A method for identifying salt tolerance of forage grass based on asexual reproduction

Abstract

The present invention discloses a method for identifying salt tolerance of forage grass based on asexual reproduction. The operating system uses a plug tray as a device, peat soil as a medium, and a bottom box as a carrier for nutrient solution and salt solution. The system is not only low-cost and easy to operate, but also can easily separate the medium-solution, which is extremely beneficial for replacing the nutrient solution and salt solution in the bottom box, and can maintain the stability of salt stress and nutrient components in the medium over a long period of time. The present invention uses the selection position of the seed stem and the treatment method after cutting as two main experimental parameters for controlling the growth consistency after cutting, significantly improving the consistency of the growth rate and growth period of the seed stem after asexual reproduction. The developed ALN counting method has clear classification, simple statistics, good data repeatability, and high correlation with multiple salt tolerance traits such as ion content, greatly improving the efficiency of salt tolerance determination. At the same time, the ALN extreme value ranges of different species under salt stress are similar and comparable, and it is an optimal universal "antenna trait" for cross-species salt tolerance comparison.

Description

Technical Field

[0001] The invention belongs to the technical field of forage germplasm resource identification, and particularly relates to a method for identifying salt tolerance of forage based on asexual reproduction. Background Art

[0002] Currently, major grain crops have limited salt tolerance, making the cultivation and ecological application of salt-tolerant forage grasses a key area of ​​saline-alkali land improvement. Major crops such as rice, wheat, corn, and soybeans struggle to achieve high and stable yields on saline soils with a salinity of 4‰, hindering the promotion of these crops in saline-alkali land agriculture. Salt-tolerant forage grasses, such as seashore paspalum, salsa grass, hybrid pennisetum, and miscanthus, can thrive on saline soils with a salinity of 4‰. Some of these grasses can even tolerate seawater irrigation, making them advantageous species for saline-alkali land improvement. They offer an effective new solution for ecological restoration and soil improvement in saline-alkali lands along my country's eastern coast and northwest.

[0003] However, most salt-tolerant forage grasses are strictly self-incompatible, resulting in no seed offspring or low seed activity, and thus rely primarily on asexual propagation through cuttings such as seed stems. The current major salt-tolerance evaluation systems all use sexually propagated seed offspring as test material, which is incompatible with the asexual propagation method of forage grasses. This results in low accuracy in salt-tolerance identification of asexually propagated forage grasses, hindering the potential for salt-tolerant forage grasses to be used in saline-alkali lands. Furthermore, in asexual propagation, the main factors affecting the consistency of forage growth after cuttings are unclear, and the growth of cutting offspring is uneven, further increasing the error in salt-tolerance identification and reducing the repeatability and accuracy of the data.

[0004] In the currently disclosed salt tolerance identification system, the salt concentration in different media is unstable, resulting in uneven salt stress intensity throughout the plant growth cycle. In the hydroponic salt tolerance identification system, it is necessary to rely on frequent solution replacement and feeding equipment to maintain the salt concentration, pH and oxygen content in the nutrient solution, which is not only cumbersome but also costly. In the salt tolerance system with soil as the medium, the soil is often irrigated by adding salt once or gradually. The salt concentration distribution in the soil and the salt stress intensity cannot be accurately controlled during the plant growth cycle.

[0005] In addition, the key indicators for salt tolerance screening are still unclear. Physiological indicators after salt stress (such as proline, malondialdehyde) and ion content (such as Na + and K + The measurement of salt tolerance traits is complex, time-consuming, and costly. Measuring phenotypic traits such as plant height and biomass is relatively simple, but is affected by many factors such as species type and leaf size. This not only fails to clearly represent the actual salt tolerance of different germplasm resources, but also makes it difficult to unify the extreme ranges of data across species. Therefore, developing "antenna" salt tolerance traits that are simple to measure, highly correlated with salt tolerance, and comparable across species is key to conducting large-scale salt tolerance screening of germplasm resources.

