Manipulation of Plant Growth Regulators on Phytochemical Constituents and DNA Protection Potential of the Medicinal Plant Arnebia benthamii

Arnebia benthamii of the family Boraginaceae is a critically endangered nonendemic plant of the Kashmir Himalayas and is used to treat a number of human diseases. The current study was based on developing an in vitro micropropagation protocol vis-à-vis induction of various secondary metabolites under in vitro conditions for the possible biological activity. A tissue culture protocol was developed for A. benthamii for the first time in the Himalayan region using varied combinations and proper media formulations, including various adjuvants: Murashige and Skoog (MS) media, growth hormones, sugars, agar, and so forth. The influence of different media combinations was estimated, and the MS + thidiazuron (TDZ) + indole 3-acetic acid (IAA) combination favors a higher regeneration potential. The higher amounts of chemical constituents were also recorded on the same treatment. The in vitro plant samples also showed a noteworthy effect of scavenging of hydroxyl radicals vis-à-vis protection from oxidative DNA damage. The in vitro raised plants are good candidates for the development of antioxidant molecules.


Introduction
Medicinal plants are vital curative agents for curing human ailments. The overexploitation of natural plant resources encouraged various programs such as conservation, micropropagation, and incremental plant architecture [1,2]. Looking at the present scenario, a significant proportion of the Northwest Himalayan (NWH) medicinal herbs, particularly Arnebia benthamii (Wall. ex G. Don) Johnston [syn. Macrotomia benthamii (Wall.) DC.], have been rendered rare and threatened over the past few years because the demand for various plant products has increased. Even though different initiatives such as habitat protection as well as Seed and Field gene banks have been started, these techniques may still prove inadequate. The tissue culture technique has become established as a very valuable methodology among the different conservation and multiplication practices presently being employed for increasing the number and improving the development of numerous economically important and threatened medicinal plants [2][3][4][5]. A. benthamii (Wall. ex G. Don) Johnston [syn. Macrotomia benthamii (Wall.) DC.] of the family Boraginaceae, locally known as Kahzaban, is a perennial medicinal herb growing in the subalpine and alpine zones of the Northwest Himalayas [6]. It is an important medicinal plant, prominent upon the list of 59 medicinal herbs that have been prioritized for conservation in NWH [6] due to their high extinction threat [7]. It is an alpine herb occurring at an altitude of 3500-4000 m, from Kashmir to Western Nepal, normally on open slopes with stony or rocky substrates. In Kashmir Valley, it is confined to certain areas such as Sinthon Top, Duksum, Karnah, Gurez, Lolab, Sonamarg, and Kargil and is rarely found [8,9]. It is classified as a critically endangered nonendemic plant of Kashmir [10,11]. A. benthamii has been listed in the Indian Red Data Book due to its overexploitation for various purposes [12,13]. Numerous secondary metabolites (i.e., shikonin and its intermediates, alkannins, other naphthoquinones, etc.) have been reported from Arnebia spp. However, "Gule Khazaban," a very expensive medicine, is derived from A. benthamii [11,13,14]. Arnebia spp. are reported to be traditional ayurvedic medicines for the treatment of diseases of the throat and tongue, fevers, and cardiac symptoms. The flowers are known to have a comforting effect on heart patients [11], while its roots have antiseptic and antibiotic properties [9]. Conservation of the existing germplasm of such a useful and threatened species is, therefore, a strategic need. Harborne and Baxter [15] have documented the anticancer properties of Arnebin 1 and Arnebin 3 isolated from the Arnebia species, and anti-HIV activity has been reported in Arnebia euchroma extracts [16].
Due to the higher incidence of microbial pathogens in foods and to control the antibiotic resistant bacterial strains by novel plant extracts [17,18], search should be continued to propagate medicinal plants by tissue culture biotechnology and to purify their medicinally active compounds for development of a novel treatment of pathogens [19]. Consequently, the study employed herein was undertaken to develop a tissue culture protocol for micropropagation of the depleted germplasm of A. benthamii. The study also endeavors to establish the effect of plant growth hormones on the induction of chemical constituents and antioxidant potential of in vitro raised plants.

