Effects of rooting powder and soil salt stress on the growth and physiological characteristics of Tamarix chinensis cuttings

Background: Vegetation restoration is a main ecological remediation technology for greening saline and alkaline soils. The objectives of this study were to determine whether aminobenzotriazole (ABT), a rooting powder, can be used to improve the physiological regulatory abilities of Tamarix chinensis under salt stress; to reveal the physiological regulatory pattern by which T. chinensis pre-treated with ABT adapts to salt stress. Results: (1) As the salt stress level increased, the cutting survival rate, height, and root length of T. chinensis gradually decreased, whereas their biomass first decreased and then increased. At salt content S c>0.9%, cutting propagation of T. chinensis was difficult, and there was a considerable decrease in its biomass. The effectiveness of ABT in improving the survival rate and growth of T. chinensis cuttings became increasingly pronounced as the salt stress level increased. (2) T. chinensis was found to adapt to salt stress through increased Chl content. However, excess salt stress inhibited Chl synthesis. ABT can be used to widen the range of tolerance of T. chinensis seedlings to salt stress during Chl synthesis. (3) T. chinensis can eliminate excess reactive oxide species (ROS) by enhancing SOD and POD activities. An excess accumulation of ROS will impede the increase in enzyme activities. ABT can help improve T. chinensis seedling enzyme system regulation and was found to be most effective at a concentration of 100 mg·L -1 . (4) ABT can reduce MDA accumulation and damage caused by membrane lipid peroxidation (MLP). ABT at a concentration of 100 mg·L -1 was found to be highly effective in reducing MDA content. Conclusions: ABT was effective in improving the survival rate and the growth and

physiological regulatory abilities of T. chinensis cuttings under salt stress. ABT enhanced the resistance of T. chinensis to salt stress. However, under high S c (>0.9%) and ABT concentration (>100 mg·L -1 ) conditions, the physiological regulatory ability of T. chinensis seedlings exposed to salt stress weakened. At S c of 0.9%, T. chinensis seedlings pre-treated with ABT at 100 mg·L -1 exhibited the most vigorous growth, highest biomass, and highest physiological and biochemical regulatory abilities.

Background
Soil salinization exerts a significant impact on sustainable agricultural development and environmental quality. Crop losses caused by salinization in irrigated areas around the world are estimated at USD 11 billion and continue to rise [1]. Soil salinization is a major environmental risk resulting from natural or human activities.
Currently, more than one billion ha of land worldwide is salinized, accounting for 30% of the total land area [2]. According to the Second National General Survey conducted by the Ministry of Agriculture of China [3], approximately 36 million ha of land in China is salinized, accounting for 4.88% of China's usable land area.
Additionally, 9.209 million ha of farmland is salinized, accounting for 6.62% of China's total farmland area. In China, salinized soils are distributed across a vast area, primarily in its northwestern, northern, northeastern, and coastal regions. Soil salinization will lead to an increase in the osmotic pressure of the soil solution and decreases in the air and water permeability and the nutrient availability in soil.
Moreover, soil salinization can also hinder normal plant growth and cause severe vegetation degradation [4]. Conventionally, there are two main approaches for ameliorating saline and alkaline soils. One approach is to directly ameliorate soils.
Engineering measures, such as constructing ditches to drain salts, pumping freshwater to reduce soil salinity (Sc), and creating raised fields, are primarily implemented to reduce Sc and improve the physical and chemical properties of soils to make them suitable for development of agricultural and forestry industries.
However, these measures are costly and have low land-use efficiency. Moreover, it is difficult to implement these measures in areas that lack freshwater resources.
The other approach is to employ conventional breeding methods and modern bioengineering measures to breed salt-tolerant crop varieties and to use advanced biotechnologies to improve the salt tolerance of plants. Vegetation restoration measures focusing on planting of local halophytic or salt-tolerant plants have become an important approach for ameliorating saline and alkaline soils [5].
