Effects of Sodium Chloride and Sodium Sulfate on Haloxylon ammodendron Seed Germination

Haloxylon ammodendron is a perennial xerophyte that can survive in extremely harsh desert conditions of Central Asia. This study evaluated the effect of salinity, and their ability to recover on seed germination of H. ammodendron, which were collected at three different desert areas, Bakanas takyr plain (H1), Gurbantüngüt Desert (H2), and Gobi Desert (H3), respectively. Seeds were treated with different concentrations of NaCl and Na2SO4 (0.00 (control), 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, and 1.40 mol/L) to detemine the germination and recover ability to salt stress. The results of the study were that H. ammodendron were more resistant to Na2SO4 than to NaCl. Regarding inhibition of seed germination H. ammodendron was in the following order: H3 > H2 > H1. Based on the tolerance and recovery, seeds can be demonstrated as follows: NaCl: H3 > H1 > H2; Na2SO4: H1 = H2 = H3. Non-germinated seeds in all salt treatments with low osmotic potential showed high recovery when transferred to distilled water, indicating that these treatments were not too toxic to affect seed viability. These results suggest that H. ammodendron can withstand high levels of salinity at three desert environments. Thus, H. ammodendron could be used to reconstruct vegetation and sustanbility development in the desert areas with high salinity.


Introduction
Soil salinization has become a worldwide resource and ecological problem, which seriously affects agricultural and forestry production and ecological reconstruction and restoration in desert areas [1]. Soil saline-alkali is a kind of environmental stress prevailing in desert areas, which seriously affects various physiological processes of plant growth and germination. Therefore, it is of great importance to research the influence of salt stress on the germination of desert plants, which will exert important theoretical and practical significance for understanding the adaptability of plants to salinity [2,3]. Most importantly, it is of great significance for the desertification control and the sustainable development of desert ecosystems.
Perennial low-level xerophytic shrubs form the bulk of the vegetation in desert areas. Haloxylon 0 ammodendron (C. A. MEY) is the main constructive species of desert vegetation and has ecological and economic meaning. Desert flora, consisting of H. ammodendron, is the most widespread type of desert vegetation in the Central Asia desert regions. These areas are, however, due to the arid climate, frequent sandstorms and aggravated salinization, whereby the biodiversity is seriously degraded, the vegetation coverage area has been seriously reduced, and the desertification area has increased significantly [4]. Over the years, due to overuse of resources (e.g., over-cutting, land reclamation, and mining), environmental degradation (reduced water resources due to development), and soil salinization, the distribution area of H. ammodendron has decreased by more than 30% and it has been classified as a threatened protected plant (National Environmental Agency, 1987) [5,6]. Consequently, H. ammodendron ecosystems warrant special attention. In this case, investigation of H. ammodendron seed germination is of great importance for the restoration and reconstruction of this species in desert areas. Given their morphological and physiological characteristics, xerophytes can tolerate long-term and severe water shortages [7]. H. ammodendron, as a Xerophyte, employs a special adaptation mechanism to survive under harsh desert conditions [7,8]. It can promote water conservation, prevent soil erosion, and has a high economic value [9]. Seeds of desert plants use a special strategy and germinate at the right time and place to maximize their chance of survival. Seed structure and environmental factors play an important role in seed germination and therefore determine the composition of desert vegetation [10]. Most of the arid regions in China experience strong environmental fluctuations and possess saline-alkaline zones [11]. Salt affects seed germination by altering the osmotic potential of cells [12]. Salinity is a major limiting portion of product productivity all over the world. Salinity reduces plant growth at all developmental degrees; however, sensitivity varies from different salt compositions. Salt tolerance is the result of a complex adaptation of salt infiltration and ionic effects during seed germination [13]. Germination is a critical stage of the plant cycle and improved tolerance of high salinity could improve the stability of plant production [14]. Water is osmotically held in salt solutions. Therefore, the salt concentration completely inhibits germination at higher levels or induces a state of dormancy at low levels. It also reduces imbibition of water because of lowered osmotic potentials of the medium and causes changes in metabolic activity [15]. Seed germination is the product of the interaction of various environmental factors, but the extent to which environmental factors affect seed germination varies with the species and ecotype. However, very few comprehensive comparative analyses have been conducted to study seed germination in H. ammodendron from different desert areas.
In this study, we aimed to investigate the effects of salt stress on the germination of H. ammodendron seeds. Seeds were collected from the Bakanas takyr plain, Gurbantüngüt Desert, and Gobi Desert, and were exposed to different concentrations of NaCl or Na 2 SO 4 . The study from the Bakanas takyr plains (Kazakhstan) of the influence of salinity on seed germination of H. ammodendron has not reported. The ability of these seeds to recover from salt stress treatments was then compared. The results of this study provide useful information indicating that the seeds of desert plants could be used to restore desertified areas and reconstruct vegetation in Central Asia.