Sterilization of Seeds and Media
Selection. Seeds were used as experimental material. Seeds soaked overnight were washed with a few drops of laboratory detergent (Labolene) and 2-3 drops of Tween 20 (surfactant) after washing under running tap water. Chemical sterilization of the seeds was achieved by treating them with 0.1% streptomycin (20 min) followed by 0.1% HgCl 2 (5 min) and 70% ethanol (45 sec). The seeds were washed 3-4 times with autoclaved double-distilled water to remove all traces of sterilant before inoculating on Murashige and Skoog (MS) basal medium [20] containing 3% (w/v) sucrose (HiMedia) and 0.8% (w/v) Difco Bacto Agar (HiMedia). Different plant growth hormones such as thidiazuron (TDZ), indole 3-acetic acid (IAA), kinetin (Kn), 6-benzylamino purine (BAP), and indole 3-butyric acid (IBA) were augmented with MS medium for various morphogenetic responses. The media were sterilized at 121 ∘ C for 20 min, and the pH of the medium was adjusted to approximately 5.5. The cultures were incubated in a culture room maintained at 25 ± 3 ∘ C with relative humidity of 60-70%. The whole plantlets after cleaning with sterile distilled water were transplanted into pots containing peat-vermiculite-sand-soil mixture (ratio 1 : 1 : 1 : 1, v/v). All the experiments were done in RBD manner. The number of shoots and their length per plant were recorded.

Analysis of Various Chemical Constituents.
In this trial, the four best plant hormonal combinations were selected based on higher shootlet formation. The four selected treatments were assigned as T1, T2, T3, and T4 and were compared with plants (T0) collected from the natural habitat. Different metabolites were analyzed.

2.5.
Alkaloids. The plant samples obtained from different cultural conditions were dried and macerated in a grinder to a powder form and 2.5 g of each plant sample was extracted with ethanol (200 ml) with 20% acetic acid added for 4 h. The filtrate was concentrated to approximately 25 ml and conc. NH 4 OH was added for precipitate formation. The precipitate was further washed with dilute NH 4 OH. The precipitate was dried and weighed, and the amount of alkaloids was determined. The filtrate was dried and weighed [21,22].

2.6.
Phenolics. The plant sample powder (1 g) was extracted with 80% ethanol (20 ml). The supernatant obtained was evaporated to dryness and was resuspended in 5 ml of H 2 O. Folin's reagent (0.5 ml) was added to different aliquots (0.1-1 ml), followed by Na 2 CO 3 (2 ml). A final volume up to 5 ml was obtained with dist. water. The tubes were kept in a water bath for 1 min and slightly vortexed. The reading was taken at 650 nm with standard catechol (2 mg%) [23].

Total
Flavonoid Content. 0.5 ml of the supernatant from the plant sample solution (2 mg/2 ml) was mixed with water (2 ml) and then 5% NaNO 2 solution (0.15 ml) was added. Then, 10% AlCl 3 solution (0.15 ml) was added and allowed to stand for 6 min followed by 4% NaOH solution (2 ml), and the final volume was made up to 5 ml with water and allowed to stand for 15 min. Absorbance was checked at 510 nm versus a water blank [25].
2.9. DNA Damage Assay. The antioxidant potential was determined by observing the DNA protection ability of the in vitro plant samples [14]. 15 l of calf thymus DNA (25 mg%) was dissolved in 20.0 mM phosphate buffer saline (pH 7.4), and to this solution, different concentrations of plant extracts were added and the mixture was incubated for 15 min at room temperature. Then, the oxidative substances (i.e., 1 l of 30 mM H 2 O, 1 l of 20 mM ferric nitrate, and 1 l of 100 mM ascorbic acid) were added, and the whole reaction was held for 1 h at 37 ∘ C. The reaction was stopped by the addition of a loading dye (40% sucrose and 0.25% bromophenol blue). Gel electrophoresis (0.7% agarose/TAE buffer) was run at 100 V for observing changes in DNA and was visualized by the Gel Doc system (Labnet, Germany).

Statistical Analysis.
The whole data set was subjected to ANOVA by SPSS software, and significance was set at < 0.005.

