A method for grading and identifying cold tolerance of tobacco seedlings in seedling stage

Abstract

The present application provides a kind of tobacco seedling cold tolerance grading identification method, comprising the following steps: S1, after sterilization of tobacco seed, sowing and cultivation, obtain tobacco seedling;S2, the obtained tobacco seedling is transplanted to nutrient medium, sets up control group and cold treatment group;S3, the tobacco seedling population subjected to low temperature stress is carried out cold injury index investigation;S4, the relative activity of multiple physiological and biochemical indexes of each tobacco material in the cold resistance candidate population is determined;S5, according to the comprehensive evaluation value, the cold resistance of tobacco material in the cold resistance candidate population is accurately sorted and finally graded;The present application effectively solves the core pain point that the existing tobacco seedling cold tolerance identification technology cannot consider high-throughput screening efficiency and identification result accuracy, overcomes the technical defects of traditional method phenotype identification subjective error, high cost of full amount of physiological index determination, multi-index evaluation system is not standard, and the technical defects that the distinguishing degree of medium cold resistance material is insufficient.

Description

Technical Field

[0001] This invention relates to a method for grading and identifying the cold tolerance of tobacco seedlings, belonging to the field of plant physiology and crop genetics and breeding technology. Background Technology

[0002] Temperature is a key environmental factor affecting plant growth, development, and geographical distribution. As an important economic crop, tobacco seedlings are extremely sensitive to low temperatures. Low temperature damage during early spring seedling raising is one of the main obstacles to high-quality and stable tobacco production in major tobacco-producing areas. Therefore, rapid and accurate identification of the cold tolerance of tobacco germplasm resources and screening of superior cold-tolerant germplasm are of great significance for tobacco cold-tolerant breeding and production practices. Currently, traditional methods for identifying cold tolerance have the following limitations: First, they rely on single morphological indicators, such as chilling injury index, plant height, and survival rate. These indicators are easily affected by subjective judgment and environmental fluctuations, and cannot reveal the intrinsic physiological resistance differences between materials, thus lacking the ability to distinguish moderately cold-tolerant materials. Second, comprehensive physiological and biochemical testing is costly. If multiple physiological and biochemical indicators (such as protective enzyme activity, osmotic regulators, and photosynthetic parameters) are measured for every material in a large-scale germplasm resource bank, the workload is enormous, the cost is high, and the efficiency is low, making it difficult to promote and apply in actual breeding. Third, the screening process lacks hierarchy. Existing methods usually treat all materials "equally," failing to utilize rapid and low-cost indicators for preliminary screening and elimination, resulting in a large waste of valuable testing resources on materials that are clearly not promising. To address the aforementioned technical issues, a method for grading and identifying the cold tolerance of tobacco seedlings is proposed. Summary of the Invention

[0003] In view of this, the present invention provides a method for grading and identifying the cold tolerance of tobacco seedlings, in order to solve or alleviate the technical problems existing in the prior art, and at least provide a beneficial alternative.

[0004] The technical solution of this invention is implemented as follows: A method for grading and identifying the cold tolerance of tobacco seedlings, comprising the following steps: S1. After disinfecting and sterilizing the tobacco seeds, sow and cultivate them to obtain tobacco seedlings; S2. The obtained tobacco seedlings were transplanted into a nutrient substrate, and a control group and a cold treatment group were set up. After being cultivated to the six-leaf-one-heart stage, they were subjected to cold stress treatment to obtain a tobacco seedling population subjected to low temperature stress. S3. Conduct a chilling injury index survey on tobacco seedling populations subjected to low temperature stress, obtain the chilling injury index of each tobacco material, and perform preliminary cold resistance classification based on the chilling injury index to screen out cold-resistant candidate populations with chilling injury index less than or equal to a preset threshold. S4. Determine the relative activity of multiple physiological and biochemical indicators of each tobacco material in the cold-resistant candidate population to obtain a physiological and biochemical indicator dataset, and perform principal component analysis on the physiological and biochemical indicator dataset to calculate the comprehensive evaluation value of each tobacco material. S5. Based on the comprehensive evaluation value, the tobacco materials in the cold-resistant candidate group are precisely ranked and finally graded according to their cold resistance strength, and the core cold-resistant germplasm is selected.

