A salt stress method for identifying salt tolerance of alfalfa

Abstract

The invention discloses a salt stress method for identifying salt tolerance of alfalfa, and relates to the technical field of salt tolerance identification. The salt stress method for identifying salt tolerance of alfalfa comprises the following steps: collecting soil leaching solutions from saline-alkali land plots with different salinity and alkali degrees, and determining the total salt content and pH value of each soil leaching solution; placing soil leaching solutions and cultivation substrates with different salt concentrations in potted containers of preset specifications, and sowing alfalfa; monitoring the growth and development of alfalfa under salt stress, and determining the index parameters of different varieties of alfalfa; and comprehensively evaluating the salt tolerance of each variety of alfalfa based on the index parameter analysis results. The present invention has the effect of significantly improving the accuracy of identification relative to the salt stress method of artificially simulated single salt and compound salt, thereby strongly supporting the accurate and rapid screening of salt-tolerant alfalfa varieties, and has important application value for ensuring high-yield, stable-yield and efficient planting of alfalfa in saline-alkali land.

Description

Technical Field

[0001] The invention relates to the technical field of salt tolerance identification, in particular to a salt stress method for identifying salt tolerance of alfalfa. Background Art

[0002] my country's saline-alkali land is widely distributed, with five saline-alkali areas, namely the eastern coastal areas, the Huanghuaihai Plain, the Northeast Plain, the northwest inland areas, and the Xinjiang and Qinghai regions. More than 30% of the saline-alkali land is distributed in the northwest, northeast, and coastal areas. After years of management and cultivation, most of the mild saline-alkali land has been converted to farmland. The remaining moderate and severe saline-alkali land has high soil salt content, poor structure, shallow groundwater and high mineralization. In addition, most areas are seriously lacking in freshwater resources, resulting in low utilization of moderate and severe saline-alkali land and poor production capacity of traditional crops.

[0003] Alfalfa is a perennial herbaceous leguminous plant and the most widely distributed cultivated forage grass in the world today. It has a cultivation history of more than 2,000 years in my country, and its main production areas are in Northwest China, North China, Northeast China, and the Yangtze and Huaihe River Basin. Alfalfa has a well-developed root system and strong nitrogen fixation ability. It is resistant to salt, alkali, drought, and barrenness. It is also rich in nutritional value. It is a high-quality forage grass favored by all kinds of livestock and poultry and is known as the "king of forage grass". Planting alfalfa in moderate and severe saline-alkali land can not only effectively solve the contradiction of "grass and grain competing for land", but also improve saline-alkali land to increase soil fertility, significantly improve the ecological environment, promote agricultural quality and efficiency and increase farmers' income, and achieve efficient utilization of saline-alkali land.

[0004] To plant alfalfa in moderate and severe saline-alkali land, in order to obtain high, stable and efficient yields, the first thing is to screen varieties with strong salt-alkali tolerance. The sensitive period of alfalfa to salt-alkali stress is mainly the germination period-seedling period. Therefore, the identification of salt tolerance of alfalfa varieties is particularly important, especially the identification of salt tolerance in the germination period-seedling period. At present, almost all international and domestic indoor salt tolerance identification of alfalfa varieties in the seedling stage uses artificially configured single salt sodium chloride solution or sodium chloride + sodium sulfate compound salt solution. In fact, the salt damage of plants such as alfalfa on saline-alkali land mainly comes from soil leaching solution, and the salt composition of soil leaching solution in saline-alkali land is relatively complex. The salt stress on alfalfa is the result of the joint action of multiple salt components. Therefore, the results of the indoor identification of single salt sodium chloride solution or chloride and sulfate compound salt solution are often quite different from the actual salt tolerance in the field. That is to say, the artificial simulation of salt stress of single salt or compound salt is not very accurate, which has caused trouble for the accurate screening of salt-alkali tolerant alfalfa varieties. In addition, the salt tolerance of alfalfa varieties can be identified at the seedling stage through field planting in saline-alkali land. The results are relatively accurate, but the time is long, generally at least 2 years or more. Therefore, it is often impossible to provide the urgently needed salt-alkali tolerant alfalfa varieties for production in a more timely manner.

[0005] Currently, no effective solution has been proposed for the problems in the related technologies. Summary of the invention

[0006] 1. Technical issues to be solved

[0007] In view of the deficiencies in the prior art, the present invention provides a salt stress method for identifying the salt tolerance of alfalfa, which has the advantage of quickly and accurately identifying the salt tolerance of alfalfa varieties at the seedling stage, thereby solving the problem that the salt stress method of artificially simulated single salt or compound salt is not accurate enough.

