (Sharma and Agrawal 2018), Decalepis salicifolia (Rodrigues et al. 2020).
The present study is focused on the: (i) development of regeneration protocol of Rumex hastatus using in vitro root explant, (ii) genetic fidelity of in vitro-derived regenerants employing PCR-based DNA markers e.g., SCoT and SRAP, and (iii) evaluation of secondary metabolites (total phenolic and flavonoid contents), quantification of luteolin and rutin, antioxidant activities (phosphomolybdenum assay and DPPH free radical scavenging assay) in both regenerants and mother plant. In addition, (iv) in vitro elicitation of luteolin and rutin contents employing various elicitors such as chitosan, phenylalanine, salicylic acid and yeast extract in root-derived callus of R. hastatus.

Plant material and selection of elite plant part
Plant material of Rumex hastatus D. Don (Fig. 1a) was collected from Mandi, Himachal Pradesh, India (76.84 ºE, 31.74 ºN) and a specimen was submitted to Herbarium of Delhi University, Delhi, India (DUH14663). The samples of fresh roots, stems and leaves of field grown R. hastatus were procured in triplicates, and then washed thoroughly under running tap water, and shade dried at room temperature (25 ± 2 °C) for 7 days (d). Dried tissue (5 g) was homogenized separately in 100 mL methanol and extracted with constant shaking at 100 rpm for 48 h at 25 ± 2 °C. The extracts were filtered through Whatman filter paper no. 1 (Whatman™, GE Healthcare UK Limited, Amersham Place, UK) and evaporated to dryness. These extracts were used for high performance liquid chromatography (HPLC) analysis. Quantification of luteolin and rutin (Sigma-Aldrich, USA) in R. hastatus through HPLC was performed with photo diode array detector of the HPLC unit (Waters, USA) using a C18 column (Supelcosil L x I.D. 250 × 4.6 mm; Sigma-Aldrich, USA). Luteolin and rutin content was determined using the modified protocol of Cristea et al. (2003), where gradient of methanol (100; v) and water: acetic acid (99: 1; v: v) was used as mobile solvents (Suppl. Table 1) and monitored at 350 nm. Peaks were identified by comparing the retention Fig. 1 Selection of elite plant part and regeneration through in vitro root derived callus of R. hastatus. a R. hastatus growing in natural habitat, b HPLC chromatogram of standard luteolin and rutin showing the peaks at retention time at 19.39 and 28.35 min, respectively, using gradient of methanol (100, v) and water: acetic acid (99:1, v:v), c graphical representation of quantity of rutin and luteolin in different parts (root, stem and leaf) of R. hastatus evaluated through HPLC, d in vitro root explants inoculated on MS medium fortified with different PGRs, e induction of callus from root explants cultured on MS medium + 5 µM TDZ, f differentiation of root callus into shoot buds on MS + 5.0 µM TDZ, after 30-d of culture (red arrows showing shoot buds), g shoot buds showing stunted growth on MS + 5.0 µM TDZ, after 30-d of inoculation, h elongated shoots on MS basal medium, after 15-d of culture, i shoots inoculated on MS + 0.1 μM IBA showing maximum number of roots, j In vitro hardening of root callus-derived regenerants (RDR) by transferring into paper cups containing soilrite-mix (75% Irish peat moss + 25% perlite), k hardened RDR growing in clay pot in botanical garden, University of Delhi, India, after 3 month of field transfer. Red scale bar = 1 cm ◂ time of standard compounds luteolin and rutin with that of the samples. The solvents used were of HPLC grade.

Establishment of aseptic cultures
Nodal explants (3-4 cm) were excised from the mother plant and surface sterilized according to the protocol given by Sharma and Agrawal (2018). Murashige and Skoog 1962 (MS) medium containing 3% sucrose and 0.8% agar-agar was used for the establishment of aseptic cultures through in vivo nodal explants. Prior to autoclaving at 121 °C and 103.4 kPa for 20 min, pH of medium was adjusted to 5.8 with 1 N NaOH or 1 N HCl. Sterilized nodal explants were then inoculated on MS medium and maintained by periodic sub-culturing of in vitro nodes after every 30-d. These cultures were incubated in a growth room at 23 ± 2 °C temperature and 50 ± 5% relative humidity under cool white fluorescent tube lights (Philips India Ltd., Kolkata, India) having the light intensity of 30-40 µmol/m 2 s with photoperiod of 16/8 h (Light/ Dark). Roots organized on the nodal explants were employed as source material for subsequent callus culture.

Induction of callus from in vitro root explants and shoot elongation
For callus induction, in vitro roots (2-3 cm in length) were inoculated on MS medium fortified with various concentrations (0.1, 1.0, 5.0 or 10.0 µM) of 6-benzylaminopurine (BAP), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ). These cultures were incubated and maintained in a growth room for 30-d. Data pertaining to percent callus induction per explant, colour and texture, and amount of callus were also observed. After the induction of an optimum greenish brown and compact callus, it was further sub-cultured on the same but fresh medium fortified with various concentrations (0.1, 10, 5.0 or 10.0 µM) of TDZ for shoot bud differentiation. Growth medium without TDZ was taken as a control. Various parameters such as percent of cultures differentiating shoot buds and an average number of shoot buds per explant were recorded after 30-d of inoculation. Such shoot buds were transferred to MS basal medium for elongation. All the used plant growth regulators were purchased from Sigma Aldrich.

