There was significant difference between different levels of salinity and genotypes for all of the studied traits, except for RWC (Table 1). The genotype × salinity interaction was significant for all traits, except for RWC and CAT (Table 1).
Effects of salinity stress on different studied traits
Callus growth traits
The salinity stress resulted to production of more brown and necrotic calli in the two studied species of Brassica (Fig. 1). Higher salinity levels resulted to more coloration in the calli colors in all genotypes (Fig. 1). According to Fig. 2A, the RGR mean for genotypes ranged from 6.79 % in br2993 genotype (B.oleracea ) to 16.94 % in cr3356 genotype (B. junceae ) . The RGR ranged from 14.06 % in control to 7.32 % under 200 mM NaCl (Fig. 3A). Addition of NaCl to B. juncea and B. oleracea led to a significant decline in CGR content, under in vitro salt stress. According to the salinity × genotype interaction, the salinity stress resulted in a decrease in RGR in all the studied genotypes, but its effect was not the same in all of them (Table 2). The genotypes of bra 2993 and bra 2828 genotypes showed more reduction in RGR at high concentrations of salinity (Table 2). The cultivar of cr3356 (B. juncea) did not show a significant decrease in RGR up to 200 mM (NaCl) (Table 2), which indicated high tolerance of this genotype to salinity stress at callus level.
This phenomenon could be resulted from water potential gradient between the nutrient medium and cell (Lokhande et al. 2010). This negative potential would result in a decrease of the growth rate of the calli in dehydrated cells under salinity stress (Golkar et al. 2020). The findings demonstrated that the reduction in callus growth was more severe in diploid genotypes rather than that of the amphidiploid ones (Fig. 2A, Table 2). There was no significant difference for the means of genotypes, salinity levels and genotype × salinity interaction for relative water content (Table 1) .
Malondialdehide (MDA) content
The highest (0.0051 MDA μg/g DW) and the least (0.002 MDA μg/g DW) content of MDA were observed at genotypes of br2993 and cr113, respectively, averaged over all salinity levels (Fig. 2B). The mean comparison for MDA changes under different salinity levels, demonstrated an increase in its content parallel with an increase in the salinity levels (Fig. 3B). Its content showed variation from 0.00328 μg/g DW in control to 0.0045 μg/g DW under 200 mM NaCl (Fig. 3B). The significant differences observed among MDA values at different levels of salinity might be attributed to the non-supplementary effects of adequate osmolites accumulation as proline in these species at salinity conditions through detoxification of ROS and the subsequent protection of membrane integrity (Ashraf and Foolad 2007). On the other hand, this type of response might be associated with the increased content in H2O2 which could be drastically enhanced under salinity stress among the plant species that undergo higher lipid peroxidation (Khan and Panda 2008).
The increased MDA content in salinity–stressed calli has been reported in other species as safflower (Golkar and Taghizadeh 2018) Nigella sativa (Golkar et al. 2020) and Acanthophyllum (Niknam et al. 2011). In diploid genotypes, the amounts of MDA showed an increase with an increase in the salinity levels except for bra2828 and bra258 under 75 mM NaCl which had a significant decrease compared to the control (Table 2). Treatments of 150 and 200 mM NaCl had the highest rates in all three genotypes which had significant differences with the control treatment (Table 2). This finding showed an increase in peroxidation and severe degradation of lipids in these genotypes under stress conditions similar to the report of B. olerace (Sahin et al. 2018). The highest content of MDA for cr3356 (0.003 μg/g DW) and cr113 (0.0024 μg/g DW) was observed in the 200 mM treatment (Table 2). The highest content of MAD (0.006 μg/g DW) for diploid genotypes was observed at 200 mM NaCl. This result could point to a selective advantage of species with amphidiploid levels to combat with deleterious effects of the salinity stress in Brassica species. Similar results in the Acanthophyllum calli show that callus of hexaploid species has a lower level of MDA than the callus of tetraploid species under the salinity stress (Niknam et al. 2011), which demonstrated a higher salinity tolerance in higher ploidy levels than that of lower ones.