[0008] (II) Technical solution

[0009] In order to achieve the above-mentioned advantage of rapid and accurate identification of salt tolerance of alfalfa varieties at the seedling stage, the specific technical scheme adopted by the present invention is as follows:

[0010] A salt stress method for identifying salt tolerance of alfalfa, comprising the following steps:

[0011] S1. Collect soil leaching solutions from saline-alkali land plots with different salinity levels, and measure the total salt content and pH value of each soil leaching solution;

[0012] S2, placing soil leaching solutions and cultivation substrates with different salt concentrations in potted containers of preset specifications, and sowing alfalfa;

[0013] S3. Monitor the growth and development of alfalfa under salt stress and determine the index parameters of different varieties of alfalfa;

[0014] S4. Analyze the index parameters of alfalfa, and conduct a comprehensive evaluation of the salt tolerance of each variety of alfalfa based on the index parameter analysis results.

[0015] Preferably, collecting soil leaching solutions from saline-alkali land plots with different salinity levels, and determining the total salt content and pH value of each soil leaching solution comprises the following steps:

[0016] S11. Select saline-alkali lands with different salinity levels from the planned alfalfa planting area, and select several groups of representative saline-alkali land plots from each saline-alkali land using a chessboard-like distribution method;

[0017] S12, using a soil leaching solution collection device to collect soil leaching solutions from each saline-alkali land plot, respectively, and putting the collected soil leaching solutions into containers for collection and marking;

[0018] S13. Evenly mix the soil leaching solutions in the containers collected from saline-alkali land plots with the same salinity, and measure the total salt content and pH value of each soil leaching solution.

[0019] Preferably, the salinity of the saline-alkali land includes mild, moderate and severe salinity;

[0020] Among them, the total content of soluble salt in the cultivated layer of slightly saline-alkali land is 0.1% to 0.2%, the total content of soluble salt in the cultivated layer of moderately saline-alkali land is 0.2% to 0.4%, and the total content of soluble salt in the cultivated layer of severely saline-alkali land is 0.4% to 0.6%.

[0021] Preferably, a soil leaching solution with different salt concentrations and a cultivation substrate are placed in a potting container of preset specifications, and sowing alfalfa comprises the following steps:

[0022] S21, selecting a potted plant container of preset specifications;

[0023] S22, dividing the potted plant containers into a stress group and a control group, wherein the stress group is added with several groups of soil leaching solutions with salt gradients, and the control group is added with distilled water;

[0024] S23, filling each potting container with vermiculite, and respectively adding soil leaching solution and distilled water into each potting container to make the water content of the vermiculite reach 20-25%;

[0025] S24. Select different varieties of alfalfa seeds and sow them inside potting containers.

[0026] Preferably, each potting container is filled with vermiculite, and soil leaching solution and distilled water are added to each potting container to make the water content of the vermiculite reach 20-25%, comprising the following steps:

[0027] S231, filling each potting container with vermiculite;

[0028] S232, adding several groups of soil leaching solutions with salt gradients into the potted plant containers of the stress group, and adding distilled water into the potted plant containers of the control group, so that the water content of the vermiculite reaches 20% to 25%;

[0029] S233. Under the premise of no outflow of water, distilled water is added by weight every day according to the evaporation of water in the vermiculite in the potting container to ensure that the water content of the vermiculite is maintained at 20% to 25% and the concentration of the soil leaching solution remains unchanged.

[0030] Preferably, monitoring the growth and development of alfalfa under salt stress and determining the index parameters of different varieties of alfalfa include the following steps:

[0031] S31. Monitoring the growth and development of alfalfa under salt stress, and measuring the index parameters of different varieties of alfalfa at the 5-leaf stage;

[0032] S32, respectively selecting the third middle leaf from the top downward from the control and each salt stress treated alfalfa seedling, measuring the fluorescence parameters of each leaf, collecting 0.3 g of the leaf and adjacent leaves, and measuring the antioxidant enzyme activity of each leaf;

[0033] S33, 10 alfalfa seedlings were randomly selected from the control and each salt stress treatment to measure plant height;

[0034] S34. After the leaf index and plant index determination were completed, the number of live seedlings of the control and each salt stress treatment was counted, and all above-ground alfalfa plants of the control and each salt stress treatment were cut, placed in an oven at 105°C for 30 minutes, and then placed at 70°C for drying and weighing.

[0035] Preferably, the leaf indicators include SPAD value, fluorescence parameters measured by a chlorophyll fluorescence meter, superoxide dismutase activity, peroxidase activity and catalase activity; the plant indicators include plant height, number of live seedlings and aboveground biomass.

