Method for rapidly identifying resistance purity of ALS herbicides in rape seeds

Abstract

The invention discloses a phenotype group-based method for rapidly identifying resistance purity of ALS herbicides in rape seeds, and relates to the technical field of biologically identifying resistance purity of rape seeds. The method comprises the following steps: sampling and disinfecting seeds, putting the seeds into plant gel containing ALS herbicides, and observing and measuring the root length of each seedling after the seeds germinate and grow; calculating the resistance purity of the ALS herbicide-resistant rape seeds according to a resistance purity calculation formula; according to the method, background influence can be eliminated, the resistance purity of the herbicide-resistant rape seeds can be rapidly and accurately identified in a high-throughput manner, and compared with a seedling-stage pesticide spraying identification method commonly used in production at present, the method can greatly shorten identification time and effectively improve the accuracy of results; compared with detection methods such as molecular marking and sequencing which are commonly used in laboratories, the method has the advantages that the technical difficulty and the detection cost of detection can be greatly reduced on the premise of ensuring the same accuracy, and the method is more suitable for field popularization and application of resistant varieties when being used for identifying the resistance purity.

Description

Technical Field

[0001] The present application relates to the field of biological identification methods, and in particular to a method for rapidly identifying the purity of rapeseed seeds' resistance to ALS herbicides based on phenomics. Background Art

[0002] Compared to other crops, rapeseed exhibits stronger hybrid vigor and significant yield increases, reaching 20% ​​to 30% or even higher. Therefore, leveraging hybrid vigor in rapeseed variety breeding significantly improves rapeseed quality and yield. Variety resistance purity is the most important quality parameter for herbicide-resistant rapeseed hybrids, directly impacting their yield and quality. After seed production, the purity of the resistant variety must be determined. Only when the purity meets a certain standard can the variety be marketed and put into large-scale production. Failure to do so will result in significant losses for growers and distributors.

[0003] Common methods for identifying seed resistance purity can be roughly divided into three categories: phenotypic, biochemical, and molecular marker. Phenotypic identification involves spraying a certain number of 3-5 leaf rapeseed seedlings in a greenhouse or field with a herbicide at a concentration twice the recommended field herbicide concentration. The survival rate is then calculated three weeks to one month after treatment, representing the resistance purity of the tested hybrid. Biochemical identification involves collecting individual leaves from non-resistant and resistant hybrid rapeseed seedlings at the 3-5 leaf stage, extracting crude ALS enzyme from the target plant, and performing in vitro enzyme inhibition tests with varying herbicide concentrations to calculate the IC50 value. The IC50 values ​​are then compared for each material to determine the resistance purity of the hybrid. Molecular marker identification involves developing molecular marker primers based on the resistance loci, extracting DNA from the materials, and then testing the DNA to calculate the resistance purity of the hybrid. Alternatively, for herbicide-resistant hybrids, Sanger sequencing can be used to directly detect the sequences of PCR products containing resistance loci, and then calculate the resistance purity of the resistant hybrids. However, phenotypic identification methods require long cycles, are time-consuming and land-intensive, and the results are easily affected by environmental factors. Furthermore, spray uniformity and resistance grading are significantly affected by the skills and experience of the identifiers, making accurate results difficult to obtain. Biochemical identification methods offer high accuracy, good reproducibility, and are unaffected by environmental factors, but they require long cycles, are time-consuming and labor-intensive, and have low identification throughput. Molecular markers and sequencing methods offer shorter cycles and high accuracy, good reproducibility, and environmental resistance, but they are expensive, complex, and require high costs and technical expertise. Currently, there are no specific identification standards for the resistance purity of herbicide-resistant rapeseed varieties. In practice, identification is primarily conducted through traditional field planting and exogenous herbicide spraying.

