Screening and evaluating method for salt tolerance threshold value in cotton seedling stage

Abstract

The invention belongs to the technical field of saline-alkali soil improvement, and particularly relates to a method for screening and evaluating a salt tolerance threshold in a cotton seedling stage. The invention discloses a method for determining a salt tolerance threshold value of cotton in a seedling stage. The method comprises the following steps: simulating stress environments with different salt concentrations; measuring the whole plant fresh weight, dry weight, plant height, root length, whole plant length, stem diameter and leaf area of the cotton seedlings after salt treatment; related indexes after salt treatment are detected, and the salt concentration tolerance degree is evaluated; and determining a salt concentration tolerance threshold value. According to the method, the relatively accurate salt tolerance threshold of the cotton seedling stage is finally determined according to the fitted model by utilizing correlation analysis and a quadratic regression equation and combining the actual growth conditions of the cotton seedlings under different salt concentrations. When the index fitting model is selected, a plurality of indexes are selected instead of a specified growth or physiological index. Compared with previous methods, the method established by the invention is a more comprehensive and accurate screening method.

Description

Technical Field

[0001] This invention belongs to the field of saline-alkali land improvement technology, and in particular relates to a method for screening and evaluating the salt tolerance threshold of cotton seedlings. Background Technology

[0002] Soil salinization has become a major obstacle to sustainable agricultural development and a significant challenge to the soil ecological environment (Van Zelm et al., 2020). Globally, approximately 1 billion hectares of soil salinization have this problem. 2 The saline-alkali land, with an annual output of 1-1.5 million hectares. 2 Salt stress is increasing at a rapid rate (Wang et al., 2011). It is projected that by 2050, more than 50% of arable land will be salinized (Kumar and Sharma, 2020). During plant growth, photosynthesis and physiological metabolism are affected by salt stress, leading to metabolic disorders during the plant's growth period, resulting in decreased biomass and crop yield, and even plant death.

[0003] Different crop species or genotypes exhibit significant differences in salt tolerance (Chaudhary et al., 2024). When salt concentration exceeds the crop's tolerance range ("threshold"), it leads to metabolic disorders in crop growth, irreversibly inhibiting survival. Even if salinity decreases or returns to normal levels later, the trend of yield decline or even death cannot be reversed (Canalejo et al., 2014). Under saline-alkali conditions, plant growth is a function of soil salinity or solution salinity (Hasanuzzaman et al., 2013). The salt concentration at which plant growth decreases by 50% is generally used as its salt tolerance threshold (Akram et al., 2010; Grieve et al., 2012). Xu et al. (2021) established a regression equation with the fresh and dry weight of the aboveground parts of sheepgrass as the dependent variable and salt concentration as the independent variable, and used the salt concentration corresponding to a 50% reduction in fresh and dry weight to the control treatment as the salt tolerance threshold. Zhang et al. (2019) used correlation and regression analysis to establish a quadratic regression equation between net photosynthetic rate and SOD enzyme activity and salt concentration. They used a 50% reduction in the selected index compared to the control as the standard and selected the lower value as the salt tolerance threshold for wolfberry. However, studies have shown that different growth and physiological indicators exhibit varying tolerance to salt stress during crop growth (i.e., different indicators may yield different salt tolerance thresholds). For example, the tolerance threshold for NaCl stress in maize photosynthesis is around 150 mmol / L, while the salt tolerance thresholds for chloroplast function and root osmotic properties are higher than 200 mmol / L (Ma Dongfang, 2013). Existing technologies suffer from unreasonable identification indicators and simplistic threshold determination methods. Some focus on or directly select biomass and morphological indicators (such as survival rate, dry and fresh weight, and plant height), while others focus on or directly select physiological and biochemical indicators (such as chlorophyll content and proline). They rely on single indicators and lack comprehensive evaluation using multiple indicators. This technology, however, uses correlation analysis and a quadratic regression equation for comprehensive evaluation using multiple indicators, resulting in more accurate results. Existing technologies suffer from deficiencies in dose-response curve construction or insufficient treatment concentrations. Many studies use only a few concentrations or directly select high-concentration salt treatments, failing to accurately determine the "inflection point." This technology, however, sets multiple salt concentration treatments (gradients) and plots the dose-response relationship to accurately define the threshold. Existing technologies also suffer from significant differences between experimental treatment conditions and crop field growth conditions. Many studies use artificially prepared salt solutions or nutrient solutions to set salt concentration gradients to screen thresholds. This technology, however, provides a crop growth environment that more closely resembles field conditions through a salt treatment method that uniformly mixes salt into the field soil.Existing technologies lack a comprehensive and unified method for determining the salt tolerance threshold of cotton, an important economic crop. This technology establishes a comprehensive and accurate method for screening the salt tolerance threshold of cotton seedlings, applicable to different cotton varieties.

