Enhanced Production of β-Caryophyllene by Farnesyl Diphosphate Precursor-Treated Callus and Hairy Root Cultures of Artemisia vulgaris L.

Artemisia vulgaris L. produces a wide range of valuable secondary metabolites. The aim of the present study is to determine the effects of various concentrations of farnesyl diphosphate (FDP) on β-caryophyllene content in both callus and hairy root (HR) cultures regeneration from leaf explants of A. vulgaris L. Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4D; 4–13 μM), α-naphthaleneacetic acid (NAA; 5–16 μM), and FDP (1 and 3 μM) was used for callus induction and HR regeneration from leaf explants of A. vulgaris L. In this study, precursor-treated (2,4D 13.5 μM + FDP 3 μM) callus displayed the highest biomass fresh weight (FW)/dry weight (DW): 46/25 g, followed by NAA 10.7 μM + FDP 3 μM with FW/DW: 50/28 g. Two different Agrobacterium rhizogenes strains (A4 and R1000) were evaluated for HR induction. The biomass of HRs induced using half-strength MS + B5 vitamins with 3 μM FDP was FW/DW: 40/20 g and FW/DW: 41/19 g, respectively. To determine β-caryophyllene accumulation, we have isolated the essential oil from FDP-treated calli and HRs and quantified β-caryophyllene using gas chromatography–mass spectrometry (GC–MS). The highest production of β-caryophyllene was noticed in HR cultures induced using A4 and R1000 strains on half-strength MS medium containing 3 μM FDP, which produced 2.92 and 2.80 mg/ml β-caryophyllene, respectively. The optimized protocol can be used commercially by scaling up the production of a β-caryophyllene compound in a short span of time.


INTRODUCTION
Artemisia vulgaris L. is an important medicinal herb that belongs to the family Asteraceae. These plants are found in several temperate countries in Europe, Asia, Northern Africa, Alaska, and North America. A. vulgaris L. has been widely used as a traditional medicine for treating a number of diseases, such as neonatal jaundice (Fok, 2002), gastric ulcers (Repetto and Llesuy, 2002), and hepatitis, as well as an analgesic agent in acupuncture therapy (Yoshikawa et al., 1996). Traditionally, it has been effective as antiviral (Tan et al., 1999), insect-repellant, fumigant (Wang et al., 2006), insecticide (Chantraine et al., 1998), antibacterial, anti-inflammatory (Tigno et al., 2000), sedative, and flavoring and perfumery agents (Da Silva, 2004). A phytochemical study has identified more than 20 flavonoids in A. vulgaris L. extracts (Lee, 1998). Williams et al. (2012) have identified 22 significant chemical components in the essential oil of A. vulgaris L.-the major components being β-caryophyllene, α-zingiberene, borneol, and α-curcumin. It also has been found that some species of the plant kingdom produce sesquiterpene β-caryophyllene (De Moraes et al., 2001).
Caryophyllene is present in Zingiber nimmonii rhizome essential oil that helps curing fungal and bacterial infection (Sabulal et al., 2006). Gertsch et al. (2008) have reported about β-caryophyllene compound that was shown to interact with the binding site cannabinoid receptor type 2 (CB2), but not to type 1 (CB1). This chemical compound is energetically anti-inflammatory and has anesthetic properties. Amiel et al. (2012) reported that the essential oil (βcaryophyllene) of Commiphora gileadensis (Balm of Gilead, mentioned in the Bible) has an anti-proliferative property against tumor cell lines. They also found that β-caryophyllene caused a strong induction of apoptosis accompanied by DNA ladder formation and caspase-3 catalytic activity in tumor cell lines. However, the plant sesquiterpenes biosynthesis pathway is considered as an isopentenyl diphosphate (IPP), which is the universal biological precursor of all isoprenoids (basic C 5 isoprene unit). It can be obtained through either mevalonate (MVA) or 2-methylerythritol 4-phosphate (MEP) pathways (Figure 1). Moreover, it has been well established that the acetyl-coenzyme A (acetyl-CoA) pathway in plants is located in the cytosol, whereas the MEP pathway is targeted to plastids inside (Croteau et al., 2000). There are studies that indicate cross-talk and possible IPP exchange between the other two paths through the cytosol and plastids (Adam and Zapp, 1998;Laule et al., 2003). There were also two independent pathways leading to IPP: the mevalonate pathway (MVA) originating from acetyl-CoA and the pyruvate-derived mevalonate-independent pathway (MEP) (Croteau et al., 2000). IPP instead contributes to the other terpenoids with its isomer dimethylallyl pyrophosphate (DMAPP). The MVA isoprenoid pathway is used by eukaryotes apart from plants, although prokaryotes with many exemptions use the MEP pathway to create IPP and DMAPP separately (Lichtenthaler, 1999;Boucher and Doolittle, 2000). Isopentenyl phosphate kinases (IPKs) generated by plant genomes are derived isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite IPP commonly used for isoprenoid/terpenoid biosynthesis (Henry et al., 2018). The objective of the present study is to enhance β-caryophyllene content in in vitro callus and hairy root (HR) cultures and to isolate the essential oil from both calli and HRs in order to understand the metabolic profile of β-caryophyllene using gas chromatography-mass spectrometry (GC-MS).