Induction of roots in the in vitro excised shoots
Root-callus derived elongated shoots (2-3 cm in length) were excised and transferred to MS medium supplemented with various concentrations (0.1, 1.0, 5.0 or 10.0 µM) of auxins such as IAA, IBA and NAA for induction of roots. These cultures were maintained in the growth room and growth parameters such as average root number and length (cm) were recorded after 30-d of inoculation.

In vitro hardening and field acclimatization
The in vitro plantlets were removed from the culture tubes and washed gently with distilled water to discard the traces of solidified growth medium and shifted to paper cups having a hole at the bottom containing 40-50 g soilrite-mix. The regenerants were then kept for two weeks in growth room at 25 ± 2 °C and 50 ± 10% humidity with photoperiod of 16 h. The regenerants were irrigated everyday with ½ strength MS medium (without sucrose) for two weeks. Further, these plantlets were acclimatized at 30 ± 5 °C and 40 ± 10% humidity in glasshouse for another two weeks and watered everyday with ¼ strength MS medium (without sucrose). Finally, these regenerants were transferred to earthen pots having garden soil and maintained in exposed environment. The survival rate was documented after 60-d of transfer to field conditions.

Genetic fidelity analysis by employing SCoT and SRAP markers
For genetic fidelity analysis, fresh leaves (1 g) of mother plant and randomly selected 10 regenerants were harvested and the genomic DNA was isolated separately following the cetyltrimethylammonium bromide (CTAB) method (Saghai-Maroof et al. 1984). A total of 34 SCoT and 52 SRAP primers were initially screened for genetic fidelity analysis of the regenerants. Based on the best amplification and reproducibility, 10 each SCoT and SRAP primers were chosen for final amplification in a thermal cycler (BioRad). All the PCR mixtures comprised 20 ng of template DNA, 2.5 µL of 10 × PCR buffer containing 15 mM MgCl 2 , 0.2 mM dNTPs, 1 U DNA Taq polymerase and 20 ng SCoT/ SRAP primers, and PCR reactions were carried out in 20 µL volumes. PCR conditions for SCoT were set as the initial denaturation step at 94 °C for 3 min, followed by 35 cycles of 94 ºC for 30 s, 50 °C for 1 min and 72 °C for 2 min and final extension was carried out at 72 ºC for 7 min. The amplified DNA products were analyzed on 1.2% (w/v) agarose gel in 0.5 × TBE buffer at 70-80 V for 1-1.5 h. However, PCR condition for SRAP was set as an initial denaturation step at 95 °C for 4 min, followed by 5 cycles of 94 °C for 1 min, 36 °C for 1 min and 72 °C for 2 min, 30 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min and final extension was performed at 72 °C for 7 min. The amplified fragments of DNA were resolved on 2% (w/v) agarose gel in 0.5 × TBE buffer at 75-85 V for 1-1.5 h. The amplified DNA fragments were visualized under UV light using gel documentation system (Alpha Innotech Corporation, San Leandro, CA). The generated pooled SCoT and SRAP markers data were scored into binary matrix and were analyzed as described by Rajput and Agrawal (2020).

Preparation of extract
Fresh roots, stems and leaves of the mother plant as well as 3-months-old acclimatized regenerants were harvested, washed thoroughly under running tap water, and dried under shade at room temperature (25 ± 2 °C) for 7-d. Dried tissue (5 g) was homogenized separately in 100 mL of respective solvent (methanol, ethyl acetate and hexane) and extracted with continuous shaking at 100 rpm for 48 h at 25 ± 2 °C. The extracts were filtered through Whatman filter paper no. 1 (Whatman™, GE Healthcare UK Limited, Amersham Place, UK) and were evaporated to dryness. These extracts were then stored at 4ºC for further experiments.

Total phenolic content (TPC)
Crude extracts prepared in various organic solvents such as methanol, ethyl acetate and hexane were used for the estimation of TPC in different parts of regenerants and mother plant. TPC was evaluated using the modified protocol of Singleton and Rossi (1965). The extracts of different plant parts were dissolved in minimum volumes of respective solvents and the final concentration of 1 mg/mL was obtained by adding methanol to the dissolved extracts. The test extracts were filtered using 0.2-µM filter (Axiva Sichem Pvt. Ltd., India) and filtrates were used for the evaluation of TPC. For estimation of TPC, 100 µL of test extracts were taken and volumes were made up to 3 mL using distilled water. To the resulting mixture, 500 µL Folin-Ciocalteu reagent was added, mixed gently and allowed to stand for 3 min in dark. Further, 2 mL of anhydrous sodium carbonate (Na 2 CO 3 ; 20% w/v) was added and incubated in dark for 1 h. Reaction mixture having methanol in place of plant extracts was taken as a negative control. Gallic acid (1 mg/mL, in methanol) in the reaction mixture without plant extracts was taken as a positive control and absorbance was taken at 650 nm using UV spectrophotometer (Beckman Coulter DUR 730). Gallic acid was used to construct the calibration curve and TPC was determined as mg of gallic acid equivalents (GAE) per gram of dry weight (mg GAE/g d.w.).