[0004] Therefore, developing an accurate, high-throughput, simple, time-saving, fast and low-cost method for identifying the resistance purity of rapeseed varieties resistant to ALS herbicides has become a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0005] In response to the above problems, the present application provides a phenotype-based method for rapid identification of the purity of rapeseed seed ALS herbicide resistance, aiming to address the shortcomings of existing technologies and establish a method for detecting the resistance purity of rapeseed herbicide-resistant seeds with simple operation, high throughput, fast speed, and accurate and stable identification results.

[0006] Specifically, this application adopts the following technical solutions:

[0007] A method for rapidly identifying the purity of rapeseed seed resistance to ALS herbicides comprises the following steps:

[0008] (1) Seed preparation: Randomly sample 150-250 seeds of rapeseed to be tested for ALS-resistant herbicide and 50 seeds of non-herbicide-resistant control samples. The sampled seeds are respectively soaked and disinfected in ethanol (e.g., 75% by volume ethanol solution) for 10 minutes, and then washed three times with distilled water before use.

[0009] (2) Gel preparation:

[0010] Pour the sterilized gel containing the ALS herbicide into a transparent plate, with the gel surface at a height of 3 / 10 of the total height of the device from the top, and allow to cool naturally before use. In the embodiment of the present application, the height of the device is preferably 10.5 cm.

[0011] The preparation method for each 200ml of plant gel is as follows: 3g of plant gel powder and 0.474g of MS culture medium are mixed, and the volume is adjusted to no more than 194ml with ddH2O. The mixture is sterilized by moist heat at 121°C for 15 minutes. When the temperature of the culture medium drops to 40-45°C, a bensulfuron-methyl solution (preferably a concentration of 1 times the recommended field herbicidal concentration) is added until the bensulfuron-methyl content reaches 6.25mg. Then, ddH2O is added to adjust the volume to 200ml.

[0012] MS medium formula: by final concentration, 1650mg / L NH4NO3, 1900mg / L KNO3, 440mg / L CaCl2·2H2O, 370mg / L MgSO4·7H2O, 170mg / L KH2PO4, 0.83mg / L KI, 6.2mg / L H3BO3, 22.3mg / L MnSO4·4H2O, 8.6mg / L ZnSO4·7H2O, 0.25mg / L Na2MoO4·2H2O, 0.025mg / L CuSO4·5H2O, 0.025mg / L CoCl2·6H2O; iron salts, 27.8mg / L FeSO4·7H2O, 37.3mg / L Na2-EDTA·2H2O, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L vitamin B6, 0.1 mg / L vitamin B1, 2.0 mg / L glycine; add ddH2O to make up the balance;

[0013] (7) Placement of seeds:

[0014] The seeds to be tested and the control seeds were placed equidistantly on the surface of the gel, with a distance of about 0.8-1 cm between the seeds, and then the top of the plate was sealed with plastic sealing film.

[0015] (8) Seed germination and growth:

[0016] Seal the plate with plastic film and place it in a tissue culture room to grow naturally for 7-10 days;

[0017] (9) Measurement and statistics:

[0018] The root length of each seedling of the identification sample and the control seed was measured. Based on the average root length of the control seeds and the longest root length H of the resistant seeds, the standard for determining the effective root length of the resistant seeds was analyzed and determined. The number of resistant seeds (R), the number of non-resistant seeds (S) and the total number of seedlings (R+S) of the identification sample were counted. The standard for resistant seeds was root length > H / 4 cm (H was the longest root length of the plate), and the standard for non-resistant seeds was root length ≤ 3H / 14 cm (H was the longest root length of the plate). At the same time, the root length of the non-resistant seeds was greater than 5 mm or the aboveground elongation was greater than 5 mm (reference was made to the control seeds to exclude seeds that did not germinate).

[0019] (6) Resistance purity calculation: Calculate the resistance purity (ρ) of rapeseed seeds resistant to ALS herbicides according to the resistance purity calculation formula:

[0020] (7) ρ = [R / (R+S)] × 100%.

