3.1. Optimum callus induction medium
Although I. amara is a significant 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 significant impact of plant growth regulators (PGRs), types of explant, photoperiod, and interactions with studied traits (data are not presented). Based on the findings, light significantly 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), photo-regulation modulated by phytochromes (Garcia et al. 2011), and/or the effect of light on the photosynthetic pathway stimulation, chloroplast differentiation, ethylene evolution, and cell division rate (Garcia et al. 2011). These findings suggest that light modulates I. amara traits in vitro by physiological changes. Similar observations with 16-h light/8-h dark photoperiod for improving callus induction and callus growth rate were previously recorded for D. polychaetum and D. kotschyi (Taghizadeh et al. 2020).
The mean interactions in a completely randomized design were compared since the interactions of PGRs and explants were significant. 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 first report regarding the optimized protocol for callus induction in I. amara. Our findings 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 influence 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 al. 2016; Taghizadeh et al. 2020). Based on the findings (Table 1), leaf explants with the enhanced MS medium with 3 mgL− 1 BAP and 1 mgL− 1 NAA demonstrated the largest CGR (0.32 mm day− 1) while leaf and stem explants at the enhanced MS medium with 3 mgL− 1 NAA and 1 mgL− 1 BAP indicated the smallest CGR (0.08 mm day− 1). Therefore, there was a positive interactive impact for NAA and BAP concentrations on the employed explant for the callus growth enhancement. PGR is a crucial factor that is responsible for CI and growth in plant cell cultures. PGR optimal concentration could rely on various factors including genotype of plant, endogenous PGR concentration of explants, and the origin of explants (Mathur and Shekhawat 2013; Taghizadeh et al. 2020). According to previous reports, auxins and cytokinins are commonly employed in the culture of plant tissues for callus induction (Garcia et al. 2011; Taghizadeh et al. 2020).
Table 1
The effects of combination of different plant growth regulators (PGRs) on the callus induction (CI) and callus growth rate (CGR) of I. amara.
Explants
|
PGRs
|
CI (%)
|
CGR (mmday− 1)
|
Leaf
|
MS + NAA (1 mg L-1) + BAP (3 mg L-1)
|
¥85.16a ± 7.89
|
0.32 a ± 0.08
|
Stem
|
MS + NAA (1 mg L-1) + BAP (3 mg L-1)
|
70.13b ± 6.75
|
0.21b ± 0.05
|
Leaf
|
MS + NAA (3 mg L-1) + BAP (1 mg L-1)
|
25.00c ± 14.02
|
0.08c ± 0.06
|
Stem
|
MS + NAA (3 mg L-1) + BAP (1mg L-1)
|
25.17c ± 9.26
|
0.1c ± 0.09
|
Leaf
|
MS + 2, 4 D (2 mg L-1)
|
-
|
-
|
Stem
|
MS + 2, 4 D (2 mg L-1)
|
-
|
-
|
Leaf
|
MS + BAP (3 mg L-1)
|
-
|
0
|
Stem
|
MS + BAP (3 mg L-1)
|
-
|
-
|
¥Data are the mean of four replicates; the values with the same superscript letters are not statistically different at P ≤ 0.05 significance level according to LSD test. |
3.2. Optimum suspension establishment medium
According to our observations, I. amara cells could not grow on the suspension media having 3% (w/v) sucrose after transferring the callus. In similar studies, Khanpour-Ardestani et al., (2015) and Taghizadeh et al., (2020) observed that high sucrose levels in the culture medium generally led to low biomass accumulations. It seems that the cell growth is stopped through high osmotic pressure and/or relatively high initial concentrations of sucrose (Mathur and Shekhawat 2013). Based on this recommendation, the sucrose concentration was reduced to 2% (w/v). The MS medium supplemented with 1 mg L− 1 NAA, 3 mg L− 1 BAP, and 2% (w/v) sucrose at a pH of 6 appeared to be optimum conditions for suspension establishment according to visual symptoms. Regarding diverse responses to hormones, it can be concluded that PGRs are critical factors that are responsible for I. amara suspension establishment in cell culture. From previous reports, the advisable concentration of these hormones is dependent on explant type, genotype, and explant origin (Mathur and Shekhawat 2013; Taghizadeh et al. 2020).
3.3. Cell growth curve
The findings represented that cell growth was extremely low in the first five 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 findings open a time window toward the use of cell suspension culture to produce phenolic compounds, flavonoids, flavonols, and anthocyanins, which acquired a pharmacological value.
3.4. Fresh weight
The results of analysis of variance showed that the chitosan elicitations, period time of elicitation, and different interactions had a significant 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 chitosan-treated 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). The stressor effects of high concentrations of chitosan (100 and 200 ppm) reduced the cell fresh weight, which could be resulted from the inhibitory effects of elicitors on cell growth and the capacity of cell osmotic adjustment, increasing the requirement for maintaining the turgor of the growing cells, consuming energy, and decreasing cell growth. Additionally, other negative effects of chitosan in higher concentrations may be related to damaged cell division and cell membrane (Rikiishi et al. 2008). Likewise, higher concentrations of chitosan in safflower reduced the callus fresh weight (Golkar et al. 2019). Similar observations were reported by Udomsuk et al. (2011) and Talukder et al. (2016) (Talukder et al. 2016) on Pueraria candollei and Plantago ovata, respectively. Thereby, 50 mg L− 1 chitosan was determined as the most favorable concentration for maintaining the cell fresh weight in I. amara.
