Method for identifying crop alkali tolerance and application thereof

Abstract

The present application belongs to the technical field of stress-resistant crop breeding, and particularly relates to a crop alkali tolerance identification method and application thereof. The identification method of the present application determines the alkali tolerance of crops at the seedling stage, which is a key stage of crop growth and development, and the alkali tolerance directly determines the seedling rate and subsequent growth basis of crops under alkali stress environment. The identification method of the present application can simultaneously determine the alkali tolerance of a large number of crop germplasms, and is rapid, simple and easy to operate. The alkali resistance of crop germplasms can be quickly and accurately determined according to the identification method of the present application. The present application provides an effective means for rapid identification of alkali-tolerant germplasms.

Description

Technical Field

[0001] This invention belongs to the field of stress-resistant crop breeding technology, specifically relating to a method for identifying crop alkali tolerance and its application. Background Technology

[0002] Soil salinization refers to the excessive accumulation of soluble salts or exchangeable sodium ions in the soil, leading to the deterioration of soil physicochemical properties and making it difficult for plants to grow. Soil salinization seriously affects agricultural production; therefore, the selection and promotion of salt-tolerant crop varieties are key to achieving the sustainable use of saline soils.

[0003] When crops are subjected to salt stress, the degree of damage depends not only on the salt concentration but also closely on the type of salt. Besides the known neutral salt Na+... + With Cl – In addition to ion imbalances caused by excessive accumulation, approximately 60% of saline soils have elevated pH levels due to their high NaHCO3 and Na2CO3 content, resulting in alkaline soils. Saline-alkaline soils limit the growth and yield of soybeans and other crops, and lead to biodiversity loss.

[0004] The root system is the primary organ for sensing and being affected by alkaline stress. High pH soil conditions inhibit root tip growth, affect root hair formation, and thus disrupt the rhizosphere microecological balance. Furthermore, when soil pH exceeds 8.5, auxin levels at the root tip decrease significantly, forcing root remodeling; altered acidic cell wall environments reduce cell wall relaxation, thereby limiting cell elongation. Simultaneously, high pH environments inhibit the activity of various cell wall modifying enzymes, hindering water absorption by roots and leading to the precipitation of metal ions such as iron, magnesium, and calcium, ultimately causing the roots to lose their normal physiological functions. In summary, alkaline stress is more harmful to crops than salt stress, and its mechanisms of action are more complex.

[0005] Currently, existing technologies have relatively complete methods for identifying salt tolerance, but methods for identifying alkali tolerance are relatively lacking. There is still a lack of unified standards for indoor alkali tolerance identification systems during the seedling stage. Existing alkali tolerance identification methods suffer from drawbacks such as complex operation, susceptibility to errors, and inability to fully reflect the alkali tolerance of plants, resulting in relatively slow progress in the identification and utilization of alkali-tolerant germplasm. Therefore, how to provide a standardized, rapid, simple, and easily applicable method for large-scale determination of the salt and alkali tolerance of germplasm has become an urgent problem to be solved in this field. Summary of the Invention

[0006] To address the lack of a standardized, rapid, simple, and large-scale method for determining the alkali tolerance of soybean germplasm in existing technologies, this invention provides a method for identifying crop alkali tolerance and its application, specifically including the following technical solution: A method for identifying crop alkali tolerance, comprising the following steps: Crops were sown in a planting substrate and divided into an alkali treatment group and a control group; An alkaline solution was added to the planting substrate of the alkali-treated group, while water was added to the planting substrate of the control group. The relative leaf area of ​​crops is counted during the seedling stage, and the alkali resistance of crops is evaluated based on the relative leaf area. The formula for calculating the relative leaf area is: leaf area of ​​the alkali-treated group / leaf area of ​​the control group.

[0007] In one embodiment, the solutes in the alkaline solution include NaHCO3 and Na2CO3, and the molar ratio of NaHCO3 to Na2CO3 is 8~10:0.1~2.

[0008] In one embodiment, the concentration of the alkaline solution, based on the total alkali content, is 30 mmol / L to 40 mmol / L.

[0009] In one implementation, the crop emergence period includes 4 to 12 days after the first crop seedling emerges.

[0010] As one implementation method, the evaluation of crop alkali tolerance includes: When the relative leaf area is ≥0.7 and <1, it is evaluated as highly alkali resistant; when the relative leaf area is ≥0.5 and <0.7, it is evaluated as alkali resistant; when the relative leaf area is ≥0.3 and <0.5, it is evaluated as sensitive; when the relative leaf area is ≥0 and <0.3, it is evaluated as highly sensitive.

[0011] In one embodiment, the planting substrate includes vermiculite, and the amount of alkali solution added in the alkali treatment group is the maximum water holding capacity of the added vermiculite; the control group is added with the same amount of water as the alkali solution group.

[0012] As one implementation, the crop is subjected to at least three biological replicates, and the leaf area of ​​the alkali treatment or the leaf area of ​​the control group is the average value of the biological replicates.

[0013] As one implementation method, both the alkali-treated group and the control group were cultured indoors; the indoor culture conditions were 12 h light / 12 h darkness, 25±2℃.

[0014] In one embodiment, the crop includes dicotyledonous plants, including soybeans.

[0015] This invention also provides the application of the identification method described above in alkali-tolerant crop breeding.