[0006] This technology addresses the defects in the current salt tolerance identification system and has developed a low-cost, easy-to-operate, and highly accurate dynamic salt tolerance evaluation system. It provides a core "antenna" indicator for salt tolerance identification and can accurately identify the salt tolerance of both asexually and sexually reproducing plants. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the first object of the present invention is to provide a method for identifying salt tolerance of forage germplasm resources based on asexual reproduction.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a method for identifying salt tolerance of forage germplasm resources based on asexual reproduction, comprising the following steps: Figure 1 As shown:

[0010] (1) Seedling hole tray and forage grass stem cuttings: Use a seedling hole tray with a bottom box and a seedling tray as the device. After slits are made on both sides of each hole in the seedling tray, fill it with peat matrix mixed with equal weight nutrient solution, immerse it in a bottom box with 4 times the weight of nutrient solution and let it absorb for 2-5 hours. Figure 1 -A. Select the grass seed stem with top leaves, cut 6-10cm downward from the starting point between the top growth point and the first stem node as the cutting material for asexual propagation, as shown in Figure 1. Figure 1 -B, Segment 2. Plant the grass cuttings in the tray substrate at a density of 1-9 stems per hole, leaving the aboveground portion at a depth of 2-5 cm. Transfer the seedling trays, along with the bottom box, to a dark environment at 4-10°C for 6-10 days before transferring them to light culture.

[0011] (2) Add salt stress and replace salt solution: When the cuttings grow to the stage of one leaf expansion, add sodium chloride solute of corresponding salt concentration directly to the bottom box solution, shake evenly at low speed on a horizontal shaker, and continue to culture under light until the end of the experiment. During this period, replace the salt solution every 4 days; before replacing the solution, first add water to each seedling hole tray until the initial weight, and empty all the solution in the bottom box after 2-5 hours of absorption. Then add nutrient solution containing corresponding salt concentration to the bottom box to the initial weight. After shaking evenly, the salt solution replacement is completed. Figure 1 -C. After 20-30 days of salt addition, the light culture was terminated and the plant height (PH), average leaf number (ALN), biomass, and ion Na + / K + / Ca 2+ Statistics of salt tolerance traits such as Figure 1 -D as shown.

[0012] (3) Forage salt tolerance curve fitting and parameter calculation: The salt tolerance traits after six sodium chloride stress treatments of 0, 100, 200, 300, 400, and 500 mM were used as data sets, and the mathematical model Sigmoid three-parameter Perform fitting to obtain y0, a, and b values, such as Figure 1 -E; where a is the maximum value of the y variable, b is the steepness of the fitting curve, and x0 represents the maximum half-activation level of the fitting curve. First, use the formula Derivative Y max Value, using the inverse function Calculate when Y max The corresponding X value on the fitting curve when it drops halfway is defined as Salt 50 , that is, the half-survival concentration of forage under salt stress, such as Figure 1 -F as shown.

[0013] In the preferred technical solution of the present application, the seedling tray is preferably a tray with 12 holes and a depth of 5 cm, the peat soil is preferably a Pindstrup (0-6 mm) soil matrix, the nutrient solution is preferably a Poly-Food water-soluble fertilizer from Haifa, Israel, prepared at a ratio of 1:1000, the seed stem length is preferably 6 cm, the post-cutting treatment conditions are preferably refrigerated at 7°C in the dark for 8 days, and the salt tolerance trait is preferably the average leaf number (ALN) for parameter calculation of the fitting curve.

[0014] In a specific embodiment, in step (1), the seed stem is selected at a position of 6-7 cm downward from the point between the top growth point and the first stem node.

[0015] In a specific embodiment, in step (1), the post-cutting treatment method is to store the cuttings in the dark at 7-8°C for 6-8 days.

[0016] The selection position of the above-mentioned seed stem nodes and the post-cutting processing method are the decisive experimental parameters for the consistency of asexual reproduction growth of forage grass.

[0017] In a specific embodiment, the ALN counting method is specifically as follows: the leaves growing from the seed stem after cutting are counted, the leaf sheath growing from the top incision is scored as 0.2, the leaf sheath extending out of the heart leaf tip is scored as 0.5, the first heart leaf is half-expanded as 0.8, and the first heart leaf is fully expanded into a true leaf as 1.0. Figure 2 -A; the second lobe is half expanded and recorded as 1.5, the second lobe is fully expanded and recorded as a true leaf as 2.0; and so on, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 as shown in Figure 2 -B. Other leaf morphologies between the scale bars are recorded as values ​​between the scale bars.

[0018] In a specific embodiment, the salt tolerance trait is average leaf number (ALN) for performing parameter calculation of the fitting curve.

[0019] Beneficial effects

[0020] The present invention develops a forage salt tolerance identification system based on asexual reproduction. The operating system uses a plug tray as a device, peat soil as a medium, and a bottom box as a carrier of nutrient solution and salt solution. The system is not only low-cost and easy to operate, but also can easily separate the medium and solution, which is extremely beneficial for the replacement of nutrient solution and salt solution in the bottom box. It can maintain the stability of salt stress and nutrient components in the medium over a long period of time, thereby providing a simple, stable and efficient experimental platform for the accurate identification of forage salt tolerance.