Researchers in China and elsewhere have investigated the use of halophytic plants to improve saline and alkaline soils. Some halophytic plants have been found to be capable of removing salts from soil by absorbing and accumulating the salts [6,7].
Halophytic plants can improve the physical and chemical properties of a soil by reducing its bulk density and improving its porosity. Improvements in the physical and chemical properties of a soil can in turn lead to an increase in its permeability and therefore facilitate salt leaching [8]. However, a main issue in implementing vegetation measures to improve saline and alkaline soils is the difficulty associated with adaptation of the planted seedlings to salt-alkali stress during the initial growth stage, resulting in slow root system growth of the seedlings and a relatively low survival rate. Hence, there is an urgent need to develop methods to improve the survival rate of halophyte seedlings during the initial planting stage and to study their physiological adaptability.
As global soil salinization becomes increasingly prominent, an increasing number of studies are being conducted to examine the physiological and ecological mechanisms by which plants respond to salt stress. These studies focus mainly on crops, vegetables, and specific halophytic plants [4,9,10]. The results of these studies are of important guidance value to the breeding of halophytic plants and their application in saline and alkaline soil amelioration and vegetation restoration.
Some researchers have found a significant increase in the superoxide dismutase (SOD) and peroxidase (POD) activities and malondialdehyde (MDA) content in the leaves of bean seedlings [11] and Jerusalem artichoke [12] with Sc. Hu et al. [13]reported an increase in the MDA content and SOD and POD activities of Panicum virgatum L. with the salt-alkali stress level. Li et al. [14] found that salt stress inhibited the growth of the branches and roots of Salix matsudana Koidz.
Additionally, they also found that low-level salt stress induced an increase in the SOD, POD, and catalase (CAT) activities, whereas high-level salt stress inhibited the activities of antioxidant enzymes. Moreover, they reported that low-level salt stress led to an increase in the MDA content and that the MDA content increased rapidly as the salt stress level increased. Hong et al. found that as the sodium chloride (NaCl) concentration increased, there was a decrease in the relative water content, chlorophyll (Chl)-a (Chla) content, total Chl (ChlT) content, and Chla/Chlb ratio in the leaves of Salix spp., but an increase in their Chlb, proline (Pro), and MDA contents [15]. Additionally, they detected an increase in the SOD activity and soluble protein content in the leaves of Salix spp. under mild salt stress and a significant decrease in these two parameters under moderate and severe salt stress. However, Zhu et al. found that as the salt stress level increased, the SOD and POD activities and MDA content in the leaves of T. chinensis cuttings first increased and then decreased [16]. Evidently, the physiological and biochemical indices of plants vary relatively significantly with the plant species and salt stress level.
T. chinensis is a shrub or small tree species belonging to the genus Tamarix in the family Tamaricaceae. T. chinensis has a developed root system and a relatively high capacity to break winds, fix sand, preserve water and soil, and ameliorate soil.
Owing to its relatively high tolerance to salts, alkalis, and droughts, T. chinensis is often used as a primary vegetation restoration tree species for restoring and rebuilding degraded ecosystems in China's western arid and desert regions, Yellow River Basin, and coastal saline and alkaline soil regions [17]. T. chinensis shrubs are the main protective shrubs for muddy coastal zones in saline and alkaline soil regions in the Yellow River Delta. These shrubs play a vital role in maintaining ecosystem stability and improving saline and alkaline soils in coastal zones. In recent years, as a result of global warming, there has been a decrease in precipitation and an increase in evaporation in the Yellow River Delta. Additionally, seawater intrusion caused by natural factors (e.g., ocean current movements) and human activities (overexploitation of underground brine resources) has intensified soil salinization and degraded the T. chinensis shrub ecosystem in the Yellow River Delta. Therefore, there is an urgent need to develop saline and alkaline soil amelioration measures that focus on T. chinensis shrub restoration. Determining the physiological and biochemical regulation processes by which T. chinensis tolerates salts is a key link and essential precondition for T. chinensis shrub restoration.