Study Site and Seed Collection
In this article, the seeds of H. ammodendron were collected from three locations: Bakanas takyr plain (Kazakhstan), Gurbantünggüt Desert (north-west China), and Gobi Desert (Inner Mongolia/northern China), respectively are hereafter referred to as H1, H2, and H3, to explore the operating of salt stress on seed germination (Table 1).

Treatments and Experimental Conditions
The experiment was carried out with four replicates of 25 seeds each on the three filter paper (Whatman N • 1) put in the Petri dishes (90 mm) diameter. Then 5 mL of various concentrations of saline to each treatment and 5 mL of distilled water were added to the control put in the LRH-250 Smart Incubator (Shanghai, China, 2014). It is marked by the appearance of roots. The germination process was carried out twice every 24 h respectively (light/darkness = 12 h/12 h) at intervals from 8 h to 16 h photoperiod. Seed germination was tested at a temperature of 5 • C/25 • C and with a concentration of 0.00 (control), 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20, and 1.40 (mol/L) NaCl or Na 2 SO 4 solutions. To compensate for the loss of moisture due to evaporation in NaCl and Na 2 SO 4 solutions, the experimental sample was weighed daily per 1000 pounds of electronic weight. After 10 days un-germinated seeds were transferred to distilled water, and the cultivation was continued for 10 days under the same conditions.

Seed Germination Index
To investigate the resistance of seeds to salt stress, various parameters were calculated including germination rate (GR), germination index (Gi), germination potential (GP), mean germination time (MGT), plant length, viability index (Vi), and germination relativity magnitude, which was assessed as peak value (PV), mean day germination (MDG), and germination value (GV). Values of Gi, Vi, MGT, PV, and GV were calculated with the following equations: where, Gt is the number of germinating seeds at time t, Dt is the germination days, and S represents the length of seedlings (cm) [19].

Recovery Germination
Final germination = (a/c) × 100 (7) where, a is the number of seeds germinated over the entire experiment; b is the number of seeds germinated in saline solution; and c is the total number of seeds used for processing. The initial germination time was recorded from the first day of seed germination. TG 50 , which indicates the time taken to reach 50% germination, was also recorded for each treatment [20].

Statistical Analysis
Before statistical analysis, Shapiro-Wilk and Kolmogorov-Smirnov tests were used to compares the observed distribution with the normal one. Also, to determine the homogeneity of variance of the data, Levene's test was performed (p > 0.05) to identify differences that occurred between individual treatments. A parried-sample t-test was carried out to determine differences among the same concentration treatment of NaCl and Na 2 SO 4 . For comparisons, a one-way ANOVA and Tukey's test was used to determine differences among treatments and then statistical tests were conducted at p < 0.05. Simple correlation with the index (Pearson correlation) was used to study the coefficients between the attributes. All statistical analyses were performed using SPSS 24 (SPSS, Los Angeles, CA, USA, 2016).

Results
Kolmogorov-Smirnov and Shapiro-Wilk tests showed that all groups were compared to the observed distribution with the normal (Appendices A and B). Results of test of homogeneity of variances among the treatments was performed using a Levene's test (Appendix C). Additionally, seed germination was compared between NaCl and Na 2 SO 4 treatments at the same concentration using a paired t-test (Appendix D). Results of the one-way ANOVA and Tukey's test of NaCl salt are shown in Tables 2  and 3 and Na 2 SO 4 salt in Tables 4 and 5 respectively. Correlation coefficients between attributes on germination in the concentration NaCl and Na 2 SO 4 are presented in Tables 6 and 7.

