Alfalfa bud stage cold resistance identification model and alfalfa cold resistance identification method

Abstract

The invention aims to provide a model and a method for identifying the cold resistance of alfalfa in the bud stage, the identification model relates to the relationship between four cold-resistant indexes and cold-resistant grade (Y) of alfalfa at 6 + / -0.1 DEG C, the four cold-resistant indexes are alfalfa germination rate (X1), alfalfa simplified vigor index (X2), alfalfa root length (X3) and alfalfa germination index (X4), and the relational expression is Y =-2.771 + 0.016 X1 + 0.061 X2 + 0.900 X3 + 0.054 X4, when Y is more than or equal to 0.70 and less than or equal to 1.00, the alfalfa variety is a strong cold-resistant variety; when Y is more than 0.40 and less than 0.70, the alfalfa variety is a medium cold-resistant variety; when Y is more than or equal to 0.00 and less than or equal to 0.40, the alfalfa variety is a non-cold-resistant variety. Experiments prove that the model has the characteristics of high accuracy, rapidness and high efficiency when being used for identifying alfalfa cold-resistant germplasm resources.

Description

Technical Field

[0001] This invention relates to the field of alfalfa breeding technology, specifically to an alfalfa bud stage cold resistance identification model and a method for alfalfa cold resistance identification. Background Technology

[0002] Seed germination is a series of ordered physiological and morphological processes that a seed undergoes from imbibition to germination. Temperature is a major limiting factor for seed germination and plant growth and development because suitable temperature conditions facilitate normal germination and growth. Seed germination is a cornerstone of crop production, influencing vegetation distribution and playing a crucial role in maintaining population stability and enhancing species diversity. The temperature requirements of seed germination reflect its ecological mechanism of environmental adaptation. When the temperature continues to rise and exceeds the optimal germination temperature, the number of germinating seeds will no longer increase; when the temperature is below the optimal temperature, respiration is inhibited, and the seed germination rate gradually slows down, until only a portion of the seeds germinate. If the ambient temperature is below the minimum temperature during seed germination, both seed germination quality and seed viability will be affected to varying degrees. Therefore, determining the temperature threshold for seed germination, especially the basal temperature—the temperature at which a particular plant seed cannot germinate below—can predict and assess the impact of temperature on seed germination rate.

[0003] Alfalfa (Medicago sativa L.) is a high-yielding, high-quality, and palatable plant with high economic value, earning it the reputation of "King of Forage." It has wide ecological adaptability and strong resistance to adverse conditions, and is widely cultivated in Northwest, North, and Northeast my country. Temperature, as a crucial environmental factor affecting alfalfa seed germination, can reflect the relative cold resistance of different alfalfa varieties within a certain range of low-temperature treatments. In northern my country, long and cold winters significantly reduce the overwintering survival rate of alfalfa varieties with weak cold resistance, leading to reduced alfalfa yields the following year. Currently, there are no reports on identifying cold-resistant alfalfa varieties, thus hindering effective breeding for highly cold-resistant alfalfa. In conclusion, understanding the response of alfalfa seeds to low temperatures during the germination stage is of significant guiding importance for alfalfa establishment and the selection of cold-resistant germplasm in northern my country. Summary of the Invention

[0004] To address the research gap in alfalfa cold resistance identification during the germination stage, this invention aims to provide a model and method for identifying alfalfa cold resistance during this period. This research can provide accurate reference data on alfalfa emergence in the field and timely sowing, and has significant guiding significance for alfalfa establishment and cold-resistant germplasm selection in northern my country.

[0005] To achieve this technical objective, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a model for identifying the cold resistance of alfalfa during its germination stage. This model involves the relationship between four cold resistance indicators and the cold resistance grade (Y) of alfalfa at 6±0.1℃. The four cold resistance indicators are: alfalfa germination rate (X1), alfalfa simplified vigor index (X2), alfalfa root length (X3), and alfalfa germination index (X4). The relationship between these four cold resistance indicators and the cold resistance grade (Y) is expressed by the following regression equation: Y = -2.771 + 0.016X1 + 0.061X2 + 0.900X3 + 0.054X4. Furthermore, when 0.70 ≤ Y ≤ 1.00, the alfalfa variety is a strongly cold-resistant variety; when 0.40 < Y < 0.70, the alfalfa variety is a moderately cold-resistant variety; and when 0.00 ≤ Y ≤ 0.40, the alfalfa variety is a non-cold-resistant variety.