[0021] The present invention uses the selection position of the seed stem and the treatment method after cutting as two main experimental parameters to control the growth consistency after cutting, significantly improving the consistency of the growth rate and growth period of the seed stem after asexual reproduction, especially during the salt addition period of one leaf expansion; through the control of the two key experimental parameters, almost all the cutting seed stems can reach one leaf expansion after 6 days of cultivation, providing a relatively consistent growth starting point for salt stress, and also significantly improving the repeatability of the determination of forage traits after salt stress, further reducing experimental errors.

[0022] The ALN counting method developed in this invention has clear classification, simple statistics, good data repeatability, and high correlation with multiple salt tolerance traits such as ion content, which greatly improves the efficiency of salt tolerance determination. At the same time, the ALN extreme value ranges between different species under salt stress are similar and comparable. It is the best universal "antenna trait" for cross-species salt tolerance comparison and has become the key to large-scale salt tolerance screening of forage germplasm resources.

[0023] The present invention takes ALN as the target salt tolerance trait, uses mathematical equations to fit the cold resistance curve and uses Salt 50 The value represents the final salt tolerance, which can more accurately and comprehensively reflect the salt tolerance curve characteristics of different forage germplasms compared with single salt stress treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a salt tolerance identification system for forage grasses based on asexual reproduction. A 12-hole plug tray with a bottom box and a transparent plastic cover was used as the device to carry 100g of peat soil and 500ml of nutrient solution ( Figure 1 -A), cuttings of seashore paspalum upright stems of Seg2 morphology were made at a density of 9 plants per hole (Step 1). Seg2 morphology was selected as follows Figure 1 -B. Cover with plastic and place in a dark place at 7℃ for 8 days (Step 2), then transfer to 26 / 18℃, 14 / 10h long daylight conditions for cultivation (Step 3). When the cuttings grow to the stage of one leaf expansion, add sodium chloride solute with the corresponding salt concentration to the solution at the bottom of the box (Step 4) to start salt stress. Replace the salt solution every 4 days (Step 5). The replacement method is as follows Figure 1-C. The experiment was terminated after 20 days of salt stress culture (Step 6). After measuring plant height and ALN traits, the roots were cleaned and the roots, stems, and leaves were separated, dried, and digested to determine biomass and Na + , K + , Ca 2+ Ion content ( Figure 1 -D). The properties were measured using the three-parameter Sigmoid model for curve fitting (Step 7), and the salt tolerance curve parameter Salt was calculated based on the curve characteristics. 50 (Step 8).

[0025] Figure 2 The chart below shows the comparison of ALN counts for forage grasses. During the one-leaf period, the number of times the leaf sheath grows out of the top incision, the number of times the leaf sheath extends out of the heart leaf tip, the number of times the first heart leaf is half-expanded, and the number of times the first heart leaf is fully expanded into a true leaf are recorded as 0.2, 0.5, 0.8, and 1.0, respectively. Figure 2 -A. The second heart lobe is half expanded and recorded as 1.5, the second heart lobe is fully expanded and recorded as a true leaf as 2.0; and so on, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 as shown. Figure 2 -B.

[0026] Figure 3 The identification process of salt tolerance of forage grass and its error-determining parameters. Figure 3 -A is the final experimental process for forage tolerance identification. Figure 3 -B is the Na of the solution in the bottom box before changing water 4 times in the experimental process + and K + Ion concentration heat map, including Na + Ion conversion to salt concentration, K + The ion concentration is the ICP peak intensity. Figure 3 -C is a comparison of the CV values ​​of plant height and ALN data by the two factors of pretreatment (S / NS) and stem node position (Seg1 / Seg2 / Seg3), S=stratification, NS=nostratification, Seg1=segment1, Seg2=segment2, Seg3=segment3; ns=notsignificant; **p<0.01 (t-test); different letters represent p<0.05 (Tukey HSD); uppercase and lowercase letters represent different significance calculations. Figure 3 -D represents the cutting seedlings after six salt stress treatments of S0, S100, S200, S300, S400, and S500 mM NaCl. The scale is 10 cm. Figure 3 -E is the fitted dynamic curve of plant height and ALN data after six salt stress treatments.