Effects of NaCl and Na 2 SO 4 Treatments on Seed Germination and Their Recovery after Soaking
High germination rates were observed in the control (distilled water) treatment, and the germination percentage decreased with increasing salinity. H1, H2, and H3 seeds, collected from Bakanas takyr plain, Gurbantüngüt Desert, and Gobi Desert, respectively, were more resistant to Na 2 SO 4 than to NaCl (Tables 2 and 3). The inhibition of seed germination by NaCl solutions was in the following order: H3 > H2 > H1 (Table 2). NaCl strongly inhibited H1 seed germination at high concentrations (1.00-1.40 mol/L). In comparison, at the highest concetration of Na 2 SO 4 (1.40 mol/L), the germination percentage of H3 seeds (74%) was higher than that of H1 and H2 seeds (Table 3). After 10 days of salt treatment, we transferred the un-germinated H1, H2, and H3 seeds to distilled water and continued to monitor their germination. After the salt stress was removed, some seeds quickly resumed germination. H1 seeds showed the highest germination upon recovery, followed by H2 seeds, and lastly H3 seeds. The germination rate of H2 and H3 seeds declined significantly when the NaCl concentration was greater than 0.60 and 0.80 mol/L, respectively. Recovery percentages were significantly higher in Na 2 SO 4 and NaCl treatmetns at all concentrations. No significant difference was detected between the final germination percentages of H1 and H3 seeds (approximately 100% in both seed types; Table 2). Germination rates of H2 and H3 seeds were the same (100%) in the presence of 1.00 mol/L Na 2 SO 4 . Final germination percentages of H1, H2, and H3 seeds treated with different concentrations of Na 2 SO 4 were similar to the control treatment (100%) ( Table 3). The TG 50 value increased significantly with an increase in NaCl and Na 2 SO 4 concentrations (Tables 2 and 3).

Germination
Attributes of H1, H2, and H3 Seeds Treated with NaCl and Na 2 SO 4 The effects of different concentrations of NaCl and Na 2 SO 4 on germination attributes of H1, H2, and H3 seeds were analyzed using a one-way ANOVA and Tukey's test. The germination rate of H1 and H3 seeds was high (93% and 100%, respectively) at NaCl concentrations ranging from 0.00 (control) to 0.60 mol/L. However, when NaCl concentration increased to 0.80 mol/L, the germination rate of H1 and H2 seeds dropped dramatically to 30% and 58%, respectively. H3 seeds were more insensitive to higher salt concentrations than H1 and H2 seeds, based on germination rates (Table 4). At low NaCl concentrations (0.00-0.10 mol/L), no significant differences were observed in Gi among the three seed types. However, the Gi of H1, H2, and H3 seeds declined with an increase in salt concentrations (0.20-0.60 mol/L). Nonetheless, H3 seeds showed high Gi values at all salt concentrations, followed by H2 and H1 seeds. In the control treatment, the GP of H1, H2, and H3 seeds was 86, 61, and 90, respectively; however, when salt concentration was increased to 0.05 mol/L, the GP of H1 and H3 seeds decreased significantly, whereas that of H2 seeds showed only a slight change. At higher NaCl concentrations (0.10-0.80 mol/L), the GP of H2 and H3 seeds dropped gradually, whereas that of H1 fluctuated between 38 and 61. When the salt concentration increased from 0.00-0.80 mol/L, the MGT of H1 and H2 seeds varied from 5.60-14.73 and 5.50-8.95 days, respectively, and that of H3 seeds varied from 2.75-10.75. At high NaCl concentrations (1.2 and 1.4 mol/L), H2 and H3 seeds took less time to germinate (1.25 and 0.5 days, respectively), whereas H1 seeds completely lost their germination. Additionally, increasing concentrations of NaCl negatively affected seed healthy index (SHI), PV, GV, and Vi. H1 seeds were more sensitive to increasing salt concentration; at a salt concentration higher than 1.00 mol/L, all germination parameters of H1 seeds were equal to zero (Table 4). In contrast, H3 seeds were the most tolerant to salt stress; all parameters (except MGT and GP) gradually decreased with the increase in Na 2 SO 4 salt concentration. Among the three seed types, H3 seeds showed the highest values of all attributes ( Table 5). The SHI of H1, H2, and H3 seeds showed no significant changed in the Na 2 SO 4 treatment when its concentration changed from 0.00 mol/L to 0.20 mol/L; however, upon further increase in concentration, the SHI of H1, H2, and H3 seeds varied from (highest-lowest) 3.32-2.46, 3.74-2.48, and 4.30-2.65, respectively. The mean germination time (MGT) increased with the increase in salt concentration until 1.00 mol/L, and then declined at 1.40 mol/L concentration. These results showed that all seeds were highly resistant to Na 2 SO 4 ; their germination rate and GP were not inhibited even at the highest concentration of Na 2 SO 4 ( Table 5).