Callus Induction
Seeds of A. vulgaris L. were collected from Johnny's Selected Seeds, Winslow, ME, United States. The botanical identity of A. vulgaris was confirmed by comparing them with respect to the reference standards at Johnny's Selected Seeds. After that, 35-day leaf explants excised from old seedlings were inoculated into Murashige and Skoog (MS) medium (Murasnige and Skoog, 1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4D) and α-naphthaleneacetic acid (NAA) (Sigma-Aldrich, Inc., St. Louis, MO, United States) for callus induction. Callus was initiated by inoculating leaf explants from a 35-day-old plant into MS medium supplemented with various concentrations and combinations of 2,4D (4.5-13 µM) and NAA (5-16 µM), with farnesyl diphosphate (FDP; 1 and 3 µM) (Sigma-Aldrich, Inc., St. Louis, MO, United States) as a precursor in the experiment, followed by a previous report by Sundararajan and Kumari (2015). Fresh weight (FW) and dry weight (DW) were recorded in precursor-treated callus after 60 days (Sivanandhan et al., 2014).

Bacterial Culture Preparation
From the overnight glycerol stock, 500 µl was taken and transferred to the Luria-Bertani (LB) medium, and the same was subcultured thrice to obtain active culture. The Gramnegative soil bacteria Agrobacterium rhizogenes (A 4 and R 1000 ) transferred to the medium were cultured overnight at 25 • C in an orbital shaker at 180 rpm. The bacterial cells were harvested by centrifugation at 5,000 rpm for 15 min at 25 • C. The pellet-containing bacterial cells were re-suspended in liquid MS medium, and the final optical density (OD) was adjusted to 0.6.

Induction and Establishment of HR Culture
In aseptic condition, leaf explants derived from 45-dayold plants were wounded with a sterile scalpel, transferred into the A. rhizogenes infection medium, and incubated for 10 min at 25 • C. After infection, the explants were dried on a sterile filter paper and placed on semisolid 1/2 MS medium supplemented with 3% sucrose and supplemented separately with 50, 100, and 150 µM of acetosyringone (AS; Sigma, United States). AS was filter-sterilized by using a 0.2-µm syringe filter 109 (Pall Corp., United States) and added to the cooled autoclaved medium. After cocultivation, explants were washed with liquid 1/2 MS containing 500 mg/L cefotaxime, FIGURE 1 | An outline for β-caryophyllene compound biosynthesis pathway operating through in vitro method of A. vulgaris plant. IDS, isopentenyl diphosphate synthase; IDI, isopentenyl diphosphate isomerase; GGPP, geranylgeranyl diphosphate; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