Currently, Chinese researchers study T. chinensis in areas such as morphology and taxonomy [18], ecological characteristics [19], cytogenetics [20], and physiological and ecological characteristics under salt [17], salt-alkali [21], and salt-drought intercross stress [16]. By contrast, researchers elsewhere, mostly treating T. chinensis as an invasive species, investigate its role in ecosystemsas well as biological control and physical removal methods for controlling its growth and propagation [22][23][24]. The physiological adaptability of T. chinensis to salt stress and its role in ameliorating saline and alkaline soils in some regions have also been examined [25]. IBA [28]. Some researchers have also examined the use of rooting powders in vegetative propagation. Research has shown that prior to cutting propagation, treating current-year low-lignified branches with a rooting powder can significantly improve their survival rate. Tan found that treating T. chinensis shoot cuttings with ABT at 200 mg·L -1 for 1 hour could increase their rooting percentage (to 84.44% on average) and biomass [29]. A combination of a rooting powder and fertilizer was found to significantly improve root activities in T. ramosissima in various soil layers [30]. However, the available studies focused primarily on propagation under single-salt or salt-free conditions, and consequently their results cannot be satisfactorily applied under field conditions. Relatively few studies have been

Experimental materials and design
Branch cuttings were harvested from the T. chinensis shrub obtained from the wild in the Shandong Changyi National Marine Ecology Special Reserve; the provenance originates from Changyi City, Shandong Province, China. We comply with the Convention on the Trade in Endangered Species of Wild Fauna and Flora. In mid-February 2018 before T. chinensis began to sprout, T. chinensis branches with a diameter of approximately 1 cm were cut and harvested. The branches were then sectioned into 15-cm-long cuttings. Each cutting was sectioned obliquely at the base and flat at the top. Four Sc stress levels were studied, namely, mild (0.3%), moderate (0.6%), and severe (0.9% and 1.2%). An Sc of ≤0.1% was selected as the control (CK). Each soil Sc was prepared based on the dry soil weight. The Sc was monitored once every 7 days, and additional salt was added to maintain the previously established Sc. An 8-cm-deep tray was placed beneath each pot. The water that leaked from each pot into the tray was poured back into the pot.
Additionally, the tray was washed, and the washing water was also poured into the pot. This procedure prevented salt loss. The bases of the cuttings were soaked at a depth of 2-3 cm in ABT solutions of various concentrations (0, 50, 100, and 200 mg·L -1 ). Subsequently, the cuttings were planted in pots filled with soil at each Sc level. Ten cuttings were planted in each pot. Three repeats were performed for each treatment. A total of 600 cuttings were planted in 60 pots. During the initial cutting propagation stage, the pots were watered with freshwater twice a day to maintain a soil water content at 60%-70% of the field capacity. After 90 days of propagation, the growth (e.g., biomass), physiological, and biochemical indices of the T. chinensis cuttings were measured and analysed.

Measurement of indices
Measurement of growth indices: Three pots were selected for each treatment. Five T. chinensis cuttings were selected from each pot to measure their growth indices.
The height of each plant was measured using a metre ruler. The aboveground and underground biomass of each T. chinensis seedling was measured by harvesting the whole plant. Each whole seedling was dug out and cleaned. Then, the branches, trunk, and root system of each seedling were identified. Each seedling was subsequently fixed at 105℃ for 30 minutes and then dried in an oven at 80℃ until a constant weight was achieved.
Measurement of physiological and biochemical indices: Three pots were selected for each treatment. Three T. chinensis cuttings were randomly selected from each pot.