Seed Germination and Shootlet Formation.
The sterilized seeds were inoculated on MS full-and half-strength medium supplemented with gibberellic acid GA 3 (1.5-20 M). Complete germination with elongated shoots was observed on 1/2 MS + GA 3 (15 M). The minimal time period required for seed germination initiation was recorded as 20 days with 78% seed germination response to the same treatment (Table 1 and   [2], Hyoscyamus niger [26], and Cichorium intybus [4,5], where TDZ was found to be effective in enhancing the shootlet formation. However, further increase in both auxin/cytokinin concentrations was not found to be favorable. Similarly, organogenesis was also noticed from the leaf-derived callus of Arnebia euchroma [27] and Bergenia ciliata [28]. The additive effect of IAA and TDZ has been seen in numerous plants such as Santolina canescens [29] and Bupleurum fruticosum [30], which confirms that even the least auxin concentrations in combination with a cytokinin positively tailored the frequency of shoot induction and plant growth. In the present study, the combined interaction of BAP and IBA resulted in maximum multiple shoot regeneration, suggesting that their interaction perhaps resulted in shifts in the endogenous synthesis of TDZ and IAA, making it either suboptimal or supraoptimal and resulting in fulfilling varying needs in the exogenous supply of both hormones [31]. Therefore, in A. benthamii, the optimum level of exogenously supplied phytohormones to maximum shoot multiplication has been registered to be 4 M TDZ and 4.5 M IAA.  (Table 3). Elongation as well as root initiation was noticed after a 6-8-week culture period. The basal zone of the elongated shoots first turned black and apparently became hard. Lower concentrations of IBA (1-2.0 M) failed to promote any desired response. However, when the concentration was raised, the basal zone of the shoots became blacker, hard, and elongated, thus giving a tap root-like appearance, followed by the initiation of lateral root initials, and a maximum of 5.3 multiple black short, thick rootlets were recorded from the main black hard tap root such as structures on 8 M of IBA and TDZ (4 M). Further increase in concentration again had no effect on rooting. When IBA was replaced with IAA (1.0-10.0 M), the basal zone of the shoots again became hard, resembling a tap root-like structure in different concentrations of IAA. This combination was fruitful for the root generation with a maximum average number of roots (8.3) noticed on 3 M of IAA and TDZ (1.5 M) ( Table 3 and Figure 1(d)). However, when the concentration was raised from 3 M to 5.0 M, no response was noticed. In some treatments, a friable light brownish callus of various degrees was also recorded at different concentrations. A protocol for the complete plantlets formation under in vitro conditions and their successful transfer into the greenhouse has been developed. The cytokinins used alone as well in combination with auxins resulted in certain desirable responses, but the most suitable plant growth regulator was found to be the combination of BAP and IBA and TDZ and IAA where maximum multiple shoots were recorded.