An increase in the content of hydrogen peroxide leads to lipid peroxidation, which ultimately leads to the destruction of cell membranes (Hossain et al. 2015). The mean comparison between different levels of salinity showed that it was ranged from 0.392 μmol/ g DW under 200 mM NaCl to 0.330 μmol/ g DW under control conditions (Fig. 3C).
The highest (0.45 μmol/ g DW) and the least (0.25 μmol/ g DW) content for H2O2 were observed at genotypes of B. oleracea br2993 and B. juncea cr113, respectively (Fig. 2C). According to the genotype × salinity interaction, H2O2 content was ranged from 0.529 (μmol/ g DW) in B. oleracea br2993 under 200 mM NaCl to 0.211 (μmol/ g DW) in B. juncea cr113 under 150 Mm NaCl condition (Table 2). Under the control condition, there was a significant difference between different genotypes (Table 2). However, this difference was not significant between B. oleracea br2828 and B. oleracea br2993 cultivars. (Table 2).
In three diploid genotypes, the amount of hydrogen peroxide was increased with increasing the salinity levels (Table 2). However, the genotype of B. oleracea br2993 had a significant reduction under 150 mM of NaCl compared to the control treatment. (Table 2). At 200 mM NaCl, three diploid genotypes showed a significant difference with the control treatment, which indicates the high sensitivity of these genotypes to salinity at high concentrations of NaCl (Table 2). Dissimilar to the diploid genotypes, amphiplipid ones (B. juncea) showed a decrease in H2O2 content with an increase in the salinity levels (Table 2). This result demonstrated at higher affinity of CAT and APX antioxidants in the amphidiploid genotypes rather than the diploid ones, in reduction of H2O2content under the salinity stress.
Protein synthesis can be affected by different environmental stresses including salt stress (Muchate et al. 2016). Several salt-induced proteins have been identified, which belong to two distinct groups: salt stress proteins that accumulate only due to salt stress, and stress-associated proteins, which accumulate in response to various environmental stresses including salinity (Ashraf and Harris 2004). Proteins accumulated in plants grown under saline conditions may provide a storage form of nitrogen which could be reutilized when stress is over and they may play a role in osmotic adjustment (Muchate et al. 2016). Averaged over all salinity levels, the highest (1.05 μg/g DW) and the least (0.68 μg/g DW) contents of total protein were observed at B. juncea cr3356 and B. oleracea br258 genotypes, respectively (Fig. 2D)
The mean of the protein content showed a significant increase from the control (0.79 μg/g DW) to 0.90 (μg/g DW) under 200 mM NaCl (Fig. 3D).
The mean comparison for salinity × genotype interaction (Table 2) indicates an increase in the total protein content with an increase in the concentration of sodium chloride in all of the genotypes in the culture medium. According to Table 2, the highest content of protein (1.12 μg/g DW) was observed at 200 mM NaCl in B. juncea cr3356 genotype, which showed a significant difference with other treatments and cultivars (Table 2). The similar results were observed in terms of increase in protein content of B. juncea genotypes, but this increase was more in B. juncea genotypes rather than in B. oleracea ones. Similar to this finding, an increase in protein content was also reported in B. oleracea calli at 100 mM NaCl (Mukhtar and Hasnain 1994) and also other species as Acanthyphyllus (Niknam et al. 2011)) and Broussonetia papyrifera (Zhang et al. 2013).
Antioxidants enzymes activity
To prevent oxidative damage, plants improve their antioxidant enzyme activities such as catalase, gayacol peroxidase, and peroxidases. On the other hand, in order to counteract the toxic effects of increasing the amount of ROS under salinity stress, various defense mechanisms including antioxidant enzyme activity are activated in the plant (Gupta and Huang 2014). Under these conditions, the enzymes inhibiting the production of ROS are increased to reduce the toxic effects of oxidative stress. In this study, a significant variation has been found regarding the anti-oxidant enzyme activities of APX and GPX. Amphidiploid genotypes generally showed higher antioxidant activity (APX and GPX) as compared to diploid ones. This suggests that high antioxidant enzyme activity has a significant role in imparting salt tolerance in these amphidiploid genotypes.