Determination of the Growth Index of the HR Cultures
Experiments were conducted to evaluate the growth rate of the hair roots grown in 1/2 MS, 1/2 MS + B 5 vitamins, 1/2 MS + FDP 1 µM, 1/2 MS + FDP 3 µM, 1/2 MS + B 5 vitamins + FDP 1 µM, and 1/2 MS + B 5 vitamins + FDP 3 µM. The growth index (GI) of all the four lines was measured initially and inoculated with 20 mg of FW bacteria-free HRs (3 weeks old). It was transferred into a 30 ml liquid medium in a 250 ml conical flask and maintained on a rotary shaker at 120 rpm in a dark room for 12 weeks. The in vitro grown HRs were subcultured in a fresh medium for 6 weeks to stimulate further growth of the hairs. Each treatment consisted of three replicates, and each replicate contained 25 explants per treatment. The GI was determined by using the following formula (Ashraf et al., 2015): Growth index (GI) = Final biomass weight − Initial biomass weight Initial biomass weight .
FW and DW of HRs were recorded after 12 weeks, according to the method of Sivanandhan et al. (2014).

Polymerase Chain Reaction Analysis
Integration of the T-DNA, which is responsible for HR formation, was confirmed by polymerase chain reaction (PCR) analysis using rolB gene and rolC gene-specific primers (Cho et al., 1998). Genomic DNA was isolated from both the control nontransformed roots and the transformed HR cultures by using the cetyltrimethylammonium bromide (CTAB) method (Khanuja et al., 1999). The following primers were used for PCR analysis: for rolB gene, forward primers-5-ATG GAT CCC AAA TTG CTA TTC CCC CAC GA-3 and reverse primers-5-TTA GGC TTC TTT CAT TCG GTT TAC TGC AGC-3 and for rolC gene, forward primers-5-ATG GCT GAA GAC GAC CTG TGT T-3 and reverse primers-5-TTA GCC GAT TGC AAA CTT GCA C-3. The PCR was carried out by amplifying with an initial denaturation at 94 • C for 5 min, followed by 35 cycles of denaturation at 94 • C for 1 min, annealing at 55 • C for 1 min rolB and rolC, and extension at 72 • C for 1 min with a final extension of 72 • C for 10 min using an Applied Biosystems Veriti Thermal Cycler (Thermo Fisher Scientific, United States). The amplified products were analyzed by electrophoresing 1.4% (w/v) of agarose gel along with 100 bp DNA marker (New England Biolabs, United Kingdom) staining with ethidium bromide.
Essential Oil Isolation and Quantification of β-Caryophyllene Content in Precursor-Treated Callus and HR by GC-MS Analysis

Isolation of Essential Oil
Calli and HRs (10 g each) were subjected to hydrodistillation for 4 h at 60 • C using a Clevenger-type apparatus. The obtained essential oil was dried over anhydrous sodium sulfate (Na 2 SO 4 ). The oil was then filtered and stored at 4 • C for further experiments.

GC-MS Analysis
The extracted sample was cleaned by passing it through a silica gel column (glass column) containing glass wool, silica gel 60 (230-400 mesh; Merck, United States), and anhydrous Na 2 SO 4 , which helps to remove high-molecular-weight polar substances and impurities that interfere with GC-MS analysis. For quantitative analysis, β-caryophyllene standard and essential oil were dissolved with ethyl acetate and analyzed using a GC-MS-QP2010 (Shimadzu, Japan) equipped with an AOC-20i autosampler. One microliter of the essential oil was injected, and the chromatographic separation of β-caryophyllene was achieved with HP5-MS column (60 m × 0.25 mm I.D. × 0.25 mm thickness; Restek, United States). Helium with a purity of 99.999% was utilized as the carrier gas at a flow rate of 10 ml/min.
The injector port, interface, and ion source temperatures were set at 260, 300, and 230 • C, respectively. The programmed oven temperature was as follows: the initial temperature was set at 120 • C for 1 min, then raised to 270 • C at 6 • C/min and finally to 320 • C at 7 • C/min, and held for 10 min. A solvent delay of 6 min was set to protect the filament from oxidation. The mass spectrometer was operated in electron impact (EI) mode at an ionization potential of 70 eV and at an emission current up to 60 mA. The quantification was performed in selected ion monitoring (SIM) mode by monitoring mass ions for each β-caryophyllene. For quantification, four different calibrators (0.125, 0.250, 0.500, and 1.00 mg/ml) were used for linearity check. It showed good correlation coefficient (r 2 = 0.9999). Scanning interval and SIM sampling rate were 0.5 and 0.2 s, respectively.