[0004] In previous studies, the third leaf from the bottom (functional leaf) of cotton has often been used as a representative leaf for assessing the physiological and photosynthetic characteristics of cotton due to its high metabolic activity and stability (the leaf position with the strongest photosynthetic capacity and most vigorous physiological activity) (Ruan Sijia et al., 2025; Xu Jing et al., 2025; Song Chen et al., 2024; Guo Jiaxin et al., 2024). The third leaf from the bottom is usually fully expanded and functionally mature, avoiding the senescence of lower leaves and the immaturity of upper leaves, thus providing a stable physiological state for measurement. This study mainly measures the growth, physiological, and photosynthetic indices of cotton under salt stress. Therefore, selecting functional leaves can most accurately reflect the active photosynthetic and metabolic state of plants under field conditions, and thus leaves from other parts were not selected for measurement. In addition, previous studies have shown that there are significant differences in the physiological responses of different leaf positions of crops under salt stress. For example, Dong Ruixiao et al. (2024) pointed out that the photosynthetic rate of the top leaves (young leaves) of sunflowers decreased less under salt stress, while the photosynthetic rate of the middle and lower mature leaves decreased significantly, and non-stomatal limitation became the main inhibitory factor. Weng Haiyong et al. (2025) believed that the sensitivity of cotton seedling leaves to salt stress showed the pattern of "upper leaf position > lower leaf position", with the top leaf being the most sensitive leaf position, and its water content decreased the most and the antioxidant enzyme activity (SOD, POD) increased the most. The research results verified that there are obvious differences in leaf position in the response of cotton to salt stress.

[0005] Cotton is the world's most important source of natural fiber. Although it is known as a "pioneer crop" for saline-alkali lands and possesses strong salt and alkali tolerance and regulation capabilities, its growth and yield are still severely affected by salt and alkali stress (Zhu et al., 2020). Studies have shown that soil salinity and alkali cause approximately 9% of the annual cotton yield reduction (Zhu et al., 2020). Furthermore, the seedling stage is a vulnerable and critical stage for cotton growth and development, easily affected by salt, which seriously threatens cotton growth and yield (Duan et al., 2022). Previous studies have focused on the mechanisms of cotton salt and alkali tolerance and the regulation of cotton salt tolerance by exogenous substances, while research on screening methods for cotton seedling tolerance thresholds is relatively limited. Analyzing differential genes in plants using salt tolerance thresholds as treatment concentrations to study salt and alkali tolerance mechanisms will lay the foundation for in-depth research on plant seedling signaling pathways and salt tolerance mechanisms under salt stress. Furthermore, in field production, understanding the salt tolerance threshold of crops during the seedling stage in advance, and preventing the crop's growing environment from exceeding its salt tolerance threshold through measures such as reducing salinity, is crucial for ensuring normal crop growth and ultimately achieving high and stable yields. Therefore, establishing a comprehensive, accurate, and thorough method for screening crop salt tolerance thresholds is of paramount importance. Summary of the Invention

[0006] Currently, there is no comprehensive, accurate, and systematic method for screening and evaluating the salt tolerance threshold of cotton seedlings. This invention utilizes correlation analysis and quadratic regression equations, combined with the actual growth of cotton seedlings under different salt concentrations, to determine a relatively accurate salt tolerance threshold for cotton seedlings based on the fitted model. When selecting indicators to fit the model, multiple indicators are chosen instead of a single growth or physiological indicator. Therefore, the method established in this invention is a more comprehensive and accurate screening method compared to previous methods.