[0005] More preferably, in step S1, the disinfection and sterilization is performed by treating with sodium hypochlorite at a mass concentration of 2% to 8% for 1 to 5 minutes; the seedling raising is carried out under conditions of temperature of 25±2℃ and relative humidity of 70% to 80%; in step S2, the cold stress treatment is performed under conditions of temperature of 4 to 8℃ and treatment time of 6 to 48 hours.

[0006] In a further preferred embodiment, the chilling injury index survey in step S3 further includes: assessing the damage level of each tobacco seedling according to a preset chilling injury grading standard. The chilling injury index is calculated using the formula: chilling injury index = Σ (number of seedlings at each chilling injury grade × chilling injury grade number) / total number of seedlings.

[0007] More preferably, the preset cold damage grading standard is: Level 0, no obvious symptoms of injury; Grade 1: The edges of the true leaves are scorched or slightly wrinkled; Grade 2, true leaves have severely wrinkled or scorched edges; Grade 3: True leaves are severely shriveled, yellowed, or mosaic-like; Level 4: True leaves with scorched and curled edges or wilted due to water loss; Level 5: The entire seedling wilts and dies, and the seedling cannot recover its growth at room temperature.

[0008] More preferably, the preliminary cold resistance classification in step S4 is as follows: materials with a cold damage index ≤ 1 are classified into a strong cold resistance candidate group, materials with a cold damage index 1 < ≤ 2 are classified into an intermediate candidate group, the materials in the strong cold resistance candidate group and the intermediate candidate group are defined as the cold resistance candidate group, and materials with a cold damage index > 2 are cold-resistant materials.

[0009] Further preferably, the multiple physiological and biochemical indicators mentioned in step S4 include: chlorophyll content, relative conductivity, malondialdehyde content, proline content, superoxide dismutase activity, and peroxidase activity.

[0010] More preferably, the relative activity of the physiological and biochemical indicators in step S4 is the ratio of the activity value of the physiological and biochemical indicators under low temperature stress to the activity value of the physiological and biochemical indicators under normal temperature control.

[0011] In a further preferred embodiment, step S4 involves performing principal component analysis on the physiological and biochemical index dataset to calculate the comprehensive evaluation value of each tobacco material. Specifically, this includes performing membership function analysis and principal component analysis on the physiological and biochemical index dataset to obtain the membership function values ​​and weights of each comprehensive index, and calculating the comprehensive evaluation value of each tobacco material based on the membership function values ​​and weights.

[0012] More preferably, the calculation formula for the membership function analysis is: ; ; Representing the The membership function value of a comprehensive index. For the first A comprehensive indicator value; For the first The maximum value of each comprehensive indicator. For the first The minimum value of each comprehensive indicator.

[0013] More preferably, the formula for calculating the comprehensive evaluation value is: ; ; in, A comprehensive evaluation value representing resilience. Representing the The membership function value of a comprehensive index. Representing the The weights of the comprehensive indicators, and the weights It is calculated from the contribution rate of each comprehensive indicator.

[0014] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: This invention effectively solves the core problem of existing tobacco seedling cold tolerance identification techniques, which cannot simultaneously achieve high-throughput screening efficiency and accurate identification results. It overcomes the technical shortcomings of traditional methods, such as large subjective errors in phenotypic identification, high costs of full-scale physiological index measurement, non-standardized multi-index evaluation systems, and insufficient differentiation of moderately cold-tolerant materials. This invention constructs a two-tiered identification system of phenotypic initial screening and physiological fine screening. First, a standardized chilling injury index is used to complete the initial grading of germplasm, which can quickly eliminate obviously cold-sensitive germplasm with excessive chilling injury indices, significantly narrowing the scope of fine screening and significantly reducing the labor and material costs of testing. This invention enables high-throughput initial screening of large-scale tobacco germplasm resources. Then, for the cold-resistant candidate populations obtained from the initial screening, a standardized comprehensive evaluation system is constructed using membership function analysis and principal component analysis, combining the relative activities of six core physiological and biochemical indicators. This eliminates the bias and subjective interference of single-indicator evaluation, accurately quantifies the intrinsic physiological resistance of germplasm, and significantly improves the distinguishability of moderately cold-resistant materials. The invention is standardized in operation and has good reproducibility, achieving an organic combination of low-cost initial screening and high-precision fine screening, providing reliable technical support for the efficient exploration of cold-resistant tobacco germplasm resources and cold-resistant genetic breeding.