Statistical Analysis
The evaluation of β-caryophyllene from various callus and HR samples of A. vulgaris L. leaf explants was performed with three replicates each. The data obtained were analyzed statistically, and observations were recorded on the frequency of both FW and DW (callus and HR cultures, respectively), as well as the number of explants responding for HR initiation growth rate. The statistical analyses were performed by ANOVA using SPSS.
The differences between means were tested for significance by Duncan's multiple range test at p = 0.05.

Callus Induction
Leaf explants were inoculated into MS medium containing 2,4D (4-13 µM) and NAA (5-16 µM) with FDP as a precursor (1 and 3 µM) individually, as per a previous study by Sundararajan and Kumari (2015). Among various concentrations tested, the highest response of callus (99 and 95.2%) was achieved in MS medium supplemented with a combination of 2,4D (9.0 µM) + FDP (3 µm), followed by MS medium supplemented with NAA (16.1 µM) + FDP (3 µM). There are various similar studies on callus induction from leaf and stem explants of A. vulgaris L. (Sujatha and Kumari, 2007;Borzabad et al., 2010a,b). The precursor-and elicitor-treated calli vary from species to species and often differ even within the same species, for example, Jatropha integerrima (Schmitt and Petersen, 2002). MS medium supplemented with 5-aminolevulinic acid (5-ALA) as a precursor 2, 5, and 10 mg/L concentration has shown an enhancement in the callus biomass and induction of rooting and shooting with a profound effect (Boitel-Conti et al., 2000). Sujatha and Dhingra (1993) reported that feeding the yeast extract elicitor in the suspension culture of J. integerrima promoted the metabolic enzyme activity and alkaloid production several folds. Other studies also reported enhanced biosynthesis of the flavonolignan silymarin compound through jasmonic acid (JA) elicitor treatment of Silybum marianum callus cell culture medium (Sanchez-Sampedro et al., 2005). In recent reports, three key genes, such as phenylalanine ammonia-lyase, chalcone synthase, and dihydroflavonol-4-reductase, were reported to be involved in the phenylpropanoid pathway. A polyphenolic compound has been used in Plantago ovata callus culture media with different growth regulators (Talukder et al., 2016). Arya and Patni (2013) reported significant enhancement of quercetin in callus culture of Pluchea lanceolata by incorporation of cinnamic acid and precursor feeding with L-phenylalanine. In the present study, FW/DW of callus 2,4D treated without precursor was observed at FW/DW: 36/22 g, whereas in precursor-treated callus the highest FW/DW: 48/28 g were noticed at the concentrations (2,4D 13.5 µM + FDP 3 µM) followed by (2,4D 9 µM + FDP µM) fresh and dry weight was exhibited (Figure 2A). Then fresh and dry weight of NAA treated callus without precursor was found to be 36/20 g, respectively, whereas in precursor-treated callus the highest FW/DW: 48/26 g was noticed at the concentrations (NAA 16.1 µM + FDP 3 µM) followed by (NAA 10.7 µM + FDP 3 µM) fresh and dry weight was exhibited ( Figure 2B). Similar reports were observed in connection with FW 500 mg withanolides production in callus culture of Withania somnifera after 4 weeks of culture (Doma et al., 2012;Sivanandhan et al., 2012). This study is in agreement with the result of a previous report on the large-scale production of artemisinin with different elicitors and precursors, such as sodium acetate, mevalonic acid, lactone, and casein acid hydrolysate, to increase artemisinin content in cell suspension culture of Artemisia annua (Baldi and Dixit, 2008). Quercetin compound was enhanced by using various precursors, such as phenylalanine and cinnamic acid, in P. lanceolata in vitro callus culture (Arya and Patni, 2013). Similar results were also observed in Psoralea corylifolia with precursor treatment in cotyledonary callus, which was obtained from FW 2,601.8 mg/g (Mohammadparast et al., 2015). A similar phenomenon was also observed with different synthetic precursors, such as phenobarbital, geraniol, transcinnamic acid, succinic acid, and tryptamine, in compact callus cluster (CCC) cultures of Catharanthus roseus with the highest biomass production of FW 10 g and DW 6 g . Phenylethanoid glycosides were enhanced by feeding precursor into the Cistanche deserticola cell culture medium, with FW 0.6 g and DW 0.4 g (Ouyang et al., 2005). In earlier studies, the intracellular amount of oleanolic acid (OA) reached its final value (DW 0.84 mg/g) after 72 h of treatment with 100 M JA, which was 9.4-fold greater than that observed in untreated control cultures. After 48 h of therapy, the inclusion of chitosan at 50 mg/L induced a 5-fold increase in OA accumulation (DW 0.37 mg/g) (Wiktorowska et al., 2010). The precursor feeding or elicitor treatment is an effective technique to improve the growth of secondary metabolites through plant cell culture (Dedaldechamp andUhel, 1999 andRao andRavishankar, 2002).