[0007] A comprehensive and accurate method for screening and evaluating salt tolerance thresholds in cotton seedlings is proposed. This method measures physiological and photosynthetic characteristics, chlorophyll fluorescence parameters, and biomass of cotton seedlings. It combines correlation analysis and quadratic regression equations to calculate the salt tolerance threshold based on the fitted model. At the same time, it refers to the actual growth of cotton seedlings under different salt concentrations and removes outliers that do not meet the requirements. Finally, it determines the more accurate salt tolerance thresholds for different cotton varieties at the seedling stage.

[0008] This invention reveals that the index selected during the screening of salt tolerance thresholds may differ from the index selected when establishing a model based on salt concentration.

[0009] For example, in this invention, the four indicators of salt-tolerant Xinluzao 53—whole plant fresh weight, whole plant dry weight, plant height, and whole plant length—showed a significant negative correlation with salt concentration and the correlation coefficient was high; while in salt-sensitive Xinluzao 60, the five indicators of whole plant length, whole plant fresh weight, leaf area, leaf width, and net photosynthetic rate showed a significant negative correlation with salt concentration and the correlation coefficient was high.

[0010] Under the conditions of this invention, different cotton varieties or crop types have different tolerances to salt stress, and cotton varieties with different salt tolerances have different salt tolerance thresholds obtained through screening.

[0011] The "salt tolerance threshold" mentioned in this invention refers to the critical value of salt tolerance that a crop can withstand. When the salt stress concentration exceeds the salt tolerance threshold of the plant, the plant's survival will be irreversibly inhibited. Even if the salinity decreases or returns to normal levels later, the trend of plant death cannot be changed.

[0012] The crop species mentioned in this invention include, but are not limited to, cotton, and the cotton varieties include, but are not limited to, Xinluzao 53 (salt-tolerant) and Xinluzao 60 (sensitive).

[0013] The screening method for the "salt tolerance threshold" described in this invention includes, but is not limited to, salt stress treatment, and can also be alkali stress treatment to obtain the "alkali tolerance threshold"; it can also be a mixed salt and alkali stress treatment to obtain the "salt and alkali tolerance threshold".

[0014] In the method described in this invention, normal garden soil is used to cultivate cotton plants to the three-leaf-one-heart stage, followed by salt stress treatment. To avoid salt stress, the salt concentration is increased every 24 hours, ultimately reaching 2, 4, 6, 8, and 10 g / kg in the soil. To maintain soil salinity and moisture, a weighing-based watering method is used daily (weighing the flowerpot to replenish evaporated water), with 200 ml of Hoagland's nutrient solution applied every 3 days. After 14 days of salt stress treatment, relevant indicators of the cotton plants are measured.

[0015] Select uniform-sized, disease-free cotton seeds, wash them several times with distilled water, disinfect them by soaking in a 5% sodium hypochlorite solution for 10 minutes, and then rinse them five times with sterilized deionized water. Sow the seeds in uniform-sized pots (each pot containing 3.5 kg of test soil). Before filling the pots with soil, line the inside of each pot with a waterproof cloth to prevent salt leakage during the subsequent salt treatment. Irrigate thoroughly after emergence. When the seedlings reach the three-leaf stage, the optimal growing conditions are: day / night temperature 28 ℃ / 23 ℃, humidity 70%, photoperiod 14 h, and light intensity 400-450 μmol·m⁻¹. -2 ·s -1 .

[0016] The method of this invention was used to detect the photosynthetic parameters, SPAD values, and chlorophyll fluorescence parameters of cotton leaves (the third leaf from the bottom) after different concentrations of salt stress treatment.

[0017] The method of this invention also detected the electrolyte leakage rate and malondialdehyde content of cotton leaves (the third to last leaf) after different concentrations of salt stress treatment.