Total flavonoid content (TFC)
Similarly, extracts prepared in three organic solvents such as methanol, ethyl acetate and hexane were taken for the determination of TFC in different parts of mother plant and regenerants. TFC was evaluated using modified protocol of Chang et al. (2002). Crude extracts were dissolved in minimum amount of respective solvents and final volumes were made by adding methanol to make the final concentration of 1 mg/mL. These extracts were filtered using 0.2-µM filter (Axiva Sichem Pvt. Ltd., India) and filtrates were used for the determination of TFC. For the analysis of TFC, 250 µL of prepared test extract was mixed to 75 µL sodium nitrite (NaNO 2 ; 5% w/v), shaken well and incubated for 6 min. In the reaction mixture, 150 µL aluminum chloride (10% w/v) and 500 µL of 1 N sodium hydroxide were added and final volume was adjusted using distilled water. Quercetin (1 mg/mL, in methanol) in the reaction mixture was considered a positive control and the absorbance was estimated at 510 nm. TFC was determined as mg of quercetin equivalents (QE) per gram of dry weight (mg QE/g d.w.) using calibration curve prepared for quercetin. All the chemicals were purchased from Thermo Fisher Scientific, Mumbai, India.

HPLC analysis of crude extract
The dried root extracts of mother plant and regenerants were used for the quantification of luteolin and rutin contents using HPLC. The HPLC analysis was performed employing the modified protocol of Cristea et al. (2003), as described previously.

Total antioxidant capacity (TAC)
Methanolic extracts of different parts of mother plant and regenerants were used for evaluation of TAC through phosphomolybdenum assay using a modified protocol from Prieto et al. (1999). In the reaction mixture, 100 µL of the sample extract was mixed with 1 mL of freshly made molybdate reagent [0.6 M sulphuric acid (Thermo Fisher Scientific, Mumbai, India), 28 mM sodium dihydrogen orthophosphate (Qualigens, Mumbai, India) and 4 mM ammonium molybdate tetrahydrate (Sigma Aldrich, USA)]. The reaction mixture was incubated at 90 °C in a water bath for 90 min. It was then allowed to cool and absorbance was recorded at 695 nm. Reaction mixture comprising methanol and ascorbic acid (20, 40, 60, 80, 100 and 200 µg/mL) were taken as negative and positive control, respectively. Total antioxidant capacity was determined by observing a calibration curve of standard ascorbic acid and calculated as ascorbic acid equivalents (AAE) in microgram per milliliter of AAE in dry weight of extract (µg/mL AAE d.w. of extract).

DPPH free radical scavenging activity
Methanolic extract of different parts of mother plant and regenerants were taken for determination of DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging activity. The DPPH free radical activity of mother plant and regenerants was measured by employing the protocol of Brand-Williams et al. (1995). The stock solution of 0.2 mM DPPH (SRL, Mumbai, India) was prepared in methanol. In addition, plant extract of different concentrations (62.5, 125, 250, 500 and 1000 µg/mL) were also prepared. In the reaction mixture, 400 µl of DPPH was added separately to 100 µL aliquots of each concentration of plant extract and 400 µL methanol followed by 30 min dark incubation and then absorbance was measured at 517 nm. Ascorbic acid (10, 20, 30, 40 and 50 µg/mL) was used as a positive control to construct a linear calibration curve. However, methanol was used as a negative control. The percentage inhibition of DPPH free radicals was calculated by using the following formula: where, Ac: absorbance of control; A ext : absorbance of the extract.

Elicitation of luteolin and rutin in root callus of R. hastatus
The greenish brown, compact, 30-d-old, root calli (90-95 mg) were inoculated on MS + 5 µM TDZ augmented with different concentrations of various elicitors such as chitosan (1, 5, 25, 50, 100 and 200 mg/L), phenylalanine (1, 5, 25, 50, 100 and 200 mg/L), salicylic acid (1, 5, 25, 50, 100 and 200 µM) and yeast extract (1, 5, 25, 50, 100 and 200 mg/L). These elicitors were prepared according to the protocol described by Singh et al. (2020b). MS + 5 µM TDZ medium without elicitor was considered as a control. Treated calli were harvested after 30-d of culture, shade dried and their dry weights (d.w.) were recorded. Extracts of dried calli were prepared using the same method as described earlier (in subsection: Plant material and selection of elite plant part). These calli were further used for the quantification of luteolin and rutin contents through HPLC using the modified protocol of Cristea et al. (2003) as mentioned previously.

Statistical analysis
Morphogenic responses of explants were observed from 12 replicates of each plant growth regulator treatment and all experiments were performed in triplicates. Percent callus induction and observations for root induction were recorded after 30-d of subculture. Quantification of bioactive compounds (total phenol content, total flavonoid content and quantity of luteolin and rutin), antioxidant assays and treatment of different elicitors were carried out in triplicates. Statistical analysis was done through one way ANOVA using Duncan's Multiple Range Test at P ≤ 0.05 employing SPSS software.

