Enhancement Production of Phenolic Compounds in The Cell Suspension Culture of Iberis Amara L.: The Effect of Chitosan Elicitation

Iberis amara L. medicinal herb is well-known for having pharmacological values although its use has been challenged by the low levels of secondary metabolites. To address this issue, this study focused on evaluating the effect of explant, photoperiod, and plant growth regulators (PGRs) to nd the optimum medium for inducing callus and establishing cell suspension in I. amara, followed by investigating the chitosan effect on some secondary metabolites. From our observations, the optimum condition for induced callus was achieved from the leaf explants in Murashige and Skoog (MS) media completed with 3 mg L − 1 6-benzylaminopurine(BAP) and 1 mgL − 1 1-naphthalene acetic acid(NAA) under 16-h light/8-h dark photoperiod. The MS enhanced with 3 mgL − 1 BAP, 1 mg L − 1 NAA, and 2% (w/v) sucrose appeared to be optimum conditions for suspension establishment. Thus, the cells were exposed to different concentrations of chitosan (200, 100, 50, and 0 mg L − 1 ) in their exponential growth stage from day 8 to 12 and day 12 to 16 following sub-cultures (T1) and sub-cultures (T2), respectively. The results showed that the 50 mgL − 1 chitosan signicantly improved the total phenol, avonoid, avonol, and anthocyanin content in the I. amara in a dose-dependent manner. The highest malondialdehyde (MDA) amount, as a result of lipid peroxidation, was observed under the 200 ppm chitosan elicitation. Overall, these novel ndings demonstrated the possibility of applying the cell suspension of I. amara treated with chitosan as a helpful approach for improving synthesizing phenolic compounds under controlled and sterile conditions without genetic modications in medicinal herbs.


Introduction
Medicinal herbs have long been known for producing secondary metabolites, which are utilized as bioactive compounds in pharmaceutical applications (Gonçalves and Romano 2018). Iberis amara L. (I. amara), as a genus of the family Brassicaceae native to southern Europe, has been used by ancient humans in order to cure rheumatism and some other diseases. This medicinal herb is also well-known bene cial for restoring health from bronchitis, asthma, and cardiac hypertrophy (Liu et al. 2020). A phytomedicine (i.e., Iberogast®) has recently been provided from the I. amara herb extract and applied to relieve the symptoms of gastrointestinal disturbances related to irritable bowel syndrome and functional dyspepsia ( Despite a large amount of information regarding the therapeutic value of I. amara, this herb meets low levels of bioactive products, which strongly depends on bio-physiological and environmental conditions (Liu et al. 2020). Therefore, plant tissue and cell suspension culture appear as a viable biotechnological tool for the production of secondary metabolites in medicinal plants. These techniques have been applied for increasing the quantity and quality of drugs and provide a promising bio-production platform for a desired natural product (Dias et al. 2016;Khvatkov et al. 2015;Yue et al. 2016). A variety of elicitors as chemical compounds stimulating the signaling pathways were suggested as an innovative tool for the improvement of natural product biosynthesis (Taghizadeh et al. 2021b). A number of elicitor compounds have so far been adopted to modulate cell metabolism and increase bioactive ingredients in cell culture media (Qiu et al. 2021; Ramirez-Estrada et al. 2016). Biotic elicitors with microorganism origins such as chitosan have been exhibited to increase the plant metabolites having therapeutic values (Qiu et al. 2021). Chitin and its deacetylated derivative (i.e., chitosan) also play a critical role in activating resistance against pathogens in plants ( Putalun et al. 2007). It is considered as a biotic elicitor that induces phytoalexin accumulations in plant tissues (Orlita et al. 2008). Elicitors  To the best of our knowledge, there are no reports on the establishment of cell suspension culture and the effect of chitosan on the accumulation of secondary metabolites in I. amara. This study is the rst one to focus on optimizing a high-throughput procedure for inducing callus and establishing cell suspension in I. amara, as well as using chitosan to enhance the content of phenols, avonoids, avonols, and anthocyanins. This understanding can lead to the introduction of a new way to improve the production yield of bene cial phenolic metabolites from undifferentiated I. amara cells.

