Optimization of Seed Germination, Callus Induction, and Secondary Metabolites Production in Allium Jesdianum: Elicitation by Methyl Jasmonate and Putrescine

Allium jesdianum is considered as the most important genera of the Liliaceae family with various pharmacological properties. The present study was carried out to determine the best pre-germination treatment in A. jesdianum. The highest seed germination (58.33%) was observed at chemical treatment including the combination of α-naphthaleneacetic acid (1 mgL − 1 ) and benzyl aminopurine (3 mgL − 1 ) on Murashige and Skoog (MS) media. The highest callus induction (86.7%) was observed at MS/2 media, which was supplemented by NAA (1 mgL − 1 ) and BAP (3 mgL − 1 ) from hypocotyl explants. Two elicitors including methyl jasmonate (MeJ) (0, 25, 50, and 100 µM) and putrescine (Pu) (0, 0.5, and 1 mM) were used to investigate their effects on lipid peroxidation, callus growth traits, total phenolics content (TPC), the contents of total avonoids (TFD), total avonols (TFL), anthocyanin, total chlorophyll, total carotenoids and on antioxidant activity through DPPH (1,1-diphenyl-2-picrylhydrazyl) method under callus culture. The results showed the superiority of MeJ over Pu for increasing the secondary metabolites and antioxidant activity in callused of A. Jesdianum, compared to the control. The highest TPC (6.02 mg GAEg − 1 FW), TFD (0.52 mg QEg − 1 FW), and TFL (0.39 mg QEg − 1 FW) were observed under 50 µM of MeJ. Meanwhile, the highest value for anthocyanin (8.99 µ mol g − 1 FW) was achieved at 25 µM of MeJ. The highest DPPH activities were observed at 50 and 100 µM of MeJ. Putrescine (0.5 mM) elicitation showed only superiority for callus growth rate (0.53 mm day − 1 ) and carotenoids (0.045 mg g − 1 DW). In this study, effective seed germination and elicitation of A. Jesdianum callus cultures were developed for the rst time. Thus, new protocols could be suitable for future studies in biotechnological aspects of this medicinal plant. and the effects of two different elicitors (MeJ and Pu) on the enhancement of different phenolics and callus related traits in A. jesdianum, by the rst. The ndings showed higher eciency of MeJ than Pu for increasing different secondary metabolites and antioxidant activity in calli of A. jesdianum. The superiority of Pu elicitation was increasing callus growth and the contents of two studied pigments (carotenoids and chlorophyll). This study suggests evaluating other different concentrations of Pu for callus elicitation. The concentration of 50 µM MeJ showed the best selective dose for enhancing the contents of phenolic compounds in A. jesdianum through callus culture. This study could lead to introducing a new way to improve the production of the benecial phytochemical compounds from undifferentiated callus cells in the threatened medicinal plant of A. jesdianum.


Introduction
Medicinal plants are traditionally considered among the main sources of secondary metabolites (SMs) and natural drugs with antioxidant properties (Yue et al. 2016). The discovery of new natural products with medicinal characteristics has been recently the subject of intense research (Yue et al. 2016). These SMs are important sources for producing different cosmetics, fragrances, avors, food additives, and pharmaceutical and industrial products (Namdeo 2007;Erb and Kliebenstein, 2020). The technique of plant cell culture is based on a set of biotechnological methods for producing valuable plant-speci c SMs on large scale, independent from environmental conditions (Vanisree et al. 2004;Smetanska 2008). In vitro culture techniques have been used for enhancing different phytochemical compounds as a promising bio-production way for desired and/or rare natural products (Dias et al. 2016; Yue et al. 2016).
In this regard, in vitro elicitation is an e cient strategy to promote plants' defense mechanism for scavenging the reactive oxygen species (ROS) (Matkowski 2008). The subsequent modulating and production of plant SMs occurs by regulating the expression of genes that are involved in producing key enzymes implicated in the biosynthesis of different defense-related compounds as phytoalexins, avones, avonoids phenolics, and other bioactive compounds (Namdeo 2007) under the elicitation process. To enhance the production of these components, chemical elicitors have been widely applied in recent years as environmentally benign techniques (Narayani and Srivastava 2017). In vitro production of worthy compounds using callus culture has been very well recognized and is being used widely for different industrial applications (Narayani and Srivastava 2017). Allium jesdianum Boiss (A. jesdianum) is an important endemic, threatened, and underutilized plant species of Iran (known locally as "Bonsorkh" and "Yazdi onion") that grows in high altitudes (1800-2600 m) of Zagros mountains in west and northwest regions of Iran (Kalantari et al. 2018;Pirbalouti 2019). This species is eaten raw or as a cooked vegetable or is used as a avor additive to fresh or cooked foods in Iran. In the eld cultivation, its propagation rate is very slow and it takes years to produce a new variety.
In folk medicine of Iran, bulbs and leaves of this plant are used for the treatment of cold, kidney problems, and rheumatic pains (Pirbalouti 2019). Also, other medical properties as antibacterial (Dorosti et al. 2017) and anticancer (Dorosti et al. 2017) are reported for this valuable species. In this regard, many attempts have been made to isolate and exploit the active medicinal compounds from it. Considering the potential value of A. jesdianum cell or tissue cultures for biotechnological application, there is an urgent need to develop strategies for the protection and conservation of this genus by the development of its fast seed germination and in vitro conservation protocols.
The present work was aimed to study (a) the effects of physical and chemical pre-treatments on seed germination of A. jesdianum under in vitro culture, (b) the optimization of an e cient procedure for callus induction of A. jesdianum, and (c) establish an e cient strategy for enhancement in commercially vital SMs of A. jesdianum under elicitation by Pu and MeJ through callus cultures.