Selection of elite plant part
Synthesis and accumulation of bioactive compounds in plants are influenced by biotic (pathogens, herbivores, etc.) and abiotic (temperature, light, water, soil, etc.) factors including geographical location, temporal and seasonal variation (Page et al. 2005), genotypes (Shamloo et al. 2017) and developmental stages (Popović et al. 2020) of the plant. These factors can significantly affect plant growth and development, even their ability to synthesize secondary metabolites, consequently leading to the change in the overall phytochemical profile (Yang et al. 2018). Therefore, before proceeding with further induction of callus and elicitation of bioactive compounds, the selection of an elite plant part yielding maximum content of luteolin and rutin is highly desirable. In the present study, significant difference in luteolin and rutin contents was seen in various in vivo plant parts viz. roots, stem and leaves of R. hastatus collected from Mandi, Himachal Pradesh, India. HPLC chromatogram of standard compounds such as rutin and luteolin showed the peaks at retention time at 19.39 and 28.35 min, respectively (Fig. 1b). HPLC analysis of plant samples revealed that roots had highest content of luteolin (747.32 ± 20.71 µg/g d.w.) and rutin (1689.43 ± 25.43 µg/g d.w.) compared to other plant parts (Suppl. Table 2; Fig. 1c). The amount of luteolin was 528.95 ± 13.75 µg/g d.w. and 322.51 ± 8.39 µg/g d.w. in leaf and stem, respectively (Fig. 1c). However, respective content of rutin in leaf and stem was 1315.00 ± 22.39 and 1193.34 ± 45.43 µg/g d.w. (Fig. 1c). As roots had maximum luteolin and rutin content compared to other plant parts, it was selected for callus induction and regeneration.

Induction of callus from in vitro roots
The root explants (Fig. 1d) were used for further in vitro regeneration through callus cultures. Initiation of callus began within two weeks of inoculation, but the variation in nature and amount of the calli were recorded after 30-d of culture. Calli induced on different auxins such as IAA, IBA and NAA were whitish, friable and non-morphogenic with numerous root hairs on the surface of growth medium (Table 1). Similarly, it was observed that 2, 4-D formed moderate amount of creamy and friable callus. Such friable calli are not ideal for differentiation into shoots, hence these calli were not further used for sub-culturing. Contrary to this, scanty to nil callus production was observed in root explants cultured on MS medium fortified with BAP. Interestingly, best callusing was observed on 5.0 and 10.0 µM TDZ after 30-d of culture (Table 1; Fig. 1e). These calli were greenish brown, compact and morphogenic which were considered as ideal for further shoot bud differentiation (Fig. 1e). TDZ, a thiadiazole-substituted phenylurea (N-phenyl-1,2,3-thidiazol-5-yl urea) has cytokinin like activity and proved to be an effective plant growth regulator in various culture systems (Mok et al. 1982;Huetteman and Preece 1993;Sharma and Agrawal 2018). TDZ promotes the conversion of purine-based cytokinins to more biologically active nucleosides and also stimulates accumulation and translocation of auxin (Mok et al. 1982; Ahmed and Anis 2012). There are several other reports regarding callus-mediated organogenesis using 5.0 µM TDZ in various plants such as Dendrobium nobile (Bhattacharyya et al. 2014), Rumex thyrsiflorus (Ślesak et al. 2015), Plumbago zeylanica (Sharma and Agrawal 2018), Pluchea lanceolata (Kher and Nataraj 2019) and Atropa acuminata (Rajput and Agrawal 2020). Besides, several other reports revealed the use of some other plant growth regulators for callogenesis.

Shoot bud differentiation and elongation
The root-derived callus obtained earlier on MS + 5.0 µM TDZ was further sub-cultured on same but fresh medium augmented with different concentrations (0.1, 1.0, 5.0 or  (Table 2). However, lower concentrations (0.1 or 1.0 µM) of TDZ and MS basal medium failed to induce any shoot buds. It was observed that the shoot buds differentiated on MS medium fortified with TDZ remained stunted till 30-d (Fig. 1g), so in order to elongate them, the callus pieces with shoot buds were sub-cultured on MS basal medium. Incidentally, these shoot buds elongated into healthy shoots within 15-d of transfer (Fig. 1h). It was observed that TDZ at higher concentrations promoted the differentiation of callus into shoot buds. However, elongation of shoot could not be obtained at the same concentrations of TDZ. Similarly, induction and differentiation of shoot buds by supplementing TDZ to growth medium have also been observed in Vitex trifolia (Ahmed and Anis 2012), Rumex thyrsiflorus (Ślesak et al. 2015), Plumbago zeylanica (Sharma and Agrawal 2018) and Atropa acuminata (Rajput and Agrawal 2020).

Induction of roots in the in vitro excised shoots
The root callus-derived shoots (2-3 cm) were then excised and inoculated separately on the MS medium fortified with different concentrations (0.1, 1.0, 5.0 or 10.0 µM) of IAA, IBA and NAA. It was recorded that 0.1 µM IBA was found to be optimum for root formation, where 100% of cultures induced an average of 12.03 ± 2.49 (Fig. 1i) roots per shoot explant having an average length of 5.75 ± 0.53 cm, after 30-d of culture (Table 3). The roots were healthy and long with numerous lateral roots and induced directly from the base of cut ends of the shoot without intervening callus. Both the number of roots and root length were reduced with increasing concentrations of IBA. However, at higher concentrations of IAA and NAA, formation of callus at the basal cut ends of the shoot was observed which inhibited the development of roots. Recently, studies on rhizogenesis also reported that IBA at lower concentrations proved to be effective for the formation of roots. Gupta et al. (2020) reported maximum mean number of roots (14.1 ± 2.9) on half-strength MS medium augmented with 0.2 mg/L IBA in Moringa oleifera. Contrarily, Kher and Nataraj (2019) observed maximum average number of roots (19.8 ± 0.9) on half-strength MS with 2 mg/L IBA within 28-d of culture in Pluchea lanceolata. Prior to this, several other investigations, too, found IBA effective for the rhizogenesis in