[0027] Figure 4Comparison of fitting curves of salt tolerance traits of forage grasses and their correlation. Figure 4 -A is the plant height, ALN, and leaf Na of seashore paspalum, cool-season and warm-season forage grasses + and K + Salt tolerance fitting curves of ion content under six salt stress concentrations. Figure 4 -B is the correlation matrix of the 11 measured phenotypes, p < 0.005; PH = plant height, Bm = biomass, R = root, S = stem, L = leaf. Figure 4 -C is plant height and ALN and leaf Na + Significant linear regression of ion content, p < 0.05.

[0028] Figure 5 To compare and verify the characteristic parameters of salt tolerance curve of forage grass. Figure 5 -A is plant height and ALN curve parameter Salt 50 Significant comparisons between different types of forage; Figure 5 -B is 48 self-pollinated progenies of seashore paspalum SP3 and Figure 5 -ALN salt tolerance parameter Salt in A 50 Comparison of PV and WS; *p < 0.05, ****p < 0.00001. PV = seashore paspalum genotype, WS = warm-season forage, CS = cool-season forage. Figure 5 -C All Seashore Paspalum (52) ALN-Salt 50 comparison. Figure 5 -D is the PCA analysis diagram of other measured traits except ALN trait of 48 self-pollinated progenies of seashore paspalum SP3. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to the examples. Reagents or instruments used without manufacturer's indication are considered to be conventional products that can be purchased on the market.

[0030] (1) Forage grass stem cuttings and seed propagation

[0031] Test Materials: Asexually propagated forage materials included seashore paspalum Adalayd and its mutant strains SP2, SP3, and SPD1. SP3 was the nationally approved variety Sunongke No. 1 (variety registration number: 605). Sexually propagated forage materials included corn grass (variety: Huafeng No. 3), pennisetum (variety: Late-maturing American Pennisetum), sweet sorghum (variety: Kaka), barley (variety: Morex), oats (variety: Baiyan No. 2), rye (variety: Dongmu 70), and ryegrass (variety: Haiyan).

[0032] The upright stem of seashore paspalum was used as the seed stem. The seed stem of the forage grass with top leaves was selected and cut 6 cm downward from the starting point between the top growth point and the first stem node (Segment 2, Figure 1 -B), as the cutting material for asexual propagation; at the same time, cut 6 cm downward from the top growth point (Segment 1, Seg 1, Figure 1 -B) and cut 6 cm from the second stem node (Segment 3, Seg 3, Figure 1 Seven kinds of forage grass seeds were sequentially washed with 7.5% hydrogen peroxide for 0.5 h, 3% hydrogen peroxide for 2 h, 2% sodium hypochlorite for 0.5 h, and sterile water for 0.5 h for later use.

[0033] A salt tolerance experimental device was constructed using a germination box with a bottom box, a 12-hole tray, and a transparent plastic cover, and Pindstrup (0-6 mm) peat soil as the soil medium. Figure 1 -A), mix 100g of dry substrate with 100ml of nutrient solution (soluble fertilizer: water at a ratio of 1:1000) to fill a 12-hole plug tray with openings on both sides, and immerse it in a bottom box filled with 400ml of the same nutrient solution to absorb for 2h.

[0034] Seashore paspalum seed cuttings were evenly placed into the holes at a density of 9 per hole, leaving 2 cm of the above-ground portion. Sterilized forage grass seeds were evenly placed on the soil surface at a density of 3 per hole, with the embryo facing upward. Covered with a transparent plastic lid, the cells were refrigerated at 7°C in the dark for 8 days (stratification, S). Afterwards, the cells were transferred to a long-day light cycle of 26 / 18°C with a 14 / 10 h photoperiod. A control cultured under light conditions without pre-treatment (non-stratification, NS) was also established.

[0035] In an experiment on the uniformity of growth of upright stem cuttings of the seashore paspalum variety Adalayd, it was found that the S treatment after 8 days of cold storage had no significant effect on the coefficient of variation (CV) of plant height traits, but it could significantly reduce the CV value of ALN, thereby significantly improving the uniformity of growth after cuttings ( Figure 3 -C). In the two-factor experiment of pretreatment (S / NS) and stem node position (Seg1 / Seg2 / Seg3), the CV values ​​of the S-Seg2 treatment were the lowest in both plant height and ALN traits, indicating that the combination of cold storage for 8 days and Seg2 stem node can significantly improve the growth consistency of forage cuttings ( Figure 3 -C), and therefore are considered as the two main test parameters for the growth uniformity of asexually propagated forage grass cuttings.