Seed collection and seed germination experiments
A. jesdianum seeds were gathered from the highland of Sabzkooh province (31° 27′ N 52° 40′ E mountain about 2,140 m height), in Chaharmahal-o-Bakhtiari region of Iran. A. jesdianum seeds were identi ed by the experts of Botanical Researcher of Department of Natural Resources, Isfahan University of Technology (IUT), Isfahan, Iran .The seeds were surface disinfected with 70% (v/v) ethanol for 90 sec and surface sterilized thoroughly with 2% (v/v) sodium hypochlorite for 15 min. The seeds were then rinsed three times with sterile water to remove the remaining amount of disinfection liquid. The sterilized seeds were then placed on to MS medium (Murashige and Skoog, 1962) 3 : Duchefa] ). Other seeds were treated with physical pre-treatments such as NaCl (1 M), cold (4°C for four weeks) and mechanical scari cation, then were cultured on MS medium supplemented with different PGRs combinations ( Table 1). The seeds were cultured on MS medium as a control group. In order to determine the effect of physical and chemical pretreatments on germination experiments were conducted in the photoperiod of 16/ 8 (light/dark) at 23 ± 2 • C for seed germination.

Germination related traits
To determine the optimal parameters of seed germination, the impacts of certain physical and chemical pretreatments on germination rate and germination percent were evaluated. The germination process was observed by emerging the rst radicle. The number of seeds germinated in each pre-treatment was recorded every two days. The germination percentage was calculated using the following equation

Physiological characteristics
Callus relative fresh weight (RFW) was according to this formulae: RFW = (FW 2 − FW 1 )/ days, where, FW 1 is the fresh weight of the callus at the initiation of the elicitation and FW 2 is the nal fresh weight of the callus at the nal day post of elicitation process. CGR was calculated at 15, 30, 45 and 60 day post callus transferring to elicitation media.

Lipid peroxidation assay
The amount of cell membrane damage was determined by measuring malondialdehyde (MDA) as the end product of peroxidation of membrane lipids. For this assay, 0.2 g of fresh calli were homogenized with 3 mL trichloroacetic acid (TCA) (0.1% w/v) (Merck, Com.). The homogenate was centrifuged for 30 min at 4000 rpm. Then the supernatant (5 mL) was collected and mixed with 4 mL of thiobarbituric acid (TBA) (0.5% w/v) and 20% TCA (w/v). Then, the sample was heated at 95°C for 25 min and then placed on ice bath. Its absorbance was measured at 532 and 600 nm with a spectrophotometer. The content of MDA was calculated by an extinction coe cient of 155 mM − 1 cm − 1 and expressed as µmol g − 1 FW (Heath and Packer, 1968).

Methanolic callus extract
At rst, 0.5 g of completely dried callus were powdered and added to 10 mL of diethyl ether The mixture was well mixed and stored in a refrigerator for 24 h. For complete evaporation of diethyl ether, the supernatant was transferred to dryer. Then 10 mL of 80% methanol were added to that puri ed supernatant and then ltered using a 0.4 µm lter.