Correlation of Germination Attributes between H1, H2, and H3 Seeds Treated with NaCl and Na 2 SO 4
The correlation between attributes of germination seeds in the concentration NaCl is presented in Table 6 and in Table 7 for Na 2 SO 4 in. There was a strong positive correlation (p < 0.001) for all seed types of H1, H2, and H3 (except type of H3 for MGT), between attributes such as GR, PV, MDG, GV, Gi, GP, SHI, and Vi. However, it should be noted type of seeds H3 (except GR) in GMT correlates negative with a negative attitude to other attributes PV, MDG, GV, Gi, GP, SHI, and Vi (Table 6). Positive, very significant correlations (p < 0.001) were found between type of seeds H1, H2, and H3 (except H1, H2 for MGT), between attributes (Table 7) such as germination rate, PV, MDG, GV, Gi, GP, SHI, and Vi. As well as all seeds type H1, H2 and H3 MGT correlated with a negative attitude to all attributes (except type of H3 for germination rate) PV, MDG, GV, Gi, GP, SHI and Vi. However, type of seeds H1, H2 correlate with a negative attitude to SHI and Vi. Thus, MGT was a distorting sign and was guilty of a false correlation between attributes except (except GR for types H1 and H2). Based on the results obtained, we can conclude that, with the exception of the distorting variable MGT, there was no significant correlation between the attributes (Table 7).