[0006] Secondly, the present invention provides a method for constructing the alfalfa sprouting stage cold resistance identification model described in the first aspect, comprising the following steps: Step 1: Germination experiments were conducted on sterilized alfalfa seeds under light at different ambient temperatures. Germination rate, germination index, root length, shoot length, seedling dry weight, and simplified vigor index were recorded. The formulas for calculating the germination rate, germination index, seedling dry weight, and simplified vigor index are as follows: Germination rate G = (Total number of germinated seeds / Number of seeds tested) × 100%; Germination index GI = ∑GT / DT, where GT is the number of germinations in T days and DT is the corresponding number of days; Seedling dry weight = average of the sum of alfalfa sprout dry weight and root dry weight; Simplified Vigor Index = Seedling Dry Weight × Germination Rate; Step 2: Based on the alfalfa germination rate, germination index, root length, shoot length, seedling dry weight, and simplified vigor index obtained in Step 1, perform correlation analysis and principal component analysis on the six indicators. Step 3: Determine the cold resistance index of alfalfa based on the analysis results of Step 2, and calculate the membership function value of each determined cold resistance index; Step 4: Perform regression analysis on the membership function values ​​of each cold resistance index obtained in Step 3 and the corresponding cold resistance index to obtain the cold resistance identification model.

[0007] Preferably, in step 1, the control conditions for the seed germination experiment include: humidity 60-80%, alternating light exposure for 12 hours and darkness for 12 hours, light intensity 10000-20000 lx; ​​and temperature 2-20℃.

[0008] Preferably, in step 1, the standard for seed germination is: when the radicle is the same length as the seed and the plumule reaches half the length of the seed, the seed is considered to have germinated; when no seeds germinate for 2-5 consecutive days, the germination rate is calculated; when the seed germinates for 8-12 days, the root length, the bud length and the dry weight of the seedling are measured.

[0009] Preferably, in step 2, the principal component analysis includes: extracting three principal components: F1, F2, and F3, wherein the eigenvector corresponding to F1 includes at least the germination rate and the simplified vigor index; the eigenvector corresponding to F2 includes at least the root length; and the eigenvector corresponding to F3 includes at least the germination index.

[0010] Preferably, in step 3, the membership function value of the cold resistance index is calculated using the fuzzy mathematical membership function method, and the calculation formula is as follows: If the measured index is positively correlated with alfalfa's cold resistance, then F ij = [X ij -X imin ] / [X imax -X imin ]; If the measured index is negatively correlated with cold resistance, then F ij =1-[X ij -X imin ] / [X imax -X imin ]; Among them, F ij Let i be the membership function value of grass species j; X ij To identify the membership value of the index of grass species i; X imax X represents the maximum value of the index belonging to the identified grass species i; imin The minimum value of index j for the identified grass species i.

[0011] Thirdly, the present invention provides the application of the alfalfa bud stage cold resistance identification model described in the first aspect in the preparation of a rapid alfalfa cold resistance identification kit.

[0012] Furthermore, the kit also includes at least the necessary reagents and tools for determining alfalfa germination rate, germination index, root length, and simplified vigor index.

[0013] Fourthly, the present invention provides a method for identifying the cold resistance of alfalfa, comprising the following steps: Step 1: Conduct a seed germination experiment on sterilized alfalfa seeds under light and at 6±0.1℃, and record the germination rate, germination index, root length, and simplified vigor index; the formulas for calculating the germination rate, germination index, and simplified vigor index are as follows: Germination rate (G) = (Total number of germinated seeds / Number of seeds tested) × 100%; Germination Index (GI): GI = ∑GT / DT, where GT is the number of germinated days in T days and DT is the corresponding number of days; Simplified Vigor Index: Simplified Vigor Index = Seedling Dry Weight × Germination Rate; Step 2: Combining the germination rate, germination index, root length, and simplified vigor index obtained in Step 1, the cold resistance level of the alfalfa variety is obtained through the identification model described in claim 1.

[0014] Preferably, in step 1, the control conditions for the seed germination experiment further include: humidity of 60-80%, alternating light exposure for 12 hours and darkness for 12 hours, light intensity of 10,000-20,000 lx, and temperature of 6 ± 0.5℃.

