Enhanced in vitro production of diosgenin in shoot cultures of Dioscorea deltoidea by elicitation and precursor feeding

Background Dioscorea deltoidea (Family: Dioscoreaceae) is a critically endangered medicinal plant widely used in traditional medicine and pharmaceutical industries for the preparation of appropriate drugs. The present study was conducted to investigate the effect of different elicitors such as salicylic acid (SA) and methyl jasmonate (MeJa) on the synthesis of diosgenin production in D . deltoidea shoot cultures. In addition, the effect of different precursors (squalene, β-sitosterol, and cholesterol) was also demonstrated. Results Results showed that precursors signicantly inuenced diosgenin production as compared to elicitors. Application of SA (200 µM) showed highest diosgenin production (0.912% DW) at 4 h incubation time, whereas MeJA (100 µM) exhibited 0.814% DW of diosgenin content at 8 h of incubation time. Among precursors, β-sitosterol at 200 µM produced maximum diosgenin content (1.006% DW) followed by 100 µM squalene (0.947% DW) harvested after 5 th day of culture. Interestingly, cholesterol showed low diosgenin production, but signicant than control cultures. Conclusions The results revealed that exposure to different elicitors and precursors have a promising application in accumulation of diosgenin in D.deltoidea shoot cultures.

restrictions [13 -19]. This technique is signi cantly vital for endangered and rare species as it stimulates biomass production without deleterious effect on natural populations [20,21]. In vitro production of bioactive compounds could be improve by using different strategies like biotic/abiotic elicitation, precursor feeding, manipulation of plant growth regulators (PGRs) and media type, concentration, carbon and nitrogen source [13,22,23]. Among these strategies, elicitation and precursor feeding, are the innovative approaches for the synthesis and production of secondary metabolites when given in suitable amounts to in vitro cultures [24,25]. Various factors like concentration and selectivity of elicitor and precursor, exposure time, culture age are important parameters affecting the successful and signi cant production of secondary metabolites [26]. Elicitors like benzoic acid, methyl jasmonate (MeJa), salicylic acid (SA), chitosan, jasmonic acid (JA) and precursors (progesterone, cholesterol, squalene, alanine, phenylalanine, mevalonic acid, shikimic acid) were used to increase the amount of phenolics, avonoids, triterpenoids, alkaloids, anthocyanins in callus cultures, cell suspension culture as well as in organ cultures of many plant families [27 -30]. In cell or organ cultures, SA and MeJa have found effective for initiation of secondary metabolites. It is considered that MeJa takes part in the pathway of signal transduction that makes speci c enzymes to catalyse biochemical reactions for the formation of compounds with low molecular weight, like polyphenols, polypeptides, terpenoids, alkaloids and quinones [31]. SA considered as key-signaling molecule and is responsible in the stimulation of defence responses in plants [32]. In D. deltoidea, diosgenin is biosynthesized by mevalonate pathway and squalene, βsitosterol, cholesterol are intermediate precursors. Cell suspensions or organ cultures precursor feeding in a metabolic pathway has produced large amount of bioactive compounds [33].
In this study, in uence of elicitors MeJa and SA, and precursors (squalene, β-sitosterol and cholesterol) were evaluated on diosgenin production from in vitro shoot cultures of D. deltoidea. To the best of our knowledge, current study is the rst attempt on the in uence of elicitors and precursors on diosgenin production in D. deltoidea shoot cultures.

