In-vitro propagation and phytochemical profiling of a highly medicinal and endemic plant species of the Himalayan region (Saussurea costus)

Efficient protocols for callus induction and micro propagation of Saussurea costus (Falc.) Lipsch were developed and phytochemical diversity of wild and in-vitro propagated material was investigated. Brown and red compact callus was formed with frequency of 80–95%, 78–90%, 70–95% and 65–80% from seeds, leaf, petiole and root explants, respectively. MS media supplemented with BAP (2.0 mgL−1), NAA (1.0 mgL−1) and GA3 (0.25 mgL−1) best suited for multiple shoot buds initiation (82%), while maximum shoot length was formed on media with BAP (1.5 mgL−1), NAA (0.25 mgL−1) and Kinetin (0.5 mgL−1). Full strength media with IAA (0.5 mgL−1) along with IBA (0.5 mgL−1) resulted in early roots initiation. Similarly, maximum rooting (87.57%) and lateral roots formation (up to 6.76) was recorded on full strength media supplemented with BAP (0.5 mgL−1), IAA (0.5 mgL−1) and IBA (0.5 mgL−1). Survival rate of acclimatized plantlets in autoclaved garden soil, farmyard soil, and sand (2:1:1) was 87%. Phytochemical analysis revealed variations in biochemical contents i.e. maximum sugar (808.32 µM/ml), proline (48.14 mg/g), ascorbic acid (373.801 mM/g) and phenolic compounds (642.72 mgL−1) were recorded from callus cultured on different stress media. Nonetheless, highest flavenoids (59.892 mg/g) and anthocyanin contents (32.39 mg/kg) were observed in in-vitro propagated plants. GC–MS analysis of the callus ethyl acetate extracts revealed 24 different phytochemicals. The variability in secondary metabolites of both wild and propagated plants/callus is reported for the first time for this species. This study may provide a baseline for the conservation and sustainable utilization of S. costus with implications for isolation of unique and pharmacologically active compounds from callus or regenerated plantlets.

Plants have been essential sources of medicine for thousands of years and nearly 80% of the world's population still relies on traditional medicine for their primary healthcare 1 . Saussurea costus is an endemic species in geographically limited places of the Himalayas, where it grows on moist slopes at altitudes of 2500-4000 m. The species is critically endangered and is listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). In addition, it is one of the 37 endangered and highly medicinal plants of the Himalayas, and has been prioritized for both in-situ and ex-situ conservation 2 . S. costus is a highly prized medicinal plant in the Kaghan valley Pakistan. Roots of S. costus have sweet and strong aromatic odor with bitter taste and are used as antiseptics as well as for treating bronchial asthma, especially of the vagotonic type. The roots of S. costus have been widely used for curing diarrhea, jaundice, stomachache, respiratory tract infections, antispasmodic agents against spasms caused by asthma, cholera, rheumatism, chronic skin diseases and leprosy 3 . Further, oil extracted from the roots (referred to as Costus oil) is used for making high grade perfumes and hair oils 4 . In addition, many studies have shown that extracts of S. costus have potent anti-cancer, anti-inflammatory and anti-ulcer properties 5 . Because of the high demands for roots, most natural populations of S. costus are on the verge of extinction 6 .
In order to avoid the future loss of endangered, endemic and rare species, conservation of plant genetic resources has long been realized as an integral part of biodiversity conservation. Plant cell and tissue culture Root initiation. To optimize root induction media, full-strength MS media was supplemented with different combination and concentrations of IAA (0.5, 1.0 mgL −1 ) and IBA (0.5, 0.1 mgL −1 ) along BAP (0.5 mgL −1 ). The time to root initiation was observed and recorded after every two days. Data on average root numbers and length were recorded after 45 days of culturing.
Preparation of solvent extraction for GC-MS. Callus subjected to different stresses (Table 1) as well as grown on CPM was shade dried and grounded to fine powder using mortar and pestle. For solvent preparation 1 g (dry weight) of powder was soaked in 10 ml of ethyl acetate for 2 days. The sample was centrifuged at 8,000 rpm for 5 min and the supernatant collected was stored at 4 °C for further analyses 18 .
Gas chromatography-mass spectrometry (GC-MS) analysis. Chemical analysis of ethyl acetate extract was carried out using gas chromatography coupled with mass spectrometry (GC-MS) with a Hewlett Packard GC-MS system (PerkinElmer precisely, Carlus 600C). The relative percentage of each component was calculated by comparing the average GC chromatogram peak to the total area. The mass detector used in this analysis was Turbo-Mass Gold-Perkin-Elmer, and the software adopted to handle mass spectra and chromatograms was a Turbo-Mass ver-5.4 19 .