[0015] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart of the method for grading and identifying the cold tolerance of tobacco seedlings according to the present invention; Figure 2 The results represent the determination of the relative activity of malondialdehyde content; Figure 3 The results represent the determination of the relative activity of peroxidase content; Figure 4 The results represent the determination of the relative activity of proline content; Figure 5 The results represent the determination of the relative activity of superoxide dismutase content; Figure 6 The results represent the determination of relative conductivity and relative activity. Figure 7 The results represent the determination of the relative activity of chlorophyll content; Figure 8 This is a dendrogram of cluster analysis for the average membership function values ​​of 6 traits. The vertical axis represents the 24 different variety numbers, and the horizontal axis represents the Euclidean distance in the cluster analysis dendrogram. Detailed Implementation

[0018] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0019] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0020] Example 1

[0021] like Figures 1-8 As shown, this embodiment of the invention uses tobacco varieties as test varieties and conducts tests under conditions of a photoperiod of 16h / 8h and a temperature of 25℃, obtaining 45 germplasm materials. This embodiment provides a method for grading and identifying the cold tolerance of tobacco seedlings, including the following steps: Step S1: Cultivation and Low Temperature Stress Treatment of Experimental Materials Tobacco germplasm resources to be identified were selected. Plump and uniform seeds were sterilized with 5% sodium hypochlorite for 3 minutes. Seedlings were raised in an artificial climate chamber with a day / night temperature of 25℃, relative humidity of 70%, a photoperiod of 16h / 8h, and a light intensity of 20000 lux. Seedlings were used for the experiment when they grew to six leaves and one true leaf. Seedlings of uniform growth were randomly divided into treatment and control groups. The treatment group was placed in a low-temperature light incubator with the following stress conditions: temperature 4℃, photoperiod of 16h light / 8h dark, light intensity of 20000 lux, and treatment duration of 24 hours. The control group was cultured synchronously at 25℃ under normal culture conditions. Each germplasm treatment was replicated three times. Step S2: Preliminary grading and screening based on the cold damage index: Specifically, after low-temperature stress, the chilling injury index of all 45 materials was investigated. The damage to plants in all treatment groups was recorded independently. The cold damage grading standards are as follows: Grade 0: No obvious damage symptoms; Grade 1: Partial scorching or slight wrinkling of true leaf margins; Grade 2: Severe wrinkling or scorching of true leaf margins; Grade 3: Severe wrinkling, yellowing, or mosaic of true leaves; Grade 4: Scorching and curling of true leaf margins or wilting due to dehydration; Grade 5: Wilting and death of the entire seedling, which cannot recover at normal temperature. Calculate the chilling injury index for each germplasm using the following formula: ; Preliminary cold resistance grading: based on the calculated cold damage index. All participating germplasm samples were divided into three levels: Category I Strong Cold Resistance Candidate Group: ≤1; Level II intermediate candidate group: 1< ≤2; Level III Cold Sensitive Elimination Group: >2; The results are shown in Table 1. There are 9 candidates in the Level I strong cold-resistant candidate group, 15 candidates in the Level II intermediate candidate group, and 21 candidates in the Level III cold-sensitive elimination group. Therefore, a total of 24 candidates were selected to enter the secondary screening.

[0022] Table 1. Cold damage index and cold resistance ranking of different tobacco varieties under low temperature stress.