Discussion
H. ammodendron is a widely distributed salt-tolerant and perennial plant species found in the deserts of north-west China and belonging to the desert component of Central Asia [21]. Salinity is one of the important factors affecting seed germination in a saline-alkaline desert environment [22]. Saline soil is produced of the growth of multiple chloride and sulfate salt subjected by NaCl and Na 2 SO 4 salts [23], high concentrations of NaCl [24] and Na 2 SO 4 [25] in the soil leads to the swelling of plants. In a previous study, TG 50 and initial seed germination time increased significantly with increasing salt concentration [26]. Strogonov and Mayer [27] separated halophytic shrubs into two groups: as chlorophyte and sulfate; Atriplex verruciferum and Ardices canescens were categorized in the chlorophyte group, and Suaeda galea and Haloxylon spp. were assigned to the sulfate group. In the current study, we examined the salt tolerance of H. ammodendron seeds by measuring their germination ability during salt stress and during the recovery period. Seeds were collected from three desert habitats, Balkash takyr plain (H1), Gurbantünggüt desert (H2), and Gobi desert (H3), and treated with different concentrations of NaCl and Na 2 SO 4 . The experiment revealed that seed germination was the highest in distilled water (control), and germination percentages decreased with the increase in salinity. Similar results have been reported for H. ammodendron [28,29], Haloxylon stocksii [30], Atriplex griffithii [31], Urochondra setulosa [32], and Suaeda salsa [33].
Germination of H1 and H2 seeds was 100% at 0.05-0.40 mol/L NaCl, and that of H3 seeds was 93% at 0.05-0.60 mol/L NaCl. In Na 2 SO 4 treatments, the GR of H1 seeds was 100% between 0.05 and 0.8 mol/L concentrations, while that of H2 and H3 seeds was 100% between 0.05 and 1.00 mol/L concentrations. When the concentration of NaCl surpassed 0.06 mol/L, the GR of H1 seeds gradually decreased and at 1.00 mol/L was completely inhibited. Cai et al. [34] showed 0.1 mol/L NaCl facilitated the germination of Ceratoides velutina seeds, and 0-0.6 mol/L NaCl showed no significant effect on the total seed germination. The results of the current study showed that the GR of H2 seeds decreased to 76% at 0.60 mol/L NaCl concentration and continued to decline gradually with increasing salt concentration, reaching 7% at 1.40 mol/L concentration. A previous study on white clover showed that the GR, GP, Gi, Vi, root length, and plant height decreased with increasing concentrations of NaCl, Na 2 SO 4 , and Na 2 CO 3 compared with the control [22]. We also showed that the GR of H3 seeds decreased to 87% at 0.80 mol/L salt concentration and continued to decrease with further increases in salt concentration, reaching 4% at 1.40 mol/L. The salt tolerance limit of Camelia sibiricum in the Alashanzuo desert, Inner Mongolia, was estimated at 0.9 mol/L for seed germination [35]. In our study, H1, H2, and H3 seeds of H. ammodendron showed stronger endurance to Na 2 SO 4 than to NaCl. The results on the inhibition of germination by saline solutions were in the following order: H1 Na 2 SO 4 > NaCl; H2 Na 2 SO 4 > NaCl; H3 Na 2 SO 4 > NaCl. Suaeda salsa seeds showed different degrees of tolerance to different salts: MgCl 2 > Na 2 SO 4 > Na 2 CO 3 > NaCl > SES (soil extract solutions) > MgSO 4 [33]. Similarly, Zehra and Saeed showed the order of salt tolerance of Haloxylon stocksii seeds, based on the percent germination: CaCl 2 > Na 2 SO 4 > NaCl > MgCl 2 > KCl [30]. Assareh at al. showed that Halostachys caspica seeds exhibit greater tolerance to Na 2 SO 4 than to NaCl [36]. However, another study showed another halophyte Salvia rosmarinus was more sensitive to NaCl than to Na 2 SO 4 [37]. In previous studies, H. ammodendron showed a lower germination rate in NaCl solution than in an isotonic Na 2 SO 4 solution [38], and Kalidium capsicum was similarly more sensitive to NaCl than to Na 2 SO 4 [39]. However, other plant species showed opposite results; for example, the germination of Prosopis strombulifera seeds was more strongly inhibited by Na 2 SO 4 than by isotonic NaCl solution [40], and Shaikh et al., [32] showed on genus of species Urochondra setulosa and Sorghum bicolor that germination was in the following order NaCl > Na 2 SO 4 . Similar results were obtained by Kaymakanova on Phaseolus vulgaris L.; in this species, Na 2 SO 4 inhibited seed germination to a greater extent than NaCl [41]. For seed germination characteristics of Halostachys capsica, as the concentration of NaCl and Na 2 SO 4 increases, the effect on many variables is in contrast [36]. In the current study, parameters such as PV, MDG, GV, Gi, SHI, and Vi showed the same values between NaCl and Na 2 SO 4 treatments at 0.20 mol/L concentration. The values of various germination attributes suggested that H3 seeds exhibit a unique salt stress response mechanism and therefore are more resistant to NaCl and Na 2 SO 4 concentrations than H1 and H2 seeds. However, when the concentrations of NaCl and Na 2 SO 4 increased, certain attributes of H3 seeds such as MGT (p > 0.05) were significantly lower than those of H1 and H2. In addition, with an increase in the concentration of NaCl and Na 2 SO 4 salts, the GP of H2 seeds decreased, although it was similar to that of H1 seeds in most treatments. In conclusion, because of the influence of NaCl and Na 2 SO 4 , H. ammodendron H3 seeds showed higher values of all attributes (except MGT) than H1 and H2 seeds. This may be because of a higher seed weight in H3 (Table 1). A previous study on seed germination in wheat (Triticum aestivum L.) that priming seed significantly (p < 0.05) the advantages of first count (FG), final count (LG), coefficient of the velocity of germination (CVG), and germination rate index (GRI) were limited, while the time expected to recover faster germination (MGT) was increased, and significant negative correlations between NaCl concentration and a positive relationship with MGT [42]. Taking into account the results discussed above, the seeds of H. ammodendron seem very resistant to salinity, as some seeds could germinate at 1.40 mol/L NaCl and Na 2 SO 4 concentrations. However, 1.00 mol/L NaCl treatment inhibited the germination of H1 seeds to a greater extent than 1.00 mol/L Na 2 SO 4 treatment. This is consistent with previous reports [33,[41][42][43], which showed that different concentrations of NaCl exert a certain inhibitory effect on seed germination.
Un-germinated seeds in salt treatments with low osmotic potential established high recovery of germination when transferred to distilled water, which implies that these treatments were not too toxic to affect seed viability. Data on salt tolerance and recovery of various types of seeds (Tables 2 and 3) suggest that H3 seeds are more resistant to NaCl than H1 and H2 seeds. In Na 2 SO 4 treatments, H1, H2, and H3 showed 100% germination recovery. As a result of the recovery, the H1 seeds compared well with H2 and H3 seeds; the GR of H1 seeds at 1.00 mol/L NaCl was higher than at concentrations 1.20 and 1.40 mol/L (97%). This is a very strong indicator of why the recovery rate of H. ammodendron seed germination at a low salt concentration is higher than that at a high concentration, although further studies are needed to verify these data. Based on the tolerance and recovery to both salts, H. ammodendron seeds can be arranged as follows: NaCl: H3 > H1 > H2; Na 2 SO 4 : H1 = H2 = H3. Seeds from all three regions showed a high GP. H. ammodendron seeds showed various interactions with NaCl and Na 2 SO 4 . Thus, H. ammodendron can be classified both as a chlorophyte and a sulfatephyte. Additionally, the different concentrations of salts tested did not result in a loss of seed viability. In fact, the seeds recovered when they were transferred to distilled water. This is because in nature, the restoration of seeds after dormancy, which was not exposed to high salinity and waited for favorable conditions. When the temperature quickly increases in spring, the snow melts to a certain extent, the water content on the soil surface increases, and salinity decreases [44]. In summer, salts leach out of the soil by flooding, and salt concentrations in the soil are maintained at a low level for a relatively long period [45]. The desert region is characterized by very little uneven rainfall and a lot of evaporation. After short periods of rain, soil moisture evaporates quickly because of high temperatures. Salt in deep soil layers is quickly carried to the surface soil by capillary action. The accumulation of salt in the surface soil creates unsuitable conditions, forcing desert plants to use different life cycle strategies to improve their chance of survival [46]. After the salt is absorbed into the seed, the seed can resume germination, but it is also important that the primary roots and buds have strong viability. High salinity can inhibit or delay the germination of most halophytes until fresh water is added to reduce stress [47].