[0018] The method of this invention also tested the whole plant fresh weight, dry weight, plant height, root length, whole plant length, stem diameter (at the cotyledon node), and leaf area (third to last functional leaf) of cotton after different concentrations of salt stress treatment.

[0019] To further improve the accuracy of the screening results, the method of the present invention also includes detecting the antioxidant enzyme activity of the third-to-last functional leaf of cotton after different concentrations of salt stress treatment.

[0020] Specifically, a comprehensive, thorough, and accurate method for screening and evaluating the salt tolerance threshold of cotton seedlings includes:

[0021] 1) Select uniform-sized, disease-free cotton seeds, wash them several times with distilled water, disinfect them by soaking in a 5% sodium hypochlorite solution for 10 minutes, and then rinse them 5 times with sterilized deionized water. Sow them in uniform-sized flowerpots (each pot containing 3.5 kg of test soil). Before filling the pots with soil, line the inside of each pot with a waterproof cloth to prevent salt leakage during later salt treatment. Water thoroughly after emergence, and wait until the seedlings reach the three-leaf stage.

[0022] 2) To avoid salt stress, a salt treatment method was adopted with increasing salt concentrations every 24 hours, ultimately achieving soil salt concentrations of 2, 4, 6, 8, and 10 g / kg simultaneously. To maintain soil salinity and moisture, a weighing-based watering method was used daily (weighing the flowerpots to replenish evaporated water), with 200 ml of Hoagland nutrient solution applied every 3 days. After 14 days of salt stress treatment, relevant indicators of the cotton were measured.

[0023] Cultivation conditions: Day / night temperature 28 ℃ / 23 ℃, humidity 70%, photoperiod 14 h, light intensity 400-450 μmol·m -2 ·s -1 .

[0024] 3) Detect the whole plant fresh weight, dry weight, plant height, root length, whole plant length, stem diameter (cotyledon junction) and leaf area (third to last functional leaf) of cotton seedlings after control and salt treatment.

[0025] 4) Detect the photosynthetic characteristics, SPAD value, and chlorophyll fluorescence parameters of the third-to-last functional leaf of cotton seedlings after control and salt treatment;

[0026] 5) Detect the electrolyte leakage rate and malondialdehyde content in the third-to-last functional leaf of cotton after control and salt treatment;

[0027] 6) Detect the antioxidant enzyme activity of the third-to-last functional leaf of cotton after control and salt treatment;

[0028] 7) Based on steps 3) to 6), perform correlation analysis between the measurement results of each indicator and the salt treatment concentration, and screen out several indicators that are significantly negatively correlated with the salt treatment concentration (confidence level < 0.01) and have a high correlation coefficient.

[0029] 8) Based on the indicators selected in step 7), establish regression equations by taking the changes of these indicators under different salt treatments as the vertical axis and the salt concentration as the horizontal axis, and take the salt concentration at which each indicator decreases by 50% as its salt tolerance threshold.

[0030] 9) Based on step 8), compare the salt tolerance thresholds calculated for each indicator with the actual growth of cotton seedlings under salt treatment concentrations. If they do not match, discard them. Finally, calculate the average value of the remaining salt tolerance thresholds; the resulting average value is the more accurate salt tolerance threshold for this cotton variety.

[0031] The present invention further provides the application of the above method in the comprehensive and accurate screening of cotton salt tolerance thresholds.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. This invention relates to a comprehensive and accurate method for screening the salt tolerance threshold of cotton seedlings. It mainly uses indicators such as physiological characteristics, photosynthetic characteristics, chlorophyll fluorescence parameters, and biomass of cotton seedlings, combined with correlation analysis and quadratic regression equations, and determines a relatively accurate salt tolerance threshold for cotton seedlings based on the fitted model.