In vitro hardening and field acclimatization
MS medium augmented with 0.1 µM IBA induced long and thick roots, which apparently increased the uptake of nutrients and facilitated the hardening and acclimatization of regenerants. In vitro rooted plantlets were successfully transferred to paper cups containing soil-rite mixture for hardening (Fig. 1j). The plants attained an average height of 68 cm after 3 month and morphologically similar to in vivo plants.
Almost 96% of root callus-derived regenerants (RDR) were effectively acclimatized and raised in pots containing garden soil in green house condition (Fig. 1k).

Genetic fidelity analysis employing SCoT and SRAP markers
Genetic fidelity of mother plant (M) and root callus-derived regenerants (RDR1-RDR10) was analyzed using SCoT and SRAP markers. Initially, a total of 34 SCoT and 52 SRAP primers were screened, out of which 10 each were selected based on scorable amplification bands on agarose gel. SCoT primers resulted in 48 reproducible scorable bands between 200 and 2200 bp, with an average of 4.8 bands per primer in RDR (Suppl. Table 3; Fig. 2a). Besides, SRAP marker analysis of RDR yielded a total of 54 distinct scorable bands between 50 and 1100 bp with an average of 5.4 bands per primer (Suppl. Table 3; Fig. 2b). The pooled SRAP and SCoT matrix data set produced a total of 102 bands, out of which 94 bands were monomorphic with an average of 4.7 bands per primer (Suppl. Table 3). The combined similarity matrix was obtained from both SRAP and SCoT markers based on Jaccard's similarity coefficients which showed the coefficient range between 0.931 to 1.000 (Suppl. Table 4). The dendrograms obtained from UPGMA showed 95% similarity between the RDR and mother plant (Fig. 2c). This high monomorphism in the banding profile obtained by both the markers confirmed the genetic stability of the micropropagated plants. Interestingly, the present investigation revealed that the plants generated through root-derived callus have very less variation. The possible reason could be due to the exogenous application of phytohormone during callus induction, shoot regeneration and prolonged culture passage or incubation period (Devarumath et al. 2002;Bhattacharyya et al. 2017). Type of explant used and regeneration pathway may also attribute towards the genetic variations among regenerants. Differentiated tissues such as root, stem and leaves generally produce more polymorphism as they undergo an intermediate callus-phase which leads to changes in the genome quantitatively and qualitatively during cell reprogramming (Leva et al. 2012). However, such genetic changes cannot be observed visually as the structural variations in the gene product do not always alter the biological activity of the genome to an extent for phenotypic expression (Bhattacharyya et al. 2017). Molecular markers such as SCoT and SRAP employed in the current study are known to be highly reproducible, reliable and efficient in detecting genetic variation in in vitro raised plants. Start codon targeted polymorphism (SCoT) markers are based on the shortconserved region adjoining the initiation codon (ATG) present in plant genes and can be correlated to functional genes and their corresponding traits (Collard and Mackill 2009). Besides, sequence-related amplified polymorphism (SRAP) markers are designed for the amplification of open reading frames which is based on two-primer (forward and reverse primers) amplification (Li and Quiros 2001). A similar study has also been reported by Bose et al. (2016) in which genetic variability was 4.38% and 7.14% in direct shoot organogenesis and callus mediated organogenesis derived plants, respectively, in Nardostachys jatamansi using SCoT and intron splice junction (ISJ) markers. The percent polymorphism between mother and micropropagated plants was also reported in Aconitum violaceum employing RAPD (3.22%) and ISSR (5.88%) markers (Rawat et al. 2013), Dendrobium nobile (3%) using RAPD and SCoT markers (Bhattacharyya et al. 2014), Plumbago zeylanica (3%) using ISSR and SCoT markers (Sharma and Agrawal 2018). In contrast, Bhattacharyya et al. (2017) found a very high polymorphism such as 6.06% and 15.62% in regenerants from direct and indirect shoot organogenesis, respectively in Rumex nepalensis by using SCoT and RAPD markers. Recently, Dhiman et al. (2021) have also been observed 8.03% polymorphism in micropropagated plants of Nardostachys jatamansi by employing SCoT and Directed Amplification of Minisatellite DNA (DAMD) markers.