Results And Discussion
Effect of elicitors on shoot biomass accumulation of D.deltoidea The effect of SA and MeJa was determined on growth rates and has different in uence on biomass accumulation in D. deltoidea shoot cultures. Different concentration of SA and MeJa (100, 200 µM) were added separately to the liquid medium at different incubation times (4-16 h) respectively on 5 th week of culture. Table 1 displays biomass production of shoot cultures treated with SA and MeJa after 10 days.
Shoots elicited with MeJa showed reduced biomass production at 4 and 16 h of incubation time but increased at 8 h. MeJa (200 µM) reduced biomass production as related to control cultures while cultures elicited with SA also showed similar pattern of biomass reduction. The results were inconsistent with Hypericum hirsutum and Hypericum maculatum shoot cultures, in which jasmonic acid (JA) at higher concentration signi cantly inhibited biomass production and SA slightly effected biomass production relative to control [34]. Recently, Jirakiattikul et al. [35] also indicated that, JA had low effect on D. membranacea shoot cultures, whereas SA had no effect. In an another study, it was found that MeJa reduced the Centella asiatica culture growth at concentration above 0.1 mM [36]. In Andrographis paniculata, biomass production was adversely effected by various concentrations of SA (10, 20, 50, 100 µM) [37]. Sivanandhan et al. [38] also reported that MeJa at 150-250 µM completely inhibited biomass production but SA showed insigni cant reduction in biomass as compared to control. Elicitor retardation has been found in other plant cell cultures also such as Salvia miltiorrhiza [39], Panax ginseng [31] and Rubia cordifolia [40]. These results suggest that growth response to different elicitors may vary among different plant species.
Effect of elicitors on diosgenin production in D.deltoidea shoot cultures The effect of SA and MeJa on the diosgenin content of D. deltoidea shoots is shown in Table 1. In order to stimulate diosgenin production, shoot cultures were exposed to elicitor treatment, which is considered as one of the effective strategies to enhance the secondary metabolite production. The present study showed remarkable differences in diosgenin production in shoot cultures treated with elicitors (MeJa and SA) at different incubation times ( Table 1). The diosgenin content was signi cantly increased in all the treated cultures as compared to control. It was found that the addition 100 µM MeJa with 8 h incubation time induced an increase in diosgenin production (0.814% DW). In this study, incubation time showed signi cant role in upregulating the diosgenin content in D. deltoidea shoot cultures; 8 h and 100 µM MeJa was observed to be optimum for the production of diosgenin while 4 h of incubation time for SA (200 µM) exhibited better yield under in vitro conditions. This suggested that elicitor used and its concentrations play an important role on the effectiveness of elicitation. In previous studies, it was reported that MeJa has increased furanocoumarin production in Ruta graveleons shoot cultures and asiaticoside production in Centella asiatica whole plant culture [41,36]. Mendoza et al. [28] reported that elicitation with 3 µM MeJa increased phenolics and avonoids content in Thevetia peruviana suspension cultures. The production of psoralen was enhanced when MeJa and SA at 100 µM was added to the suspension culture of Psoralea corylifolia [42]. Therefore, MeJa have been well established as key signal compounds and positively involved in the signal transduction pathway that leads to secondary metabolite production [43,35]. Diosgenin production was also favoured by SA which is widely studied signaling molecule to trigger secondary metabolite production in plants [44]. SA is generally produced in plants and accumulated at pathogen attack sites, and then spreads to several plant parts triggering the biosynthetic pathway for accumulation of secondary metabolite production [45]. This is an agreement with Diwan and Malpathak [41] and Coste et al. [34] who reported that 200 µM SA increased the furanocoumarin production in Rutagraveolens shoot cultures and hypericin and pseudohypericin content in Hypericum maculatum shoot cultures. However, 50 µM SA signi cantly reduced the diosgenin accumulation in micro-tubers of Chlorophytum borivilianum [46]. In Hypericum perforatum, the hyperforin production was increased in shootlet meristem cultures when treated with 1mM of SA [47]. In in vitro shoot cultures of Swertia paniculata SA promoted amarogentin, swertiamarin and mangiferin yield at optimal concentrations [48]. In the present study, the diosgenin production attained through elicitation of D. deltoidea shoot cultures was higher than reported in eld plants (0.197% DW). Considering the diosgenin accumulation observed in the elicited shoots, D. deltoidea is undoubtedly a potential industrial plant for the production of valuable bioactive compounds.
Effect of precursors on biomass accumulation in D.deltoidea shoot cultures Precursors such as squalene, cholesterol and β-sitosterol correlated to the biosynthesis of diosgenin were supplemented in shoot cultures of D.deltoidea showed signi cant reduction in biomass accumulation ( Table 2). The precursors treated shoots remained in range of 0.517 to 1.559 g of DW. Squalene showed insigni cant biomass reduction when cultures were harvested after 5 th day but biomass accumulation was signi cantly reduced in both concentrations when harvesting was done after 10 th day. In case of cholesterol, there was biomass reduction at both concentrations. β-sitosterol at 100 µM concentration showed insigni cant biomass reduction after 5 th day of harvesting, whereas 200 µM concentration of βsitosterol greatly affected biomass accumulation. Sivanandhan et al. [27] reported that squalene and cholesterol reduced biomass accumulation in Withania somnifera suspension cultures. In another report, it was found that biomass production was decreased due to the addition of squalene and cholesterol in shoot cultures of Digitalis purpurea [12].
Effect of precursors on diosgenin production in D.deltoidea shoot cultures GC-MS analysis con rmed that β-sitosterol signi cantly in uenced the diosgenin content when compared to other precursors. Our results indicated that 100 µM of squalene induced upregulation of diosgenin production (0.947% DW) for 5 days followed by 200 µM of squalene (0.636% DW) for 5 days as compared to control (0.319% DW). It was found that diosgenin content was signi cantly reduced with the increase of harvesting time. Squalene (200 µM) yielded lowest diosgenin content (0.412% DW) after 10 day of exposure time. In addition, β-sitosterol also greatly affected diosgenin content in D.deltoidea shoot cultures. Our results suggest that β-sitosterol (200 µM) was critical for diosgenin synthesis and maximum content (1.006% DW) was found on 5 th day of exposure time followed by β-sitosterol 100 µM (0.782% DW). After 10 days of time diosgenin content was signi cantly reduced as exposure period play an important role in diosgenin production. Cholesterol showed less effect on diosgenin production as compared to squalene and β-sitosterol. Cholesterol (200 µM) for 5 days showed optimum diosgenin content (0.635% DW) followed by cholesterol (100 µM) for 5 days (0.562% DW). In all the cases 10 days harvesting time showed least diosgenin production. Squalene at 6 mM produced highest withanolides in Withania somnifera cell suspension culture [28]. In shoot cultures of Digitalis purpurea, cardiotonic glycosides production was greatly affected by squalene and cholesterol but less effective than progesterone [12].