[0016] The beneficial effects of this invention are as follows: This invention provides a method for identifying crop alkali tolerance, comprising the following steps: sowing crops in a planting substrate, dividing them into an alkali treatment group and a control group; adding an alkali solution to the planting substrate of the alkali treatment group and adding water to the planting substrate of the control group; calculating the relative leaf area of ​​the crops at the seedling stage, and evaluating the alkali tolerance of the crops based on the relative leaf area; the formula for calculating the relative leaf area is: leaf area of ​​the alkali treatment group / leaf area of ​​the control group. This invention's identification method measures the alkali tolerance of crops at the seedling stage, a crucial stage in crop growth and development, where alkali tolerance directly determines the seedling rate and subsequent growth foundation under alkali stress. Using this invention's identification method, alkali tolerance can be simultaneously measured on a large number of crop germplasms, and the method is rapid, simple, and easy to operate. The judgment method according to this invention can quickly and accurately determine the alkali resistance of crop germplasm. This invention provides an effective means for the rapid identification of alkali-tolerant germplasm. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 Legend for leaf area and root system analysis systems: A is the ImageJ recognition and analysis image; B is the black and white binary image of leaf area; C is the root system recognition and analysis image; D is the black and white binary image of root system. Figure 2 Phenotypic characteristics of the aboveground parts of eight materials after 10 days of treatment with alkaline solutions of different concentrations; Figure 3 The phenotypes of eight materials treated with different concentrations of alkaline solution for 10 days were shown; where A is seedling rate; B is plant height; C is leaf area; D is aboveground fresh weight; and E is aboveground dry weight. The data were obtained through three biological replicates, with the error bar set as standard error (n=3). express P The significance level is <0.05. express P The significance level is <0.01; Figure 4 The underground phenotypes of eight materials after 10 days of treatment with alkaline solutions of different concentrations were determined. Among them, QN5 was Qinong No. 5, ZH357 was Zhonghuang 357, TN943 was Tongnong 943, SZH101 was Suizhonghuang 101, and TF31 was Tiefeng 31. Figure 5 The phenotypes of eight materials treated with different concentrations of alkaline solution for 10 days were shown. Among them, A is the fresh weight of the underground part; B is the dry weight of the underground part; C is the root length; D is the root surface area; E is the root volume; and F is the number of root tips. The data were obtained by three biological replicates, and the error bar is the standard error (n=3). express P The significance level is <0.05. express P The significance level is <0.01; Figure 6 Correlation analysis was conducted on seedling growth indicators under different concentrations of alkaline solution treatment; where A was 30 mmol / L; B was 35 mmol / L; and C was 40 mmol / L. express P The significance level is <0.05. express P The significance level is <0.01; Figure 7 This serves as an evaluation standard for the alkali tolerance of soybeans during the seedling stage. Figure 8 The differences in emergence phenotypes among eight soybean germplasms under salt-alkali stress; Figure 9 The differences between individual soybean germplasm plants in eight soybean accessions after 10 days of treatment under salt-alkali stress; Figure 10 Phenotypic results of eight soybean germplasm accessions after 10 days of salt-alkali stress treatment; where A represents root length and B represents average root diameter; data were obtained from three biological replicates, with the error bar representing standard error (n=3). express P The significance level is <0.05. express P The significance level is <0.01; Figures 8-10 Among them, G552 is Gong 552, ZH37 is Zhonghuang 37, CN52 is Changnong 52, ZH685 is Zhonghuang 685, HK60 is Heike 60, TN943 is Tongnong 943, JDD2 is Jidou 2, and TN15 is Tongnong 15. Detailed Implementation

[0019] This invention provides a method for identifying crop alkali tolerance, comprising the following steps: Crops were sown in a planting substrate and divided into an alkali treatment group and a control group. An alkali solution was added to the planting substrate of the alkali treatment group, and water was added to the planting substrate of the control group. The relative leaf area of ​​the crops was counted at the seedling stage, and the alkali tolerance of the crops was evaluated based on the relative leaf area. The formula for calculating the relative leaf area is: leaf area of ​​alkali treatment group / leaf area of ​​control group.

[0020] In one embodiment, the solutes in the alkaline solution include NaHCO3 and Na2CO3. In another embodiment, the molar ratio of NaHCO3 to Na2CO3 is 8-10:0.1-2. In yet another embodiment, the molar ratio of NaHCO3 to Na2CO3 can be any one of 8:0.1, 9:0.1, 10:0.1, 8:1, 9:1, 10:1, 8:2, 9:2, and 10:2, or an intermediate value between any two of these values. In a preferred embodiment, the molar ratio of NaHCO3 to Na2CO3 is 9:1. This invention, regarding the composition and ratio of the alkaline solution, can more realistically simulate the conditions of soda-alkali soil. Multiple alkaline components can produce a combined stress effect, causing serious damage to crops. A single-component alkaline solution cannot demonstrate the true damage of saline-alkali soil to plants. The limitations of this invention can screen for germplasm with resistance potential in real saline-alkali soil.

[0021] In one embodiment, the alkaline solution is prepared from sodium bicarbonate and sodium carbonate in a molar ratio of 9:1, with the total alkali content of the two solutes as the measurement standard, and a concentration gradient of 30 mmol / L to 40 mmol / L. The total alkali content refers to the total molar content of the two solutes, sodium bicarbonate and sodium carbonate. In one embodiment, the concentration of the alkaline solution, based on the total alkali content, can be any one of 30 mmol / L, 35 mmol / L, and 40 mmol / L, or an intermediate value between any two points. In a preferred embodiment, the concentration of the alkaline solution, based on the total alkali content, is 35 mmol / L. During the experiments of this invention, it was found that alkali treatments at concentrations of 35 mmol / L and 40 mmol / L can be used for identifying alkali tolerance in soybeans during the seedling stage. However, an alkali concentration of 40 mmol / L generally causes severe damage to the root system, leaf area, and other indicators of different germplasms, manifesting as root browning and a decrease in leaf area, among other damages. At this concentration, it is actually not conducive to distinguishing between alkali-tolerant and sensitive germplasms. Therefore, the present invention's limitation on the concentration of the alkaline solution comprehensively considers both stress intensity and applicability for identification. This not only reflects the crop germplasm's resistance to alkaline conditions but also avoids inaccurate evaluations caused by excessively high alkaline concentrations. As a preferred embodiment, after considering both stress intensity and applicability for identification, 35 mmol / L was ultimately determined as the suitable alkaline concentration for identifying soybean alkali tolerance at the seedling stage.

[0022] In one implementation, the crop emergence period is 4 to 12 days after the emergence of the first crop seedling. In another implementation, the crop emergence period can be any one of the following, or the midpoint between any two points: 4, 5, 6, 7, 8, 9, 10, 11, and 12 days after the emergence of the first crop seedling. In a specific embodiment, the crop emergence period is 10 days after the emergence of the first crop seedling. In another implementation, leaf area photography can be taken 10 days after the first crop seedling emerges, once the true leaves have unfolded. In another implementation, the emergence of the first crop seedling can begin after the emergence of the first soybean seedling in all treatment groups. In another implementation, the leaf area is the sum of the areas of all leaves in a top-down photograph of the crop after the true leaves have unfolded. In another implementation, the relative leaf area is detected using ImageJ.