[0015] Preferably, in step 1, the standard for seed germination is: when the radicle is the same length as the seed and the plumule reaches half the length of the seed, the seed is considered to have germinated; when no seeds germinate for 2-5 consecutive days, the germination rate is calculated; when the seed germinates for 8-12 days, the root length of the seedling is measured.

[0016] Compared with the prior art, the present invention has the following technical effects: This invention investigated the cold resistance of nine alfalfa varieties from both domestic and international sources: Zhaodong, Gongnong No. 2, Longmu 801, Gretel, WL525, Saidi, Aohan, WL168, and Golden Queen. Germination experiments were conducted on these nine alfalfa varieties under different temperatures (2℃, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, and 20℃). All alfalfa materials showed a decreasing trend in seed germination rate, germination index, simplified vigor index, root length, shoot length, and seedling dry weight. The final results indicate that 6℃ is the critical temperature for alfalfa germination; above this temperature, the germination rate of all varieties exceeded 50%. Principal component analysis determined that germination rate (X1), simplified vigor index (X2), root length (X3), and germination index (X4) can be used as the main indicators for evaluating cold resistance during the budding stage. Membership function evaluation showed the following order of cold resistance among varieties: Longmu 801 > Zhaodong > Gongnong 2 > WL168 > Aohan > Jin Huanghou > Grete > Saidi > WL525. A regression equation for cold resistance identification was established using regression analysis: Y = -2.771 + 0.016X1 + 0.061X2 + 0.900X3 + 0.054X4. This regression equation can be used for the identification of cold-resistant alfalfa germplasm resources and for screening cold-resistant alfalfa materials. Screening cold-resistant alfalfa germplasm resources suitable for planting in cold regions and establishing a method for identifying cold resistance during the budding stage of alfalfa can provide a basis for the rapid and accurate identification of cold-resistant alfalfa germplasm resources. Attached Figure Description

[0017] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 The germination rate of alfalfa seeds at different temperatures is shown in Example 1 of this invention. Detailed Implementation

[0018] In the description of this invention, cold resistance is a genetic trait formed by plants through long-term adaptation to low temperatures, which means that plants can continuously resist or adapt to low temperatures.

[0019] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1

[0022] This embodiment studies the cold resistance of alfalfa buds at the following nine varieties, as detailed below: I. Experimental Materials Nine alfalfa materials from both domestic and international sources were collected, including four domestically approved varieties (Longmu 801, Gongnong 2, Zhaodong alfalfa, and Aohan alfalfa) and five imported varieties (WL168, Golden Queen, Greet, Saidi, and WL525). The variety names, dormancy levels, and source information of all tested alfalfa materials are shown in Table 1.

[0023] Table 1. Alfalfa materials and their sources tested

[0024] The above-mentioned varieties and materials can also be obtained from national seed banks and enterprises. This invention does not limit the source of the varieties and materials.

[0025] II. Operation Process and Index Measurement Select 300 uniform, plump, and appropriately sized alfalfa seeds of various varieties, sterilize them with 0.1% KMnO4 for 15 minutes, and then rinse them 4-5 times with sterile water. Place a double layer of filter paper on a petri dish (110 mm in diameter), arrange 100 alfalfa seeds neatly on the filter paper, and place it in a light incubator (light incubator conditions: humidity 80%, alternating light for 12 hours and darkness for 12 hours, light intensity 15000 lx). Germination experiments were conducted in triplicate, with each treatment performed at constant temperatures (2℃, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃, 18℃, and 20℃, with temperature fluctuations not exceeding 0.5℃). From 8:00 to 9:00 AM daily, distilled water was added to the petri dishes based on the amount of water evaporated, and the number of germinated seeds was observed and recorded. The germination standard was defined as follows: when the radicle was equal in length to the seed and the plumule reached half the length of the seed, germination was considered complete. Data recording was stopped and the germination rate was calculated after three consecutive days of no further germination. Ten seedlings were collected after 10 days of germination at each temperature, and root and shoot lengths were measured. The measurement indicators and calculation methods are as follows: (1) Germination rate: After treatment for 1 day, the number of germinated seeds was counted and recorded every day.

[0026] Germination rate (G) = (Total number of germinated seeds / Number of seeds tested) × 100%, unit: %.