[0023] Step S3: Determine the activity of physiological and biochemical indicators of tobacco after cold treatment: From the cold-resistant candidate populations identified in step S2 and from each germplasm of the normal-temperature control group, functional leaves at the same leaf position were selected, wrapped in aluminum foil, and immediately flash-frozen in liquid nitrogen. These leaves were then transferred to an ultra-low temperature freezer at -80°C for storage and were used to determine the activity of physiological and biochemical indicators. Chlorophyll content, relative conductivity, malondialdehyde, proline, superoxide dismutase, and peroxidase activity were measured using Solarbio and Addison reagent kits. To eliminate differences in the activity of basic physiological and biochemical indicators among germplasms, the relative activity of each physiological and biochemical indicator under low-temperature stress was calculated as follows: Figures 2-7As shown in Table 2, this example then analyzed the coefficient of variation (COP) of chlorophyll content, relative conductivity, malondialdehyde (MDA), proline, superoxide dismutase (SOD), and peroxidase (SOD) activities in 24 tobacco samples under normal and cold stress conditions. The results are shown in Table 2. Compared with normal conditions, the COP of some physiological indicators changed significantly under cold treatment. The COP of relative conductivity decreased from 76.72% in the control group to 62.38%; the COP of peroxide (POD) activity decreased from 51.05% in the control group to 39.01%; the COP of proline (Pro) content decreased from 50.25% to 44.24%; and the COP of superoxide dismutase (SOD) activity decreased from 53.81% to 49.70%. The COPs of relative conductivity and SOD activity both decreased by more than 10 percentage points. However, the COP of chlorophyll content showed the opposite trend, increasing from 23.68% in the control group to 29.32%. The coefficient of variation for malondialdehyde (MDA) content was the most stable, changing almost unchanged from 30.72% to 30.85%. Under cold stress, the coefficients of variation for various physiological indicators ranged from 29.32% to 62.38%, indicating significant differences in the physiological responses of different tobacco germplasm resources under low-temperature stress, suggesting rich genetic variation potential within the population. Furthermore, the effects of cold stress on different physiological indicators varied: the coefficients of variation for relative conductivity, POD, Pro, and SOD decreased, suggesting that the responses of these indicators tended to be consistent across germplasm, possibly regulated by stress-induced conserved physiological pathways; while the coefficient of variation for chlorophyll content increased, indicating strong genotype specificity in its response. Therefore, it is difficult to comprehensively evaluate the cold tolerance of tobacco using any single indicator; a comprehensive analysis of these physiological traits is needed to clarify the intrinsic relationship between each indicator and cold tolerance, and thus screen reliable indicators for identifying cold tolerance.

[0024] Table 2. Analysis of the coefficient of variation results for various physiological indicators.

[0025] Next, a correlation analysis was performed on the cold tolerance coefficients of six physiological and biochemical indicators—relative conductivity, malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), proline, and chlorophyll—in 24 tobacco seedling control and treatment samples. The results are shown in Table 3. The table shows that MDA is significantly negatively correlated with POD and chlorophyll, with correlation coefficients of -0.57** and -0.48**, respectively; POD is significantly positively correlated with chlorophyll and SOD, with correlation coefficients of 0.68** and 0.37*, respectively; and the correlation coefficient between SOD and Pro is 0.01, indicating no significant correlation between them. This suggests overlap in cold tolerance information among the various physiological and biochemical indicators. Therefore, the activity of any single physiological and biochemical indicator cannot be used as a standard for judging tobacco cold tolerance. Further dimensionality reduction analysis is needed to extract the main components of tobacco cold tolerance from the six physiological and biochemical indicators.