[0023] As one implementation method, a relative leaf area ≥0.7 and <1 is evaluated as highly alkali-tolerant; a relative leaf area ≥0.5 and <0.7 is evaluated as alkali-tolerant; a relative leaf area ≥0.3 and <0.5 is evaluated as sensitive; and a relative leaf area between ≥0 and <0.3 is evaluated as highly sensitive. This invention has found through experiments that under alkali treatment conditions of 35-40 mmol / L, relative leaf area shows a significant positive correlation with relative root length, relative root surface area, relative root volume, relative root tip number, relative underground fresh weight, and relative underground dry weight. Therefore, the decrease in leaf area is selected as the core germplasm for evaluating crop alkali tolerance. In existing technologies, alkali tolerance of crops is usually determined by statistically analyzing indicators such as the fresh weight, dry weight, and root length of the aboveground and underground parts. However, crop roots are very fragile, and become even more fragile after alkali stress, making it easy to damage the integrity of the plant and cause errors during the separation process. This invention, through experimentation, has shown that the decrease in leaf area can significantly represent the alkali tolerance of crops, and is easy to statistically analyze with minimal error. The limitations of the alkali tolerance evaluation indicators in this invention solve the problems of time-consuming, labor-intensive, and error-prone alkali tolerance identification in existing technologies.

[0024] In one embodiment, the planting substrate includes vermiculite. In another embodiment, the amount of alkali solution added to the alkali treatment group is equal to the maximum water-holding capacity of the added vermiculite; the control group contains the same amount of water as the alkali solution group. This invention selects vermiculite as the planting substrate. Vermiculite has a porous structure with excellent water absorption and retention; its light and loose texture and high porosity provide sufficient oxygen for crop roots, preventing root suffocation and rot. Vermiculite is less prone to compaction when used as a planting substrate over a long period, which is beneficial for seedling root extension and root matting. Furthermore, vermiculite has poor thermal conductivity, which buffers diurnal temperature differences, ensuring stable substrate temperature and protecting seedling roots from low and high temperature stress, resulting in uniform emergence and growth.

[0025] In one implementation, the crop is subjected to at least three biological replicates, and the leaf area trait is taken as the average of the biological replicates. Limiting the biological replicates can reduce errors caused by chance.

[0026] In one implementation method, both the alkali-treated group and the control group were cultured indoors. The indoor culture conditions were 12 h light / 12 h darkness, 25 ± 2 ℃. In another implementation method, during indoor culture, water was added every 3 days. In another implementation method, using 8 cm × 8 cm × 8 cm flowerpots, the amount of water added was 50-100 mL / time / 3 days. This invention focuses on the critical growth stage of seedling emergence, using vermiculite seedling cultivation in a controlled indoor environment for identification. This reduces interference from external factors such as temperature and precipitation in the natural environment, achieving stability and repeatability of the identification results and improving the practicality of the identification method.

[0027] In one embodiment, the crop includes a dicotyledonous plant. In one embodiment, the dicotyledonous plant includes soybean.

[0028] Existing technologies disclose technical solutions for identifying crop salt and alkali tolerance using germination rate, taproot length, and root biomass as core indicators. However, in practice, calculating taproot length and root biomass requires meticulous, time-consuming, and labor-intensive operations, and can easily damage the integrity of the plant's root system, making rapid identification of large-scale germplasm difficult. Existing technologies also disclose techniques for treating soybeans with salt and alkali stress using salt and alkali solutions during the germination period, using seed germination rate as an indicator to evaluate soybean salt and alkali tolerance. However, seed germination refers to the process by which a crop initiates germination using its own reserves. Seedling establishment, on the other hand, is the independent growth and development of seedlings under stress conditions after germination. Seed germination and subsequent seedling establishment are two independent physiological stages. A seed's ability to germinate using its own stored nutrients under stress does not mean that the seed can continue to tolerate environmental stress while continuously relying on environmental nutrients. Therefore, the germination rate of a crop under stress conditions cannot directly reflect its seedling potential, nor can it systematically reflect the crop's tolerance to stress factors. This invention, through experiments, screened out alkali tolerance indicators that effectively reflect the seedling potential of soybeans under alkaline treatment conditions, are easy to identify, and have small errors, and provided accurate alkali tolerance judgment criteria. It solves the problem in existing technologies of lacking accurate, simple, rapid, and easily operable methods for alkali tolerance identification.

[0029] This invention also provides the application of the identification method described above in the breeding of alkali-tolerant crops.

[0030] In one embodiment, the application in alkali-tolerant crop breeding includes selecting alkali-tolerant crops using the identification method described in this invention for use in alkali-tolerant crop breeding. In one embodiment, the alkali-tolerant crop is a highly alkali-tolerant or alkali-tolerant crop.

[0031] As one implementation method, the identification method of the present invention can also be combined with existing salt tolerance identification methods to screen crops that have both salt and alkali tolerance properties.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for identifying crop alkali tolerance and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1: A method for identifying crop alkali tolerance The crop was sown in vermiculite and divided into an alkali treatment group and a control group. Each of the alkali treatment group and the control group was subjected to three biological replicates. An alkaline solution was added to the planting substrate of the alkaline treatment group until the vermiculite reached its maximum water-holding capacity; the alkaline solution contained NaHCO3 and Na2CO3 in a molar ratio of 9:1, and the concentration of the alkaline solution was 35 mmol / L based on the total alkali content.

[0034] Add the same amount of water as the alkali-treated group to the planting substrate of the control group; Leaf area was measured 10 days after the first crop emerged. The method was as follows: after the soybean true leaves unfolded, the soybean seedlings were photographed from above, and the sum of the leaf areas in the photos was measured using ImageJ software. The average of three biological replicates was taken as the leaf area, and the relative leaf area of ​​the crop was calculated. The alkali tolerance of the crop was evaluated based on the relative leaf area. The formula for calculating the relative leaf area is: leaf area of ​​the alkali-treated group / leaf area of ​​the control group.