Induction and Establishment of HR Culture
The transformation efficiency of A. rhizogenes required a different time and duration for the root induction from leaf explants of A. vulgaris L. ( Table 1). The highest transformation frequency with a direct root induction from the wounding site was observed in leaf explants infected with AS 150 µM (A 4 and R 1000 ) strains after 10-15 days of culture (Figure 3). The results of the present study were similar to Persicaria minor HR induction treatments with AS at 100 and 200 µM, which suggested that phenolic compounds play a vital role in the induction medium, particularly at wounded sites (Danphitsanuparn et al., 2012;Ashraf et al., 2015). It was reported that AS stimulated A. rhizogenes T-DNA to bring out HR from the wounded cell tissue (Farshad Ashraf et al., 2013). HR induction by different bacterial strains has been documented earlier in different plant species (Zehra et al., 1999). In C. roseus, six different A. rhizogenes strains were used for HR induction (Toivonen et al., 1989). Strain induced HR with the help of Rauwolfia serpentina leaf explants with a transformation frequency of 70% for strain when compared with strain LBA 9402, with a frequency of 45% (Goel et al., 2010). Tao and Li (2006) reported HR induction in Torenia fournieri leaf explant with higher transformation efficiency at 65% than that in A 4 strain. Wu et al. (2003) earlier reported that 100 µM of AS is the best concentration for enhancing the genetic transformation in Nicotiana tabacum var. Previous studies indicated that the A. rhizogenes strain, which influenced HR induction in various explant parts, was showing HR induction (Wu et al., 2003;Pietrosiuk et al., 2006;Thimmaraju et al., 2008). In the present study, exogenous  application of AS along with naturally produced phenolics from wounded leaf explants increased the transformation frequency by vir-genes induction.