[0021] Furthermore, the rapeseed seeds used for identification in step 1) above are sampled using the quartering method. Specifically, the sample is first poured onto a smooth, flat tabletop or glass plate and mixed thoroughly using a sample divider. The sample is then spread into a square of equal thickness and scored diagonally with a sample divider to form four triangles with opposite corners. Two of the triangles with opposite corners are then randomly selected as samples. Finally, the remaining sample is mixed again and the above method is repeated until the weight of the remaining two triangles is approximately equal to one-fourth of the sample weight (1,000-seed weight).

[0022] Furthermore, the rapeseed seeds used for identification in the above step 1) were placed on 5 plates, 50 seeds per plate, i.e., 5 repetitions; and the control seeds were placed on 1 plate.

[0023] Preferably, the growth conditions of the tissue culture room in step 4) are: temperature 19° C. to 22° C., humidity 58%, light intensity 2300 lux, and photoperiod 14 h light / 10 h dark.

[0024] In this application, the resistant rapeseed seeds used for identification are generally ALS-resistant rapeseed seeds with practical production value, such as Ning R101, Ning R201, Huinong Oil, Su R001, Jindi Oil No. 1, and Hongyou 789, that can tolerate 3-4 times the recommended field herbicide concentration; or resistant germplasm materials such as M342 and EM28. The control line used in the examples is the sensitive material N131. In specific implementations, other rapeseed varieties or lines that are not resistant to ALS herbicides can also be used as controls.

[0025] Compared with the existing rapeseed herbicide-resistant seed resistance purity identification technology, this application scheme has the following beneficial effects:

[0026] 1) This application first discovered a specific concentration of bensulfuron-methyl (1 / 16 times the recommended herbicide concentration) that is optimal for safely and effectively distinguishing between resistant and non-resistant rapeseed. Based on this concentration, a method for rapidly identifying the resistance purity of rapeseed seeds resistant to ALS herbicides was proposed. This method is consistent with the results of existing conventional resistance purity identification methods, such as field planting, molecular markers, and sequencing, demonstrating the successful application of the present method in identifying the resistance purity of rapeseed seeds resistant to herbicides.

[0027] 2) The identification method proposed in this application offers advantages such as simplicity, high throughput, rapid speed, accurate and stable identification results, and strong versatility, greatly simplifying the process of identifying the resistance purity of new varieties and combinations. Furthermore, it is economical, environmentally friendly, highly reproducible, and easily scalable.

[0028] 3) This application uses plant gel as a supporting matrix, which is clearer and more transparent, and can make the observation results more accurate and clear during the root identification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0030] Figure 1 These are the plate phenotypes of Su R001 (A), Jindi Oil No. 1 (B), and sensitive material N131 (C) used in Example 1.

[0031] Figure 2 These are the plate phenotypes of M342 (A), EM28 (B) and the sensitive material N131 (C) used in Example 2.

[0032] Figure 3 This is the field phenotype of Su R001, Jindiyou No. 1 and N131 after exogenous spraying of 1 times the recommended field concentration of bensulfuron-methyl at the three to five leaf stage.

[0033] Figure 4 M342, EM28 and N131 were sprayed with 1 times the recommended field concentration of bensulfuron-methyl at the three to five leaf stage, and the field phenotypes were observed one month later. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] The rapeseed herbicide-resistant hybrid varieties Su R001 and Jindiyou No. 1, resistant conventional germplasms M342 and EM28, and sensitive conventional germplasm N131 involved in the following examples were all provided by the rapeseed team of Jiangsu Academy of Agricultural Sciences. The resistant hybrid varieties used in this example are all existing resistant varieties, as disclosed in the document "Evaluation of cross-resistance and resistance gene analysis of rapeseed germplasm M196 to ALS herbicides" (Lu Xinyu et al., Journal of Plant Genetic Resources, 2024, 25(12): 2069-2080).

[0036] Reagents and devices involved in the embodiment:

[0037] (1) Disinfectant: 75% (volume concentration) ethanol.