[0035] The method for evaluating crop alkali tolerance is as follows: when the relative leaf area is ≥0.7 and <1, it is evaluated as highly alkali tolerant; when the relative leaf area is ≥0.5 and <0.7, it is evaluated as alkali tolerant; when the relative leaf area is ≥0.3 and <0.5, it is evaluated as sensitive; when the relative leaf area is ≥0 and <0.3, it is evaluated as highly sensitive.

[0036] Example 2: Screening of Soybean Alkali Tolerance Indicators Taking eight soybean varieties—Qinong 5 (QN5), Williams 82 (W82), Peking (Pek), Zhonghuang 357 (ZH357), Tiefeng 31 (TF31), NY27-38, Tongnong 943 (TN943), and Suizhonghuang 101 (SZH101)—as examples, a screening test on soybean alkali tolerance indicators was conducted to examine the tolerance of different soybean varieties to alkali solution concentrations and alkali tolerance indicators.

[0037] Select a number of plump soybean seeds and sow the eight soybean varieties mentioned above in flowerpots measuring 8 cm × 8 cm × 8 cm. Vermiculite was used as the planting substrate in each flowerpot. Nine soybean seeds were sown in each flowerpot. The sown soybeans were divided into three alkaline solution treatment groups and a control group. Each treatment for each soybean variety was performed at least three times biologically replicated.

[0038] The concentrations of the three alkaline solution treatment groups were 30, 35, and 40 mmol / L, respectively, based on the total alkali concentration. The pH values ​​of the solutions were measured, and the pH values ​​of all three groups were approximately 8.8. The alkaline solution was a mixture of NaHCO3 and Na2CO3 in a molar ratio of 9:1.

[0039] The amount of alkaline solution used in each alkaline solution treatment group was 200 mL per flowerpot.

[0040] The control group was given the same amount of water as the alkaline solution treatment group.

[0041] After treatment, all flowerpots were placed in the artificial climate chamber of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, for cultivation under the following conditions: 12 h light / 12 h dark, 25 ± 2 ℃. Each flowerpot was watered with 85 mL every 3 days after sowing.

[0042] Timing began from the emergence of the first soybean seedling (cotyledon hook emerging from the soil) in all treatment groups, with the number of seedlings observed daily. On day 10 after the first soybean seedling emerged, plant growth was observed by taking aerial photographs of the soybean seedlings. The sum of the leaf areas of the soybeans in the photos was measured using ImageJ software, and the average leaf area of ​​each plant was taken as the result. Illustrations of the photographs taken and the ImageJ-processed images are shown below. Figure 1 As shown in Figures A through B.

[0043] The height of the seedlings was measured 10 days after the first soybean seedling emerged, and the fresh weight of the above-ground and underground parts was also measured. The specific procedure was as follows: The soybean roots were carefully removed from each pot, rinsed thoroughly with clean water, and each plant's root system was placed in a scanner. The Wanshen LA-S root analysis system was used to calibrate and identify the plant's root system, and measurements of root length, root surface area, root volume, and other traits were performed. The results are shown below. Figure 1As shown in C~D.

[0044] Subsequently, the aboveground and underground parts of soybean were dried in an oven at 65℃ for 4 days, and the dry weight of the aboveground and underground parts was measured separately. The relative values ​​of growth indicators such as relative seedling rate (RSSR), relative leaf area (RLA), relative root length (RRL), relative aboveground fresh weight (RSFW), relative underground fresh weight (RRFW), relative aboveground dry weight (RSDW), and relative underground dry weight (RRDW) were calculated, and correlation analysis was performed.

[0045] Microsoft Excel 2021 was used to process and analyze the data, while Origin 2021 and GraphPadPrism 10 were used for correlation analysis and graphing. The specific formulas are as follows: Relative seedling survival rate (RSSR): RSSR = (Number of seedlings in the alkali-treated group / Number of seedlings in the control group) × 100%; Relative leaf area (RLA): RLA = average leaf area of ​​alkali-treated group / average leaf area of ​​control group; Relative plant height (RPH): RPH = Average plant height of alkali-treated group / Average plant height of control group; Relative shoot fresh weight: RSFW = Fresh shoot weight of alkali-treated group / Fresh shoot weight of control group; Relative shoot dry weight: RSDW = Dry shoot weight of alkali-treated group / Dry shoot weight of control group; Relative root fresh weight (RRFW): RRFW = Fresh weight of root in alkali-treated group / Fresh weight of root in control group; Relative root dry weight: RRDW = Dry weight of root underground in alkali-treated group / Dry weight of root underground in control group; Relative root length (RRL): RRL = Average root length of alkali-treated group / Average root length of control group; Relative root surface area (RRSA): RRSA = Average root surface area of ​​the alkali-treated group / Average root surface area of ​​the control group; Relative root volume (RRV): RRV = Average root volume of the alkali-treated group / Average root volume of the control group; Relative root average diameter (RRAD): RRAD = average root diameter of alkali-treated group / average root diameter of control group; Relative root tip number (RRTN): RRTN = Average root tip number in the alkali-treated group / Average root tip number in the control group; Salt tolerance index (SI); Where: t i Assign values ​​to the seedling condition of the salt-treated group; n i C represents the number of plants in each category within the salt treatment group; i The seedling condition of the control group is assigned a value; Ni is the number of plants of each category in the control group.

[0046] The seedling condition values ​​for each group were calculated using the methods published in the reference "Liu Xiexiang, et al. Establishment of a method for identifying salt tolerance in soybean seedlings and screening of salt-tolerant germplasm [J]. Acta Agronomica Sinica, 2020, (Issue 1)".

[0047] The emergence status of soybeans is displayed on Figure 2 The emergence rate, plant height, leaf area, aboveground fresh weight, and aboveground dry weight of the above-mentioned eight soybean varieties were statistically analyzed. Figure 3 And in Tables 1-5. Phenotypic characteristics of the underground root system are as follows: Figure 4 As shown in the figure. The statistical results of the fresh weight, dry weight, root length, root surface area, root volume, and number of root tips of the underground parts are as follows: Figure 5 As shown in Tables 6-11.