[0036] Preferably, analyzing the index parameters of alfalfa and comprehensively evaluating the salt tolerance of each variety of alfalfa based on the index parameter analysis results comprises the following steps:

[0037] S41. Determine the correlation between the measured indexes of alfalfa and salt tolerance, the measured indexes including leaf indexes and plant indexes;

[0038] S42. If the measured index of alfalfa is positively correlated with salt tolerance, the membership function value of each measured index is calculated using the positive correlation membership function;

[0039] S43. If the measured index of alfalfa is negatively correlated with salt tolerance, the membership function value of each measured index is calculated using the negative correlation membership function;

[0040] S44, using the standard deviation coefficient method to calculate the standard deviation coefficient of each measurement index, and calculating the weight coefficient of each measurement index based on the standard deviation coefficient;

[0041] S45, calculating the comprehensive evaluation value of each measurement indicator based on the weight coefficient of each measurement indicator;

[0042] S46. Determine the salt tolerance of each variety of alfalfa based on the comprehensive evaluation value, and classify the salt tolerance of alfalfa into grades.

[0043] Preferably, the calculation formula of the standard deviation coefficient is:

[0044]

[0045] Where V j Represents the standard deviation coefficient of the jth measurement index;

[0046] X ij represents the membership function value of the jth measured index of the i-th variety of alfalfa;

[0047] represents the average value of the jth measured index;

[0048] n represents the total number of measured indicators.

[0049] Preferably, the calculation formula of the comprehensive evaluation value is:

[0050]

[0051] In the formula, D represents the comprehensive evaluation value;

[0052] μ(x j ) represents the membership function value of the jth measurement index;

[0053] x j represents the jth measurement index parameter;

[0054] W j represents the weight coefficient of the jth measurement index;

[0055] n represents the total number of measured indicators.

[0056] (III) Beneficial effects

[0057] Compared with the prior art, the present invention provides a salt stress method for identifying salt tolerance of alfalfa, which has the following beneficial effects:

[0058] The salt stress method for identifying the salt tolerance of alfalfa provided by the present invention can realize the accurate and rapid identification of the salt tolerance of alfalfa varieties in the seedling stage, and the variety identification accuracy rate reaches more than 96%. Compared with the salt stress method of artificially simulated single salt and compound salt, it has the effect of significantly improving the identification accuracy, thereby strongly supporting the accurate and rapid screening of salt-alkali tolerant alfalfa varieties, and has important application value and promotion value for ensuring high-yield, stable yield and efficient planting of alfalfa in saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0060] Figure 1 The present invention is a flowchart of a salt stress method for identifying salt tolerance of alfalfa according to an embodiment of the present invention. DETAILED DESCRIPTION

[0061] To further illustrate each embodiment, the present invention provides drawings, which are part of the disclosure of the present invention and are mainly used to illustrate the embodiments. They can be used in conjunction with the relevant descriptions in the specification to explain the specific principles of the embodiments. With reference to these contents, ordinary technicians in this field should be able to understand other possible implementation methods and advantages of the present invention.

[0062] According to an embodiment of the present invention, a salt stress method for identifying salt tolerance of alfalfa is provided.

[0063] The present invention is further described with reference to the accompanying drawings and specific embodiments. Figure 1 As shown, according to the salt stress method for identifying salt tolerance of alfalfa in an embodiment of the present invention, the salt stress method for identifying salt tolerance of alfalfa comprises the following steps:

[0064] S1. Collect soil leaching solutions from saline-alkali land plots with different salinity levels, and measure the total salt content and pH value of each soil leaching solution.

[0065] The steps of collecting soil leaching solutions from saline-alkali land plots with different salinity levels and determining the total salt content and pH value of each soil leaching solution include:

[0066] S11. Select saline-alkali lands with different salinity levels from the planned alfalfa planting area, and select several groups of representative saline-alkali land plots from each saline-alkali land using the chessboard method.

[0067] Specifically, in the planned alfalfa planting area, three types of saline-alkali land, namely light, moderate and severe, were selected, and three representative plots were selected from the light, moderate and severe saline-alkali lands, for a total of nine plots, and the area of ​​each plot was not less than 1 mu.

[0068] Specifically, the total soluble salt content of the cultivated layer soil in slightly saline-alkali land is 0.1% to 0.2%, the total soluble salt content of the cultivated layer soil in moderate saline-alkali land is 0.2% to 0.4%, and the total soluble salt content of the cultivated layer soil in severely saline-alkali land is 0.4% to 0.6%.

[0069] S12. Using a soil leaching solution collection device, collect soil leaching solutions from each saline-alkali land plot, and put the collected soil leaching solutions into containers, collect them, and mark them.