Identification of compounds. Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST). The spectrum of a component was compared with the spectrum of the known components stored in the NIST library. Similarly, name, molecular weight and structure of the components of the test materials were ascertained 19 .
Statistical analysis. Statistical analysis was performed with Statistic 8.1 (Trial version). Results were presented as mean ± standard error (SE), and the data was analyzed by one way Analysis of variance (ANOVA) at 0.05% confidence level (p < 0.01). All in-vitro propagation treatments had 5 replications whereas; the phytochemical analyses had three replications for each treatment.

Results and discussion
Callus induction. Callus response was influenced by hormonal combinations as well as the type of explant used. The callus response varied i.e. 80-95%, 78-90%, 70-95% and 65-80% for seeds, leaf, petiole and root explants, respectively ( Fig. 1A-H). Similarly, explants were grown on MS media alone (as control) for 14 days and no callus induction or regeneration was observed and therefore, these results are not included. Maximum  Table 2). It was also noted that subculture of callus into new media increased the callus biomass. Maximum callus growth from seed (1.86 g), leaf (1.65 g), petiole (1.42 g) and root (1.14 g) were record at 2, 4-D (0.5 mgL −1 ) and Kinetin (1.0 mgL −1 ) after twenty-eight days of culture (Table 2). Higher concentration of 2,4-D reduced callus induction and it was observed that the colour changed to brown with hard texture, followed by necrosis. Although 2,4-D is a synthetic plant growth regulator, its role in callus induction is highlighted for S. costus. Previous studies have also reported the efficacy of exogenous 2,4-D in other medicinal plants. Hassan et al. (2009) 20  Shoot bud initiation. Auxiliary buds induction was observed after 15 to 20 days of culturing ( Fig. 2A,B).
The earliest shoot bud initiations were observed on media agitated with BAP (2 mgL −1 ), NAA (1 mgL −1 ) and GA3 (0.25 mgL −1 ). Higher concentration of BAP resulted in earlier buds induction. The analysis revealed BAP had a marked influence on the rate of induction. Similarly, BAP in low concentration, the induction rate was 64% and the lateral buds sprouted late. In addition, new buds were relatively thinner and delicate. ANOVA showed that shoot bud initiation was highly significant among the treatments (Table 3). Previous studies have also indicated that high level of BAP and low GA 3 induced greater response to shoot buds initiation 22 . Similarly, BAP here was most effective for bud induction. GA 3 contributes to the initiation and elongation of auxiliary buds and expansion of leaves 23 . Further, GA 3 regulates the growth and development of plants, mainly by stimulating mitotic division and cell elongation 24 . It was found that high level of GA 3 effectively increased stem length, while lower GA 3 concentration inhibited potato shoot growth 25 . Further, GA 3 has long been used to break dormancy and to stimulate shoot elongation in different species of magnolias 26 . In line with the previous reports, it was also observed that BAP in combination with GA 3 was important for bud initiation, reducing time for buds initiation as well as resulted in stronger buds 27 .

Shoot bud proliferation.
Full strength media augmented with BAP (0.1 mgL −1 ), NAA (0.25 mgL −1 ) and Kinetin (0.25 mgL −1 ) proved best for shoot bud proliferation and elongation (Fig. 2C). Significant differences were observed in multiplication rate and numbers of shoots between T7, T8 and T9, although T13 is significantly   Table 3. Influence of different plant growth regulators on buds initiation and Range analysis. Vigorous and green buds (+ + +); healthy buds (+ +); weak bud ( +). Each Value represents the mean ± SE of five replicates. Significant deference at P ≤ 0.05, x -± Sd-average ± Standard deviation, x ± SE -average ± Standard error.  shoot length was recorded for media fortified with BAP (1.5 mgL −1 ), NAA (0.25 mgL −1 ) and Kinetin (0.5 mgL −1 ). While, the minimum shoot length was recorded in media with BAP (0.5 mgL −1 ), NAA (0.25 mgL −1 ) without Kinetin (Fig. 2D,E). ANOVA revealed significant variation in T15 compared to T13 and T14 ( Table 4). The addition of even smaller amounts of BAP or NAA help inducing adventitious shoot formation by increasing propagation coefficient 32 . Other researchers have also reported that highest shoot length (3.73 ± 0.14 cm) of S. rebaudiana was observed on MS media supplemented with BAP (2.0 mgL −1 ) and IAA (0.25 mgL −1 ) after 15 days of culturing 33 . Additionally, higher concentrations of BAP reduces shoot length, which is in agreement to the known literature 34 .  (Table. 5). ANOVA showed that TI6 was significantly different, while T17 and T18 had no significant variation (Table 5). IBA is a highly stable and potential auxin for roots induction 35 . Maximum numbers of roots (6.76) were recorded on full strength media supplemented with BAP (0.5 mgL −1 ), IAA (0.5 mgL −1 ) and IBA (0.5 mgL −1 ) (Fig. 2E). On the contrary, least number of roots per plant (3.84) were formed on media supplemented with IAA (1 mgL −1 ). Statistical analysis revealed that T16 and T18 varied significantly ( Table 5). The in-vitro derived shoots on MS medium were supplemented with a range of concentrations of two auxins (IAA and IBA) for 75 days, it was observed that the lower concentrations of BAP (0.5 mgL −1 ) in combination with IBA (1 mgL −1 ) resulted in a higher root length (2.53 cm), while IAA (1mgL −1 ) and IBA (1 mgL −1 ) alone induced roots length of (1.5 cm) and (2.27 cm) respectively. Results showed that IAA in comparison to IBA reduced roots length when compared at the same concentration (Table 4). Statistical analysis showed that root length at T16 was significantly different from T17 and T18. Cheepala et al. (2004) 36 reported that IAA is a widely used auxin for rooting in A. stenosperma and A. villosa. In several other plants species the promoting effect of IBA in rooting has also been reported 37 . In contrast, induction of rooting of G. scabra was obtained on NAA (0.3 mgL −1 ) and IAA (0.1 mgL −1 ) containing media 38 . Similarly higher percentage of rooting were obtained in half strength MS media with NAA (1.0 mgL −1 ), were as full strength medium with NAA (1.5 mgL −1 ) was the best media for rooting 10 . Bekheet (2013) 39 has indicated that addition of IAA, IBA or NAA (1 mgL −1 ) induced rooting of in-vitro grown P. dactylifera. However, in the present study, IAA in combination with IBA was found to be the most efficient in multiple shoots induction, followed by IBA alone.

Acclimatization.
The ultimate success of all in-vitro micro propagation endeavors heavily relies on the higher survival rates of such plantlets. Direct field transfers of the plantlets do not allow acclimatization of the in-vitro generated plants as they fail to establish successful interactions with the soil microbes and/or to sustain the environmental conditions 40 . Here, well rooted micro propagated plantlets were transferred into plastic pots containing autoclaved garden soil, farmyard soil, and sand (2:1:1) as shown in Fig. 2F-H. The plants were then acclimatized in the growth room at 27 °C temperature for 2 weeks followed by another 3 weeks at room temperature under laboratory conditions. Finally, 35-40 days old plantlets were transferred to nursery where, morphological anatomical and growth characteristics were observed (results not shown) and survival efficiency recorded. Out of 92 plantlets, 80 (87%) could successfully acclimatize and the relatively low mortality rate here is likely to be due to the biohardening of the micropropagated plants achieved prior to their nursery transfer. Similarly, we have given water to the plantlets after 6 days interval and that too very close to the roots and have avoided leaves. This approach has been previously reported beneficial for in-vitro raised plants 41 and the survival rate could be raised significantly higher if biotization of the explants is attempted 42 .
Phytochemical variation. Total sugar contents. Total sugar contents revealed significant variation with treatments. Maximum sugar contents (808.326 µM/ml) was observed in callus cultured on CPM-4 supplemented with 60 gL −1 sucrose, while the lowest sugar contents (16.64 µM/ml) was noted in wild plants (Fig. 3A). Accumulation of sugars contents in different parts of plants increases in response to a variety of environmental stresses 43 . The accumulation of total sugars is associated with adaptation of plants to various environmental stresses 44 . The results shown here are in agreement to earlier findings where salinity increased total sugar contents in leaves of in-vitro propagated P. euphratica. Similarly, addition of NaCl (250 mmoll −1 ) increased sugars contents by 2.7 times 45 . In calli of M. arborea total sugars account for about 90% of the total dry weight and there were no significant differences. The remarkable differences between the embryogenic and non-embryogenic calli of M. arborea, was the amount of sugar found in embryogenic calli 46 . A similar trend with total sugars ac- www.nature.com/scientificreports/ cumulation was also detected in P. kurroa 47 . In line results are also shown for the total sugars in selected calli of D. caryophyllus subjected to different concentrations of culture filtrate that were significantly higher than those of non selected calli 48 .