[0048] Table 1. Seedling survival rate (%) of 8 materials after 10 days of treatment with different concentrations of alkaline solution.

[0049] Table 2. Plant height (cm) of eight materials after 10 days of treatment with different concentrations of alkaline solution.

[0050] Table 3. Leaf area (cm²) of 8 materials after 10 days of treatment with different concentrations of alkaline solution. 2 )

[0051] Table 4. Fresh weight (g) of the aboveground parts of eight materials after 10 days of treatment with alkaline solutions of different concentrations.

[0052] Table 5. Dry weight (g) of the aboveground parts of eight materials after 10 days of treatment with alkaline solutions of different concentrations.

[0053] Table 6. Fresh weight (g) of the underground parts of eight materials after 10 days of treatment with alkaline solutions of different concentrations.

[0054] Table 7. Dry weight (g) of the underground portion of eight materials after 10 days of treatment with alkaline solutions of different concentrations.

[0055] Table 8. Root length (cm) of eight materials after 10 days of treatment with different concentrations of alkaline solution.

[0056] Table 9. Root surface area (cm²) of 8 materials after 10 days of treatment with different concentrations of alkaline solution. 2 )

[0057] Table 10 Root volumes (cm³) of eight materials after 10 days of treatment with different concentrations of alkaline solution. 3 )

[0058] Table 11. Number of root tips (units) of 8 materials after 10 days of treatment with alkaline solutions of different concentrations.

[0059] Note: In Tables 1-11, there are 3 sets of values ​​for each type of soybean, representing biological replicates of 3 flowerpots; each value is the average value of each germinated soybean plant in that flowerpot.

[0060] Depend on Figure 2 It can be seen that all 8 soybean samples were able to germinate normally.

[0061] Depend on Figure 3 As shown in Tables 1-5, there was no significant difference in the emergence rate between the alkali-treated group and the water control group, indicating that alkali stress treatment at concentrations of 30-40 mmol / L had no significant effect on the number of soybean seedlings.

[0062] Under 30 mmol / L alkaline treatment, the plant height and aboveground fresh weight of the eight materials were not significantly different from those of the control group. The leaf area and aboveground dry weight of QN5, W82, Pek, ZH357, TF31, and NY27-38 under alkaline treatment were not significantly different from those of the control group. The leaf area of ​​SZH101 and TN943 was significantly lower than that of the control group, and the aboveground dry weight of SZH101 was also significantly reduced.

[0063] Under 35 mmol / L alkali treatment, the alkali tolerance of different experimental materials showed significant differences. Except for TF31, Pek, and NY27-38, the plant height of the other five germplasms was significantly reduced; the aboveground fresh weight and dry weight of ZH357 were significantly lower than those of the control. Leaf area changes among different germplasms also showed significant differences. Except for QN5, Pek, TF31, and NY27-38, the leaf area of ​​the other four materials decreased significantly, with ZH357 and TN943 showing the largest decreases.

[0064] Under 40 mmol / L alkaline treatment, the plant height, aboveground fresh weight, and aboveground dry weight of QN5, SZH101, W82, ZH357, and TN943 were significantly lower than those of the control; except for NY27-38, the leaf area of ​​the other 7 germplasms was significantly reduced compared with the control.

[0065] Depend on Figure 4 It can be seen that as the alkali concentration increased, the underground parts of all eight germplasm materials decreased significantly.

[0066] Depend on Figure 5 As shown in Tables 6-11, under alkali treatment of 30 mmol / L, except for QN5 and NY27-38, the root length and root tip number of the other materials were significantly lower than those of the control group. The root surface area and root volume of SZH101, W82, and Pek were significantly lower than those of the control group. The fresh weight of the underground part of TF31 was significantly lower than that of the control group, but the dry weight of the underground part was not significantly different from that of the control group.

[0067] Under alkali treatment at 35 mmol / L, compared with the control, except for NY27-38, the root length, root surface area and root tip number of the other germplasms were significantly reduced. The fresh weight of the underground parts of SZH101, W82, ZH357, TF31 and TN943 was significantly reduced compared with the control, but the dry weight of the underground parts of W82, ZH357 and TF31 was not significantly different from the control.

[0068] Under 40 mmol / L alkaline treatment, the root length of all eight germplasms was significantly reduced compared to the control. Except for NY27-38, the fresh weight of the underground part, dry weight of the underground part, root surface area, root volume, and number of root tips of the other materials were significantly reduced compared to the control.

[0069] In summary, compared with the 30 mmol / L alkaline solution treatment, the 35 mmol / L and 40 mmol / L alkaline solution treatments further aggravated root damage and significantly inhibited growth and development, manifested as browning and necrosis of the taproot, yellowing, and a significant reduction in the number of lateral roots. The 40 mmol / L alkaline stress severely inhibited the normal growth and development of the aboveground parts and roots of soybean seedlings. Both 35 mmol / L and 40 mmol / L alkaline treatments can be used for identifying alkali tolerance in soybean seedlings. Considering both stress intensity and applicability, at a concentration of 40 mmol / L, the excessively high alkali concentration caused some damage to the plant phenotype, making it impossible to effectively distinguish between relatively alkali-tolerant and sensitive soybean germplasm. Therefore, 35 mmol / L was ultimately determined as the suitable alkali concentration for identifying alkali tolerance in soybean seedlings at the seedling stage.

[0070] Correlation analysis was performed on the alkali tolerance coefficients of 11 traits under three alkali concentration treatments using Origin 2021 software. The results are as follows: Figure 6 As shown.

[0071] Depend on Figure 6 It is evident that under 30 mmol / L alkali treatment, relative root length was significantly positively correlated with relative root surface area, relative root volume, and relative root tip number; relative aboveground fresh weight was significantly positively correlated with relative underground fresh weight and relative aboveground dry weight. Under 35 mmol / L alkali treatment, relative leaf area was significantly positively correlated with relative root length and relative underground dry weight; under 40 mmol / L alkali treatment, relative leaf area was significantly positively correlated with relative root length, relative root surface area, relative root tip number, relative underground fresh weight, and relative underground dry weight.