[0026] Table 3 Correlation analysis of physiological and biochemical indicators of cold tolerance

[0027] Step S4: Principal component analysis and secondary screening of cold resistance comprehensive evaluation value: Six physiological and biochemical indicators were measured for 24 varieties. Since chlorophyll content, relative conductivity, malondialdehyde, proline, superoxide dismutase, and peroxidase activity have both positive and negative indicators (higher positive values ​​indicate greater cold resistance, and lower negative values ​​indicate greater cold resistance), membership function analysis and principal component analysis were performed on the physiological and biochemical indicator dataset to obtain the membership function values ​​and weights of each comprehensive indicator. Based on the membership function values ​​and weights, a comprehensive evaluation value for each tobacco material was calculated. The following formula was used for standardization to eliminate the influence of dimensions: The formula for calculating membership function analysis is: ; ; Representing the The membership function value of a comprehensive index. For the first A comprehensive indicator value; For the first The maximum value of each comprehensive indicator. For the first The minimum value of a comprehensive indicator; The formula for calculating the comprehensive evaluation value is: ; ; in, A comprehensive evaluation value representing resilience. Representing the The membership function value of a comprehensive index. Representing the The weights of each comprehensive indicator, and the weights It is calculated from the contribution rate of each comprehensive indicator; To compare the contributions of different indicators to cold resistance, the six physiological and biochemical indicators were first converted into membership function values, then principal component analysis was performed on each membership function value, and finally the comprehensive evaluation value was calculated and ranked, as shown in Table 4.

[0028] Table 4. 24-hour (CI), membership function (U), (D) values ​​and overall evaluation of tobacco germplasm.

[0029] Step S5: Select tobacco varieties with cold resistance based on the grading and screening results. Based on the comprehensive evaluation value The 24 materials were ranked by their cold resistance and then subjected to hierarchical clustering. The distance matrix of the 24 materials was calculated using Euclidean distance, and centroid clustering was used to group the materials into three categories, such as... Figure 8 As shown, Category 1 consists of 20 samples, representing intermediate tolerance, accounting for 83.3% of the total materials; Category 4 consists of 3 samples, representing cold tolerance, accounting for 12.5% ​​of the total materials; and Category 3 consists of 1 sample, representing strong tolerance, accounting for 4.1% of the total materials.