[0070] Specifically, a soil leaching solution collection device is used to collect soil leaching solutions from 0 to 50 cm soil layers in each plot. The number of collection points in each plot is determined according to the amount of soil leaching solution used. The soil leaching solutions collected from light, moderate and heavy saline-alkali plots are respectively put into different containers and marked.

[0071] S13. Evenly mix the soil leaching solutions in the containers collected from saline-alkali land plots with the same salinity, and measure the total salt content and pH value of each soil leaching solution.

[0072] Specifically, the collected soil leaching solutions are brought back to the laboratory, and the soil leaching solutions collected from lightly, moderately, and heavily saline-alkali land plots are respectively placed into different large containers and mixed, and the total salt content is determined and marked for later use.

[0073] S2. Put soil leaching solutions and cultivation substrates with different salt concentrations into potting containers of preset specifications, and sow alfalfa.

[0074] The method of placing soil leaching solutions and cultivation substrates with different salt concentrations in a potted container of preset specifications and sowing alfalfa includes the following steps:

[0075] S21, selecting a potted plant container of preset specifications;

[0076] S22, dividing the potted plant containers into a stress group and a control group, wherein the stress group is added with several groups of soil leaching solutions with salt gradients, and the control group is added with distilled water;

[0077] S23, filling each potting container with vermiculite, and respectively adding soil leaching solution and distilled water into each potting container to make the water content of the vermiculite reach 20-25%.

[0078] Wherein, step S23 comprises the following steps:

[0079] S231, filling each potting container with vermiculite;

[0080] S232, adding several groups of soil leaching solutions with salt gradients into the potted plant containers of the stress group, and adding distilled water into the potted plant containers of the control group, so that the water content of the vermiculite reaches 20% to 25%;

[0081] S233. Under the premise of no outflow of water, distilled water is added by weight every day according to the evaporation of water in the vermiculite in the potting container to ensure that the water content of the vermiculite is maintained at 20% to 25% and the concentration of the soil leaching solution remains relatively unchanged.

[0082] S24. Select different varieties of alfalfa seeds and sow them inside potting containers.

[0083] Specifically, in actual application, soil leaching solutions with different salt concentrations are placed in potted containers of preset specifications to sow alfalfa, which mainly includes:

[0084] Choice of potted container: It is advisable to use a porcelain flower pot with a height of no less than 20cm, a bottom diameter of no less than 10cm, and a mouth diameter of no less than 12cm.

[0085] Fill with vermiculite: Fill the pot with vermiculite.

[0086] Add soil leaching solution: Divide the flower pots into a stress group and a control group, and add the corresponding solution into each potting container to make the vermiculite water content reach 20% to 25%. The specific stress treatment process is as follows:

[0087] According to the total salt content and pH value of the soil leaching solution, the stress group was set to three gradients, namely mild stress (0.1%-0.2%), moderate stress (0.2%-0.4%) and severe stress (0.4%-0.6%). The mild stress added a soil leaching solution with a total salt content of 0.15%, the severe stress added a soil leaching solution with a total salt content of 0.32%, and the severe stress added a soil leaching solution with a total salt content of 0.51%. The solution added to the control group was distilled water.

[0088] Sowing: Select alfalfa seeds with a purity of more than 98% and a germination rate of more than 95%. Plant 5 pots for each treatment and sow 20 seeds in each pot.

[0089] Stress management: Under the premise of no outflow of water, distilled water is added by weight every day according to the evaporation of vermiculite water in the potting container to ensure that the water content of vermiculite is maintained at 20%-25% and the concentration of soil leaching solution remains relatively unchanged.

[0090] S3. Monitor the growth and development of alfalfa under salt stress and determine the index parameters of different varieties of alfalfa.

[0091] Among them, monitoring the growth and development of alfalfa under salt stress and determining the index parameters of different varieties of alfalfa include the following steps:

[0092] S31. Monitoring the growth and development of alfalfa under salt stress, and measuring the index parameters of different varieties of alfalfa at the 5-leaf stage;

[0093] S32, respectively, from the control and each salt stress treatment of alfalfa seedlings, select the third middle leaf from the top downward, after measuring the fluorescence parameters of each leaf, collect about 0.3g of the leaf and surrounding leaves and bring them back to the laboratory to measure the antioxidant enzyme activity of the leaves;

[0094] S33, randomly select 10 alfalfa seedlings from the control and each salt stress treatment to measure plant height (measure all surviving plants if there are less than 10 plants);

[0095] S34. After the leaf index and plant index determination were completed, the number of live seedlings of the control and each salt stress treatment was counted, and all above-ground alfalfa plants of the control and each salt stress treatment were cut, placed in an oven at 105°C for 30 minutes, and then placed at 70°C for drying and weighing.