GPX participates in numerous physiological processes and characterized as an electron donor (Ahire, et al., 2013).The content of GPX for genotypes ranged from 0.32 μmol/mg protein in B. juncea (cr3356) to 0.007μmol/mg protein in genotype of B. oleracea br258 genotype (Fig. 4A). The content of GPX showed an increasing trend from control ( 0.084 μmol/mg protein) to 0.12 (μmol/mg protein) under 200 Mm NaCl (Fig. 5A). This increase under in vitro salinity culture was similar to previous reports on Bacopa monnieri (Ahire et al. 2013) and Spinacia oleracea (Muchate et al. 2019). Comparison of salinity × genotype interaction for GPX activity showed that the highest activity (0.39 μmol/mg protein) was observed at 200 mM NaCl for B. juncea cr3356 genotype (Table 2), but the lowest activity (0.002 μmol/mg protein) was related to the genotype of bra2993 at 200 mM NaCl, which was not significantly different from the control treatment in the same genotype. According to Table 2A in diploid cultivars (B. oleracea br2993 and B. oleracea br258) no significant difference was observed for GPX activity between different salinity levels.
The GPX activity in B. oleracea br2993 was increased up to the level of 150 mM NaCl and then decreased sharply at 200 mM NaCl, which was not significantly different from the control treatment (Table 2). However, in the two amphiploid genotypes, GPX activity showed a significant increase with an increase in the concentration of NaCl (Table 2). The results of the previous reports show that in the amphiploid species of Brassica, the activity of GPX was higher than that of the diploid species under non-salinity stress (Menget al., 2011).
Peroxidases are a set of glutathione reductase ascorbate cycle enzymes that can convert oxygenated water to water by removing H2O2 (Das and Roychoudhury 2014). APX is the most important reducing substrate that carries the dismutase of H2O2 to water. The mean comparison of genotypes showed a variation from 0.28 μmol/mg protein in B. juncea cr3356 to 0.07μmol/mg protein in B. oleracea br258 (Fi.g 4B). The APX content varied from 0.11 μmol/mg protein under 75 mM NaCl to 0.17 μmol/mg protein under 200 mM NaCl (Fig. 5B). Comparison of salinity× genotype interaction on the activity of APX showed that the highest activity of APX (0.432 μmol per minute per gram of protein) belonged to the treatment of 150 mM NaCl on B. juncea cr3356, which was not significantly different from 200 mM NaCl treatment (Table 2). Also, the lowest activity of APX was related to 150 and 200 mM NaCl on B. oleracea br258, which did not have significant difference with the control and 75 mM NaCl treatments (Table 2). With an increase in the salinity levels, the activity of APX significantly decreased in B. oleracea br258, but in other genotypes, this trend was not significantly observed (Table 2). As the duration of exposure to salinity stress increases, the activity of APX showed more increase in the amphiplipoid genotypes, rather than the diploid genotypes. This finding was similar to the finding in different ploidy levels of turnip (Meng et al. 2011) and the calli of eggplant (Yasar et al. 2006)and shoot culture in Spinacia oleraces (Muchate et al. 2019) under salinity stress.
Plants are endowed with H2O2-metabolizing enzymes such as CAT and APX (Das and Roychoudhury 2014; Sofo et al. 2015). Averaged over all the salinity levels, the highest CAT activity ( 0.041 μmol/mg protein) was related to B. juncea cr3356 genotype which shows a significant difference with other cultivars (Fig. 4C). No significant difference was observed for catalase activity for diploid genotypes. The CAT activity showed an increase from the control ( 0.02 μmol/mg protein) to 0.031 μmol/mg protein under 150 mM NaCl (Fig. 5C) that was not significanly different with other salinity levels including 75 mM and 200 mM (Fig. 5C). The considerable increase in CAT activity observed in the calli of different Brassica species under salinity stress could sustain electron flows that are the main producers and targets of the ROS action in Brassica species (Sofo et al. 2015). The main sites of presence of catalase are peroxisomes which convert H2O2 to H2O and O2 (Sofo et al. 2015). According to the non-significant effect of salinity × genotype interaction for CAT activity, it can be concluded that trend responses of different genotypes was similar at different salinity levels.