Normally grown, mature leaves of the T. chinensis cuttings in the same area were collected and tested to determine their physiological and biochemical indices. The photosynthetic pigment content in each leaf was measured per unit fresh weight using the ethanol-acetone soaking procedure [31]. SOD activity was measured by nitroblue tetrazolium photoreduction [32]. POD activity was measured by guaiacol colorimetry [33]. The MDA content was measured by thiobarbituric acid colorimetry [34]. At least three repeated measurements of each index were obtained and subsequently averaged. cutting growth vary relatively significantly. An increase in the growth regulator concentration will prevent gymnosperms from rooting. Some research has also found that increasing the growth regulator concentration will not increase the rooting percentage but can improve the quality of the root system. This phenomenon is related to the difference in the type and concentration of hormones needed and the hormone sensitivities among tree species [35]. A rooting powder (e.g., ABT) can be used to facilitate growth of the root system of T. chinensis cuttings. By increasing and regulating the content of endogenous hormones and the activities of important enzymes in T. chinensis seedlings, ABT stimulates cell division and growth in the inner root sheath, strengthens root system development, facilitates vigorous growth, significantly reduces cell membrane damage caused by salt stress, and increases salt stress adaptability. In this study, under mild salt stress (Sc≤0.3%), the ABT concentration showed no significant impact on the survival rate of T. chinensis cuttings. As Sc increased, ABT at a suitable concentration increased the survival rate. However, as the salt stress level continued to increase, ABT became ineffective. A relatively high soil salinity generally causes osmotic stress in plants and disrupts their nutrient ion balance, thereby affecting physiological and biochemical processes such as growth, photosynthesis, osmotic adjustment substance synthesis, and lipid metabolism [36], ultimately limiting their growth rate and biomass accumulation. In this study, there was a significant decrease in the biomass, root length, and height of T. chinensis under salt stress. T. chinensis may reduce biomass accumulation and utilize more resources and energy to respond to high salinity-induced damage [36], which suggests that plants under stress are able to respond to adverse external conditions by altering their biomass allocation pattern. By reducing the proportion of biomass allocated to root systems, some plants reduce salt absorption and salt transport to their aboveground portions [37]. Some plants acquire more water and nutrients by increasing the proportion of biomass allocated to their root systems. This mechanism increases plant growth ability and salt dilution in plant cells [38]. In this study, T. chinensis seedlings pre-treated with ABT at 100 mg·L -1 had relatively long root systems and the highest biomass at Sc of 0.9%, and their aboveground and belowground portions showed consistent growth.

Effects of salt stress on Chl contents in T. chinensis cuttings
Chl is an important substance in plant photosynthesis. The Chl content can reflect the ability of plants to assimilate substances. Under salt stress, plant Chl contents gradually decreased as Sc increased. Some research has shown that as Sc increases, the Chl content in plants either first increases and then decreases or gradually increases, and the plants exhibit relatively high salt tolerance [39]. In this study, as Sc increased, Chla, Chlb, and ChlT contents in the leaves of T. chinensis cuttings pre-treated with ABT at various concentrations all initially decreased, then increased, and then decreased again. This pattern may result from the low Scs, which was conducive to T. chinensis growth and did not activate its resistance mechanism to salt stress. As the salt stress level increased, the Chl content began  [11]. Under salt stress, there is a threshold for the ROS level tolerable to plant cells. Below this threshold, plants are able to remove ROS by increasing the activities of antioxidant enzymes. Beyond this threshold, the activities of antioxidant enzymes will be inhibited, resulting in an excess accumulation of ROS and leading to plant tissue damage [28]. In this study, as the salt stress level increased, SOD activity first increased and then decreased in the leaves of the T. chinensis cuttings pre-treated with ABT at various concentrations.
This result suggested that as the salt stress level increased, ROS began to accumulate in the leaf cells of T. chinensis, and T. chinensis removed excess ROS by increasing the SOD activity to adapt to salt stress. However, under relatively high salt stress, the ROS that formed exceeded the regulation ability of SOD.
Consequently, SOD activity was inhibited and therefore decreased. This SOD pattern is consistent with that in T. austromongolica and T. chinensis [21] in saline and alkaline habitats and in the leaves of Pennisetum alopecuroides (L.) Spreng [43] under NaCl stress.