[0036] (2) Pasture salt stress process

[0037] When the cuttings and germinated seeds have grown to the one-leaf stage (seaside paspalum requires 6 days of cultivation, cool-season forage grasses require 4 days, and warm-season forage grasses require 2 days), salt stress is added by dissolving the corresponding sodium chloride solute into the nutrient solution in the bottom box. Five salt stress treatments of 100, 200, 300, 400, and 500 mM sodium chloride are set, and no sodium chloride addition (0 mM) is used as the control.

[0038] The salt solution in the bottom box is replaced every four days. The specific replacement method is: before replacing the solution, first add water to each seedling tray bottom box until it reaches the initial weight. After 2-5 hours of absorption, empty all the solution in the bottom box, and then add nutrient solution containing the corresponding salt concentration to the bottom box to the initial weight. The salt solution replacement is completed. + and K + Ion content determination Figure 3 -B shows Na under salt stress + Elements and macronutrients K that represent nutritional conditions + The conditions remained stable without significant fluctuations throughout the salt stress cycle, indicating that this experimental setup and its associated liquid exchange method can stably maintain the salt concentration and nutrient levels in the medium.

[0039] The fitting curve coefficients of the phenotypic indicators of the seashore paspalum variety Adalayd under salt stress for 24 days (Day After Salt, DAS) at different salt concentrations are as follows: Figure 3 -F indicates that the fitting coefficients of the two phenotypic indices under salt stress reached their maximum values ​​after 20 days of DAS. After 20 days of DAS, the light culture was terminated and the phenotypic and ion content traits were statistically analyzed.

[0040] After measuring the height and average leaf number (ALN) of the grass, the roots of the grass under different salt concentrations were cleaned ( Figure 3 -D) and the roots, stems, and leaves were separated and dried to determine the dry weight. After digesting the grass sample with 5 ml of nitric acid as the medium, the solution was filtered to a constant volume with a 0.45 μm filter head and the Na + , K + , Ca 2+ Average leaf number (ALN) counting method: count the leaves growing from the seed stem after cutting. The leaf sheath growing from the top incision is recorded as 0.2, the leaf sheath extending out of the heart leaf tip is recorded as 0.5, the first heart leaf is half-expanded as 0.8, and the first heart leaf is fully expanded into a true leaf as 1.0. Figure 2 -A; the second lobe is half expanded and recorded as 1.5, the second lobe is fully expanded and recorded as a true leaf as 2.0; and so on, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 as shown in Figure 2 -B. Other leaf morphologies between the scale bars are recorded as values ​​between the scale bars.

[0041] (3) Forage cold resistance curve fitting and parameter calculation

[0042] The phenotypic data and ion content after 6 treatments of 0, 100, 200, 300, 400, and 500 mM sodium chloride during salt stress period are used as the Y variable data set, and the corresponding 0, 100, 200, 300, 400, and 500 mM sodium chloride are used as the X variable data set. The mathematical model Sigmoid three-parameter Perform fitting, such as Figure 3 -E. Representative indicators include plant height, ALN, leaf Na + and K + The ion content can be fitted into a dynamic curve, such as Figure 4 -A. Only the fitting curve of ALN trait can use the same Y-axis scale among different forage species. The data extreme value ranges of other traits cannot be unified, indicating that only ALN is a universal phenotypic trait across species. In the correlation matrix of 11 traits, plant height and ALN have significant high correlation with the ion content representing salt tolerance, such as Figure 4 -B. Plant height and ALN and leaf Na + The linear regression of ion content is as follows Figure 4 -C shows the two and the leaf Na representing the salt stress level + There was a significant negative correlation between the ion content.

[0043] To further obtain the three parameters a, b and x0 values ​​of the mathematical model Sigmoid, first use the formula Derivative Y max Value, then use the inverse function Calculate when Y max The corresponding X value on the fitting curve when it drops halfway is defined as Salt 50 Salt 50 The larger it is, the stronger its salt tolerance is.

[0044] (4) Screening of core “antenna” curve indicators for forage salt tolerance

[0045] Salt of the salt tolerance fitting curves of plant height and ALN traits 50 Calculations were performed to compare parameters of different forage types. Figure 5 -A. Salt calculated from the fitting curve of plant height 50 The differences among different species were significantly higher than those of ALN, and the Salt calculated from the ALN curve was 50 There were significant differences only between seashore paspalum and other forages, and no significant differences between cool-season and warm-season forages, which was more consistent with the actual salt tolerance of forages.