[0034] 2. This invention selects multiple indicators for comprehensive evaluation when screening salt tolerance thresholds for cotton seedlings, rather than specifying a single growth or physiological indicator. Simultaneously, it combines the values ​​calculated by the model with the actual growth of cotton seedlings under different salt concentrations, eliminating outliers, thereby establishing an accurate method for screening salt tolerance thresholds for cotton seedlings. Attached Figure Description

[0035] Figure 1 The figure shows the effect of salt stress on biomass accumulation in cotton seedlings in Example 1, where a is the fresh weight of the whole plant, b is the dry weight of the whole plant, c is the plant height, d is the root length, e is the total plant length, and f is the stem diameter.

[0036] Figure 2The figure shows the effect of salt stress on the antioxidant enzyme activity and malondialdehyde content of cotton seedlings in Example 1, where a represents superoxide dismutase activity, b represents peroxidase activity, c represents catalase activity, and d represents malondialdehyde content.

[0037] Figure 3 The graph shows the effect of salt stress on the electrolyte leakage rate and SPAD value of cotton seedlings in Example 1, where a is the electrolyte leakage rate and b is the SPAD value.

[0038] Figure 4 The figure shows the effect of salt stress on the photosynthetic characteristics of cotton seedlings in Example 1, where a is the net photosynthetic rate, b is the transpiration rate, c is the stomatal conductance, and d is the intercellular CO2 concentration.

[0039] Figure 5 The figure shows the effect of salt stress on the chlorophyll fluorescence parameters of cotton seedlings in Example 1, where a is the potential photochemical efficiency and b is the maximum quantum yield.

[0040] Figure 6 The graph shows the correlation between various indicators of cotton seedlings under salt stress and salt concentration, as well as the correlation analysis between various indicators in Example 1. In the graph, a represents the Xinluzao 53 variety, and b represents the Xinluzao 60 variety.

[0041] Figure 7 The graph shows the quadratic regression equation established between the salt treatment concentration and the corresponding index of Xinluzao 53 in Example 1, where a is the fresh weight of the whole plant, b is the dry weight of the whole plant, c is the plant height, and d is the length of the whole plant.

[0042] Figure 8 The graph shows the quadratic regression equation established between the salt treatment concentration and the corresponding index of Xinluzao 60 in Example 1, where a is the total plant length, b is the total plant fresh weight, c is the leaf area, d is the leaf width, and e is the net photosynthetic rate. Detailed Implementation

[0043] Example 1

[0044] 1. Using the salt-tolerant cotton variety "Xinluzao 53" and the salt-sensitive cotton variety "Xinluzao 60" as research materials, uniformly sized and disease-free cotton seeds were selected. The seeds were washed several times with distilled water, disinfected by soaking in a 5% sodium hypochlorite solution for 10 minutes, and then rinsed five times with sterilized deionized water. They were then sown in uniformly sized pots. A pot experiment was conducted using non-saline-alkali field topsoil (0-20 cm) as the substrate, with each pot containing 3.5 kg of non-saline-alkali field topsoil. The physicochemical properties of the non-saline-alkali field topsoil are shown in Table 1. Before filling the pots with soil and sowing, a waterproof cloth was placed inside each pot to prevent salt leakage during subsequent salt treatment. The seedlings were thoroughly irrigated at emergence. Once the seedlings reached the three-leaf stage, different concentrations of salt were applied.

[0045] Table 1 Physicochemical properties of soil samples

[0046]

[0047] The pot experiments were conducted in a greenhouse at the College of Agriculture, Shihezi University. Greenhouse conditions were: day / night temperature 28℃ / 23℃, humidity 70%, photoperiod 14 h, and light intensity 400-450 μmol·m⁻². -2 ·s -1 .