Although catalase activity increased with increasing salinity levels, but in genotypes of cr2993 and br2828, catalase level decreased at 200 mM NaCl (Table 2). This finding was different with other reports in the callus of melon under salinity stress (Kusvuran 2012) and shot cultures of Spinacia oleracea (Muchate et al. 2019). In general, as the chromosome number increased, DNA content per cell and enzyme activity per cell increased either (Yildiz 2013).
Proline, as an important buffer in maintenance of osmotic homeostasis, exhibited significant dose-dependent concentration increases upon salt treatment (Sofo et al. 2015). The mean comparison of genotypes averaged over all salinity levels for proline showed that it varied from 139.8 μg/g DW in B. oleracea br2993 to 503. 4 (μg/g DW) in B. juncea cr3356 (Fig. 4D). In Brassica calli, the proline content averaged over all the genotypes was found to be increased dramatically with increasing the salt from 175.5 μg/g DW in control to 406.7μg/g DW in 200 mM (NaCl) (Fig. 5D). Our findings indicating proline accumulation in response to increasing salt concentration have been confirmed in various in vitro salt stress systems (Lokhande et al. 2010; Niknam et al. 2011; Golkar et al. 2017; Golkar and Taghizadeh 2018; Muchate et al. 2019). The mean interaction for genotypes × salinity showed that the highest amount of proline ) 639.77μg/g DW) was related to B. juncea cr113 genotype under 200 mM NaCl that had no significant difference with B. juncea cr3356 genotype under 200 mM NaCl (Table 2), while the lowest one (47.70 μg/g DW) was observed at B. oleracea br2828 under 150 mM NaCl, (Table 2). No steady trend was observed for proline changes in different genotypes in response to an increase of the salinity levels (Table 2). So it could be compromised that the role of proline accumulation in tolerance to salt stress remains controversial, and implied that the enhanced proline levels are stress-effected rather than being a factor in stress tolerance. According to the findings, the increase of proline was greater in amphiploid genotypes rather than in diploid ones. Similar to this finding, Chandra and Dubey (2010) reported more efficient antioxidant system in hexaploids rather than diploids in Cenchrus. Higher chromosome number and gene expression caused an increase in the concentration of particular secondary metabolites and chemicals that are responsible for better defense mechanism in polyploids than in diploids (Yildiz 2013).
Finally it could be stated that understanding the physiological and biochemical factors that lead to salt tolerance is a very important issue in the selection of stress-tolerant plants (Gupta and Huang 2014). However, callus culture under salinity stress technique offers a means for focusing only on those biochemical and physiological indicators inherent to cells that contribute to adaptation to salt stress (Rai et al. 2011; Golkar and Taghizadeh 2018). It is possible that defense against oxidative stress is organized differently in Brassica species with differing ploidy levels. This study evaluated, for the first time, the effects of salinity stress on biochemichal changes in two different species of Brassica with various ploidy levels.
The findings obtained from this research confirmed that polyploid species in Brassica (Ashraf and McNeilly 2004) and other plant species as sugar cane (Yildiz 2013) could generally withstand salinity stress better than their respective diploid ones. But this research implied that this conclusion at callus level is a novelty finding. Moreover, with regard to the role of polyploidy in the evolution and speciation of the Brassica genus, the higher ability for the salt tolerance observed in B. juncea, might have been due to the greater polyploidy level in this species compared to B. oleracea. It could be suggested that the higher salt tolerance of amphidiploids as (B. juncea) has been compromised from the genomes of A (B. campestris) and C (B.oleracea L.) (Ashraf et al. 2001). In agreement with our results, higher ploidy levels in wheat (Chandra and Dubey 2010) and Achanthyphyllus (Niknam et al. 2011)) were considerably more salt-tolerant than in lower ploidy levels.