Under experimental analysis in our study, assessment of stress tolerance in the presence of NaCl was based on the determination of soluble carbohydrates, free proline and Na+/K+ as well as SOS1, SERK1 and WEE1 expression. The results indicated that the soluble sugar accumulation rate of alfalfa callus was highly increased by 80–110 mM NaCl with the treatment of long term period. Soluble sugar accumulation also slightly increased with the 50 mM NaCl with prolonged exposure time, which was in agreement with the change in the cell growth rate when compare to control callus (Fig. 4). In our cases, the results verified that the accumulation of soluble sugars is dependent on increasing salt levels and this was confirmed by the statistical analysis. Moreover, the period of treatment had great influence on callus stressed at 80 mM, which accumulated more sugars than all other treatments. The function of soluble sugars in in vitro acclimization is explosive, and even their accumulation can be harmful in the lower salt levels such as 50 mM (Fig. 4). The results of Arefian et al. (2014) are evident that a considerable collection of sugar weakens the osmotic potential of cells and decreases injury of turgidity in resistant genotypes of chickpea. Tested genotypes higher concentrations NaCl treated alfalfa callus indicated a remarkably increased of sugar contents. It is proved that higher concentrations NaCl induced the sugar metabolism; thus eleminating NaCl severity.
In alfalfa callus, proline content was irregularly detected under salt stress acclimization and the effects of acclimization on proline accumulation are negatively correlated to the salt-tolerance ability. Although the proline content of tested callus subjected to 50 mM showed statistically significant increase in the short term (50 mM) salt treated callus compared with controls, there was a declining trend between the groups in all treatments (Fig. 3). These results are in agreement with the result obtained in two tobacco cultivars under salt stress (Wang et al. 2013). Previous studies indicated that a positive relationship between proline rates and callus cultures by salt stress is present in many plant species. Patnaik et al. (1997) used palmarosa callus cells produced from cultured nodal explants as their experimental resources and different dosage of NaCl to select quickly growing callus; NaCl-resistant callus cells indicated adaptive mechanisms including increased proline content. The results are in disagreement with other reports that suggested the increase in proline content under increasing salinity in callus cultures. However, our results are very far from expected situation due to the degrees of proline decreased with increasing NaCl concentration and exposure time. It is possible that 50 mM NaCl induced the proline activity, thereby affecting acclimization capacity, callus are properly growing in lower NaCl concentration in vitro conditions.
The degree of salt resistance appeared to be directly correlated to the SOS1 expression levels of the genotypes, as determined by RT-PCR analysis, suggesting that the resistance is manifested by a Na+/K + content resistance mechanism (Fig. 6). Expression of SOS1 was induced by NaCl in callus, where high levels of SOS1 expression were detected in callus cells of 50 and 110 mM treatments (Fig. 5). This explains that increased SOS1 expression supported a mechanism that led to withstand stress severity in the presence of NaCl. In terms of the result of the SOS1 gene expression, there was considerable variation between the three genotypes because of the effects of the physiological alterations in conditions of exogenous NaCl. For example, Elçi and Ömer Bey genotypes exhibited similar values of SOS1 gene expression, but Na+/K+ content did not show similar values in the lower NaCl. In contrast, the Muş ecotype did not show the expected expression rates in the callus after treatment with salt stress. Interestingly, Muş genotype exhibited the highest expression level of SOS1 in 0 mM NaCl and a lowest expression level at 80 mM NaCl. The reduction in SOS1 gene expression level in the Muş genotypes ranged from 10,71 to 6,34 in the 50 mM NaCl treatment (Fig. 6). Similarly, the proline level detected in the same genotype showed significantly reduced from that detected in control callus. This suggests that the significant reduction in callus of alfalfa genotype is not solely a Na+/K+ effect of exogenous NaCl, but that several regulation factors exist in the callus cells (Luo et al 2020). It is conceivable that 50 mM NaCl promoted physiological activity; thereby adjusting Na+/K+ exclusion capacity, SOS-mediated pathway restricts Na+ amount by epidermal cells in the cytosol. This result was also detected and resolved to maintain optimal cytosolic Na+/K+ homeostasis in barley (Shi et al. 2002) and arabidopsis (Chen et al.2008) subject to salt stress. Darko et al. (2015) showed that in wheat/barley addition lines, the treatments of salt could induce various genes related to Na+ uptake and transport was not linked with the salt resistance of the genotypes.