Conclusions
This work demonstrated that the elicitation practice using exogenous elicitors could signi cantly improve the pharmacologically active diosgenin content in shoot cultures of D.deltoidea as compared to control.
The results of the current study also suggest that type, concentrations and exposure time of different elicitors and precursors used in this study, remarkably stimulate the diosgenin content in D. Deltoidea shoot cultures. Among the different elicitors used, SA signi cantly in uenced the concentration of diosgenin in D. Deltoidea shoot cultures. The results revealed that the use of β-sitosterol act as potent precursor and elicited highest amount of diosgenin as compared to squalene and cholesterol.
The study provides valuable insights into the potential manipulation of precursors on a large scale for the production of diosgenin. In addition, current ndings also provide a reference for enabling the scale-up production of valuable compounds by the aid of bioreactor system. Moreover, further studies are crucial to design metabolic engineering approaches that would enhance the synthesis of valuable bioactive compounds in in vitro cultures.

Explant selection and sterilization
Healthy shoots were obtained from mother plants kept in greenhouse. The explants were rinsed in running tap water (10 min), followed by tween-20 (10 min) and nally washed with distilled water thrice. Surface sterilization of explants was performed inside the Laminar Air Flow Chamber with 0.1 % (w/v) of mercuric chloride (HgCl 2 ) for 3 min and then thoroughly rinsed with sterilized distilled water in order to remove the HgCl 2 traces. Sterilized nodal segments were carved into proper size (1.5-2.0 cm) before inoculation.

Culture conditions and shoot initiation
Nodal segments were inoculated onto the Murashige and Skoog (1962) medium supplemented with 3% sucrose, 0.8 % agar (w/v) and BA (2.0 mg/l) and IBA (1.5 mg/l) for direct shoot organogenesis. The pH of the medium was adjusted to 5.8 with 1 N NaOH or 1 N HCl. The medium was autoclaved at 121ºC for 15 minutes. The cultures were maintained at (25±2 °C) with photoperiod (16 h light/8 h dark) and photosynthetic photon ux (PPF) of 40-50 µmol m -2 s -1 provided by cool white uorescent tubes. After 21 days plantlets were transferred into liquid MS medium with similar composition of PGRs for 5 week and then used for further process. Optimal harvest time was evaluated in terms of biomass accumulation at 7 weeks of culture in liquid media when plant biomass reached a maximum level of 1.95 g DW (6.6 g FW) and further increase or decrease in harvest time has led to reduction of biomass accumulation.

Biomass quanti cation
Fresh weight of control shoots and treated shoots were recorded after harvesting. In vitro harvested shoots were freeze-dried and lyophilized and dry weight measurement was recorded.

Sample preparation
The shoots were pulverized into a ne powder after drying and 1 g DW of ne biomass powder of each set was macerated with aqueous ethanol (50% v/v) for 24 h at room temperature. The extract was ltered through Whatman lter paper No 1 and dried with help of rotary evaporator at 40 ºC. 20 ml of HCL (10%) was mixed to the dried residue and hydrolysed at 98 ºC for 1 h. After cooling, chloroform (15 ml) was added two times for washing and collective mixture was extracted and isolated, lower layer i.e.
chloroform layer was collected and other 20 ml chloroform was used to extract upper layer. Chloroform layers were combined and concentrated to dryness. An appropriate amount of methanol was added to residue and nal concentration was ltered through 0.2 µM syringe lter and preserved in refrigerator (4°C) for further analysis.

GC-MS analysis
An Agilent 7890A Gas chromatography coupled to a 5875C mass spectrometer detector (XL MSD) with triple axis and mass hunter work station software (USA) was used for the analysis of diosgenin. Declarations Ethics approval and consent to participate: Not applicable.

Consent for publication: Not applicable
Availability of data and materials: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: Lovely Professional University gave nancial support but has no role in the study design, performance, data collection and analysis, decision to publish, or preparation/writing of the manuscript.
Authors'contributions: RN did the experiment, SG help in writing the paper, AD helps in collection of plant samples, AJ helped in GC-MS analysis, PD helped in statistical analysis, VK and TM did literature survey while DKP conceived the idea, and supervised the work. All authors read and approved the nal Manuscript  Each value represents mean ± SD of three replicates. Within a column, means followed by the same letter are not significantly different (P ≤ 0.05) according to Tukey Test.