3.5. Lipid peroxidation
MDA is a product of lipid peroxidation that indicates free-radical accumulations and oxidative stress. Improved membrane permeability and disrupted membrane integrity were possible reasons for the induction of oxidative stress (Taghizadeh et al. 2020). Further, the content of MDA increased remarkably in the cells that were elicited with 100 and 200 ppm chitosan compared with the control (Fig. 3b). The largest increase in the MDA amount to 3.93 and 3.87 µmol. g− 1 FW was observed under 200 ppm chitosan-treated cells on the 12-16th day and 8-12th elicitation, indicating a nearly 2-fold increase in comparison with that of the control cells. In the present research, the increment of MDA content (Fig. 3b) indicated that high chitosan concentrations might have a direct impact on the cell membrane functions and structures. The oxidative stress also induced disruption in the integrity of the membrane, leading to increased permeability of the cell membrane (Taghizadeh et al. 2019). Consequently, the cells could not hinder the peroxidation of membrane lipids. The increment of MDA content is in agreement with the decrease in fresh weight that was observed under 200 ppm chitosan-treated cells. In fact, high concentrations of chitosan caused the development of free oxygen radicals (reactive oxygen species, ROSs) and oxidative stress, and thus could directly disrupt cell membrane structures and eventually, caused cell death and fresh weight losses. The lipid peroxidation levels demonstrated no significant difference in control and cells with 50 ppm chitosan elicitation. The observed differences in the values of MDA at 50 ppm chitosan were not significant probably due to the supplementary impacts of higher accumulations of phenolics at chitosan elicitation via ROS detoxification and membrane integrity protection. The effectiveness of secondary metabolites (e.g., phenolics) in the deceleration of lipid peroxidation is attributed to their free radical-scavenging ability (Taghizadeh et al. 2019). The lipid peroxidation might also mediate signal transduction resulting in the increased generation of secondary metabolites. Other works indicated an increase of lipid peroxidation in Acer pseudoplatanus L. cultured cells under the chitosan elicitation (Malerba and Cerana 2015).
3.6. 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, 50 mg L− 1 chitosan on the 12-16th day after inoculation led to a greater increase in the total phenol accumulation when compared to other treatments and control. The total phenol compounds significantly increased approximately 1.22 fold from the control condition (1.22 mg GA g− 1 DW) up to 34.1 (mg GA g− 1 DW) when calli were treated with 50 mg L− 1 chitosan (Fig. 3c). Many studies demonstrated the strong relationship between plant secondary metabolism and plant defense responses (Taghizadeh et al. 2021a). The effectiveness of secondary metabolites such as phenolic compounds in the deceleration of lipid peroxidation is attributed to their free radical-scavenging ability (Taghizadeh et al. 2019). Phenolic compounds could act as Fenton reaction inhibitors and iron chelators or could directly eliminate free radicals and reduce oxidative damage (Manquián-Cerda et al. 2016; Maqsood et al. 2014). The higher phenolic compounds might protect plant cells under the chitosan elicitation and show interference with the signaling cascade in plant cell responses. Consistent with the findings of the present research, it was reported that the phenol content derived from phenylpropanoid pathways is induced as a result of in vitro chitosan application in Coleus aromaticus (Govindaraju and Arulselvi 2018) and Carthamus tinctorius (Golkar et al. 2019). 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 chitosan-treated I. amara (Khan et al. 2003).
3.7. Flavonoid content
Flavonoid accumulation in plants has a major role in protecting plants encountered with biotic and abiotic damages (Alrawaiq and Abdullah 2014). Flavonoids are the key components of the antioxidant system with subgroups as flavonols and anthocyanins (Falcone Ferreyra et al. 2012). The treatment of I. amara cells on the 12-16th day after inoculation with 50 mg L− 1 chitosan resulted in a significant increase in the flavonoid accumulation (0.94 mg rutin. g− 1 DW) in contrast to other treatments and the control (Fig. 3d). In other words, the 50 mg L− 1 chitosan caused an increase of about 2.19 folds flavonoid content rather than a non-elicited callus. Similarly, the increased content of flavonoid compounds was reported in Plantago ovata (Talukder et al. 2016), Carthamus tinctorius (Golkar et al. 2019), and Pueraria candollei using chitosan (Udomsuk et al. 2011). The increase in flavonoid content derived from the chitosan elicitor suggests a higher rate of flavonoid production owing to the possible positive effect of this elicitor on the expression of the gene coding enzymes engaged in the flavonoid biosynthesis (Talukder et al. 2016). In fact, increased phenolic compounds and flavonoids may protect plant cells against ROS generation in response to biotic and abiotic elicitors, and it will interfere with the signaling cascade involved in plant adaptation to environmental stresses. For instance, an improved flavonoid accumulation was reported due to the increased expression of the gene-coding PAL enzyme in the Coleus aromaticus plant (Govindaraju and Arulselvi 2018). Likewise, Chen et al. (2009) concluded that chitosan increases the expression of genes responsible for flavonoid and phenylpropanoid biosynthesis in soybean sprouts (Chen et al. 2009). Overall, the increased flavonoid content in I. amara may be related to the direct effect of 50 mg L− 1 chitosan on gene expression, transcription factors, and activity of enzymes that are involved in the phenylpropanoid pathway.