Proline content. Since, callus promoting media was used as a control; the stresses imposed increased proline content in callus from 1.63 to 48.14 mg/g F.Wt. The variability in proline content among the different treatments were highly significant as shown in  www.nature.com/scientificreports/ Total phenolic compounds. Phenolics compounds represent a diverse array of plant secondary metabolites, which are predominantly used as powerful scavengers of free radicals (Pietta, 2000) 60 . Here, highest phenolic contents (642.72 mgL −1 ) accumulated in calli cultured on CPM when compared to wild or in-vitro propagated plantlet. Similarly, lowest levels of phenolic compounds (420 mgL −1 ) were recorded in plants collected from wild (Fig. 5A). Increase in phenolic compounds accumulation (37% and 34%) was observed in callus treated with 100 mgL −1 yeast extract and 50 mgL −1 salicylic acid 24 . These finding are supported by those given in El-Nabarawy et al. (2015) 61 , where the culture medium supplemented with low concentration of yeast extract increased phenolic accumulation in micro propagated plants. Furthermore, Gorni and Pacheco (2016) 62 have reported that A. millefolium treated with 0.5 and 1.0 mM salicylic acid significantly increases phenolic contents. A slight increase in total phenolic content was found in callus treated with glycine (200 mgL −1 ), yeast extracts (500 mgL −1 ) and salicylic acid (100 mgL −1 ). This increase of phenolic contents in callus cultures was related to mitochondrial activity; that is, while the cell dehydrogenase activity (FADH2/NADH) and the cytochrome C-oxidase decrease, the production of phenolic compounds increases 63 . On the other hand, variation in total phenolics within the mother source plant, micropropagated plants and callus subjected to different stresses may be attributed to changes in the levels of various phytohormones or other endogenous physiological pathways that occur in plant 64 . Also synthetic plant growth regulators used during the micro propagation pathways make a significant contribution in the production of secondary metabolites within the in-vitro cultured cells and tissues by controlling the expression of genes involved in the synthesis of secondary metabolites such as shikimate and flavonoids 65 .
Total anthocyanin. Anthocyanin contents were detected in the form of Pelargonidin-3-glucoside per kilogram of fresh sample. In the current analyses, in-vitro propagated plant possessed highest amounts of anthocyanin (32.39 mg/kg) followed by wild (31.84 mg/kg) whereas, lowest amount of anthocyanin was recorded in callus   Table 6. The major components in the CPM extract were Propanic acid, 2-methyl-,3,7-dimethyl-2,6-octadienyl ester and Selina-3,7(11)-diene. The analysis of GC-MS chromatogram showed peaks of various phytochemical constituents present in ethyl acetate CPM extracts (Fig. 6).
In contrast, major components identified in CPM-1 were Nonadecane, 2,6,10,14,18-pentamethyl, Nonadecane,2,6,10,14-tetramethyl and 6-tetradecane sulfonic acid, butyl ester (see Table 6, Fig. 7). In CPM-2, major phytocomponents were Octacosane,1-Iodo, Octadecane-2,6,10,14-tetramethyl, Nonadecane, 2,6,10,14,18-pen-  Table 6). While, the phytocomponents such as Octadecane-2,6,10,14-tetramethyl and Hentriacontane were present in all the tasted samples. High amount of Octadecane-2,6,10,14-tetramethyl was observed in CPM-2, while the amount of Hentriacontane was higher in CPM-1. Previously, Gwari et al. derivative and L-(-)-Sorbofuranose and pentakis (trimethylsilyl) ether. This great variation in phytocomponents of S. costus may be attributed to factors related to ecotype, chemotype, phenophases and the variations in environmental conditions such as temperature, relative humidity, irradiance and photoperiod. Moreover, the genetic background may also affect the chemistry of secondary metabolites of plants 72 . Furthermore, exposure to various type stresses may result in drastic epigenetic modifications thereby, changing the transcriptional activities and the overall transcriptomic profile 73 . Recently it has been shown that stable phenotypes can be generated through epigenetic modifications and thereby increasing the success and survival of plants in their natural habitats. Although, we have not studied any such epigenetic modifications here, but these are very likely targets and are important consideration to be included in future studies.

Conclusion
Efficient protocols for large scale callus induction of four explants (seeds, leaf, petiole and internodes) as well as micro propagation from auxiliary buds of S. costus were developed. Callus formation was greatly influenced by type of explant used and maximum callus tissue with minimum time taken was record for seed explants. The best response to direct organogenesis was observed on media fortified with BAP (2.0 mgL −1 ), NAA (1.0 mgL −1 ) and GA3 (0.25 mgL −1 ). Micropropagated plantlets suffer high mortality due to their slow acclimatization to ex-vitro conditions. In spite of the prior limited success with Asteraceae members in inducing roots during tissue culture and acclimatization; here, the regenerated plantlets had 87% of survival rate. We argue this survival rate could be further improved through biotization of micro propagated plants with endophytic bacteria and fungi. Here, phytochemical characterization and variability in metabolites such as total sugars, proline, flavonoids, ascorbic acid, phenolics and anthocyanin is recorded from callus, wild as well as micro propagated plantlets. It is also demonstrated that S. costus callus is rich source of various bioactive compounds as indicated in the GC-MS