[0048] 2. Six salt treatment experiments were conducted: control (CK, ultrapure water without salt), and treatments with NaCl concentrations of 2, 4, 6, 8, and 10 g / kg. To avoid salt stress, the prepared NaCl solution was applied to the normal soil in multiple applications every 24 hours. (This refers to treatments with a higher salt concentration set to avoid salt stress in plants; the salt concentration was gradually increased every 24 hours to reach the final set concentration. For example, if each pot contained 1 kg of soil and the salt concentration was set at 8 g / kg, 2 g of salt would be dissolved and applied on the first day, 24 hours later, and so on, until the final concentration of 8 g / kg was reached on the fourth day.) All treatments simultaneously reached the final salt concentration of 2, 4, 6, 8, and 10 g / kg, with g / kg based on the soil level. There were a total of six treatments, with five replicates for each treatment. After reaching the final concentration, the soil was weighed and watered daily (the weight of the flowerpot was used to replenish the evaporated water) to maintain the preset salt concentration for 14 days (200 ml of Hoagland nutrient solution was applied every 3 days). After 14 days of salt stress treatment, the cotton was tested for relevant indicators. The specific watering procedure was as follows: weigh and water on day 1, day 2, day 3, day 4, and day 8, and watering with 200 ml of nutrient solution. Five samples were taken from each treatment soil, and the total salt content of the soil in different treatments was determined using a water-to-soil ratio of 5:1 (Table 2).

[0049] Table 2. Salt content of the prepared soil

[0050]

[0051] Data were processed using Microsoft Excel 2003 and analyzed using SPSS 20.0. Statistical analysis was performed using one-way ANOVA, and Duncan's multivariate range comparison was used to test for significance (p<0.05). Graphs were generated using Origin 2018 and RStudio-3.6.2.

[0052] 3. Determination of relevant indicators for cotton

[0053] (1) Determination of growth indicators of cotton seedlings

[0054] After 14 days of salt stress treatment in step 2, carefully remove the cotton seedlings from the pots, wash the soil off the roots with clean water, and measure the plant height, root length, leaf length, and leaf width with a ruler. Measure the third-to-last functional leaf from the same location, and determine the leaf area (cm²). 2 The calculation formula is: leaf length × leaf width × 0.75 (leaf area coefficient); the stem diameter (at the cotyledon node) is measured using vernier calipers, and the fresh weight is measured using a balance with a strength of 0.1%.

[0055] The results are shown in Table 3 and Figure 1 It is known that:

[0056] As shown in Table 3, the leaf area of ​​Xinluzao 53 was significantly higher under the 2 g / kg salt treatment than other treatments, increasing by 16.37% compared to the control (CK), and then gradually decreased. The leaf area of ​​Xinluzao 53 was the smallest under the 10 g / kg salt treatment, decreasing by 10.72% compared to the CK. The leaf area of ​​Xinluzao 60 was the largest under the 2 g / kg salt treatment, increasing by 3.41% compared to the CK, and then gradually decreased. The leaf area of ​​Xinluzao 60 was significantly lower under the 10 g / kg salt treatment than other treatments, decreasing by 32.49% compared to the CK.

[0057] Depend on Figure 1 It can be seen that with increasing salt concentration, the fresh weight and dry weight of cotton seedlings gradually decreased. The fresh weight and dry weight of Xinluzao 53 were significantly lower than the control (CK) under the 6 g / kg NaCl treatment, decreasing by 24.98% and 43.20%, respectively; the fresh weight and dry weight of Xinluzao 60 were significantly lower than the CK under the 2 g / kg NaCl treatment, decreasing by 15.13% and 22.17%, respectively. With increasing salt concentration, the plant height and root length of cotton seedlings also showed a similar trend. The root length of Xinluzao 53 was significantly lower than the CK under the 10 g / kg NaCl treatment, decreasing by 17.52%; the root length of Xinluzao 60 was significantly lower than the CK under the 6 g / kg NaCl treatment, decreasing by 17.36%. With increasing salt concentration, the total length of cotton seedlings gradually decreased.

[0058] Table 3. Effects of salt stress on leaf area of ​​cotton seedlings

[0059]

[0060] (2) Determination of antioxidant enzyme activity and malondialdehyde content in cotton seedling leaves

[0061] Potassium phosphate buffer (5 mL, 50 mM, pH 7.8) was added to the leaf samples, and the mixture was homogenized in an ice bath. The homogenate was centrifuged at 10,000 × g for 20 min at 4 °C, and the supernatant was used to determine the enzyme activity by spectrophotometry; the malondialdehyde content was determined by the thiobarbituric acid method.