3.8. Flavonol content
The highest amount of flavonol (i.e., 0.8 mg rutin per dry weight) was detected in the cell suspension supplemented with 50 mg L− 1 chitosan on the 12-16th day (Fig. 3e), reflecting the favorable effect of low concentrations of the chitosan elicitor on the flavonol content in I. amara cells. Conversely, the lower level of chitosan (50 mg L− 1) was found to further accumulate flavonols compared to its higher levels. These observations are in line with the results of Talukder et al. (2016) and Govindaraju and Arulselvi (2018) on Coleus aromaticus and Coleus aromaticus, respectively. Moreover, flavonol production increased in the cell suspension of the chitosan-treated Cocos nucifera herb (Chakraborty et al., 2009). Despite such reports regarding the beneficial effects of chitosan on flavonol content, the mechanism of action and biosynthetic or signaling pathways affected by this hormone remain unknown.
3.9. Anthocyanin content
Given that anthocyanins have been well-known for anti-inflammatory, anti-cancer, anti-microbial, and antioxidant properties, many studies have focused on these natural products. In the current experiment, the highest amount of anthocyanin (i.e., 12.27 µmol per dry weight), as a subgroup of flavonoids, was detected in the cell suspension treated with 50 mg L− 1 on the 12-16th day (Fig. 3f). Our findings on the chitosan-derived increased content of secondary metabolites corroborate with those of Park et al. (Park et al. 2019) and Govindaraju and Arulselvi (2018) on Fagopyrum esculentum and Coleus aromaticus, respectively. The change in anthocyanin content can be attributed to the modulated activity of transcription factors responsible for the anthocyanin biosynthesis pathway, which is affected by the chitosan elicitor and/or partially by the applied plant growth regulators in the callus induction/suspension establishment media (Kim et al. 2006). Although the exact mode of action of chitosan is complicated, our findings verified the dependence of the elicitation process in vitro on elicitor concentrations. Some other factors including callus age, culture medium, plant species and genotype, and elicitor type and its exposure time can thus have a significant effect on elicitation processes (Philibert et al. 2017).
According to our data, the contents of the total phenolics, flavonoids, flavonol, MDA, and anthocyanins in chitosan-treated cells on the 12-16th day (T2) were significantly higher than those of cells treated with chitosan on 8-12th elicitation (T1). These observations suggest that the antioxidant system is significantly stimulated by increasing the age of cells elicited by chitosan.
3.10. Trait correlations
Table 2 provides the obtained correlations among the evaluated traits. The total phenolic compounds indicated significant positive correlations with flavonoid 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, flavonoid and anthocyanin might provide protection for plant cells against stresses and show interference with the signaling cascades in plant responses. Furthermore, such a rise could be due to the elicitation of their biosynthetic pathways, the improved enzymatic activity, and the expression of pertinent genes. There was a significant negative correlation between MDA and the total phenolic compounds (-0.784**), flavonoid (-0.456*), anthocyanin (-0.722**), and fresh weight (-0.619**), implying that increases in all these metabolites caused a decrease in the MDA level. As mentioned earlier, MDA is a cytotoxic product of lipid peroxidation that indicates free-radical accumulations. Chitosan affects plant cells via inducing oxidative stress and increasing activity, lifetime, and concentrations of free radicals. The possible causing factor for the induction of oxidative stress disrupted membrane integrity, improved lipid peroxidation and cell toxicity, and then caused cell death (Taghizadeh et al. 2020). In other words, these compounds have possibly a vital role as free radical scavenging in I. amara cells subjected to chitosan elicitation. In this condition, cell fresh weight decreased by an increase in MDA. Therefore, this correlation represented the strong antioxidant property of total phenolic, flavonoid, and anthocyanin in ROS scavenging or detoxifying the harmful effects of elicitation.
Table 2
Correlation coefficients among bioactive components in the I. amara studied under different concentration of chitosan.
|
Total Phenolic
|
Flavonoid
|
Flavonol
|
Antocyanin
|
Fresh weight
|
MDA
|
Total Phenolic
|
|
|
|
|
|
|
Flavonoid
|
|
|
|
|
|
|
Flavonol
|
|
|
|
|
|
|
Antocyanin
|
|
|
|
|
|
|
Fresh weight
|
|
|
|
|
|
|
MDA
|
|
|
|
|
|
|
** and * Significant at 1% and 5% levels of probability, respectively. |