[0072] Figure 6 The correlation analysis of the three alkali treatments showed that relative leaf area and relative root traits exhibited a synchronous increase and decrease trend. At low alkali concentrations (30 mmol / L), changes in root-related traits were not significantly correlated with leaf area. However, with increasing alkali concentration, the correlation between root traits and leaf area gradually strengthened and reached a significant level. In the eight germplasm accessions screened in this invention, alkali stress was observed to have varying degrees of impact, indicating that inhibited root development under high-concentration alkali stress may be an important reason for the overall stunted growth and significant reduction in leaf area. Based on the above correlation analysis, relative leaf area can serve as an effective indicator for evaluating soybean alkali tolerance, and it can be measured non-destructively and rapidly, making it suitable for large-scale screening of alkali tolerance in germplasm resources.

[0073] In summary, through experiments, this invention found that 35 mmol / L alkali treatment can be used for identifying alkali tolerance in soybeans during the seedling stage, comprehensively considering both stress intensity and applicability. Furthermore, at this concentration, relative leaf area showed a significant positive correlation with relative root length and relative underground dry weight. Since relative leaf area is significantly correlated with other alkali tolerance indicators of crops, and this indicator is easy to observe, the relative leaf area of ​​soybeans under 35 mmol / L alkali treatment was used as an evaluation index for soybean alkali tolerance.

[0074] Among the eight soybean germplasms mentioned above, Qinong 5 exhibited significant alkali tolerance, while Tongnong 943 showed significant alkali sensitivity. Therefore, Qinong 5 and Tongnong 943 were used as alkali-tolerant and alkali-sensitive controls, respectively. Relative leaf area was used as the phenotype for evaluating alkali tolerance, and the relative leaf area value was used as the alkali tolerance coefficient. Combining the decrease in soybean leaf area under alkali stress, the degree of leaf wrinkling, and the growth status of true leaves, and using 0.3, 0.5, and 0.7 as critical values, the alkali tolerance coefficient was divided into four levels, as shown in Table 12. The alkali tolerance phenotypes of each level of soybean are as follows: Figure 7 As shown.

[0075] Table 12 Phenotypic characteristics of soybean seedling stage under alkali stress

[0076] This invention uses the relative leaf area at the soybean seedling stage as the core indicator for identifying alkali tolerance. This indicator clearly, accurately, and reliably reflects the soybean's tolerance to alkaline environments. Furthermore, the identification process is simple and unlikely to damage the soybean plant, thus minimizing the risk of inaccurate results. In summary, this invention provides a simple, rapid, easy-to-operate, and accurate indicator for identifying plant alkali tolerance.

[0077] Example 3: Validation of Salt-Tolerant Soybean Germplasm Identification Method This embodiment prepared 28 test materials, including 1 wild soybean NY27-38 and 27 cultivated soybeans. The test materials are shown below: Qinong 5, Jiyu 303, Shanning 33, Guochuangdou 78, Zhonglongdou 120, Wandou 43, Yundou 1, Zhonghuang 204, Zhonghuang 663, Zhonghuang 37, Tongnong 943, Dongsheng 140, Zhonghuang 357, Qihuang 34 (Qihuang34), Zhonghuang205, Peking, Suizhonghuang101, Williams82, Tiefeng31, NY27-38, Zhonghuang685, Heike60, Jidadou2, Tongnong15, Dongnongdou254, Dongnongdou255, Gong552, Changnong52.

[0078] The soybean germplasm was provided by the Soybean Genetic Resources Research Group of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. Salt and alkali tolerance of the tested materials was determined. The aforementioned Gong 552 is also known as G552.

[0079] 1. Methods for determining salt tolerance of crops during the seedling stage: Select 54 plump seeds from each soybean variety for later use.

[0080] Prepare 28 sets of flowerpots, each for planting one of the 28 soybean varieties mentioned above. Each set contains 6 replicates of flowerpots, each measuring 8 cm × 8 cm × 8 cm. Add vermiculite to the flowerpots as a seedling substrate, and then sow 9 seeds in each flowerpot.

[0081] After sowing, 24 flowerpots were placed in a large blue box for the following treatment: In each group of 6 flowerpots, 3 served as the control group and the other 3 as the salt treatment group; the control group flowerpots were placed in one large blue box, and the salt treatment group flowerpots were placed in another large blue box. The control group flowerpots were treated with water (200 mL per pot), while each pot in the salt treatment group was watered with an equal volume of a 150 mmol / L NaCl solution to maximize the water holding capacity of the vermiculite.

[0082] After treatment, the flowerpots were placed in an artificial climate chamber for cultivation under the following conditions: 12 hours of light / 12 hours of darkness, 25±2℃. Each flowerpot was watered with 85 mL every 3 days. On the 10th day after sowing, the salt damage was investigated using a single-plant classification and recording method. Individual plants were divided into four categories, assigned values, and the salt tolerance index of each material was calculated to determine the salt tolerance level and salt tolerance of the crop.

[0083] The single-plant classification method and the calculation method of salt tolerance index are described in the literature “Liu Xiexiang, et al. Establishment of salt tolerance identification method for soybean seedling stage and screening of salt-tolerant germplasm. Acta Agronomica Sinica, 2020, 46(01): 1–8.”

[0084] The salt tolerance level and salt tolerance results of each soybean material at the emergence period are shown in Table 2.

[0085] 2. Methods for determining alkali tolerance of crops during seedling emergence: Select 108 plump seeds from each soybean variety for later use.

[0086] Prepare 28 sets of flower pots, each used to plant one of the 28 soybean germplasms mentioned above. Each set of flower pots contains 6 pots measuring 8 cm × 8 cm × 8 cm. Add vermiculite to the flower pots as a seedling substrate, and then sow 9 seeds in each flower pot. Three flower pots in each set serve as the control group, and three flower pots serve as the alkali treatment group.

[0087] After sowing, flowerpots from the control group and the alkali-treated group were placed in a large blue box. The control group was treated with water, with each flowerpot receiving 200 mL of water. The alkali-treated group simulated soda-salt soil conditions. The reagent for the alkali-treated group was a 35 mmol / L alkali solution prepared by mixing NaHCO3 and Na2CO3 in a 9:1 molar ratio, based on the total alkali concentration.