PCR Analysis
Molecular confirmation of the transformed HRs was achieved by PCR analysis. The presence of rolB gene fragment (780 bp) and rolC gene fragment (540 bp) was confirmed by using genespecific primers, and successful transformation was confirmed by the presence of rolB and rolC gene fragments. The results of PCR study indicated that the rolB and rolC gene fragments were incorporated into the A. vulgaris L. HR cultures. It indicates that the presence of the incorporation of the T-DNA into the genome of A. vulgaris L. HRs has been successful (Figure 8 and Supplementary Figure 5). Many studies concluded that the HR culture metabolite production depends on rol gene expression (Tiwari et al., 2007;Bulgakov, 2008). The rol genes play a major role in the pathway that leads to the increased production of secondary metabolites. In many cases, the rol gene of strain is involved in the parthenogenesis of the HR disease and establishment of transgenic plants with simple developmental and morphological alteration (Jouanin et al., 1987). The establishment of the root inducing (Ri) disease in HRs is interrelated with the expression of the rolA, B, C, and D loci in the majority assay of plant species (Taylor et al., 1985;Cardarelli et al., 1987). Along with them, rolB plays the most important role, whereas rolA, rolC, and rolD encourage the root development synergistically (Aoki and Syôno, 1999). The growth model of rolB-induced HR is characterized by high-level growth, branching, and plagiotropism (Capone et al., 1989). Genetically mediated transformation by Ri T-DNA of A. rhizogenes has been established to be an effective indirect way of accumulating and producing high levels of secondary metabolites in plant cells (Guillon et al., 2006). In a confirmation study in agreement with A. rhizogenes, there is a type of Gram-negative well-known soil-borne bacterium that causes HRs in many plants. Root loci (rol) genes harbored by the Ri plasmid of this bacterium are integrated into the host plant genome, causing HRs. rol genes are a reflection to affect the growth and development of transformed roots and induce secondary metabolite synthesis by turning on the mediated transcription defense genes (Ono and Tian, 2011).