Plants reduce salt stress-induced damage by increasing POD activity. The causes of the increase in POD activity include not only ROS production but also cell membrane damage and changes in the calcium ion concentration. At various Scs, POD activity in the leaves of T. chinensis cuttings pre-soaked in water was relatively high, suggesting that the relatively large amount of H 2 O 2 produced in leaf cells of T. chinensis seedlings that were not pre-treated with ABT resulted in increased POD activity to remove H 2 O 2 . Evidently, the damage (MLP) induced by salt stress was three times greater in T. chinensis seedlings that were not pre-treated with ABT than in T. chinensis seedlings in the three ABT pretreatment groups. Li et al. found that as the salt stress level was increased, POD activity in T. austromongolica and T. chinensis first increased and then decreased [21]. They also found that POD activity in the leaves of T. austromongolica and T. chinensis reached a maximum at Sc of 1.2%, which was 3.5 and 3.6 times greater than in the control group, respectively. A similar pattern was also found in this study. POD activity in the leaves of T. chinensis seedlings pre-treated with ABT at 200 mg·L -1 first increased and then decreased with increasing salt stress. At Scs of 0.3%-0.9%, there was a significant increase in POD activity in the leaf cells. The antioxidant enzyme system decomposed the H 2 O 2 produced by SOD through disproportionation reactions by increasing POD activity. Under high salt stress (Sc≥1.2%), excess ROS were produced in leaf cells, exceeding the removal threshold of POD. As a result, the accumulating ROS damaged the enzyme system, resulting in a decline in POD activity. Compared with pretreatment with ABT at 50 and 100 mg·L -1 , the sensitivity of POD activity was relatively low in the leaves of T. chinensis seedlings pre-treated with ABT at 200 mg·L -1 , and the ability of POD in these T. chinensis seedlings to regulate salt tolerance was also relatively low. Thus, MDA can be used as an important index for evaluating the extent of membrane system damage under stress [44]. The MDA content in plant leaves varies relatively significantly with their salt tolerance. As salt stress increases, there is a continuous increase in MDA content in the leaves of plants with relatively low salt tolerance, such as T. chinensis tissue culture seedlings [45]. In comparison, with increasing salt stress, the MDA content in the leaves of plants with relatively high salt tolerance first decreases and then increases [32]. In this study, the MDA content in the leaves of T. chinensis cuttings first increased and then decreased as the salt stress level increased. This finding is consistent with those reported by Li et al. showing the effects of MLP in the seedling leaves of six gramineous forages [46], but T. chinensis seedling photosynthesis was enhanced by increasing the content of Chl to adapt to salt stress at suitable levels. However, under excessively high salt stress, Chl synthesis was disrupted, resulting in a decline in Chl content. Increasing the ABT concentration could strengthen the tolerance of T. chinensis seedlings to salt stress during Chl synthesis.
As the salt stress level increased, T. chinensis seedlings showed reduced ROSinduced damaged through increases in SOD and POD activities. Excess ROS accumulation inhibited the increase in enzyme activities. More significant changes in MLP in the cell membranes of T. chinensis seedlings resulted in higher MDA accumulation. ABT helped enhance the regulatory ability of the enzyme system of T. chinensis cuttings and significantly reduced the damage caused by low salt stress in cell membranes. The enzyme activities were highest in the T. chinensis seedlings pre-treated with ABT at 100 mg·L -1 , and the cell membranes in these seedlings sustained the least significant oxidative damage. The following conditions were found to be suitable for vegetative propagation of T. chinensis: Sc≤0.9% and ABT≤100 mg·L -1 . At Sc of 0.9%, T. chinensis seedlings pre-treated with ABT at 100 mg·L -1 showed the most vigorous growth, had the highest biomass, and exhibited relatively high physiological regulatory ability and salt adaptability.  Effects of salt stress on SOD activity in the leaves of T. chinensis cuttings with various conce Figure 5 Effects of salt stress on POD activity in the leaves of T. chinensis cuttings with various conce Figure 6 Effects of salt stress on MDA content in leaves of T. chinensis cuttings with various concentr