[0046] The 48 self-pollinated progenies of the SP3 mutant of seashore paspalum were used as research materials. Figure 3 -A complete salt tolerance process was tested to determine the phenotypic traits such as plant height, ALN, dry weight and ion content ( Figure 1 -D), and found that 48 self-pollinated offspring of ALN-Salt 50 There was no significant difference between Adalayd, SP2, SP3 and SPD1, but it was significantly higher than that of cold and warm season forages ( Figure 5 -B); the extreme genotypes PV17 and PV74 ( Figure 5 -C), also appears in PCA results other than ALN ​​data ( Figure 5 -D), further confirming that ALN and its curve parameter Salt50 can be used as core indicators for salt tolerance screening.

[0047] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A method for identifying salt tolerance of forage germplasm resources based on asexual reproduction, characterized in that: The method comprises the following steps: (1) Seedling hole tray and forage grass stem cuttings: Use a seedling hole tray with a bottom box and a seedling tray as a device, make slits on both sides of each hole in the seedling tray, fill it with peat substrate mixed with equal weight nutrient solution, immerse it in a bottom box with 4 times the weight of nutrient solution and let it absorb for 2-5 hours; select forage grass stems with top leaves, and cut 6-10 cm downward from the starting point between the top growth point and the first stem node as the cutting material for asexual reproduction; plant forage grass cuttings in the hole tray substrate with a seed stem density of 1-9 roots per hole and a depth of 2-5 cm on the aboveground part. Transfer the seedling hole tray together with the bottom box to a dark place at 4-10°C for 6-10 days, and then transfer it to light culture; (2) Adding salt stress and replacing salt solution: When the cutting seedlings grow to the stage of one leaf expansion, add sodium chloride solute of corresponding salt concentration directly to the bottom box solution, shake evenly at low speed on a horizontal shaker, and then continue to culture under light until the end of the experiment. During this period, replace the salt solution every 4 days; before replacing the solution, first add water to each seedling hole tray until the initial weight, and empty all the solution in the bottom box after 2-5 hours of absorption. Then add nutrient solution containing corresponding salt concentration to the bottom box to the initial weight, and shake evenly to complete the salt solution replacement; terminate the light culture 20-30 days after adding salt and conduct statistics on the salt tolerance of the average number of leaves; (3) Forage salt tolerance curve fitting and parameter calculation: The salt tolerance traits after six sodium chloride stress treatments of 0, 100, 200, 300, 400, and 500 mM were used as data sets, and the mathematical model Sigmoid three-parameter Perform fitting to obtain y0, a, and b values; where a is the maximum value of the y variable, b is the steepness of the fitting curve, and x0 represents the maximum half-activation level of the fitting curve; first use the formula Derivative Y max Value, using the inverse function Calculate when Y max The corresponding X value on the fitting curve when it drops halfway is defined as Salt 50 , that is, the half-survival concentration of forage under salt stress; In step (1), the seed stem is selected by cutting 6-7 cm downward from the area between the top growth point and the first stem node; In the step (2), the average leaf number counting method is specifically as follows: counting is performed based on the morphology of leaves grown from the seed stem after cutting, with the leaf sheath growing out of the top incision being recorded as 0.2, the leaf sheath extending out of the heart leaf tip being recorded as 0.5, the first heart leaf being half-expanded being recorded as 0.8, the first heart leaf being fully expanded into a true leaf being recorded as 1.0; the second heart leaf being half-expanded being recorded as 1.5, the second heart leaf being fully expanded into a true leaf being recorded as 2.0; and so on.

2. The method for identifying salt tolerance of forage germplasm resources based on asexual reproduction according to claim 1, characterized in that: The seedling tray is a tray with 12 holes and a depth of 5 cm.

3. The method for identifying salt tolerance of forage germplasm resources based on asexual reproduction according to claim 1, characterized in that: The peat matrix is ​​a Pindstrup soil matrix.

4. The method for identifying salt tolerance of forage germplasm resources based on asexual reproduction according to claim 1, characterized in that: The nutrient solution was prepared with Poly-Food water-soluble fertilizer from Haifa, Israel at a ratio of 1:1000.

5. The method for identifying salt tolerance of forage germplasm resources based on asexual reproduction according to claim 1, characterized in that: In the step (1), the post-cutting treatment method is to store the cuttings in the dark at 7-8°C for 6-8 days.

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