Determination of total phenolic content
Based on the Folin-Ciocalteau reagent method described by Singleton et al. (1999) total phenolics content (TPC) was determined. In brief, 0.2 g fresh weight of celli were homogenized with 3 ml CH 3 OH (Merck. Com) and centrifuged at 4000 rpm for 25 min. Then, methanolic extract (0.5 mL) was mixed with 2.5 mL of Folin-Ciocalteau reagent (Sigma-Aldrich, Com), followed by the addition of 2.0 mL of 7 % Na CO solution. The mixture was disposed in the dark for 90 min at room temperature. The absorbance of the supernatant was recorded at 765 nm against the reagent blank by a spectrophotometer. The amount of TPC was then quanti ed by the method of calibration curve using gallic acid (GA 3 ) (Sigma-Aldrich, Com) as standard.

Determination of total avonoids and total favonols content
Total avonoids (TFD) and total avonols (TFL) contents were determined spectro-photo metrically using the method of Miliauskas et al. (2004). In brief, 0.2 g fresh calli was ground in 3 mL of methanol and centrifuged at 4000·rpm for 25 min. Then, supernatant was used to assay TFD and TFL contents. For estimation of TFL, 0.5 mL of methanolic extract, 0.5 mL of AlCl 3 (2% w/v) solution and 1.5 mL of sodium acetate (5% w/v) were mixed and after 90 min of incubation at room temperature, the absorbance of mixture was measured at 445 nm. For total avonoid 0.5 mL of methanolic extract, 125 µL of AlCl 3 solution (%10 w/v) and 125 µL of CH 3 COOK (1M) were mixed and the samples were kept at room temperature for 30 min. A spectrophotometer was used to measure the absorbance of the reaction mixture at 415 nm with. The contents of total avonoids and avonols were expressed as mg quercetin (QE) equivalents per gram of fresh mass (mg QE g − 1 FW).

Determination of anthocyanin
At the rst step, 2 mL acidi ed methanol (1% HCI) was used for homogenizing the fresh calli (0.2 g) at room temperature (Hara et al. 2003). After one day, the total extract was centrifuged for 25 min at 4000 rpm. Then, the content of anthocyanin was determined by spectrophotometer at wave length of 511 nm based on the extinction coe cient of Raphanusin (33000 M − 1 cm − 1 ).

Determination of photosynthetic pigments
Carotenoids and chlorophyll contents were determined according to the method of Lichtenthaler and Buschmann (2001). Acetone (80%) (Merck, Com.) was used for these pigments extraction from fresh calluses. For carotenoids, absorption spectra of the extracts were measured at 470, 652 and 665 nm and its concentrations were calculated according to the Lichtenthaler equations.

DPPH radical scavenging activity
Radical scavenging activities of sa ower calli were determined by DPPH assay (Golkar and Taghizadeh, 2018). The methanolic extract (20 µL) was added to 1 mL of 50 µM DPPH (Sigma, Aldrich) solution in methanol. The prepared extracts ranged from 0 to 250 µg mL − 1 .The mixtures were mixed and incubated in the dark condition for 20 min. The reduction of DPPH absorption was measured at 515 nm. The positive control was ascorbic acid (Sigma, Aldrich). The DPPH radical scavenging activity was calculated using the equation: Inhibition concentration (inhibition percentage: IP %) = (absorbance controlabsorbance sample)/(absorbance control) × 100.

Statistical analysis
This study elicitation section was carried out as a factorial based on a completely randomized design with six replications. The germination and callus induction experiments were carried out as a completely randomized design with six replications. The analysis of variance done using SAS software version 9.3 (SAS Institute, 2011). Then, mean comparisons (± standard deviations) were carried out using Fisher's least signi cant difference (LSD 5% ) test.

Seed germination
The optimization of germination is considered as a major gap in A. jesdianum. To the best of our knowledge, no previous study has reported the optimization in germination through seed in A. jesdianum. Some studies have optimized germination in some Allium species (Kamenetsky and Gutterman 2000). Some treatments such as scari cation and darkness helped break the dormancy of some genus as A. suworowii, A. a atunense, and A. altissimum at cold temperatures (Kamenetsky and Gutterman 2000). This study, for the rst time, evaluated the effects of different treatments to triggering germination of A. Jesdianum under in vitro culture. Different treatments including mechanical (scari cation), chemical (different combination of plant growth regulators), and thermal conditions (cold temperature) were applied to optimize the germination ratio in A. jesdianum ( Table 1). Evaluation of germination under different treatments showed extensive variation in GP in about 30-45 days after initiating the treatment.
There was a statistically signi cant difference (P < 0.01) in GP and GR between different pre-germination treatments (Table S1). According to the data in Table 1  scari cation and cold-water stress were found to be effective for germination in seeds of A. jesdianum; however, chemical treatments had the most effect on breaking seed dormancy. Therefore, from the above ndings, it can be inferred that dormancy of the seeds of A. jesdianum was probably associated with the enclosed covering around the embryo. The seeds under control condition gave a similarly high GP (52.17%) but seeds took a long time to germinate, similar to germination in Parkia biglobosa (Abbas et al. 2018). It seems that the ecological background of the natural habitat for seed collection region and harvesting time play predominant roles in seed germination capacity in A. jesdianum. The optimized protocol could be used for rapid propagation of this plant under in vitro conditions.