[0027] (2) Germination Index (GI): GI = ∑GT / DT (GT is the number of germinations on day T; DT is the corresponding number of days).

[0028] (3) Root length and shoot length: Measure the root length and shoot length of each seedling with vernier calipers and calculate the average value in cm.

[0029] (4) Seedling dry weight: The sum of the dry weight of alfalfa sprouts and the dry weight of roots. Calculate the average value. Unit: g.

[0030] (5) Simplified Vigor Index: Simplified Vigor Index = Seedling Dry Weight × Germination Rate.

[0031] III. Results and Analysis (1) Seed germination characteristics Combine Table 2 and Figure 1The germination rate of different alfalfa seeds generally decreased with decreasing temperature. At 12–20℃, the germination rates of different alfalfa varieties showed little difference; at 6–10℃, the germination rates of each material ranged from 72% to 96%; at 4℃, the germination rates of each material ranged from 11% to 66%, with only some varieties exceeding 50%; at 2℃, the germination rates of each variety ranged from 7% to 26%, and all were less than 50%. These results indicate that above 6℃, the germination rates of the tested materials were all above 50%, while below 6℃, they decreased to varying degrees. Therefore, 6℃ can be considered a critical germination point for alfalfa, suggesting that temperatures below the critical germination temperature inhibit seed germination and can be utilized in future production practices.

[0032] Table 2. Changes in alfalfa seed germination rate at different temperatures.

[0033] Note: In the table, the same lowercase letter in the same column indicates that the difference is not significant. P> 0.05), the same below.

[0034] As shown in Table 3, the germination index of each variety decreased with decreasing temperature. At 12–20℃, the germination index ranged from 36.51 to 48.72; at 6–10℃, it ranged from 14.79 to 41.14; and at 2–4℃, it ranged from 1.11 to 9.36. These results indicate that the germination index of alfalfa seeds under low-temperature (2–4℃) treatment was significantly lower than that under other temperature treatments.

[0035] Table 3. Changes in alfalfa seed germination index at different temperatures.

[0036] The simplified vigor index is an important indicator for determining seed germination quality. As shown in Table 4, the simplified vigor index of all alfalfa varieties generally showed a downward trend as the temperature decreased. At 10–20℃, the simplified vigor index of each variety ranged from 9.96 to 13.40; at 2–4℃, the simplified vigor index ranged from 0.35 to 5.12; and at 6℃, the simplified vigor index ranged from 4.66 to 8.89. The simplified vigor index of all varieties decreased significantly under low-temperature treatment (2–4℃).

[0037] Table 4. Changes in simplified vigor index of alfalfa seeds under different temperatures.

[0038] As shown in Table 5, low temperature inhibited the growth of seed radicles. At 12–20℃, the root length of each alfalfa variety ranged from 1.587 to 3.331 cm. Subsequently, the root length of each alfalfa material showed a decreasing trend with decreasing temperature: 1.227–1.900 cm at 8–10℃; 0.593–0.863 cm at 6℃; and 0.132–0.501 cm at 2–4℃. Overall, when the temperature was below 6℃, the root length of each alfalfa variety was significantly lower than that at 8–20℃, indicating that seeds have a certain tolerance range for low temperatures. Below this range, the decomposition of nutrients and physiological activities within the seed are affected, limiting the elongation and growth of the radicle.

[0039] Table 5. Changes in alfalfa seed root length at different temperatures

[0040] As shown in Table 6, the shoot length of alfalfa decreased with decreasing temperature. At 12–20℃, the shoot elongation of all materials was normal, with roughly the same length, ranging from 3.987 to 4.094 cm. At 6℃, the shoot length of each alfalfa variety ranged from 1.657 to 2.090 cm. When the temperature dropped to 2–4℃, no embryos grew in any of the alfalfa materials due to the excessively low temperature, indicating that the embryo is more sensitive to low temperatures than the radicle. Below 6℃, seeds can germinate and develop radicles, but low temperatures inhibit shoot elongation, demonstrating that the critical temperature of 6℃ is crucial for seed germination and the growth of both the radicle and the embryo.