[0096] Among them, the measurement indicators of alfalfa include leaf indicators and plant indicators. Leaf indicators include SPAD value, fluorescence parameters measured by chlorophyll fluorescence meter, superoxide dismutase activity, peroxidase activity and catalase activity; plant indicators include plant height, number of live seedlings and aboveground biomass.

[0097] Specifically, in practical applications, monitoring the growth and development of alfalfa under salt stress and measuring the index parameters of different varieties of alfalfa mainly include:

[0098] The relevant indexes were measured when alfalfa was at the 5-leaf stage.

[0099] Three alfalfa seedlings were randomly selected for each variety control and salt stress treatment, and the SPAD value, initial fluorescence (Fo), maximum fluorescence yield (Fm), and maximum photochemical efficiency (Fv / Fm) of the third middle leaf were measured from the top to the bottom. After the fluorescence index measurement was completed, about 0.3 g of the measured leaf and surrounding leaves were collected and brought back to the laboratory, and the superoxide dismutase activity (SOD), peroxidase activity (POD), and catalase activity (CAT) of the leaves were measured using activity detection kits.

[0100] Ten alfalfa seedlings were randomly selected for each variety control and salt stress treatment, and the plant height (PD) was measured using a ruler.

[0101] After the determination of leaf and plant indices, the number of live seedlings of the control and salt stress treatments was counted, and all the above-ground plants of alfalfa of the control and salt stress treatments were cut, put into kraft paper bags and placed in an oven. After being sterilized at 105°C for 30 minutes, they were placed in an environment of 70°C and dried to constant weight. The dry weight was weighed to an accuracy of 0.001g.

[0102] S4. Analyze the index parameters of alfalfa, and conduct a comprehensive evaluation of the salt tolerance of each variety of alfalfa based on the index parameter analysis results.

[0103] The steps of analyzing the index parameters of alfalfa and comprehensively evaluating the salt tolerance of each alfalfa variety based on the index parameter analysis results include:

[0104] S41. Determine the correlation between the measured indexes of alfalfa and salt tolerance, the measured indexes including leaf indexes and plant indexes;

[0105] S42. If the measured index of alfalfa is positively correlated with salt tolerance, the membership function value of each measured index is calculated using the positive correlation membership function.

[0106] Specifically, the calculation formula of the positive correlation membership function is:

[0107] μ(x j )=(xj -x min ) / (x max -x min );

[0108] In the formula, μ(x j ) represents the membership function value of the jth measurement index, where j = 1, 2, ..., n;

[0109] x j represents the jth measurement index parameter;

[0110] x min represents the minimum value of the jth measurement index parameter;

[0111] x max Indicates the maximum value of the jth measurement index parameter.

[0112] S43. If the measured index of alfalfa is negatively correlated with salt tolerance, the membership function value of each measured index is calculated using the negative correlation membership function.

[0113] Specifically, the calculation formula of the negative correlation membership function is:

[0114] μ(x j )=(x max -x j ) / (x max -x min );

[0115] S44. Calculate the standard deviation coefficient of each measurement index using the standard deviation coefficient method, and calculate the weight coefficient of each measurement index based on the standard deviation coefficient.

[0116] The calculation formula of the standard deviation coefficient is:

[0117]

[0118] Where V j Represents the standard deviation coefficient of the jth measurement index;

[0119] X ij represents the membership function value of the jth measured index of the i-th variety of alfalfa;

[0120] represents the average value of the jth measured index;

[0121] n represents the total number of measured indicators.

[0122] The calculation formula of the weight coefficient is:

[0123]

[0124] Where Wj represents the weight coefficient of the jth measurement index;

[0125] V j Represents the standard deviation coefficient of the jth measurement index;

[0126] n represents the total number of measured indicators.

[0127] S45. Calculate the comprehensive evaluation value of each measurement index based on the weight coefficient of each measurement index, determine the salt tolerance of each alfalfa variety, and rank the salt tolerance of each alfalfa variety according to the comprehensive evaluation value.

[0128] The calculation formula of the comprehensive evaluation value is:

[0129]

[0130] In the formula, D represents the comprehensive evaluation value;

[0131] μ(x j ) represents the membership function value of the jth measurement index;

[0132] x j represents the jth measurement index parameter;

[0133] W j represents the weight coefficient of the jth measurement index;

[0134] n represents the total number of measured indicators.

[0135] S46. The salt tolerance of each alfalfa variety is determined based on the comprehensive evaluation value, and the salt tolerance of alfalfa is graded. The salt tolerance grading standards for alfalfa varieties are shown in Table 1.