[0038] (2) MS culture medium formula: macroelements, 1650mg / L NH4NO3, 1900mg / L KNO3, 440mg / LCaCl2·2H2O, 370mg / L MgSO4·7H2O, 170mg / L KH2PO4; trace elements, 0.83mg / L KI, 6.2mg / L H3BO3, 22.3mg / L MnSO4·4H2O, 8.6mg / L ZnSO4·7H2O, 0.25mg / L Na2MoO4·2H2O, 0.025mg / L CuSO4·5H2O, 0.025mg / L CoCl2·6H2O; iron salts, 27.8mg / L FeSO4·7H2O, 37.3mg / L Na2-EDTA·2H2O; organic substances, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L vitamin B6, 0.1 mg / L vitamin B1, 2.0 mg / L glycine; make up the balance with ddH2O and sterilize (moist heat sterilization at 121°C for 15 min) before use;

[0039] (3) Preparation of bensulfuron-methyl mother solution: Add ddH2O to bensulfuron-methyl powder (Jiangsu Kuaida Agrochemical Co., Ltd., formulation: wettable powder, 10% active ingredient) to prepare a bensulfuron-methyl mother solution with a concentration of 1 times the recommended field herbicidal concentration, place it in a clean bench for ultraviolet light sterilization for 24 hours, and set aside;

[0040] (4) 1.5% plant gel (containing herbicide): 3g plant gel powder (purchased from Shanghai Yuanye Biotechnology Co., Ltd., plant cell culture grade plant gel), 0.474g MS culture medium, mix well and dilute to 194ml / plate with ddH2O (in practice, no more than 194ml), sterilize with moist heat at 121℃ for 15min; when the culture medium temperature drops to 40-45℃, add bensulfuron-methyl mother solution until the bensulfuron-methyl content is 6.25mg bensulfuron-methyl (1 / 16 of the field recommended concentration), and then add ddH2O to dilute to 200ml;

[0041] (5) The flat panel device system used in the embodiment is the flat panel device disclosed in patent CN116794032A, which has a height of 10.5 cm. In specific implementations, devices / flat panels made of other transparent materials can also be used to culture and observe the rooting of rapeseed seeds, or the roots can be directly observed by adding absorbent paper to a culture dish and then pulling it out.

[0042] Example 1 Rapid Identification of ALS-Resistant Rapeseed Seed Purity

[0043] The ALS-resistant rapeseed varieties used in this example are Su R001 and Jindiyou No. 1, and the control material is N131.

[0044] 1. Resistance identification steps:

[0045] Seed preparation: 250 seeds of rapeseed to be tested for ALS-resistant herbicide resistance and 50 seeds of non-herbicide-resistant control samples were randomly sampled. The sampled seeds were disinfected by soaking in ethanol for 10 minutes, washed three times with distilled water and then set aside.

[0046] Rapeseed seeds for identification are sampled using the quartering method. This involves first pouring the sample onto a smooth, flat tabletop or glass plate and mixing it thoroughly with a sample divider. Then, spread the sample into a square of equal thickness and use a sample divider to score the sample diagonally, dividing it into four triangles with opposite corners. Then, randomly select two of the triangles as samples. Finally, mix the remaining sample again and repeat the above method until the weight of the remaining two triangles approximates to 1 / 4 of the sample weight (1,000-seed weight).

[0047] Gel preparation: Pour 1.5% plant gel into the flat-plate device system used for identification, with the height of the gel surface from the top of the device being 3 / 10 of the entire device height. Cool naturally and set aside.

[0048] Seed placement: Place the seeds to be tested evenly spaced on the gel surface, approximately 0.8-1 cm apart. Place 50 seeds per plate. Seal the top of the plate with plastic film. Place five plates per sample, i.e., replicate five times. Place one plate for control seeds.