Quantification of β-Caryophyllene in Callus Culture
The essential oil isolated from A. vulgaris L. callus was quantified with various concentrations of the β-caryophyllene standard compound. The calibration curve (SIM method) was determined to be utilized. GC-MS of β-caryophyllene compound retention time peak was observed at 7.34 s. In the present study, extracts of callus samples were analyzed through GC-MS by the scan mode method, and the highest β-caryophyllene compound was observed in without precursortreated callus 2,4D 13.5 µM (1.61 mg/ml) and precursortreated callus 2,4D 13.5 µM + FDP 3 µM (2.38 mg/ml), followed by NAA 16.1 µM (2.28 mg/ml) (Supplementary  Figures 1, 2 and Table 2). These results were supported by a previous report (Arya and Patni, 2013), where enhanced quercetin compound (0.23 mg/ml) in precursor-treated callus was noticed. Treatment of A. absinthium callus with benzyl adenine (BAP) and NAA (2 mg/L), along with amino acids, such as valine and cysteine (12.5 mg/L), resulted in higher content of artemisinin compound (Zia et al., 2007). Enhanced trigonelline content was noticed through thin-layer chromatography, gas-liquid chromatography, and GC-MS analysis in nicotinic acid used as precursor in in vitro callus culture (Mathur and Kamal, 2012). Similarly, casein hydrolysate precursor feeding in cell suspension culture at 50, 75, and 100 mg/L concentrations resulted in the highest scopoletin compound, as revealed by high-performance thinlayer chromatography analysis (Abyari et al., 2016). Various explants of Simmondsia chinensis (leaves, nodes, internodes, shoot apices, and cotyledons) were used for callus induction from MS media along with various growth regulators [NAA, BAP, indole-3-acetic acid (IAA), and isopentenyl adenine (2ip)], and the final quantitative estimation of essential oils was observed at the highest amount of oil content at a 9-week-old callus culture (Aftab et al., 2008). A similar phenomenon has been observed in several studies Ouyang et al., 2005;Sivanandhan et al., 2012). In this study, calli are compared with previous reports that were treated with different precursors/elicitors as shown in Table 3.
The attained values, similar to those reported from different A. rhizogenes mediated transformations of several medicinal plant species, have been studied for the production of essential oil by both transformed and non-transformed root cultures (Santos et al., 2002(Santos et al., , 2005Szoke et al., 2004;Sujatha et al., 2013). The results from the present study agreed with the previous reports on various precursor and elicitor treatments for root induction. Some plants of vincamine biosynthesis were established through Vinca minor HR clone that was determined with various elicitor (acetyltransferase) and precursor treatments (cyclooxygenase inhibitor); the highest vincamine compound was observed at 17% (Verma et al., 2014). Enhanced tanshinones production in Salvia miltiorrhiza HR culture was used with different elicitors  combined with yeast extract + Ag + and Ag + + methyl jasmonate (MeJA) for yeast extract + Ag + + MeJA, and elicitation metabolite profiling was analyzed by ultra-performance liquid chromatography (UPLC) (Cheng et al., 2013). The use of MeJA and other signaling compounds for elicitation of transforming root cultures is opening a new way for a possibly profitable in vitro secondary metabolite production (Ahlawat et al., 2014). A soluble recombinant enzyme can catalyze the covalently bonded metal ion-dependent conversion of FDP to β-caryophyllene, sesquiterpene olefin contained in A. annua essential oil, was given by heterologous gene expression in Escherichia coli. Moreover, all through the initial stages, the β-caryophyllene synthase overexpressed in certain plant tissues and triggered in old ones in response to fungal elicitor suggests a role besides β-caryophyllene in host defense (Cai et al., 2002). Recently, Souret et al. (2002) have cloned three genes from A. annua that affect terpenes' biosynthesis: 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), and synthase from squalene (SQS) to use a library of cDNA made from hairy A. annua roots. Previously, overexpression of farnesyl pyrophosphate synthase in A. annua leads to high artemisinin aggregation levels by converting IPP and DMADP into diphenylphosphinic acid pentafluorophenyl ester (FDPP) (Banyai et al., 2010). Cloning and characterization of A. annua 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (AaHDR) involved in enhanced artemisinin production via improved artemisinin biosynthetic pathway precursor development through the MEP pathway were being reported in A. annua. Tryptophan alkaloid compound enhanced through precursor feeding in HR culture has been identified (Hughes et al., 2004). The effect of precursor-treated HR suspension culture has produced a high level of glucosinolate compound of Nasturtium montanum and Cleome chelidonii (Songsak and Lockwood, 2004). Robins et al. (1990Robins et al. ( , 1991 also reported hyoscyamine compound biosynthesis during metabolic pathways as amino acid precursors. A. rhizogenes enables the high production of these secondary metabolites for extensive industrial applications (Srivastava and Srivastava, 2007;Suza et al., 2008;Swarna and Ravindhran, 2012) that are useful as pharmaceuticals, cosmetics, and food additives (Signs and Flores, 1990;Guillon et al., 2006). In this study, HR culture has been compared with some previous reports that were treated with different precursors/elicitors (Table 3).

CONCLUSION AND PERSPECTIVES
This study, to the best of our knowledge, is the first attempt in reporting in vitro precursor treatment for enhancement of β-caryophyllene content by using callus and HR culture of A. vulgaris L. Although the biosynthesis pathway of secondary metabolites has not been well explored, the terpenoid biosynthesis pathway is well known for the production of β-caryophyllene via FDP. In this study, we aimed to enhance the output by using FDP as a precursor, and our findings proved that by using precursor-treated cultures, we can improve β-caryophyllene production. In conclusion, β-caryophyllene enhancement was successfully done with precursor-treated HR culture of A. vulgaris L. after 12 weeks in combined treatment culture of 1/2 MS + FDP 3 µM (2.92 mg/ml, respectively). These findings could encourage and provide support to synthesize new active natural bioactive compounds as an alternative to synthetic repellent and insecticides.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
SB and BR conceived the idea presented. SB developed the study, performed the experimental work, and wrote the main manuscript. RB developed the GC-MS methodology and performed the analysis. AM and DS analyzed the data interpretation. SS and GP revised the manuscript contents. Finally, all authors have read and approved the manuscript.

FUNDING
The authors acknowledge the University Grants Commission, New Delhi, India for providing financial assistance (UGC letter no. F.No.42-922/2013