[0062] The results are as follows Figure 2 As shown, antioxidant enzyme activity reflects the crop's ability to scavenge reactive oxygen species (ROS) under salt stress; the stronger the antioxidant enzyme activity, the stronger the ability to scavenge ROS. Malondialdehyde (MDA) content is an indicator of the degree of oxidative damage in plants; higher MDA content indicates more severe oxidative damage. With increasing salt concentration, the antioxidant enzyme activity in cotton seedlings showed a trend of first increasing and then decreasing. With increasing salt concentration, the MDA content in cotton seedlings showed a gradual increasing trend, and the overall increase in MDA content in Xinluzao 53 was less than that in Xinluzao 60. Specifically, under NaCl treatments of 8 and 10 g / kg, the MDA content in Xinluzao 53 was significantly lower than that in Xinluzao 60, by 32.59% and 35.27%, respectively.

[0063] (3) Determination of SPAD value of cotton seedling leaves and electrolyte leakage rate in cells

[0064] SPAD values ​​were measured using the third-to-last functional leaf of representative plants under each treatment, and the results were obtained using a SPAD-502 Plus chlorophyll meter. Electrolyte leakage rate in leaf cells was expressed as relative conductivity and measured using the conductivity method.

[0065] The results are as follows Figure 3 As shown, with increasing salt concentration, the electrolyte leakage rate of cotton seedling leaves gradually increased, while the SPAD value of the leaves gradually decreased. At a salt treatment concentration of 6 g / kg, the SPAD value of Xinluzao 53 was significantly lower than that of the control (CK), decreasing by 27.91%; at a salt treatment concentration of 2 g / kg, the SPAD value of Xinluzao 60 was significantly lower than that of the CK, decreasing by 8.00%.

[0066] (4) Determination of photosynthetic characteristics and chlorophyll fluorescence parameters of cotton seedlings

[0067] The photosynthetic characteristics and chlorophyll fluorescence parameters were measured between 08:30 and 10:30 in the morning. The third-to-last functional leaf was selected. The photosynthetic characteristics were measured using a LI-6400 portable photosynthesis meter (LI-COR innc, Lincoln, Nebraska, USA). The chlorophyll fluorescence parameters were measured using a portable pulse amplitude modulation fluorometer (Diving-PAM, Walz, Germany).

[0068] The results are as follows Figure 4 , 5 As shown:

[0069] With increasing salt concentration, the net photosynthetic rate of cotton seedling leaves gradually decreased. The decrease was more pronounced in Xinluzao 60, and at salt concentrations of 2 g / kg, 8 g / kg, and 10 g / kg, its net photosynthetic rate was significantly lower than that of Xinluzao 53, decreasing by 13.31%, 22.83%, and 21.52%, respectively. Transpiration rate and stomatal conductance showed a trend of first decreasing and then increasing. The stomatal conductance of Xinluzao 60 varied considerably, and at salt concentrations of 2 g / kg, 8 g / kg, and 10 g / kg, its stomatal conductance was significantly higher than that of Xinluzao 53, exceeding it by 20.37%, 9.80%, and 66.60%, respectively.

[0070] With increasing salt concentration, the potential photochemical efficiency of cotton seedlings showed a trend of first increasing and then decreasing. The potential photochemical efficiencies of both Xinluzao 53 and Xinluzao 60 reached their maximum values ​​at a salt concentration of 4 g / kg, increasing by 24.74% and 46.57% respectively compared to the control (CK). The maximum photochemical efficiency of cotton seedlings also showed a trend of first increasing and then decreasing.

[0071] (5) Analysis of salt tolerance thresholds in cotton seedlings

[0072] The results are as follows Figure 6 , 7 As shown in Figure 8:

[0073] Figure 6 Correlation analysis showed that four indicators of Xinluzao 53—whole plant fresh weight, whole plant dry weight, plant height, and whole plant length—exhibited highly significant negative correlations with salt concentration, and the correlation coefficients were relatively high. Five indicators of Xinluzao 60—whole plant length, whole plant fresh weight, leaf area, leaf width, and net photosynthetic rate—exhibited highly significant negative correlations with salt concentration, and the correlation coefficients were relatively high.