Phytochemical evaluation of mother plant and regenerants
Amongst all the plant extract used, the highest phenolic content was observed in methanolic root extract of RDR (89.24 ± 1.24 mg GAE/g d.w.), followed by methanolic root extract of mother plant (74.57 ± 4.94 mg GAE/g d.w.) (Fig. 3a). Contrary, the lowest phenolic content of 1.49 ± 0.22 mg GAE/g d.w. was seen in the hexane leaf extract of mother plant (Fig. 3a). Similarly, maximum flavonoid content was also observed optimum in methanolic root extract of RDR (384.11 ± 3.22 mg QE/g d.w.), followed by mother plant (341.11 ± 1.70 mg QE/g d.w.) (Fig. 3b). The results indicated that regenerants had the highest phenolic and flavonoid content as compared to mother plant. Polyphenols exhibit high solubility in polar solvents such as   (Barchan et al. 2014;Do et al. 2014). The maximum phenolic and flavonoid contents were observed in methanolic root extract of RDR compared to extract of other plant parts. Since methanolprepared root extracts produced the highest levels of TPC and TFC in regenerants and mother plant of R. hastatus, it was selected for further analysis and quantification of luteolin and rutin content through HPLC. Maximum content of luteolin was observed in RDR (1042.15 ± 19.30 µg/g d.w.), followed by mother plant (747.32 ± 20.71 µg/g d.w.) (Fig. 3c). Similarly, maximum quantity of rutin was seen in RDR (1914.49 ± 45.99 µg/g d.w.), followed by mother plant (1689.43 ± 25.43 µg/g d.w.) (Fig. 3c). The HPLC analysis thus has revealed that a significant higher amount of luteolin and rutin present in root extract of regenerants. Significantly high quantity of secondary metabolites has been reported due to endogenous physiological changes, organ type or hormonal contents during culture conditions in micropropagated plants (Surveswaran et al. 2010). Addition of synthetic plant growth regulators alters the expression of genes involved in the biosynthesis of secondary metabolites resulting in the significantly higher production of these compounds under culture conditions (Pourebad et al. 2015). The above results are in consonance with the findings of Bose et al. (2016) and Bhattacharyya et al. (2017), where micropropagated plants of Nardostachys jatamansi and Rumex nepalensis, respectively, possessed a significantly higher amount of phenolics and flavonoids as compared to the mother plant. Micropropagated plants of Rumex cyprius showed enhanced quantity of phenols as well as luteolin and rutin content (Al Khateeb et al. 2017).

Analysis of antioxidant activity of mother plant and regenerants
Of all the different organic solvents tried, methanolic extract found to be the most effective in terms of maximum TPC, TFC and luteolin and rutin content. Hence, methanolic extract of different parts of R. hastatus was selected for further determination of antioxidant activity as evaluated through phosphomolybdenum and DPPH free radical scavenging assays.

Total antioxidant capacity (TAC)
Phosphomolybdenum (PM) assay is based on the ability of a reducing agent (antioxidant) to convert molybdenum (VI) into the phosphomolybdate (V) complex, which is evaluated spectrophotometrically at 765 nm (Ahmed et al. 2015). In the present investigation, higher antioxidant capacity was seen in methanolic root extract of RDR (300.05 ± 8.45 µg/ mL AAE d.w. of extract), followed by mother plant (280.71 ± 10.70 µg/mL AAE d.w. of extract) (Fig. 3d). Leaf and stem extract showed less but significant antioxidant capacity. Higher antioxidant activity in these regenerated plants corresponds to the presence of higher amount of secondary metabolites as compared to the mother plant. Plants produce a wide range of secondary metabolites such as phenols, flavonoids, tannins, alkaloids, etc. which act as a source of natural antioxidants with multiple biological activities.
The strong antioxidant activity of phenols and flavonoids is attributed to their ability to play the role of free radical scavengers which prevent the production of reactive oxygen species (Panche et al. 2016). Higher antioxidant activity in micropropagated plants can be directly correlated to the occurrence of higher amount of phenolics and flavonoids compared to mother plant (Sen et al. 2013;Piątczak et al. 2014). Methanolic leaf extracts of micropropagated plants of Rehmannia glutinosa have showed strong antioxidant activity (Piątczak et al. 2014). Kousalya and Bai (2016) also reported that methanolic extract of callus derived from in vitro regenerants of Canscora decussate possessed potent antioxidant activity. Recently, Rebelo et al. (2022) also studied that in vitro micropropagated plants had high activity of antioxidant enzymes compared with the mother plants of Tuberaria lignosa.

DPPH free radical scavenging activity
DPPH assay is the most widely accepted method to determine free radical scavenging activity due to its stability, rapidity, sensitivity and less expensive. In the presence of an antioxidant molecule, the violet solution of DPPH is converted to DPPH-H (diphenylhydrazine) with discoloration. This discoloration implies the radical scavenging activity of the plant extract (Aksoy et al. 2013). A dose-dependent increase in the free radical scavenging activity with increasing concentrations of extracts was recorded. The maximum radical scavenging activity was shown by the RDR (93.81 ± 0.50%), followed by the mother plant (87.31 ± 0.21%) at 1000 µg/ml of methanolic root extract (Fig. 3e). Leaf extract showed significantly moderate DPPH free radical scavenging activity. However, stem extract showed the least DPPH free radical scavenging activity in both mother plant and regenerants. Thus, the results revealed that regenerants showed strong free Fig. 2 Genetic fidelity analysis of root callus-derived regenerants (RDR). a amplification profiles of RDR and mother plant obtained after PCR analysis using SCoT primers. i SCoT 4; ii SCoT 6; iii SCoT 26; iv SCoT 27, b amplification profiles of RDR and mother plant obtained after PCR analysis using SRAP primers. i Em1/Me6; ii Em3/Me1; iii Em2/Me12; iv Em4/Me5, c Unweighted Pair Group Method with Arithmetic mean (UPGMA) dendrogram based on Jaccard's similarity indices obtained from SCoT and SRAP markers data set showing 95% similarity between the mother plant (M) and root callus-derived regenerants (RDR1-RDR10). L ladder, M mother plant, RDR root callus-derived regenerants ◂ 1 3 radical scavenging activity compared to the mother plant. The lower free radical scavenging activity of mother plant extract might be due to the presence of lower amount of phenols and flavonoids (Bagnazari et al. 2018). Similar results were also obtained in Nardostachys jatamansi (Bose et al. 2016), Rumex nepalensis (Bhattacharyya et al. 2017) and Atropa acuminata (Rajput and Agrawal 2020), where micropropagated plants possessed strong radical scavenging activity compared to the mother plant.
A positive correlation has been reported between TPC and DPPH free radical scavenging activity of plant extract (Li et al. 2009).