[0030] Example 2

[0031] This invention also provides a method for grading and identifying the cold tolerance of tobacco seedlings, based on the grading and identification of cold tolerance of 30 local tobacco germplasms. The specific implementation steps are as follows: 1. Preparation of experimental materials: The test materials were 30 local tobacco germplasm resources from Guizhou Province, all of which came from the tobacco germplasm resource bank of Guizhou Provincial Tobacco Science Research Institute and were local varieties with potential for application in production. 2. Standardized culture and low-temperature stress treatment of experimental materials: Seed disinfection and sterilization: Select plump and undamaged seeds for each test germplasm, soak them in a 3% sodium hypochlorite solution for 5 minutes for disinfection, rinse them 5 times with sterile water after disinfection to completely remove residual disinfectant, and complete the seed disinfection and sterilization. Standardized seedling raising: Disinfected seeds are sown into seedling trays, which are filled with seedling substrate. The seedling substrate is a mixture of vermiculite and peat moss in a 1:1 volume ratio. After sowing, the seedlings are placed in an artificial climate chamber. The seedling environment is set as follows: temperature 25±2℃, relative humidity 75%, light cycle 16h light / 8h dark, light intensity 20000 lux, and the substrate is kept moist under normal management. Seedling grouping and low temperature stress: After 45 days of seedling culture, when the seedlings grew into six-leaf and one-heart seedlings, seedlings with uniform growth were selected and randomly divided into a normal temperature control group and a low temperature treatment group. Each group was repeated 3 times, with 15 seedlings per replicate. The low temperature treatment group was placed in a 6℃ low temperature light incubator with the same light conditions as the seedlings and the stress treatment time was 48h. The normal temperature control group was cultured synchronously under normal conditions at 25℃. 3. Preliminary cold resistance grading and screening based on the cold injury index: After the low-temperature stress ended, the damage level of individual seedlings was assessed according to the 0-5 level chilling injury grading standard, which was completely consistent with that in Example 1. The number of plants at each level was counted and the chilling injury index of each germplasm was calculated. Preliminary grading was carried out according to the same grading rules as in Example 1. In the end, among the 30 tested germplasms, there were 6 strong cold-resistant candidate groups with a chilling injury index ≤ 1, and 10 intermediate candidate groups with a chilling injury index 1 < chilling injury index ≤ 2. These were combined to obtain a total of 16 cold-resistant candidate germplasms. The remaining 14 germplasms with a chilling injury index > 2 were cold-intolerant materials and were eliminated. 4. Determination of physiological and biochemical indicators of cold-resistant candidate populations: For the control and treatment groups of 16 cold-resistant candidate populations, functional leaves at the same leaf position were collected, flash-frozen in liquid nitrogen and stored at -80℃. Following the same method as in Example 1, six indicators were measured: chlorophyll content, relative conductivity, malondialdehyde content, proline content, superoxide dismutase activity, and peroxidase activity. The relative activities of each indicator were calculated, and a physiological and biochemical indicator dataset was constructed. 5. Calculation of comprehensive cold resistance evaluation value based on principal component analysis: Data standardization was performed using the membership function formula identical to that in Example 1. Principal component analysis was then conducted on the standardized data, and three principal components with a cumulative contribution rate ≥85% were extracted as comprehensive indicators. The weights of each comprehensive indicator were calculated according to the formula, and the comprehensive evaluation values ​​of cold tolerance for the 16 germplasm accessions were finally obtained. The formula for calculating membership function analysis is: ; ; Representing the The membership function value of a comprehensive index. For the first A comprehensive indicator value; For the first The maximum value of each comprehensive indicator. For the first The minimum value of a comprehensive indicator; The formula for calculating the comprehensive evaluation value is: ; ; in, A comprehensive evaluation value representing resilience. Representing the The membership function value of a comprehensive index. Representing the The weights of each comprehensive indicator, and the weights It is calculated from the contribution rate of each comprehensive indicator; 6. Final cold tolerance grading and core germplasm selection: Based on the comprehensive evaluation value The cold tolerance of 16 germplasm accessions was ranked. Through systematic cluster analysis, the germplasm was finally divided into 3 strongly cold-tolerant germplasm accessions, 11 moderately cold-tolerant germplasm accessions, and 2 cold-sensitive germplasm accessions. Three core cold-tolerant local germplasm accessions were screened out, and the cold tolerance classification and identification of all germplasm accessions were completed. This verified the universality and stability of the method of the present invention under different stress conditions and different germplasm populations.

[0032] In operation, this invention involves: firstly, disinfecting and sterilizing tobacco seeds with sodium hypochlorite; then, raising seedlings at 25±2℃ and 70%–80% relative humidity; and finally, dividing the seedlings into a normal temperature control group and a low-temperature treatment group when they reach the six-leaf-one-heart stage. The treatment group is then subjected to low-temperature stress treatment at 4–8℃ for 6–48 hours. Subsequently, the chilling injury phenotype of the seedlings in the treatment group is investigated, and the chilling injury index of each germplasm is calculated according to a 0–5 grade standard. Based on this, a preliminary grading is completed, eliminating cold-intolerant materials with a chilling injury index >2 and screening out cold-resistant candidate populations with a chilling injury index ≤2. Next, six core physiological and biochemical indicators of the seedlings in the two candidate populations are measured, and the relative activity of the indicators is calculated to construct a dataset. Through membership function analysis and principal component analysis, a comprehensive evaluation value of the cold resistance of each germplasm is calculated. Finally, based on the comprehensive evaluation value, the cold resistance of the germplasm is ranked and hierarchically clustered to achieve the final grading of the cold resistance of tobacco germplasm and to screen out the core cold-resistant germplasm.