[0088] After treatment, all the large blue boxes were placed in the artificial climate chamber of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, for cultivation under the following conditions: 12 h light / 12 h dark, 25 ± 2 ℃. Each pot was watered with 85 mL of water every 3 days after sowing.

[0089] The alkali resistance of 28 soybean varieties was rated according to the alkali resistance rating criteria established in Table 12 of Example 2. The evaluation results are shown in Table 13.

[0090] 3. Methods for identifying salinity in crop seedlings The salt-tolerant near-isogenic line 820-3 (carrying the salt-tolerant haplotype GmSALT3) and the salt-sensitive near-isogenic line 820-4 (carrying the sensitive haplotype Gmsalt3), previously bred by the applicant, were used as the salt-tolerant control and the salt-sensitive control, respectively. These two near-isogenic lines are disclosed in the prior art “Guan R, et al. Salinity tolerance in soybean is modulated by natural variation in GmSALT3[J]. The Plant Journal, 2014, 80(6): 937-950.”

[0091] Twenty-seven plump seeds were selected from each soybean variety, and 28 sets of flowerpots were prepared for planting each of the 28 soybean varieties. Each set consisted of three replicates of flowerpots, each measuring 8 cm × 8 cm × 8 cm. Vermiculite was added to the flowerpots as a seedling substrate, and then nine seeds were sown in each flowerpot.

[0092] Twenty-four small flowerpots were placed in a large blue box. On days 9 and 12 after sowing, each flowerpot was treated with 85 mL of 200 mmol / L NaCl. On day 15 after NaCl treatment, the salt damage level of each material was investigated, and the degree of leaf necrosis was assessed according to a 5-level classification method. The results are shown in Table 13.

[0093] The five-level classification method refers to the existing technology "Liu Xiexiang, et al. Establishment of salt tolerance identification method for soybean seedling stage and screening of salt-tolerant germplasm [J]. Acta Agronomica Sinica, 2020, (No. 1)."

[0094] Table 13 Salt and alkali tolerance of 28 soybean germplasms

[0095] As shown in Table 13, among the 28 germplasms, 2 germplasms exhibited the characteristics of Level 1 high alkali tolerance, 10 germplasms exhibited the characteristics of Level 2 alkali tolerance, 7 germplasms exhibited the characteristics of Level 3 sensitivity, and 9 germplasms exhibited the characteristics of Level 4 high sensitivity.

[0096] In addition, the salinity of 28 soybean germplasm accessions at the emergence stage was determined using 150 mmol / L NaCl solution, and the salt tolerance at the seedling stage was determined using 200 mmol / L NaCl solution. A total of 13 highly salt-tolerant materials at the emergence stage (Grade 1) and 12 highly salt-tolerant materials at the seedling stage (Grade 1) were identified.

[0097] This shows that there are significant differences in the effects of salt stress and alkali stress on soybean seedling growth during the emergence period. Among the 28 germplasm accessions mentioned above, Qinong 5, Jiyu 303, Shanning 33, Peking, Suizhonghuang 101, Heike 60, and Dongnongdou 254 exhibit both salt and alkali tolerance during the emergence period.

[0098] Based on the above identification results, eight soybean varieties with different salt and alkali tolerances were selected as research subjects: G552, ZH37, CN52, ZH685, HK60, TN943, JDD2, and TN15. Correlation analysis of salt and alkali tolerance was conducted. The results are as follows: Figure 8 , Figure 9 Figure 10 As shown in Tables 14 and 15.

[0099] Table 14 Root length (cm) of 8 soybean germplasms after 10 days of treatment under salt-alkali stress.

[0100] Table 15. Average root diameter (mm) of eight soybean germplasms after 10 days of treatment under salt-alkali stress.

[0101] Depend on Figures 8-10 As shown in Tables 14-15, salt-alkali stress not only inhibits the aboveground growth of soybeans but also significantly affects root morphology, with varying degrees of damage among different materials. Salt stress significantly inhibits soybean growth rate, leading to a decrease in seedling rate and plant height. Alkali stress primarily inhibits aboveground morphogenesis and leaf development, causing wrinkled and deformed leaves, rod-shaped growth, and reduced photosynthetic area, thereby affecting plant nutrient accumulation and growth. Furthermore, both salt and alkali stress significantly inhibit the total root length of soybeans, but the inhibitory effect of alkali stress is more pronounced. Under salt stress, except for ZH685, the average root diameter of all materials was not significantly different from the control group. However, under alkali stress, except for HK60, the average root diameter of the other five materials was significantly higher than the control. These results indicate that under alkali stress, soybeans can compensate for the loss of root surface area due to reduced lateral roots by increasing the average root diameter, thereby maintaining a certain capacity for water and nutrient absorption and sustaining plant growth.

[0102] In summary, there is no significant correlation between salt tolerance and alkali tolerance in soybeans at the seedling stage, indicating that soybeans may have differentiated stress regulation mechanisms to cope with neutral and alkaline salt stress. Regarding the setting of stress conditions, this invention simulates the main components of saline-alkali soil by mixing NaHCO3 and Na2CO3 in a 9:1 molar ratio as an alkaline solution sample and determining an appropriate stress concentration gradient. The technical solution of this invention not only solves the problem of significant differences between single salt stress and natural saline-alkali environments in the field, but also effectively distinguishes the differences in alkali tolerance among different soybean germplasms through precise concentration gradient settings, overcoming the limitations of existing technologies that only address single alkaline salt stress.

[0103] Furthermore, this invention focuses on the critical growth stage of seedling emergence, employing a vermiculite seedling cultivation method under controlled indoor conditions for identification. This reduces interference from external factors such as temperature and precipitation in the natural environment, ensuring the stability and reproducibility of the identification results. Simultaneously, this method has a shorter identification cycle, meeting the needs of large-scale screening of soybean germplasm resources for alkali tolerance. In addition, this invention simultaneously applies salt and alkali stress treatments to soybean seedlings during the emergence stage, clarifying the phenotypic differentiation characteristics of salt-tolerant and alkali-tolerant soybean germplasm. This effectively compensates for the deficiency of "emphasizing salt over alkali" in soybean stress tolerance identification work, providing technical support for the screening of alkali-tolerant soybean germplasm and research on alkali tolerance mechanisms.