[0136] Table 1: Classification standards for salt tolerance of alfalfa varieties

[0137]

[0138] The salt stress method for identifying salt tolerance of alfalfa provided by the present invention can realize accurate and rapid identification of salt tolerance of alfalfa varieties in the seedling stage, and the variety identification accuracy rate reaches more than 96%, which is more than 40% and 30% higher than the salt stress methods of artificially simulated single salt and compound salt, respectively, and strongly supports the accurate and rapid screening of salt-alkali tolerant alfalfa varieties, and has important application value for ensuring high-yield, stable and efficient planting of alfalfa in saline-alkali land. In addition, the salt stress method for identifying salt tolerance of alfalfa provided by the present invention is accurate, rapid and effective, easy to operate, and can be applied in the eastern coastal areas of my country, the Huanghuaihai Plain, the Northeast Plain, the Northwest Inland, and the saline-alkali areas of Xinjiang and Qinghai. The use of the saline-alkali soil leaching solution independently developed by the present invention to carry out salt stress of alfalfa in the seedling stage is the key to realizing accurate and rapid identification of salt tolerance of alfalfa varieties.

[0139] The salt stress method for identifying salt tolerance of alfalfa provided by the present invention is further described below in conjunction with specific embodiments:

[0140] Example 1: Collection of leachate from saline-alkali soil

[0141] Step 1. Select saline-alkali land with different degrees of salinity: In the planned alfalfa planting area, select three types of saline-alkali land: light, moderate and severe.

[0142] Step 2. Determine representative plots: Select 3 representative plots from the three types of saline-alkali land: mild, moderate, and severe, for a total of 9 plots, and the area of ​​each plot shall not be less than 1 mu.

[0143] Step 3. Collect soil leaching solution: Use a soil leaching solution batch collection device to collect soil leaching solution from 0 to 50 cm soil layer in each plot. The number of collection points for each plot is determined according to the amount of soil leaching solution used. The soil leaching solutions collected from light, moderate and severe saline-alkali plots are respectively put into different containers and marked.

[0144] Step 4. Mix the soil leaching solution, determine the total salt content and pH: Bring the collected soil leaching solution back to the laboratory, put the soil leaching solutions collected from light, moderate and severe saline-alkali land plots into different large containers, mix them, determine the total salt content and pH, mark them, and set them aside.

[0145] Example 2: Salt stress and salt tolerance assessment of alfalfa at the seedling stage

[0146] Step 1. Choose a potted container: It is advisable to use a porcelain flower pot with a height of no less than 20cm, a bottom diameter of no less than 10cm, and a mouth diameter of no less than 12cm.

[0147] Step 2: Fill with vermiculite: Fill the flowerpot with vermiculite and keep it ready for use.

[0148] Step 3: Add soil leaching solution: Divide the flower pots into a stress group and a control group, and add the corresponding solution into each potting container to make the vermiculite water content reach 20% to 25%. The specific stress treatment process is as follows:

[0149] According to the total salt content and pH value of the soil leaching solution, the stress group was set to three gradients, namely mild stress (0.1%-0.2%), moderate stress (0.2%-0.4%) and severe stress (0.4%-0.6%). The mild stress added a soil leaching solution with a total salt content of 0.15%, the severe stress added a soil leaching solution with a total salt content of 0.32%, and the severe stress added a soil leaching solution with a total salt content of 0.51%. The solution added to the control group was distilled water.

[0150] Step 4: Sowing: Select alfalfa seeds with a purity of more than 98% and a germination rate of more than 95%. Plant 5 pots for each treatment and sow 20 seeds in each pot.

[0151] Step 5. Stress management: Under the premise of no outflow of water, distilled water is added by weight every day according to the evaporation of water in the vermiculite in the potting container to ensure that the water content of the vermiculite is maintained at 20% to 25% and the concentration of the soil leaching solution remains relatively unchanged.

[0152] Step 6. Determination of index parameters: Determination of relevant indexes is carried out when alfalfa is at the 5-leaf stage.

[0153] Three alfalfa seedlings were randomly selected from each variety and treatment, and the SPAD value, initial fluorescence (Fo), maximum fluorescence yield (Fm), and maximum photochemical efficiency (Fv / Fm) of the third middle leaf were measured from the top to the bottom. After the fluorescence index measurement was completed, about 0.3 g of the measured leaves and surrounding leaves were collected and brought back to the laboratory, and the superoxide dismutase activity (SOD), peroxidase activity (POD), and catalase activity (CAT) of the leaves were measured using an activity detection kit. Ten alfalfa seedlings were randomly selected from each variety and treatment, and the plant height (PD) was measured using a ruler (all surviving plants were measured when there were less than 10 plants).