In vitro elicitation of luteolin and rutin compounds with various elicitors
The root calli raised on MS + 5 µM TDZ were augmented with different elicitors such as chitosan (0, 1, 5, 25, 50, 100 and 200 mg/L), phenylalanine (0, 1, 5, 25, 50, 100 and 200 mg/L), proline (0, 1, 5, 25, 50, 100 and 200 mg/L), salicylic acid (0, 1, 5, 25, 50, 100 and 200 µM) and yeast extract (0, 1, 5, 25, 50, 100 and 200 mg/L) for the elicitation of luteolin and rutin contents after 30-d of treatment and analyzed through HPLC.   Figure 1b). A decline in rutin and luteolin content was observed beyond 25 mg/L and 50 mg/L of chitosan concentrations, respectively. Chitosan (1,4-linked glucosamine) is a deacetylated derivative of chitin, which is the component of many pathogenic fungi (Katiyar et al. 2015). It is a potent elicitor having bio-degradable properties and also known for inducing the defense responses through the production of secondary metabolites in plants (Muxika et al. 2017;Hidangmayum et al. 2019). Besides, chitosan causes transient accumulation of secondary messenger molecules such as cAMP, Ca 2+ and NO, ion uptake and transportation, activation of NADPH oxidase and G-protein, mitogen-activated protein kinases (MAPKs) phosphorylation and salicylic acid-related signaling pathways (Ramirez-Estrada et al. 2016;Singh et al. 2020b). Chitosan had positive effect on callus growth upto 50 mg/L concentration in R. hastatus. Similar reports demonstrated the positive effect of chitosan on callus biomass in Rumex vesicarius (Sayed et al. 2016), Fagonia indica (Khan et al. 2019), Cullen corylifolium (Singh et al. 2020a) and Plumbago zeylanica (Singh et al. 2020b). However, chitosan at higher concentrations is inhibitory for callus growth and cells shifted their energy expenditure to the production of secondary metabolites (Golkar et al. 2019). There are several studies that reported elicitation of various bioactive compounds using chitosan as an elicitor. The increased production of formononetin (12.45-fold) and calycosin (6.17fold) has been achieved with 100 mg/L chitosan in hairy root cultures of Astragalus membranaceus (Gai et al. 2019). Similarly, Singh et al. (2020a) have also reported elicitation in contents of daidzein (11.2-fold) and psoralen (7.2-fold) at 50 mg/L chitosan and genistein increased to 6.21-fold by augmenting 100 mg/L chitosan to callus cultures of Cullen corylifolium. In another report of Singh et al. (2020b), 4.58-fold increment in plumbagin yield has been achieved with 50 mg/L chitosan compared to control in root callus cultures of Plumbago zeylanica. The content of lepidine has been enhanced up to 19.87-fold with 250 mg/L chitosan over control in callus cultures of Lepidium sativum (Bakhtiari and Golkar 2021). In addition, Bavi et al. (2022) have also been reported that chitosan at 40 mg/L promoted the synthesis of cafeic acid, benzoic acid, 4-hydroxy benzoic acid, epicatechin, and apigenin in the cell suspension culture of Zataria multifora. Chitosan caused oxidative burst which induced the expression of genes pertaining to MAPKs cascades which activates the transcription factors involved in the biosynthesis of secondary metabolites (Gai et al. 2019).

Phenylalanine
A slight increment in callus biomass production as well as in luteolin and rutin contents were seen in root calli after treatment with increasing concentrations (0, 1, 5, 25, 50,  Figure 1d). Subsequently, a sharp decline in content of luteolin and rutin beyond 100 mg/L phenylalanine concentration was observed. Phenylalanine is an aromatic amino acid, which acts as a precursor molecule for the synthesis of numerous secondary metabolites (Yang et al. 2020).
To enhance the production of secondary metabolites, various precursors of their biosynthetic pathways have already been employed in in vitro cultures. An increase in flavonoid content (1.6-fold) and antioxidant capacity have been reported in lentil sprouts treated with phenylalanine (Świeca et al. 2014). Similarly, Kumar et al. (2018) have also reported that phenylalanine at 200 mg/L concentration improved the conessine content (7.0-fold) in green bark-derived callus cultures of Holarrhena antidysenterica. When phenylalanine was supplemented with lactalbumin hydrolysate led to 3.5-fold increment in anthocyanin yield compared to control in cell suspension cultures of Panax sikkimensis roots and also upregulation of enzyme activity and gene expression of phenylalanine ammonia lyase (PAL) and UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT) was observed at same treatment (Biswas et al. 2020). The results of current study suggest the use of in vitro elicitation supported by precursor feeding and it is an effective, reliable and cheap tool for ameliorating the bioactive compounds.