Callus induction
Despite the role of A. jesdianum as an important medicinal plant, no research has focused on using in vitro cultures in this neglected species. In the present investigation, callus initiation from explants (hypocotyl and seeds) were observed within 14-18 days on all medium compositions (Fig. 1). The induced calli grew into yellow-to-greenish color with a semi-friable structure on four different media. The growth of calli were fast after their induction.
Analysis of variance showed a signi cant effect of treatments on CI and CGR (Table S2). Callus induction in all tested PGRs and explants were effective ( Table 1). The effects of different PGRs (2,4-D, NAA and BAP) and explants (seed and hypocotyl) on callus induction (%) and callus growth rate of A. jesdianum is presented at Table 2. As seems, this is the rst report regarding the optimized protocol for callus induction in A. jesdianum. According to Table 2, MS/2 media supplemented with 1 mgL − 1 NAA and 3 mgL − 1 BAP were recognized as the best combination for highest callus induction ( 83.92%) in seeds explants of A. jesdianum (Table 2), which showed no signi cant difference with MS/2 media supplemented with NAA (1 mg L − 1 ) + BAP (3 mgL − 1 ) from hypocotyl explants ( Table 2). The least value for CI (42.01%) was denoted to MS/2 media which was supplemented by 2 mgL − 1 BAP and 1 mgL − 1 2,4-D ( Table 2). These ndings showed no callus induction on complete MS media for this species. So it could be resulted that lower concentrations of macro and micro elements was better for cells differentiation from explants in this genus. The good response of hypocotyl explants could be due to homogenous cell nature and easy availability of growth substances to each cell of homogenous tissues, hypocotyl explants showed high potential for callus induction. The combined effects of auxins and cytokinins have been considered as essential factors for callus induction in A. jesdianum. Comparison of four different treatment implied that higher concentrations of BAP (as cytokinin group) than 2,4D and NAA (as auxins), could be promotive for more frequency in callus initiation and its growth in A.jesdianum.  (Table 2), but the lowest one (0.18 mm day − 1 ) was observed at MS/2 media which was supported by 2 mgL − 1 BAP + 1 mgL − 1 2,4D ( Table 2). This implied at a positive interactive effects of these concentrations of BAP and 2,4D on used explant for enhancing the callus growth.

Callus growth traits
When studying the callus growth, the responses of relative fresh weight and callus growth rate were evaluated to different doses of MeJ and Pu. Overall, a decrease in RFW was observed in response to most of the treatments compared to the control ( Table 3). The least content for RFW (0.60) was observed at 100 µM concentration of MeJ rather than control (0.42) ( Table 3). The stressor effects of both used elicitors (especially at higher concentrations) reduced the RFW. This is probably due to the inhibitory effects of elicitors on cell growth and capacity of cell osmotic adjustment, which increase the requirement for maintaining turgor of the growing cells, consuming energy, and decrease in callus growth  (Table 3). For CGR, all elicitations resulted in a signi cant reduction compared to the control, except for 0.5 (mM) of Pu (Table 3). This result indicates the bene ciary effects of lower concentrations of Pu (0.5 mM) on motivating callus growth. Such a response is consistent with ndings of cell growth and callus diameter in Hypericum perforatum (Abdollahipoor et al. 2017). It is well established that Pu stimulates not only callus cell extension but also its cell division. The nonsigni cant effects of MeJ on callus growth were also reported on cultures of Zanthoxylum stenophyllum Hemsl (Biondi et al. 2004). Elicitation by these elicitors changed the color of calli to yellow-to-pale brown and pale brown after the elicitation period. The calli under Pu were more turbid. The elicitation process diminishes the friable structure of the calluses to an approximate extent.