[0154] After the leaf index and plant index determination were completed, the number of live seedlings of each treatment was counted, and all the above-ground alfalfa plants of each treatment were cut, put into kraft paper bags and placed in an oven. After being sterilized at 105°C for 30 minutes, they were placed in an environment of 70°C and dried to constant weight. The dry weight was weighed to an accuracy of 0.001g.

[0155] Step 7. Comprehensive evaluation of salt tolerance: After obtaining the above index parameters, the comprehensive salt evaluation (D value method) is used to comprehensively evaluate the salt tolerance of each alfalfa variety at the seedling stage.

[0156] The control experiment of the salt stress method for identifying salt tolerance of alfalfa provided by the present invention is as follows:

[0157] In this experiment, 15 alfalfa varieties, including Zhongmu No. 1, Zhongmu No. 3, and Zhongmu No. 4, were identified for their salt tolerance at the seedling stage. Three salt gradients were set for each identification method, namely, mild salinity (salt content 0.15% ± 0.0001), moderate salinity (salt content 0.32% ± 0.0003), and severe salinity (salt content 0.51% ± 0.0002). The specific control experimental results are shown in Table 2.

[0158] Control Example 1: Sodium chloride single salt stress;

[0159] Control Example 2: sodium chloride + sodium sulfate compound salt stress;

[0160] Control Example 3: Salt stress method for identification of salt tolerance of alfalfa;

[0161] Control example 4 (CK): salt stress in field cultivation in saline-alkali land.

[0162] Table 2: Accuracy of salt tolerance identification of alfalfa seedlings under different salt stresses

[0163]

[0164]

[0165] The calculation formula for the accuracy of salt tolerance identification of alfalfa at the seedling stage under different salt stresses is: R=QV / QT, wherein: R represents the accuracy of salt tolerance identification of alfalfa at the seedling stage under different salt stresses compared with the identification in saline-alkali land field cultivation; QV represents the number of salt-alkali tolerant varieties that fall into the salt stress method of saline-alkali land field cultivation by the artificial simulation salt stress method; QT represents the number of salt-alkali tolerant varieties identified by the artificial simulation salt stress method.

[0166] According to Table 2, the salt tolerance identification method under sodium chloride single salt stress has an accuracy rate of 67.50% for the salt tolerance identification of alfalfa varieties at the seedling stage; the salt tolerance identification method under sodium chloride + sodium sulfate composite salt stress has an accuracy rate of 72.12% for the salt tolerance identification of alfalfa varieties at the seedling stage; and the salt tolerance identification method for alfalfa provided by the present invention has an accuracy rate of 96.67% for the salt tolerance identification of alfalfa varieties at the seedling stage.

[0167] In summary, with the aid of the above-mentioned technical scheme of the present invention, the salt stress method for identifying the salt tolerance of alfalfa provided by the present invention can realize the accurate and rapid identification of the salt tolerance of alfalfa varieties in the seedling stage, and the variety identification accuracy rate reaches more than 96%. Compared with the salt stress method of artificially simulated single salt and compound salt, it has the effect of significantly improving the identification accuracy, thereby strongly supporting the accurate and rapid screening of salt-alkali tolerant varieties of alfalfa, and has important application value for ensuring high-yield, stable yield and efficient cultivation of alfalfa in saline-alkali land.

[0168] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A salt stress method for identifying salt tolerance of alfalfa, characterized in that: The salt stress method for identifying salt tolerance of alfalfa comprises the following steps: S1. Collect soil leaching solutions from saline-alkali land plots with different salinity levels, and measure the total salt content and pH value of each soil leaching solution; The method of collecting soil leaching solutions from saline-alkali land plots with different salinity levels and determining the total salt content and pH value of each soil leaching solution comprises the following steps: S11. Select saline-alkali lands with different salinity levels from the planned alfalfa planting area, and select several groups of representative saline-alkali land plots from each saline-alkali land using a chessboard-like distribution method; S12, using a soil leaching solution collection device to collect soil leaching solutions from each saline-alkali land plot, respectively, and putting the collected soil leaching solutions into containers for collection and marking; S13, mixing the soil leaching solutions in each container collected from saline-alkali land plots with the same salinity, and measuring the total salt content and pH value of each soil leaching solution; S2, placing soil leaching solutions and cultivation substrates with different salt concentrations in potted containers of preset specifications, and sowing alfalfa; The method of placing soil leaching solutions and cultivation substrates of different salt concentrations in a potted container of preset specifications and sowing alfalfa comprises the following steps: S21, selecting a potted plant container of preset specifications; S22, dividing the potted plant containers into a stress group and a control group, wherein the stress group is added with several groups of soil leaching solutions with salt gradients, and the control group is added with distilled water; S23, filling each potting container with vermiculite, and respectively adding soil leaching solution and distilled water into each potting container to make the water content of the vermiculite reach 20-25%; The method of filling each potting container with vermiculite and respectively adding soil leaching solution and distilled water into each potting container to make the water content of the vermiculite reach 20-25% comprises the following steps: S231, filling each potting container with vermiculite; S232, adding several groups of soil leaching solutions with salt gradients to the potted plants of the stress group, and adding distilled water to the potted plants of the control group, so that the water content of the vermiculite reaches 20%-25%; S233. Under the premise of no outflow of water, distilled water is added by weight every day according to the evaporation of water in the vermiculite in the potting container to ensure that the water content of the vermiculite is maintained at 20%~25% and the concentration of the soil leaching solution remains unchanged; S24, selecting different varieties of alfalfa seeds and sowing them inside the potting container; S3. Monitor the growth and development of alfalfa under salt stress and determine the index parameters of different varieties of alfalfa; S4. Analyze the index parameters of alfalfa, and conduct a comprehensive evaluation of the salt tolerance of each variety of alfalfa based on the index parameter analysis results.