[0074] Figure 7 , 8 The quadratic regression equation was established with NaCl concentration as the abscissa and the changes in the whole plant fresh weight, whole plant dry weight, plant height, and whole plant length of Xinluzao 53 as the ordinate. The NaCl concentration at which the corresponding indicators decreased by 50% was used as its salt tolerance threshold. Figure 7 The actual salt tolerance thresholds for Xinluzao 53 were 0.9135%, 1.0158%, and 1.1053%, respectively. The average value of 1.06% was taken as the final salt tolerance threshold for the Xinluzao 53 variety. Similarly, using the changes in total plant length, total fresh weight, leaf area, leaf width, and net photosynthetic rate of Xinluzao 60 as the ordinate, a regression equation was established (…). Figure 8The actual salt tolerance thresholds for Xinluzao 60 were 0.8442% and 0.8588%, respectively. The average value of 0.85% was obtained as the final salt tolerance threshold for the Xinluzao 60 variety.

[0075] In summary, this invention establishes a comprehensive and accurate screening method for the salt tolerance threshold of cotton seedlings by using physiological indicators, photosynthetic characteristics, and biomass, employing correlation analysis and quadratic regression equations, and combining these with the actual growth of seedlings. The salt tolerance threshold of cotton seedlings is then determined based on the fitted model.

[0076] This invention provides a comprehensive, accurate, and thorough method for screening and evaluating salt tolerance thresholds in cotton seedlings. This method will lay the foundation for future in-depth research on plant signaling pathways and salt tolerance mechanisms under salt-alkali stress. Furthermore, this method will help farm managers understand the salt tolerance thresholds of crops in advance, and prevent crop growth environments from exceeding their tolerance thresholds through salinity reduction and other appropriate measures. This provides valuable guidance for ensuring normal crop growth and ultimately achieving high and stable yields.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for determining the salt tolerance threshold of cotton seedlings, characterized in that, Includes the following steps: (1) Simulate stress environments with different salt concentrations; (2) Determine the whole plant fresh weight, dry weight, plant height, root length, whole plant length, stem diameter and leaf area of ​​cotton seedlings after salt treatment; (3) Detect relevant indicators after salt treatment and evaluate salt concentration tolerance; (4) Determination of salt concentration tolerance threshold.

2. The method according to claim 1, characterized in that, The salt concentration mentioned in step (1) is the final concentration; Among them, the salt treatment method was adopted with increasing salt concentrations every 24 hours, which ultimately resulted in salt concentrations in the soil reaching 2, 4, 6, 8, and 10 g / kg, respectively.

3. The method according to claim 1, characterized in that, In step (2), the stem thickness refers to the cotyledon junction, and the leaf area refers to the third-to-last functional leaf.

4. The method according to claim 1, characterized in that, The relevant indicators mentioned in step (3) include the photosynthetic characteristics, SPAD value, and chlorophyll fluorescence parameters of the third-to-last functional leaf of cotton seedlings after salt treatment; Electrolyte leakage rate and malondialdehyde content in the third-to-last functional leaf of cotton after salt treatment; Antioxidant enzyme activity in the third-to-last functional leaf of cotton after salt treatment.

5. The method according to claim 4, characterized in that, The salt tolerance evaluation in step (3) is to use the changes of relevant indicators under different salt treatments as the vertical axis and the salt concentration as the horizontal axis to establish regression equations, and take the salt concentration when each indicator decreases by 50% as its salt tolerance threshold.

6. The method according to claim 5, characterized in that, The method for determining the salt tolerance threshold in step (4) is as follows: calculate the average value of the salt tolerance threshold, and the average value obtained is the more accurate salt tolerance threshold for the cotton variety.

7. The application of the method for determining the salt tolerance threshold of cotton seedlings as described in any one of claims 1-6 in the screening and evaluation of salt tolerance thresholds in cotton seedlings.

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