[0049] Seed germination and growth: Place the plates in a tissue culture chamber under the following growth conditions: 19°C–22°C, 58% humidity, 2300 lux light intensity, and a 14-hour light / 10-hour dark photoperiod. Identify the seeds after 7 days of natural growth (generally, identification can be performed after 7–10 days of growth, when visual inspection of the root length of resistant seeds shows a statistically significant difference compared to control seeds).

[0050] After 7 days, the growth of the flat-plate roots of the three materials Su R001, Jindi Oil No. 1, and N131 were as follows: Figure 1 As shown in A, B, and C.

[0051] Measurement and statistics: Measure the root length of each seedling of the identification sample and control seeds, as shown in Table 1. Based on the average root length of the control seeds and the longest root length H of the resistant seeds, analyze and determine the standard for determining the effective root length of the resistant seeds. Statistically calculate the number of resistant seeds (R), non-resistant seeds (S), and the total number of seedlings (R+S) in the identification sample. 2. Method for calculating the resistance purity of rapeseed herbicide-resistant seeds, the steps are as follows:

[0052] A) measuring the 1.5% phytogel level in the plate apparatus to 7 cm according to the herbicide-resistant seed resistance identification method described in 1 above, and counting the number of resistant seeds (R), the number of non-resistant seeds (S), and the total number of seedlings (R+S) on each plate;

[0053] B) The formula for calculating resistance purity is: ρ = [R / (R+S)] × 100%.

[0054] Table 1 Maximum root length (H) of the tested varieties, corresponding values ​​of 3H / 14 and H / 4, and resistance purity of the varieties

[0055]

[0056] One biological replicate of the Su R1001 plate assay was Figure 1 As shown in middle A, the average resistance purity identification results of five biological replicates were 91%.

[0057] A biological replicate of Jindiyou No. 1 plate identification is as follows Figure 1 As shown in B, the average resistance purity of five biological replicates was 89%.

[0058] One biological replicate of the N131 plate assay was Figure 1 As shown in middle C, the average value of the non-antibody purity identification results of five biological replicates was 100%.

[0059] Resistance purity verification: Referring to the method disclosed in the literature "Evaluation of cross-resistance and analysis of resistance genes of rapeseed germplasm M196 to ALS herbicides. Journal of Plant Genetic Resources, 2024, 25(12): 2069-2080", the same batch of "Su R001", "Jindi Oil No. 1" and "N131" seed samples were simultaneously subjected to field planting identification, molecular marker identification and sequencing identification. Among them, field planting identification used seedlings in the 3-5 leaf stage to bensulfuron-methyl exogenously sprayed (the spraying amount was in accordance with the concentration recommended in the product manual), and the number of surviving resistant seedlings was statistically investigated one month later. The resistance purity calculation formula is: Field planting identification seed resistance purity = (number of surviving resistant plants / total number of surveyed plants) × 100%. The field growth of the three materials is shown in Figure 2. Figure 2 shown.

[0060] Molecular marker identification utilizes the differences in DNA bands between resistant and non-resistant varieties, referring to the method disclosed in the literature "Synergistic mutations of two rapeseed AHAS genes confer high resistance to sulfonylureaherbicides for weed control." The resistance purity is calculated as follows: molecular marker identification seed resistance purity = (number of resistant seed band patterns / total number of seeds tested) × 100%;

[0061] Sequencing identification utilizes the differences in peak patterns of resistance sites identified in resistant species and those in non-resistant species, and is performed with reference to the method disclosed in the document "Development and Application of CAPS Markers for the M342 Herbicide Resistance Gene." The resistance purity calculation formula is: Sequencing identification seed resistance purity = (number of resistant species peak patterns / total number of seeds tested) × 100%. The identification results of the four methods were analyzed and compared to verify the accuracy of this method in resistance purity identification.

[0062] The results of the resistance purity verification showed that the same batch of "Su R1001", "Jindi Oil No. 1" and "N131" seed samples were used for field planting and identification, and the resistance purity results were 89% and 87% respectively. The non-resistant purity of N131 was 100%.

[0063] The molecular marker identification showed that the purity of resistance strains was 92% and 90% respectively, while the purity of non-resistant strain N131 was 100%.