In Arabidopsis, SERK1, SERK3 and SERK4 participated in stress resistance and are activated by brassinosteroid (Albrecht et al. 2008). The rice SERK1 have been reported to be stimulated in ABA signaling which promotes oxidative stress (Hu et al. 2005). Li et al. (2017) reported that response to the salt stress stimulates the expressions of SERK1 and SERK3 in barley. Our results displayed that SERK1 exhibits high levels of expression at 50 mM of the salt stress, similarly to what had already been reported by Pérez-Núñez et al. (2009). SERK1 is a gene that has been well-known and used as a marker gene for somatic embryogenesis formation (Mahdavi-Darvari et al. 2015; Montalvo et al. 2020). Moreover, the observations obtained at 50 mM of the salt stress are in agreement with the induction of embryogenic calluses (Fig. 1). The same basal response content was detected for SERK1 in Elçi and Ömer Bey genotypes under normal and NaCl conditions. Muş displayed a strong expression of SERK1 in the acclimated state with respect to non-acclimated state. It is likely that the degree of SERK1 expression is associated with the degree of embryogenic callus activation which is linked to the optimizations by 2,4-D, a known somatic embryogenesis inducer. It is clearly detected from Elçi and Ömer Bey genotypes that SERK1 induced undifferentiated cells and strong stability. This results displayed that embryogenic induction capacity is a genotypic trait and suggested the presence of gene or supression of genes participated in embryogenic callus initiation (Gandonou et al. 2005). Our results in alfalfa propose that the somatic cell induction of callus is accompanied by 50 mM NaCl, which could be caused by the synergistic functions of the endogenous auxins and of the 2,4-D present in the medium and could support the embryogenic callus initiation. The remarkable rise in the expression of SERK1 at 50 mM and the decrease in its expression under 80 and 110 mM conditions seem to confirm this hypothesis (Fig. 7). This could be attributed to fact that NaCl at lower dosage acted like an auxin and promoted the regular growth of callus on media, which in turn improved the overall embryogenic capacity as detected in rice SERK1 (Hu et al. 2005). This gene is inducible by exogenous treatment including abscisic acid, salicylic acid and jasmonic acid which is SERK1 in two rice cultivars to lead an enhancement in host resistance against blast fungus (Hu et al. 2005). Somatic cells induce callus differentiation rapidly and then lose mitotic and morphological changes in alfalfa callus (Fig. 1). The adverse impact of long-term treatments with a high dosage of NaCl has been reported and observations are also consistent with the results obtained in callus of triticale by Yazıcılar et al. (2021). The increase in the dosage of NaCl in the present study is an indicative of a decreased SERK1 gene expression response stress severity that resulted in reduction cell viability. These results indicate that the NaCl on controlling cell differentiation could be a necessary step to precisely modulate the activity of somatic embryogenesis-related genes in alfalfa, which is in agreement with the earlier findings required for somatic embryogenesis (Nolan et al. 2003; Elmaghrabi et al. 2013; Luo et al 2020).
WEE1 was expressed in the alfalfa callus tissue, suggesting that it plays a role in cell cycle besides its involvement in DNA replication. Similarly, After applying the salt stress, 50 mM NaCl of long-term salt stress displayed a clear increase of WEE1 expression. It’s expression in the presence of NaCl was remarkable at 50 mM and 110 mM rather than 80 mM concentration (Fig. 8). Elmaghrabi et al. (2017) detected high expression of WEE1 (Medicago truncatula L.) in the PEG treatments, thus verifying the regulation of the cell cycle, which is also confirmed as necessary to defend the cells from DNA damage induced by the PEG-related osmotic stress treatment. Our results do not support the claim that there has been reversible DNA injury due to the higher dosage NaCl exposed on callus. It is likely that the level of salt stress tolerance is independent with the level of WEE1 expression, which is correlated to the regulation in plant cell cycle progression. Previous researchers showed that WEE1 had been characterized as a role in plant genome endoreduplication, considering its expression in endoreduplication tomato and maize cells (Salomonsson et al. 1993; Sunarpi et al. 2005). These results are in agreement with our findings, which demonstrated that there are some specific correlations between WEE1 and on ploidy identification of alfalfa. The amount of nuclear DNA was the highest in Muş (6.50 pg-1 C) and had almost similar values with Ömer bey (6.02 pg-1 C), whereas Elçi (5.08 pg-1 C) genotype had low nuclear DNA amounts (Fig. 2). Barow and Meister (2003) revealed that an inversely correlation exists between the endoreduplication and genome size and, in most conditions, the species with a small genome display higher levels of endoreduplication. Our findings partially confirm this phenomenon; Muş ecotype displayed the highest peak value in endoreduplication followed by Elçi and Ömer Bey which is also confirmed by the presence of diploid genome with basic chromosome number 2n = 2x = 16. Muş (6.50 pg-1 C) callus somaclones indicated higher genome sizes Muş (6.50 pg-1 C) than that of the leaf (3.80 pg-1 C) somaclones. This can be explained that a longer period of in vitro cultivation seemed to increase somaclonal variations, which involves the interaction of auxin, can act directly to induce polyploidy events. These findings are consistent with those published by De Schutter et al. (2007) in the study on ploidy distribution profile of Arabidopsis thaliana in various tissues on cell cycle regulation of WEE1. Their results demonstrated that WEE1 expression levels could induce responses to types of stress stimuli and inhibit plant growth by arresting dividing cells in the G2 phase of the cell cycle after DNA stress. This can be explained as the WEE1, which involves in the regulation of programme of endoreduplication, can act as a main regulator of the mitosis to endocycle transition.