Plant materials and optimization of germination medium
I. amara seeds were provided from the Gol Daroo Co. and then transferred to the lab for determining their best germination medium. To sterilize, the seeds were treated with 70% alcohol for 60 s as well as 2 % NaOCl solution for 20 min, and eventually washed in sterile water three times. After sterilization being carried out, the seeds were placed on MS base (Murashige and Skoog 1962) medium containing 5 mg L − 1 GA3, 0.8% (w/v) agar and 3% (w/v) sucrose at pH 6. Eventually, the seeds were incubated at 25 ± 1°C under the 16 h light/8 h dark photoperiod in the growth chambers.

Optimization of callus induction
For callus induction, the explants of I. amara were obtained from in vitro grown plants on the MS medium. Four weeks after the growth of buds in vitro in the completely sterile conditions, two explants including 6-7 mm stem and leaf sections were prepared and placed on MS medium with four treatments as follows: 2 mg L − 1 2,4 D; 1 mg L − 1 NAA + 3 mg L − 1 BAP; 1 mg L − 1 BAP + 3 mg L − 1 NAA; 3 mg L − 1 BAP.
Explants were cultured on MS medium without PGRs were considered as a control. The explants were incubated at 25 ± 1°C and humidity 60% under 16h light/8h dark photoperiod in the growth chamber.

Relative Callus growth rate
We measured the callus growth rate (CGR) and callus induction percent (CI). For CI calculation, the relation below was used for measuring callus induction: [(n/N) × 100], following completion of sixth week from the callus induction initiation. "N" represents the cultured explants' total number and "n" denotes the callus explants' total number. For calculating CGR, the means of CGRs (mm day − 1 ) at every two weeks was used in a two-month period (Afshar and Golkar 2016).

Suspension culture establishment
To determine an optimum cell suspension culture, friable callus (2.5 g) was moved into 100 mL asks comprising 25 mL MS medium containing 1 mg L − 1 NAA + 3 mg L − 1 BAP augmented by 2% (w/v) sucrose. Then incubated in darkness on gyratory shakers at 125 rpm. The subculture of cell suspension was carried out at steady intervals of 14 days on the fresh media.

The curve of cell growth
After being isolated from the suspension culture through ltration, 2.5 g of fresh cells were cultured in 25 mL MS within 100 mL asks. Cell growth was followed with asks harvested at two days intervals from the day of subculture (0th day) until cell weight was stable (24th day). The weight of the samples was measured and recorded by a digital scale. According to the plotted growth curve, the best time to harvest cells was selected.

Elicitor treatment
To elicit the biosynthetic pathway of natural products in I. amara, the chitosan with 50k Da molecular weight (Sigma-Aldrich, Germany) was dissolved in 3% (v/v) acetic acid (0.1 M) through mild heating and continuing mixing at 55°C for 8 h. The eventual concentration of chitosan was adjusted to 10 mg. mL − 1 . To further dissolve chitosan, the solution was mixed and autoclaved for 20 minutes at 121°C, and kept at 4°C. In the exponential growth stage, we exposed the cells to chitosan at the varying concentrations (0, 50, 100, and 200 mg L − 1 ), which was performed from day 8 to 12 following sub-cultures (T1) and day 12 to 16 following sub-cultures (T2). Some empirical studies and reviewed literature were used as the basis When elicitations were nished, we harvested the cells from the suspension cultures by ltration. For this purpose, a Buchner funnel was utilized with a nylon mesh in frozen and vacuum state in liquid N 2 , and it was maintained at -80°C for subsequent biochemical surveys.

Cell growth assay
In order to measure the cell growth, cells were collected after treatment and rinsed with distilled water twice. The fresh weight was then measured using a digital scale.

Cells extraction process
Firstly, cells were perfectly dried, and then 200 of them were ground in liquid N. Afterward, the cells were homogenized with methanol (3 mL), followed by centrifuging for 15 min at 10000 rpm. The supernatant was then gathered and maintained at -20°C for the subsequent analysis.

Total phenol content Determination
To estimate total phenol content, the methanolic extract (0.5 ml) was supplemented with 1.5 ml of 10% Folin-Ciocalteu reagent and 1.5 ml of 15% sodium carbonate, and eventually kept at 25°C for 90 min. The solution absorbance was read at 725 nm by using a spectrophotometer. The total content of phenolic compounds was estimated according to the gallic acid standard in mg gallic acid equivalent per g of cell dry weight (Taghizadeh et al. 2019).