[0041] Table 6. Changes in alfalfa seed sprout length at different temperatures

[0042] As shown in Table 7, the dry weight of alfalfa seedlings generally decreased with decreasing temperature. At 2–4℃, the dry weight of seedlings for each variety ranged from 0.047 to 0.080 g. The dry weight of seedlings for each variety at low temperatures (2–4℃) was significantly lower than at other temperatures, which may be related to the inhibitory effect of low temperature on the synthesis and decomposition processes of seed contents.

[0043] Table 7. Changes in dry weight of alfalfa seedlings at different temperatures

[0044] Example 2 Based on the cold resistance data of the nine alfalfa varieties in Example 1, this example determines the cold resistance index of alfalfa during the budding stage, as follows: As shown in Table 8, correlation analysis was performed on the six indicators measured during the alfalfa germination period using SAS 9.0 software. There was a highly significant positive correlation between seed germination rate and germination index and simplified vigor index (P<0.01), with correlation coefficients of 0.824 and 0.933, respectively. There was also a significant positive correlation between simplified vigor index and germination index and seedling dry weight, with correlation coefficients of 0.674 and 0.924, respectively. Root length and shoot length also showed a highly significant positive correlation (0.797, P<0.01). These results indicate that alfalfa materials with higher germination rates have higher seed vigor, which is more conducive to the elongation and growth of the radicle and plumule.

[0045] Table 8 Correlation analysis of alfalfa sprouting stage indicators

[0046] Note: *Significantly correlated at the 0.05 level; **Highly significantly correlated at the 0.01 level.

[0047] As shown in Table 9, this embodiment further utilizes SAS 9.0 software for principal component analysis. Principal component analysis transforms multiple germination indicators into several comprehensive indicators to compensate for the shortcomings of single indicators in evaluating cold resistance. Six single indicators were measured during the germination period of the tested materials. Based on the principle of a cumulative contribution rate ≥ 85%, this study extracted three principal components (F1, F2, F3). The first principal component (F1) has an eigenvalue of 3.53 and a contribution rate of 58.9%. The indicators with larger values ​​in its corresponding eigenvector are germination rate and simplified vigor index. The second principal component (F2) has an eigenvalue of 2.12 and a contribution rate of 35.5%. Its corresponding eigenvector mainly reflects root length. The third principal component (F3) has an eigenvalue of 0.22 and a contribution rate of 3.7%. The indicator with the larger value is the germination index. The cumulative contribution rate of these three principal components reaches 98.1%, transforming the six single indicators into three comprehensive indicators (F1, F2, F3), which essentially encompass most of the information of the measured indicators.

[0048] Table 9 Principal Component Analysis

[0049] Example 3 Based on the principal component analysis results in Example 2, the root length, germination rate, simplified vigor index, and germination index of alfalfa at 6℃ were determined to be important indicators for screening cold-resistant germplasm materials. This example further determined the cold resistance identification model for alfalfa during the germination period.

[0050] By applying the fuzzy mathematical membership function method, the measured indicators are comprehensively evaluated, and the membership function values ​​of each indicator are calculated: If the measured index is positively correlated with alfalfa's cold resistance, then F ij = [X ij -Ximin ] / [X imax -X imin ]; If the measured index is negatively correlated with cold resistance, then F ij =1-[X ij -X imin ] / [X imax -X imin ]; Among them, F ij Let i be the membership function value of grass species j; X ij To identify the membership value of the index of grass species i; X imax X represents the maximum value of the index belonging to the identified grass species i; imin The minimum value of index j for the identified grass species i.

[0051] The membership function values ​​of each alfalfa species were summed to obtain the average membership function value. The higher the average value, the stronger the cold resistance of the alfalfa. The calculation results are shown in Table 10. The comprehensive evaluation value of each variety is its root length, germination rate, simplified vigor index, and germination index. Fij The average value was calculated. Finally, the cold resistance of alfalfa varieties was compared, and the order of cold resistance from strongest to weakest was: Longmu 801 > Zhaodong > Gongnong 2 > WL168 > Aohan > Jin Huanghou > Grete > Saidi > WL525. Strongly cold-resistant varieties: Longmu 801, Zhaodong, Gongnong 2; moderately cold-resistant varieties: WL168, Aohan, Jin Huanghou; non-cold-resistant varieties: Grete, Saidi, WL525. The cold resistance levels were divided according to the Fij values ​​calculated in Table 10: strongly cold-resistant varieties 0.70–1.00 (inclusive); moderately cold-resistant varieties 0.40–0.70 (exclusive); non-cold-resistant varieties 0.00–0.40 (inclusive).