Root Hardening and Field
The in vitro raised plantlets were successfully transferred and kept in a growth chamber present in the laboratory at the requisite temperature and humidity. Initially, caps of the culture vials were removed and culture vials containing in vitro born plantlets of A. benthamii were kept in the incubation room for 1 week to slowly reduce the high humidity conditions within the culture vials. After Note. Data scored after 12 weeks of culture period. Data represented as mean ± SD ( = 10); + = low intensity callus formation; ++ = moderate intensity callus formation; +++ = high intensity callus formation. Data was statically analyzed using Duncan multiple range test by SPSS 17.0 software. The values followed by different superscripts are statically significant with each other at < 0.05. TDZ: thidiazuron; IAA: indole 3-acetic acid; Kn: kinetin; BAP: 6-benzylamino purine; IBA: indole 3-butyric acid.
these plantlets were transferred from incubation to normal room conditions where in vitro plantlets were deflasked, agar was washed under tap water with the help of mild brushing. The plantlets were then potted in small plastic cups containing an autoclaved sand : soil : peat : vermiculite (1 : 1 : 1 : 1) mixture (Figure 1(e)). The potted plantlets were placed in a growth chamber at a temperature of 20-25 ∘ C with humidity of 60-70% for one to two weeks followed by their transfer to a greenhouse chamber. These plantlets were then shifted to the greenhouse and were kept there for more than one month. Then, the plantlets were transferred to a net house. The plantlets were continuously monitored, and they showed a response in their growth and development.  (Table 4 and Figure 1(f)). The tissue culture technique is being increasingly exploited for clonal multiplication and in vitro conservation of valuable germplasm threatened by extinction. An efficient procedure for in vitro multiplication is an essential prerequisite for employing in vitro techniques for germplasm conservation. The present investigation carried out on shoot tips of A. benthamii offers a potentially efficient protocol for mass propagation and conservation of this medicinal herb. There is only one published report on in vitro studies of A. benthamii [32], and the current study uses extensive trials pertaining to the use of growth hormones in   addition to successful hardening of plantlets as well. Rooting was observed from multiple shoots and initiation of lateral roots and maximum 8.3 multiple black short thick rootlets was recorded from the main black hard tap root-like structure on 3 M of IBA and TDZ (4 M). In earlier reports on A. euchroma, rooting was noticed under the influence of IBA [27,32,33], and hence our results are in accordance with these. In the present case, IAA was found to be most effective for rooting compared to IBA and NAA. The efficacy of IAA in rooting may be due to its faster uptake than NAA in the present studies.
In this experimental line, the four best plant hormonal combinations were selected based on higher shootlet formation. The four selected treatments were assigned as T1 = BAP ( Figure 2. In the current study, the highest induction of secondary metabolites was observed in T3, obtaining plants with higher amounts of phenols (390 g/g) followed by flavonoids (260 g/g) and alkaloids (235 g/g), which may be due to the influence of the specific plant growth regulator used in the media [34]. PGR has a significant impact on regulation of biosynthetic pathways in plants for synthesis of various metabolites. In addition, the in vitro raised plants were recorded to have a higher content of some essential compounds such as phenolic substances, alkaloids, and flavonoids. The results are in line with the findings of various researchers who also report that the PGRs may also influence the production of secondary metabolites under in vitro conditions [35,36]. For example, the accumulation of alkamide and CADs in Echinacea angustifolia increased upon addition of cytokines to the culture medium. The amplified biosynthesis of metabolites is allied with tissue differentiation, that is, a clump formation in cell suspensions or the formation of more complex structures [34], which may explain the accumulation of high amounts of metabolites in cultured shoots in our study. However, tannins (123.5 g/g) and terpenoids (120 g/g) were found to be higher in T0 plants ( Figure 2). The overall discrepancy between the field and in vitro plants may be due to a number of factors such as seasonal variation [32], plant-to-plant variation in chemical content, and variation in agroclimatic conditions [37]. It appears to be pertinent to accept the in vitro system which may serve as an alternative source of metabolites and thus may be exploited for efficient generation of such substances throughout the year, which are pharmacologically promising but are severely limited in production [14,38].

Biological Activity.
The oxidative substances completely cause DNA degradation (Figure 3). The scavenging of radicals in different ethyl acetate extracts of in vitro raised plants of Arnebia benthamii protecting the DNA against damage by ascorbic acid (standard) was determined. In the current BioMed Research International 7 Figure 3: Protective effect of DNA through scavenging of radicals by A. benthamii extracts. Lane 1: native calf thymus (ct) DNA + reaction mixture + T1 plant samples (500 g/ml). Lane 2: native calf thymus (ct) DNA + T2 plant samples (500 g/ml). Lane 3: native calf thymus (ct) DNA + reaction mixture + T3 plant samples (500 g/ml). Lane 4: native calf thymus (ct) DNA + reaction mixture + T4 plant samples (500 g/ml). Lane 5: native calf thymus (ct) DNA + reaction mixture. Lane 6: native calf thymus DNA. Lane 7: native calf thymus DNA + ascorbic acid (500 g/ml) + reaction mixture. study, methanol extracts of all samples obtained from various media combinations under in vitro conditions showed significant scavenging of the OH radicals and vis-à-vis protection from DNA damage in ct DNA (Lanes 1-4). The current findings are in continuation of the previous reports on the DNA protective effect of field grown plants of A. benthamii [14,39] suggesting the role of some bioactive compounds in scavenging of hydroxyl radicals [13]. This is the first report depicting the DNA damage protection ability of in vitro raised plants, which might be due to the presence of various secondary metabolites as determined above.

Conclusions
In this study, the first ever-complete protocol for the in vitro regeneration and acclimatization under field conditions of A. benthamii, a critically endangered medicinal plant of NWH, was developed, leading to a conservation plan for the endangered species. The in vitro raised plants, particularly the PGR specific growth, had a significant impact on the presence of volatile/nonvolatile metabolites. The biological efficacy of plant extracts allows their use in drug formulations against possible oxidative DNA damage.

Conflicts of Interest
The authors declare that they have no conflicts of interest.