Flavonoid and avonol content Determination
Total avonol and avonoid contents were determined via the aluminum chloride procedure, as elucidated by Tahsili et al., (2014). For total avonol, 3 mL of sodium acetate, 1 mL of methanolic extract, and 1 mL of aluminum chloride were mixed together. The absorbance for each mixture was determined at 445 nm. For total avonoid, 250 µL of potassium acetate, 1 mL of methanolic extract, and 250 µL of aluminum chloride were mixed and remained at 25 ± 1°C for 30 min to read the absorbance at 415 nm. Rutin was utilized as the standard for the calibration curve. The content of total avonol and avonoid was presented as mg rutin per g of dry weight.

Anthocyanin content Determination
To measure the anthocyanin content, according to Hara et al. (2003), 200 mg of dried cells was ground in 3 ml of acidic methanol (99: 1 ratio of ethanol to acetic acid), and then the resulted extract was carefully centrifuged at 12000 rpm for 20 min. The supernatant was placed in the dark overnight after ltration and its absorption was recorded by using a spectrophotometer at 511 nm. To calculate the concentration of anthocyanins, M − 1 cm − 1 33000 extinction coe cient was exerted and the anthocyanin accumulation was presented in nmol per g of dry weight.

Lipid peroxidation determination
MDA was measured for determining the cell membrane damage and lipid peroxidation. MDA was the end product of membrane lipids' peroxidation. Therefore, 0.1% trichloroacetic acid (TCA) solution was used for homogenizing the samples. We centrifuged the homogenate at 10000 rpm, and then collected supernatant. The process was followed by mixing supernatant (500 µL) with 0.5% thiobarbituric acid (TBA) (2 mL) and 20% TCA. Then, the sample was warmed for 30 min at 95°C, and it was swiftly chilled in the ice bath. A spectrophotometer was used for measuring the sample's absorbance at 600 and 532 nm. The extinction coe cient used for quantifying the amount of MDA was 155 mM − 1 cm − 1 , and it was presented as µmol g − 1 FW (Heath and Packer 1968).

Statistical analysis
The current study was performed as a factorial experiment in a completely-randomized design (CRD) with three repeats. Analysis of variance and mean comparison (LSD at P ≤ 0.05) were accomplished through SAS software V. 9.1 (SAS Institute Inc.). The differences among means were represented as mean ± standard error.

Optimum callus induction medium
Although I. amara is a signi cant medical herb, there is no study on using in vitro cultures of this plant. The signs of callus production were observed approximately 12-14 days after transferring stem and leaf explants to medium compositions.
The results of the analysis of variance (ANOVA) indicated the statistically signi cant impact of plant growth regulators (PGRs), types of explant, photoperiod, and interactions with studied traits (data are not presented). Based on the ndings, light signi cantly affected I. amara callus growth (CGR, CI) as compared to dark conditions. In addition, visual symptoms revealed that the induction, formation, and growth of callus in 16-h light/8-h dark photoperiod was highly better than the conditions of absolute darkness. As a result, further analyses were applied only on 16-h light/8-h dark photoperiod. According to previous reports, this phenomenon (i.e., light effect) may be associated with the crosstalk between light signaling and IAA hormone (Rikiishi et al. 2008 The mean interactions in a completely randomized design were compared since the interactions of PGRs and explants were signi cant. Table 1 presents the effects of different PGRs (MS, MS + 2 mg L − 1 2,4 D, MS + 1 mg L − 1 NAA + 3 mg L − 1 BAP, MS + 1 mg L − 1 BAP + 3 mg L − 1 NAA, and MS + 3 mg L − 1 BAP) and explants (leaf and stem) on callus induction (%) and callus growth rate of I. amara. As previously mentioned, this is the rst report regarding the optimized protocol for callus induction in I. amara. Our ndings showed no callus induction on the MS medium, as a control, for two explants. Based on the results, 2 mg L − 1 2,4 D and 3 mg L − 1 BAP represent an inhibitory in uence on callus production thus they were excluded from further analyses. The completed MS medium with 3 mg L − 1 BAP and 1 mg l − 1 NAA was found to be the appropriate culture medium for the highest callus induction (85.16%) in the leaf explant of I. amara (Table 1). The least values for CI (27.17%) and (25%) were denoted to leaf and stem explants at the MS medium supplemented by 3 mgL − 1 NAA + 1 mgL − 1 BAP (Table 1). Leaf explants were detected to be suitable for callus induction so that they induced large calluses in the culture medium ( Fig.  1). As observed in the recent work, the callus induction from the leaf/stem explant relied on the combination and concentration of PGRs. Accordingly, the optimal level of exogenous PGRs is vital for CI in I. amara. Four treatments were compared, and it was shown that BAP concentrations (as cytokinin) were higher compared to NAA and 2,4D (as auxins), which might promote a higher frequency in CI and growth in I. amara. Similar observations with NAA and BAP hormones for improving callus induction were previously recorded in some herbs (Hajati et