[0064] Sequencing identification showed that the purity of the resistance products was 91% and 88% respectively, and the purity of N131 non-resistance product was 100%.

[0065] Table 2 Comparison of seed resistance purity of herbicide-resistant varieties Su R001 and Jindiyou No. 1 identified by different methods

[0066]

[0067] Note: The sensitive material N131 was used as the non-resistant control group.

[0068] The above resistance purity verification results are shown in Table 2. The identification results of the method for rapid identification of ALS-resistant rapeseed seed resistance purity (plate method) provided in this example are highly consistent with field planting identification, molecular marker identification and sequencing identification.

[0069] Example 2

[0070] In this example, the herbicide-resistant rapeseed germplasms M342 and EM28 were used as research objects, and the sensitive conventional material N131 was used as a control. The test seeds were provided by the rapeseed team of Jiangsu Academy of Agricultural Sciences.

[0071] 1. Seed resistance identification steps are the same as in Example 1.

[0072] 2. Identification of the resistance purity of herbicide-resistant germplasm materials, the steps are as follows:

[0073] A) Referring to the method disclosed in Example 1, the agar liquid level in the plate apparatus was measured at 7 cm, and the number of resistant seeds (R), the number of non-resistant seeds (S), and the total number of seedlings (R + S) on each plate were counted; the rapeseed root growth is shown in Table 1.

[0074] B) Calculation formula of resistance purity: same as Example 1.

[0075] One biological replicate of the M342 plate assay was Figure 3 As shown in A, the average resistance purity of three biological replicates was 90%.

[0076] One biological replicate of the EM28 plate assay was Figure 3 As shown in B, the average resistance purity of three biological replicates was 60%.

[0077] One biological replicate of the N131 plate assay was Figure 3 As shown in middle C, the average value of the non-antibody purity identification results of three biological replicates was 100%.

[0078] Resistance purity verification: steps are the same as in Example 1

[0079] The results of resistance purity verification showed that the same batch of M342, EM28 and N131 seed samples were used for field planting and identification, and their growth conditions were as follows: Figure 4 The resistance purity results were 91% and 62% respectively, and the non-resistant purity of N131 was 100%;

[0080] The molecular marker identification showed that the purity of resistance strains was 89% and 59% respectively, while the purity of non-resistant strain N131 was 100%.

[0081] Sequencing identification showed that the purity of the resistance products was 91% and 60% respectively, and the purity of N131 non-resistance product was 100%.

[0082] Table 3 Comparison of seed resistance purity of herbicide-resistant materials M342 and EM28 identified by different methods

[0083]

[0084] Note: The sensitive material N131 was used as the non-resistant control group.

[0085] The results of resistance purity verification are shown in Table 3. The identification results of a method for rapid identification of rapeseed seed resistance purity to ALS herbicides (plate method) are highly consistent with field planting identification, molecular marker identification and sequencing identification.

[0086] The above examples disclose a method for rapidly identifying the resistance purity of ALS-resistant rapeseed seeds. This method is also used to identify the resistance purity of resistant rapeseed varieties, resulting in a method for rapidly identifying the resistance purity of ALS-resistant rapeseed seeds (referred to as the plate method). The above examples used four different methods, including the plate method, field planting, molecular markers, and sequencing, to identify the resistance purity of the same batch of herbicide-resistant seeds. The results showed that the identification results from the four methods were consistent, demonstrating that the resistance purity identification method provided in the above examples can be successfully applied to the identification of the resistance purity of herbicide-resistant rapeseed seeds. This method overcomes the shortcomings of existing rapeseed resistance purity identification technologies, such as long testing cycles, time-consuming and labor-intensive processes, high costs, and expensive instrumentation. It offers advantages such as simple operation, high throughput, rapid speed, accurate and stable identification results, and strong versatility, significantly simplifying the resistance purity identification process for new varieties and new combinations. Furthermore, its economical and environmentally friendly nature, high repeatability, and ease of implementation provide a technical guarantee for reducing risks during the promotion and application of herbicide-resistant rapeseed hybrid varieties. The invention solves the problem of low efficiency of the existing method for identifying the purity of herbicide-resistant rapeseed.