[0052] Table 10 Comprehensive Evaluation of Membership Functions

[0053] By comprehensively considering alfalfa germination rate, simplified vigor index, root length, germination index, and the mean of the membership function, stepwise regression analysis was further performed using SAS 9.0 software to obtain the relationship between the membership function values ​​and alfalfa germination rate, simplified vigor index, root length, and germination index, i.e., the relationship between alfalfa germination period cold resistance grade and each index. The following cold resistance identification regression equation was established: Y = -2.771 + 0.016X1 + 0.061X2 + 0.900X3 + 0.054X4, where Y represents the comprehensive evaluation value of cold resistance under the regression analysis method (i.e., the membership function value, also known as the cold resistance grade), and X1, X2, X3, and X4 represent the germination rate, simplified vigor index, root length, germination index, or their average values ​​at 6±0.5℃, respectively. This regression equation can be used to evaluate alfalfa cold resistance and can be used for screening alfalfa cold-resistant materials.

[0054] Table 11 Regression Analysis of Alfalfa Sprouting Stage Indicators

[0055] Note: *The difference is significant at the 0.05 level; **The difference is significant at the 0.01 level.

[0056] Example 4

[0057] Based on the alfalfa cold tolerance identification model obtained in Example 3: Y = -2.771 + 0.016X1 + 0.061X2 + 0.900X3 + 0.054X4, this example validated the cold tolerance model for more alfalfa varieties, as detailed below: I. Cold resistance results obtained using model statistics 300 seeds of each of 12 alfalfa varieties (Longmu 803 alfalfa, Longmu 806 alfalfa, Prairie No. 2 alfalfa, Reindeer alfalfa, Adina alfalfa, Suntory alfalfa, Qianjing alfalfa, 420 alfalfa, WL363 alfalfa, Qingta alfalfa, WL656 alfalfa, and WL903 alfalfa) were selected and sterilized with 0.1% KMnO4 for 15 min, followed by rinsing with sterile water 4-5 times. Double-layered filter paper was placed on a petri dish (110 mm in diameter), and 100 alfalfa seeds of each variety were neatly arranged on the filter paper. The dish was then placed in a light incubator (light incubator conditions: 80% humidity, 12 h light, 12 h darkness, light intensity 15000 lx). Each treatment was repeated three times. Germination experiments were conducted under constant temperature (6℃). From 8:00 to 9:00 AM daily, distilled water was added to the petri dishes using a weighing method based on the amount of water evaporated. The number of germinated seeds was observed and recorded. The germination standard was defined as follows: when the radicle was the same length as the seed and the plumule reached half the length of the seed, germination was considered complete. Data recording was stopped and the germination rate was calculated when no more seeds germinated after three consecutive days. The measurement indicators and calculation methods are as follows: (1) Germination rate (G) = (total number of germinated seeds / number of seeds tested) × 100%, unit: %.

[0058] (2) Germination index (GI) = ∑GT / DT (GT is the number of germinations on day T; DT is the corresponding number of days).

[0059] (3) Root length: Measure the root length of each seedling with a vernier caliper and calculate the average value in cm.

[0060] (4) Seedling dry weight: The sum of the dry weight of alfalfa sprouts and the dry weight of roots. Calculate the average value. Unit: g.

[0061] (5) Simplified Vigor Index: Simplified Vigor Index = Seedling Dry Weight × Germination Rate.

[0062] Based on the data from Tables 2-5 and 10 above, Table 12 is compiled as a data comparison table.

[0063] Table 12 Overall Evaluation of Alfalfa Variety Comparison Group

[0064] Substituting the average values ​​of germination rate, simplified vigor index, root length, and germination index of the 12 varieties at 6±0.5℃ into the equation: Y=﹣2.771+0.016X1+0.061X2+0.900X3+0.054X4, the results are shown in Table 13 below: Table 13 Overall Evaluation of Alfalfa Variety Validation Group

[0065] Longmu 803 alfalfa, Longmu 806 alfalfa, Prairie No. 2 alfalfa, and Reindeer alfalfa are in the range of 0.70 to 1.00 for strongly cold-resistant varieties; Adina alfalfa, Suntory alfalfa, Qianjing alfalfa, and 420 alfalfa are in the range of 0.40 to 0.70 for moderately cold-resistant varieties; WL363 alfalfa, Qingta alfalfa, WL656 alfalfa, and WL903 alfalfa are in the range of 0.00 to 0.40 for non-cold-resistant varieties.