[0104] Salt stress and alkali stress are two major abiotic stresses that inhibit soybean growth and development in saline-alkali soils. Both significantly inhibit plant growth by disrupting ion balance and inducing oxidative stress. However, due to significant differences in the nature and mechanism of action of these stresses, the degree, manifestation, and response strategies of plants under salt stress and saline-alkali slopes are quite different. For example, under high pH alkali stress (pH > 10), no signs of seed germination were observed, while under moderate pH alkali stress (9 < pH < 10), seeds could only complete the radicle breaking through the seed coat and could not develop further. However, seeds treated with 0.6% and 1.2% NaCl salt stress could complete radicle emergence, indicating that salt stress inhibited radicle development less than alkali stress. The results of this invention also confirm that salt stress and alkali stress have significant differences in their effects on soybean growth and development during the emergence period. Salt stress mainly inhibits soybean emergence rate and growth rate, with relatively minor damage to the root system; alkali stress, on the other hand, has a significant inhibitory effect on root growth and development. The above differences are closely related to the distinct mechanisms of salt and alkali stress. Under salt stress, high concentrations of NaCl lead to increased soil osmotic pressure, hindering water absorption during soybean seed germination. Simultaneously, NaCl... + and Cl - Accumulation within the plant body leads to ion toxicity to cells, manifesting as decreased germination rate, leaf yellowing, and reduced plant height. High concentrations of carbonate ions (CO3-) in alkaline soils further exacerbate this problem. 2- ) and bicarbonate ions (HCO3) -This leads to increased soil alkalinity, damages the integrity of root cell membranes, inhibits root cell division and differentiation, affects the formation of lateral root primordia, and reduces soil Fe content. 3+ Ca 2+ Mg 2+ The availability of nutrients hinders root nutrient absorption, resulting in short, thick roots and sparse lateral roots. Insufficient nutrient supply also impairs the growth of the aboveground parts, leading to a decrease in leaf area. This invention clarifies the organ-specific response characteristics of soybean seedlings to these two stresses, providing new insights into the physiological mechanisms of soybean salt-alkali tolerance.

[0105] Example 4 Verification of Salt and Alkali Tolerance of Soybean Germplasm In this embodiment, 41 soybean germplasms were prepared, and their salt and alkali tolerance was determined according to the salt and alkali tolerance determination method described in Example 3. The results are shown in Table 16.

[0106] Gong552, Qinong5, Jiyu303, Shanning33, Shengdou139, Guocha ngdou78), Shidou45, Zhonglongdou120, Qingtiandou819, Qinong115, Wandou43 Wandou43), Yundou1, Heyan51, Zhonghuang204, Zhonghuang663, Nongqingdou249, Zhonghuang37, Tongnong943, Dongsheng140, Wangdou311, Zhonghuang357 Zhonghuang357, Zhonghuang392, Qingnongdou2308, Qihuang57, Qihuang34, Zhongyandou1, Jiyu3012, Zhonghuang205, Peking, Jiyu3545, Suizhonghuang101, Williams82, Tiefeng31, NY27-38, Changnong52, Zhonghuang685, Heike60, Jidadou2, Tongnong15, Dongnongdou254, Dongnongdou255.

[0107] Table 16 Salt and alkali tolerance of 41 soybean germplasms

[0108] As can be seen from the above, the salt and alkali tolerance determination method described in Example 3 of this application can accurately and reproducibly determine the salt and alkali tolerance of soybean germplasm, and the test results of different batches show good stability. This invention provides an accurate and simple method for determining salt and alkali tolerance.

[0109] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for identifying crop alkali tolerance, characterized in that the steps include... include: Crops were sown in a planting substrate and divided into an alkali treatment group and a control group; An alkaline solution was added to the planting substrate of the alkali-treated group, while water was added to the planting substrate of the control group. The relative leaf area of ​​crops is counted during the seedling stage, and the alkali resistance of crops is evaluated based on the relative leaf area. The formula for calculating the relative leaf area is: leaf area of ​​the alkali-treated group / leaf area of ​​the control group.

2. The identification method as described in claim 1, characterized in that, The solutes in the alkaline solution include NaHCO3 and Na2CO3, and the molar ratio of NaHCO3 to Na2CO3 is 8~10:0.1~2.

3. The identification method as described in claim 1 or 2, characterized in that, The concentration of the alkaline solution, based on the total alkali content, is 30 mmol / L to 40 mmol / L.

4. The identification method as described in claim 1, characterized in that, The crop emergence period includes 4 to 12 days after the first crop seedling emerges.

5. The identification method as described in claim 1, characterized in that, The evaluation of crop alkali tolerance includes: When the relative leaf area is ≥0.7 and <1, it is evaluated as highly alkali resistant; when the relative leaf area is ≥0.5 and <0.7, it is evaluated as alkali resistant; when the relative leaf area is ≥0.3 and <0.5, it is evaluated as sensitive; when the relative leaf area is ≥0 and <0.3, it is evaluated as highly sensitive.

6. The identification method as described in claim 1, characterized in that, The planting substrate includes vermiculite, and the amount of alkali solution added in the alkali treatment group is the maximum water holding capacity of the added vermiculite; the control group is added with the same amount of water as the alkali solution group.

7. The identification method as described in claim 1, characterized in that, The crop was subjected to at least three biological replicates, and the leaf area of ​​the alkali treatment or the leaf area of ​​the control group was the average of the biological replicates.

8. The identification method as described in claim 1, characterized in that, Both the alkali-treated group and the control group were cultured indoors; the indoor culture conditions were 12 h light / 12 h darkness, 25±2℃.

9. The identification method according to any one of claims 1 to 8, characterized in that, The crop includes dicotyledonous plants, including soybeans.

10. The application of the identification method according to any one of claims 1 to 9 in alkali-tolerant crop breeding.

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