2. The salt stress method for identifying salt tolerance of alfalfa according to claim 1, characterized in that: The salinity of the saline-alkali land includes mild, moderate and severe salinity; Among them, the total soluble salt content of the cultivated layer soil in slightly saline-alkali land is 0.1%~0.2%, the total soluble salt content of the cultivated layer soil in moderate saline-alkali land is 0.2%~0.4%, and the total soluble salt content of the cultivated layer soil in severely saline-alkali land is 0.4%~0.6%.

3. The salt stress method for identifying salt tolerance of alfalfa according to claim 1, characterized in that: The method of monitoring the growth and development of alfalfa under salt stress and determining the index parameters of different varieties of alfalfa comprises the following steps: S31. Monitoring the growth and development of alfalfa under salt stress, and measuring the index parameters of different varieties of alfalfa at the 5-leaf stage; S32, respectively selecting the third middle leaf from the top downward from the control and each salt stress treated alfalfa seedling, measuring the fluorescence parameters of each leaf, collecting 0.3 g of the leaf and adjacent leaves, and measuring the antioxidant enzyme activity of each leaf; S33, 10 alfalfa seedlings were randomly selected from the control and each salt stress treatment to measure plant height; S34. After the leaf index and plant index determination were completed, the number of live seedlings of the control and each salt stress treatment was counted, and all above-ground alfalfa plants of the control and each salt stress treatment were cut, placed in an oven at 105°C for 30 minutes, and then placed at 70°C for drying and weighing.

4. The salt stress method for identifying salt tolerance of alfalfa according to claim 3, characterized in that: The leaf indicators include SPAD value, fluorescence parameters measured by chlorophyll fluorescence instrument, superoxide dismutase activity, peroxidase activity and catalase activity; The plant indicators include plant height, number of live seedlings and aboveground biomass.

5. The salt stress method for identifying salt tolerance of alfalfa according to claim 4, characterized in that: The step of analyzing the index parameters of alfalfa and comprehensively evaluating the salt tolerance of each variety of alfalfa based on the index parameter analysis results comprises the following steps: S41, determining the correlation between the measured index and the salt tolerance of alfalfa, wherein the measured index includes a leaf index and a plant index; S42. If the measured index of alfalfa is positively correlated with salt tolerance, the membership function value of each measured index is calculated using the positive correlation membership function; S43. If the measured index of alfalfa is negatively correlated with salt tolerance, the membership function value of each measured index is calculated using the negative correlation membership function; S44, using the standard deviation coefficient method to calculate the standard deviation coefficient of each measurement index, and calculating the weight coefficient of each measurement index based on the standard deviation coefficient; S45, calculating the comprehensive evaluation value of each measurement indicator based on the weight coefficient of each measurement indicator; S46. Determine the salt tolerance of each variety of alfalfa based on the comprehensive evaluation value, and classify the salt tolerance of alfalfa into grades.

6. The salt stress method for identifying salt tolerance of alfalfa according to claim 5, characterized in that: The calculation formula of the standard deviation coefficient is: ； In the formula, Indicates j The standard deviation coefficient of each measurement indicator; Indicates i The first j The membership function value of the measurement index; Indicates j The average value of the measured indicators; n Indicates the total number of measured indicators.

7. The salt stress method for identifying salt tolerance of alfalfa according to claim 6, characterized in that: The calculation formula of the comprehensive evaluation value is: ； In the formula, D Indicates the comprehensive evaluation value; Indicates j The membership function value of the measurement index; Indicates j Measured index parameters; Indicates j The weight coefficient of each measurement indicator; n Indicates the total number of measured indicators.

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