[0066] II. Field Cold Resistance Verification The experimental site is located in the Fularji Research Base, Qiqihar City, Heilongjiang Province, at E123.41°N 47.15°. The highest temperature is 38.5°C, the lowest is -39.5°C, the average annual temperature is 3°C, the accumulated temperature (≥10°C) is 2722.1°C, the average annual precipitation is approximately 480 mm, and the frost-free period is around 132 days. Twelve alfalfa varieties were used (Longmu 803 alfalfa, Longmu 806 alfalfa, Prairie No. 2 alfalfa, Reindeer alfalfa, Adina alfalfa, Suntory alfalfa, Qianjing alfalfa, 420 alfalfa, WL363 alfalfa, Qingta alfalfa, WL656 alfalfa, and WL903 alfalfa).

[0067] The experimental plot area is 15m² 2 (3m×5m), row spacing 30cm, seeding rate 15kg / hm -2 A completely randomized block design was used, with three replicates. Ten rows were planted per plot, with a 60cm spacing between plots. Pre-sowing land preparation was meticulous to ensure uniform seed germination. During the experiment, routine field management was implemented, including timely weeding and pest and disease control. Irrigation was conducted once before winter, with an irrigation quota of 225–375 m³. 3 / hm 2 Overwintering rate survey: Three sample sections, each 1m long, were randomly selected from each plot. The total number of plants in the sample sections was counted before overwintering and again the following year after the plants turned green. Overwintering rate (%) = number of surviving plants after greening / total number of plants before overwintering × 100%. The results are shown in Table 14. Table 14. Overwintering rate statistics from field cold resistance experiments

[0068] Longmu 803 alfalfa, Longmu 806 alfalfa, Prairie No. 2 alfalfa, and Reindeer alfalfa had a wintering rate of 89%–99% in cold-resistant varieties; Adina alfalfa, Suntory alfalfa, Qianjing alfalfa, and 420 alfalfa had a wintering rate of 68%–77% in moderately cold-resistant varieties; and WL363 alfalfa, Qingta alfalfa, WL656 alfalfa, and WL903 alfalfa had a wintering rate of 20%–44% in non-cold-resistant varieties. Therefore, the model of this invention has high accuracy in identifying the cold resistance of alfalfa.

[0069] This invention also provides a rapid cold-resistance identification kit for alfalfa. The kit uses the identification model Y = -2.771 + 0.016X1 + 0.061X2 + 0.900X3 + 0.054X4, with the following criteria: when 0.70 ≤ Y ≤ 1.00, the alfalfa variety is strongly cold-resistant; when 0.40 < Y < 0.70, the alfalfa variety is moderately cold-resistant; and when 0.00 ≤ Y ≤ 0.40, the alfalfa variety is not cold-resistant. The kit also includes necessary reagents and tools for seed germination, germination rate determination, simplified vigor index, root length measurement, and germination index measurement of alfalfa.

[0070] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. A model for identifying cold tolerance at the seedling stage of alfalfa, characterized by, The identification model relates to the relationship between 4 cold resistance indexes of alfalfa at 6±0.1 DEG C and a cold resistance grade (Y), wherein the 4 cold resistance indexes are alfalfa germination rate (X1), alfalfa simplified vigor index (X2), alfalfa root length (X3), and alfalfa germination index (X4), and the relationship between the 4 cold resistance indexes and the cold resistance grade (Y) is represented by the following regression equation: Y=-2.771+0.016X1+0.061X2+0.900X3+0.054X4, and when 0.70<=Y<=1.00, the alfalfa variety is a strong cold resistance variety; when 0.40 2. The method for constructing a cold tolerance identification model for alfalfa sprout stage according to claim 1, characterized in that, The method comprises the following steps: Step 1, the sterilized alfalfa seeds are subjected to seed germination experiment under different environmental temperatures under illumination, and germination rate, germination index, root length, bud length, seedling dry weight, and simplified vigor index are counted; the calculation formulae of the germination rate, the germination index, the seedling dry weight, and the simplified vigor index are as follows: Germination rate G=(total number of germinated seeds / number of tested seeds) x 100%; Germination index GI=∑GT / DT, wherein GT is the number of germinated seeds on T days, and DT is the corresponding number of days; Seedling dry weight=average value of the sum of alfalfa bud dry weight and root dry weight; Simplified vigor index=seedling dry weight x germination rate; Step 2, according to the alfalfa germination rate, germination index, root length, bud length, seedling dry weight, and simplified vigor index obtained in step 1, correlation analysis and principal component analysis are performed on the 6 indexes; Step 3, alfalfa cold resistance indexes are determined according to the analysis result of step 2, and the membership function values of the determined cold resistance indexes are calculated; Step 4, regression analysis is performed on the membership function values of the cold resistance indexes obtained in step 3 and the corresponding cold resistance indexes, so as to obtain the cold resistance identification model.