Lipid peroxidation
During the elicitation process, some processes such as lipid peroxidation of membranes lead to enhancing antioxidant enzyme activity and the activation of SMs production (Das and Roychoudhury 2014 Table 3). The MDA content showed a 2.94-fold increase (P < 0.05) in the calluses exposed to 1 mM (Pu) compared with the control cultures. In agreement with these previous studies, exposure to MeJ signi cantly increased the MDA content in Panax ginseng.  Table 3). The highest (6.02 mg GAEg − 1 FW) and the least (2.86 mg GAEg − 1 FW) contents of TPC were observed at treatments of 50 mM MeJ and control treatments, respectively (Table 3). In callus cultures treated with 25, 50, and 100 µM of MeJ, the TPC showed 1.32-, 2.10-, and 1.01-fold increases compared to control, respectively (Table 3). When treated with 50 µM MeJ, callus cultures induced maximum corresponding TPC levels of 6.02 mg GAEg − 1 FW, while 100 µM MeJ resulted in inhibited levels with the value 2.89 mg GAEg − 1 FW on callus culture (Table 3) (Table 3). For Pu elicitation, only the lower concentration level (0.5 mM) showed a signi cant difference with control (Table 3). This nding was like the positive effects of MeJ increase in the total avonoids of Hypericum perforatum (Wang et al. 2015) and  (Table 3). This nding was similar to the elicitation effects of Pu on anthocyanin production of Daucus carota under callus culture (Sudha et al. 2003). Therefore, at higher concentrations, both elicitors had an inhibitory or non-signi cant effect on Ant content in A. jesdianum. Among these two elicitors, MeJ acted as a better inducer of anthocyanin biosynthesis in A. jesdianum callus cultures than Pu. The novel results from the present study show that callus cultures of A.jesdianm under elicitation could be considered as an e cient way for these valuable pigments production.
A  Table 3). The highest (0.27 mg g − 1 DW) and the least (0.002 mg g − 1 DW) contents for ChlT were for 50 µM (MeJ) and 1 mM Pu, respectively (  (Fig. 2). It was observed that A. jesdianum calluses had a dosedependent DPPH radical scavenging activity under MeJ elicitation (Fig. 2). This result suggests A. jesdianum is a good free radical scavenger under elicitation. Similarly, the increase in antioxidant activity of callus cells (e.g., DPPH method) was observed under MeJ elicitation in different cell culture techniques (Ali et al. 2007;Ho et al. 2020).

Correlation between different traits
The simple correlation among studied traits is presented in Table 4. A negative and signi cant correlation was found between RWC with TFD (-0. 46 ** ) and TFL (-0.35 ** ). The lipid peroxidation content as MDA showed a negative and signi cant correlation with RWC (-0.64 ** ) and CGR (-0.53 ** ) ( Table 5). This nding shows the importance of cell wall integrity on retaining cell water content and its growth under cellular conditions. The antioxidant activity (DPPH method) showed positive and signi cant correlation with TFL (0.43 ** ), TPC (0.33 ** ), and ChlT (0.37 ** ). The antioxidant activity through the DPPH method was found to be mainly TPC (0.33 ** ) and TFL (0.43 ** ), depending on callus cultures treated with different elicitors. Furthermore, a positive correlation between TPC and TFL with DPPH suggests that the elicitation process increased the antioxidant activity of A. jesdianum through more accumulation of TPC and TFL in callus culture.

Conclusion
Plant cell cultures have been perceived as an attractive method for producing SMs under controlled conditions. This study optimized seed germination, callus induction, and the effects of two different elicitors (MeJ and Pu) on the enhancement of different phenolics and callus related traits in A. jesdianum, by the rst. The ndings showed higher e ciency of MeJ than Pu for increasing different secondary metabolites and antioxidant activity in calli of A. jesdianum. The superiority of Pu elicitation was increasing callus growth and the contents of two studied pigments (carotenoids and chlorophyll). This study suggests evaluating other different concentrations of Pu for callus elicitation. The concentration of 50 µM MeJ showed the best selective dose for enhancing the contents of phenolic compounds in A. jesdianum through callus culture. This study could lead to introducing a new way to improve the production of the bene cial phytochemical compounds from undifferentiated callus cells in the threatened medicinal plant of A. jesdianum. Abbreviations BAP : 6-Benzylaminopurine; 2,4-D: 2,4-Dichlorophenoxyacetic acid; FW: Fresh weight; DW: Dry weight; NAA: α-Naphthaleneacetic acid; MS: Murashige and Skoog; MeJ: Methyl jasmonate; Pu: Putrescine; TPC: total phenolics content; TFD: total avonoids; TFL: total avonols; DPPH: 2,2-diphenyl-1-picrylhydrazyl.