Conclusions
In this study, we examined the germination of H. ammodendron seeds collected from the Bakanas takyr plain, Gurbantünggüt Desert, and Gobi Desert. The results obtained indicate that the germinability of the seed is progressively reduced with the increase in the saline concentration, and H3 seeds exhibit a unique salt stress response mechanism and are more resistant to NaCl and Na 2 SO 4 concentrations than H1 and H2 seeds due to their higher seed weight. Based on the tolerance and recovery, seeds can be ordered as follows: NaCl: H3 > H1 > H2; Na 2 SO 4 : H1 = H2 = H3. However, the different concentrations of salts tested did not result in a loss of seed viability, in fact, seeds restored when transferred in distilled water. This indicates that seeds from three desert areas all hold a high recovery ability and these treatments were not too toxic to affect seed viability. In conclusion, the study on germination seeds of great importance to providing a scientific and theoretical basis for large-scale restoration, improvement, and protection of H. ammodendron forests. This can also provide sustainable development in arid and saline regions, and the vegetation restoration and reconstruction of damaged ecosystems in Central Asia.
Author Contributions: Available data collection, writing up and gap assessment and design were done by Z.Z.; methodology, formal analysis, visualization, and writing-original draft preparation: C.S., A.K., and D.S.; while editing and proofing, as well as supervision of the whole work during this project, were performed by X.X.; correspondence Y.W. All authors have read and agreed to the published version of the manuscript.