Salicylic acid
When root callus was exposed to various concentrations (0, were also observed at 25 µM salicylic acid over their respective controls (Table 4; Suppl. Figure 1e, f). However, it was also observed that a sharp decrease in the callus growth and bioactive compounds accumulation at higher concentrations (> 25 µM). Salicylic acid act as an endogenous signaling molecule that induces the defense system in plants through activation of phenylalanine ammonia lyase synthesis pathway and other major antioxidant enzymes involved in metabolic pathways ultimately resulting to enhanced yield of secondary metabolites (Rahimi et al. 2014;Fesel and Zuccaro 2016). Supplementing the growth medium with salicylic acid led to the increased production of luteolin (9.13-fold) and rutin (2.62-fold) in R. hastatus root callus cultures. Similarly, several studies have also documented where salicylic acid improved the synthesis of secondary metabolites in various plant taxa. The increased contents of hypericin (7.98-fold) and pseudohypericin (13.58-fold) have been achieved in shoot cultures of Hypericum hirsutum treated with 50 µM salicylic acid (Coste et al. 2011). Cell suspension cultures of Gymnema sylvestre supplemented with 200 µM salicylic acid have elicited 4.9-fold gymnemic acid content (Chodisetti et al. 2015). Plumbagin accumulation has also been enhanced to 3.4-fold with 25 µM salicylic acid added to root callus cultures of Plumbago zeylanica (Sharma and Agrawal 2018). Enhancement in quantity of psoralen, daidzein and genistein up to 14.8-fold, 4.8-fold and 5.5-fold has been achieved in cotyledon callus cultures of Cullen corylifolium fortified with 5 µM salicylic acid (Singh et al. 2020a). Rajan et al (2020) have reported that 1 mM salicylic acid enhanced the production of rhamnetin to 2.7-fold compared to control in cell suspension culture of Vernonia anthelmintica.

Yeast extract
A gradual increase in luteolin and rutin content as well as callus biomass proliferation were analyzed with increasing doses (0, 1, 5, 25, 50, 100 and 200 mg/L) of yeast extract. Increment of 27.48% (197.44 ± 12.08 mg d.w.) in callus biomass was observed at 100 mg/L yeast extract over control (154.87 ± 12.37 mg d.w.) (  Figure 1 h). However, a slight decrease in the callus growth production along with luteolin and rutin content were observed at 200 mg/L yeast extract. Yeast extract is a mixture of chitin, N-acetylglucosamine oligomers, β-glucan, amino acid residues, glycopeptides, and vitamins (Pitta-Alvarez et al. 2000;Baenas et al. 2014). El-Sharabasy et al. (2012) reported that callus biomass production has been promoted by amino acid residues and glycopeptides. Besides, plant cells having pattern recognition receptors for chitin and β-glucan act as microbe-associated molecular patterns, and modulates the expression of certain defense-related genes consequently resulting in enhanced synthesis of secondary metabolites (Jumali et al. 2011;Fesel and zuccaro 2016). Saponin content was increased to 20-fold with yeast extract treatment in cell cultures of Panax ginseng (Lu et al. 2001). Enhanced production of plumbagin up to 3.5-fold was achieved with yeast extract in root cultures of Drosera burmanii (Putalun et al. 2010). Yeast extract was also observed to enhance total isoflavonoids (4.5-fold) compared to control in hairy root cultures of Pueraria candollei (Udomsuk et al. 2011). Maqsood and Abdul (2017) have also reported that yeast extract added to the growth medium improved vinblastine and vincristine productivity by 22.74% and 48.49%, respectively, over control in Catharanthus roseus. Sharma and Agrawal (2018) have reported increased synthesis of plumbagin to 6.5-fold with yeast extract in root callus cultures of Plumbago zeylanica. Application of yeast extract to cell cultures of Trigonella foenum-graecum has also led to 1.66fold increased content of diosgenin (İlgar et al. 2021). In a recent report of Bavi et al. (2022) where yeast extract at a very high concentration (1200 mg/L) has increased the contents of phenylpropanoids gallic acid, vanillin, salicylic acid, catechin, rutin and terpenoids carvacrol and thymol in cell suspension culture of Zataria multifora.

Conclusion
The current investigation, for the first-time, reported regeneration protocol using in vitro root explants of R. hastatus, a medicinally important plant of Western Himalayas. Genetic fidelity analysis revealed high similarity between root callusderived regenerants and mother plant and their true-to-type nature was confirmed through monomorphic banding profile of molecular markers such as SCoT and SRAP. Regenerants showed high total phenolic and flavonoid contents along with strong antioxidant activity compared to mother plant. Besides, the present study has also revealed that phenylalanine and chitosan significantly enhanced luteolin and rutin content in root callus cultures of R. hastatus by 17.42-fold and 2.74-fold, respectively. Thus, the current study supports that in vitro elicitation can effectively enhance the production of bioactive molecules. The developed protocol can be successfully utilized for the conservation and commercial propagation of this traditional medicinal plant. Further, R. hastatus can also be utilized for its pharmaceutically and therapeutically active compounds through genetic manipulation or elicitation through root-derived callus using various biotic or abiotic elicitors.