[0033] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for grading and identifying the cold tolerance of tobacco seedlings, characterized in that, Includes the following steps: S1. After disinfecting and sterilizing the tobacco seeds, sow and cultivate them to obtain tobacco seedlings; S2. The obtained tobacco seedlings were transplanted into a nutrient substrate, and a control group and a cold treatment group were set up. After being cultivated to the six-leaf-one-heart stage, they were subjected to cold stress treatment to obtain a tobacco seedling population subjected to low temperature stress. S3. Conduct a chilling injury index survey on tobacco seedling populations subjected to low temperature stress, obtain the chilling injury index of each tobacco material, and perform preliminary cold resistance classification based on the chilling injury index to screen out cold-resistant candidate populations with chilling injury index less than or equal to a preset threshold. S4. Determine the relative activity of multiple physiological and biochemical indicators of each tobacco material in the cold-resistant candidate population to obtain a physiological and biochemical indicator dataset, and perform principal component analysis on the physiological and biochemical indicator dataset to calculate the comprehensive evaluation value of each tobacco material. S5. Based on the comprehensive evaluation value, the tobacco materials in the cold-resistant candidate group are precisely ranked and finally graded according to their cold resistance strength, and the core cold-resistant germplasm is selected.

2. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 1, characterized in that: In step S1, the disinfection and sterilization is performed by treating with sodium hypochlorite at a mass concentration of 2% to 8% for 1 to 5 minutes; the seedling raising is carried out under the conditions of a temperature of 25±2℃ and a relative humidity of 70% to 80%; in step S2, the cold stress treatment is performed under the conditions of a temperature of 4 to 8℃ and a treatment time of 6 to 48 hours.

3. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 1, characterized in that: The chilling injury index survey in step S3 further includes: assessing the damage level of each tobacco seedling according to the preset chilling injury grading standard. The chilling injury index is calculated as follows: chilling injury index = Σ (number of seedlings in each chilling injury grade × chilling injury grade number) / total number of seedlings.

4. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 3, characterized in that: The preset cold damage grading standard is as follows: Level 0, no obvious symptoms of injury; Grade 1: The edges of the true leaves are scorched or slightly wrinkled; Grade 2, true leaves have severely wrinkled or scorched edges; Grade 3: True leaves are severely shriveled, yellowed, or mosaic-like; Level 4: True leaves with scorched and curled edges or wilted due to water loss; Level 5: The entire seedling wilts and dies, and the seedling cannot recover its growth at room temperature.

5. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 1, characterized in that: The preliminary cold resistance classification in step S4 is as follows: materials with a cold damage index ≤ 1 are classified as strong cold resistance candidate group, materials with a cold damage index 1 < cold damage index ≤ 2 are classified as intermediate candidate group, the materials in the strong cold resistance candidate group and the intermediate candidate group are defined as the cold resistance candidate group, and materials with a cold damage index > 2 are cold-resistant materials.

6. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 1, characterized in that: The multiple physiological and biochemical indicators mentioned in step S4 include: chlorophyll content, relative conductivity, malondialdehyde content, proline content, superoxide dismutase activity, and peroxidase activity.

7. A method for grading and identifying the cold tolerance of tobacco seedlings according to claim 1 or 6, characterized in that: The relative activity of the physiological and biochemical indicators mentioned in step S4 is the ratio of the activity value of the physiological and biochemical indicators under low temperature stress to the activity value of the physiological and biochemical indicators under normal temperature control.

8. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 1, characterized in that: In step S4, principal component analysis is performed on the physiological and biochemical index dataset to calculate the comprehensive evaluation value of each tobacco material. Specifically, this includes: performing membership function analysis and principal component analysis on the physiological and biochemical index dataset to obtain the membership function values ​​and weights of each comprehensive index, and calculating the comprehensive evaluation value of each tobacco material based on the membership function values ​​and weights.

9. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 8, characterized in that: The calculation formula for the membership function analysis is as follows: ； ； Representing the The membership function value of a comprehensive index. For the first A comprehensive indicator value; For the first The maximum value of each comprehensive indicator. For the first The minimum value of each comprehensive indicator.

10. The method for grading and identifying the cold tolerance of tobacco seedlings according to claim 8, characterized in that: The formula for calculating the comprehensive evaluation value is as follows: ； ； in, A comprehensive evaluation value representing resilience. Representing the The membership function value of a comprehensive index. Representing the The weights of the comprehensive indicators, and the weights It is calculated from the contribution rate of each comprehensive indicator.

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