Cell growth curve
The ndings represented that cell growth was extremely low in the rst ve days (the delayed phase). The exponential growth of these cells was recorded from the 6th to 16th day after inoculation (the logarithmic phase). The highest fresh weight of cells was observed on the 16th day, which increased approximately four folds. Then, the cells entered the dormant phase for about four days and the cell growth stopped (the dormant phase). Eventually, the weight of the cells decreased and the cells entered the death phase ( Fig. 2). Therefore, the times for treatment application were considered at the beginning (8-12th day) and middle (12-16th day) of the logarithmic growth stage. These ndings open a time window toward the use of cell suspension culture to produce phenolic compounds, avonoids, avonols, and anthocyanins, which acquired a pharmacological value.

Fresh weight
The results of analysis of variance showed that the chitosan elicitations, period time of elicitation, and different interactions had a signi cant effect on all the studied traits (data known show). Based on the results, the maximum fresh weight (10.3 g) was detected in both control cells and 50 mg L − 1 chitosantreated cells on the 12-16th day after the harvest (Fig. 3a). The minimum fresh weight (6.22 g) was detected in 200 mg L − 1 chitosan-treated cells on the 8-12th day (Fig. 3a).

Total phenol content
According to previous reports, elicitors can enhance the level of phenolic compounds through a rapid increment in the activity of key enzymes responsible for biosynthetic pathways, including PAL (Govindaraju and Arulselvi 2018). Thus, the interaction effect of different concentrations and treatment intervals of chitosan were investigated to retest this output in I. amara (Fig. 3c). Based on the evidence,  Khan et al. (2003) also proved an about 50% augmentation in the total phenol amount in the soybean plants following chitosan treatment, displaying a positive correlation between PAL activity and the total phenol content. Considering that PAL is a vital enzyme in the phenylpropanoid biosynthetic pathway, it seems that its overactivity resulted in more phenol accumulations in chitosantreated I. amara (Khan et al. 2003). According to our data, the contents of the total phenolics, avonoids, avonol, MDA, and anthocyanins in chitosan-treated cells on the 12-16th day (T2) were signi cantly higher than those of cells treated with chitosan on 8-12th elicitation (T1). These observations suggest that the antioxidant system is signi cantly stimulated by increasing the age of cells elicited by chitosan. Table 2 provides the obtained correlations among the evaluated traits. The total phenolic compounds indicated signi cant positive correlations with avonoid and anthocyanin (0.702 ** and 0.762 ** ) suggested at synchronization pathways for biosynthesis and gathering these compounds in chitosan and normal elicitation ( Table 3). According to these results, with higher phenolic compounds, avonoid

Conclusion
In this study, the optimum in vitro conditions were obtained for inducing callus and establishing the cell suspension of the I. amara medicinal herb. The optimum medium for callus induction was provided from leaf and stem explants in the MS medium supplemented by 3 mg l − 1 BAP and 1 mg l − 1 NAA. The MS medium supplemented by 3 mg L − 1 BAP, 1 mg L − 1 NAA, and 2% (w/v) sucrose at a pH of 6 was found suitable for achieving rapid-growing suspension cells. Interestingly, 50 mg l − 1 chitosan remarkably improved the phenol, avonoid, avonol, and anthocyanin content in the I. amara medicinal herb in a dose-dependent way. As a result, chitosan (50 ppm) was detected as an e cient elicitor for enhancing secondary metabolites in I. amara and probably other medicinal herbs. The results of the present work can pave the way for improving the generation of bene cial medicinal compounds from undifferentiated cells in a vulnerable medicinal herb called I. amara.  Growth curve of suspension-cultured I. amara cells during 24 days of incubation in MS medium augmented with 1 mg L-1 NAA and 3 mg L-1 BAP.

Figure 3
Effect of treatment intervals and chitosan concentrations on fresh weight (a); MDA content (b); phenolic compounds (c); avonoid (d); avonol (e); anthocyanin (f) of I. amara cells. T1: A group of cells treated from 8th to 12th day after inoculation, T2: A group of cells treated from 12th to 16th day after