3. The method for constructing the alfalfa sprouting stage cold resistance identification model according to claim 2, characterized in that, In step 1, the control conditions of the seed germination experiment include: humidity of 60-80%, alternating light for 12 h / dark for 12 h, and illumination intensity of 10000-20000 lx; and the temperature is selected to be 2-20 DEG C.

4. The method for constructing a cold tolerance identification model for alfalfa sprout stage according to claim 2 or 3, characterized in that, In step 1, the standard of seed germination is that when the radicle is equal in length to the seed and the plumule reaches half of the seed, the seed is germinated; the germination rate is counted when no seed germinates continuously for 2-5 days; and the root length, the bud length, and the seedling dry weight are measured when the seed germinates for 8-12 days.

5. The method for constructing the alfalfa sprouting stage cold resistance identification model according to claim 2, characterized in that, In step 2, the principal component analysis includes: extracting 3 principal components F1, F2, and F3, wherein the characteristic vector corresponding to F1 at least includes the germination rate and the simplified vigor index; the characteristic vector corresponding to F2 at least includes the root length; and the characteristic vector corresponding to F3 at least includes the germination index.

6. The method for constructing the alfalfa sprouting stage cold resistance identification model according to claim 5, characterized in that, In step 3, the membership function value of the cold resistance index is calculated by using the membership function method of fuzzy mathematics, and the calculation formula is as follows: If the measured index is positively correlated with the cold tolerance of alfalfa, then F ij = [X ij -X imin ] / [X imax -X imin ]; If the measured index is negatively correlated with cold tolerance, then F ij = 1 - [X ij - X imin ] / [X imax - X imin ]; Wherein, F ij is the index membership function value of the i grass species j; X ij is the identified index membership value of the i grass species j; X imax is the identified index membership maximum value of the i grass species j; X imin is the identified index membership minimum value of the i grass species j.

7. The application of the alfalfa bud stage cold resistance identification model of claim 1 in the preparation of an alfalfa cold resistance rapid identification kit.

8. A method for identifying cold tolerance in alfalfa, characterized by, The method comprises the following steps: Step 1, the seed germination experiment of the sterilized alfalfa seed is carried out under the condition of illumination and 6±0.1℃, and the germination rate, germination index, root length and simplified vigor index are counted; the calculation formula of the germination rate, the germination index and the simplified vigor index is: Germination rate (G)=(total number of germinated seeds / number of tested seeds) x 100%; Germination index (GI): GI=∑GT / DT, wherein GT is the number of germinated seeds on T day, and DT is the corresponding number of days; Simplified vigor index: simplified vigor index=dry weight of seedling x germination rate; Step 2, the cold tolerance grade of the alfalfa variety is obtained through the identification model of claim 1 in combination with the germination rate, germination index, root length and simplified vigor index obtained in step 1.

9. The method of authentication of claim 8, wherein, In step 1, the control condition of the seed germination experiment further includes: humidity 60~80%, alternating light 12h / dark 12h, light intensity 10000~20000lx, and temperature 6±0.5℃.

10. The method of authentication according to claim 8 or 9, characterized in that, In step 1, the standard of seed germination is that when the radicle is equal to the seed and the plumule reaches half of the seed, the seed germination is achieved, and the germination rate is counted when there is no germinated seed for 2~5 days continuously; the root length is measured by taking the seedling when the seed germination lasts for 8~12 days.

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