[0087] The above description is only one embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for rapidly identifying the purity of rapeseed seeds resistant to ALS herbicides, characterized in that: The specific steps are as follows: (1) Seed preparation: After sampling the rapeseed seeds to be tested and the control seeds without resistance, they were disinfected with ethanol and then washed three times with distilled water for later use; (2) Gel preparation: Pour the plant gel into a transparent plate, with the gel surface at a height of 3 / 10 of the total height of the device from the top, and allow to cool naturally before use. The preparation method for each 200ml of plant gel is as follows: 3g plant gel powder, 0.474g MS culture medium, mix well, and then dilute to no more than 194ml with ddH2O. Sterilize with moist heat at 121℃ for 15 minutes. When the culture medium temperature drops to 40-45℃, add bensulfuron-methyl solution to a bensulfuron-methyl content of 6.25mg, and then add ddH2O to dilute to 200ml. The MS culture medium formula is as follows: based on the final concentration, 1650 mg / L NH4NO3, 1900 mg / L KNO3, 440 mg / L CaCl2·2H2O, 370 mg / L MgSO4·7H2O, 170 mg / L KH2PO4, 0.83 mg / L KI, 6.2 mg / L H3BO3, 22.3 mg / L MnSO4·4H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O; iron salts, 27.8 mg / L FeSO4·7H2O, 37.3 mg / L Na2-EDTA·2H2O, 100 mg / L inositol, 0.5 mg / L niacin, 0.5 mg / L Vitamin B6, 0.1 mg / L vitamin B1, 2.0 mg / L glycine; add ddH2O to make up the balance; (3) Placement of seeds: The seeds to be tested and the control seeds are placed on the surface of the gel respectively, and then the top of the plate is sealed; (4) Seed germination and growth: Place the plate in a tissue culture room and grow naturally for 7-10 days; (5) Measurement and statistics: Measure the root length of each seedling of the identification sample and the control seed. Based on the average root length of the control seeds and the longest root length H of the resistant seeds, analyze and determine the standard for determining the effective root length of the resistant seeds. Count the number of resistant seeds R, the number of non-resistant seeds S, and the total number of seedlings R+S in the identification sample. The criterion for resistant seeds is root length > H / 4 cm, where H is the longest root length on the plate; the criterion for non-resistant seeds is root length ≤ 3H / 14 cm, where H is the longest root length on the plate, and the root length of non-resistant seeds is greater than 5 mm or the aboveground elongation is greater than 5 mm; (6) Calculation of resistance purity: The resistance purity ρ of rapeseed seeds resistant to ALS herbicides was calculated according to the resistance purity calculation formula: ρ=[R / (R+S)]×100%.

2. The method for rapidly identifying the purity of rapeseed seed resistance to ALS herbicides according to claim 1, characterized in that: In step (3), 150-250 rapeseed seeds to be tested are sampled, and 50 control seeds are sampled.

3. The method for rapidly identifying the purity of rapeseed seed resistance to ALS herbicides according to claim 1, characterized in that: In step (3), the distance between seeds on the gel surface is 0.8-1 cm.

4. The method for rapidly identifying the purity of rapeseed seed resistance to ALS herbicides according to claim 1, characterized in that: In step (4), the growth conditions in the tissue culture room are: temperature 19°C to 22°C, humidity 58%, light intensity 2300 lux, and photoperiod 14 h light / 10 h dark.

5. The method for rapidly identifying the purity of rapeseed seed resistance to ALS herbicides according to claim 1, characterized in that: The sampling in step (1) refers to sampling by quartering method.

6. The method for rapidly identifying the purity of rapeseed seed resistance to ALS herbicides according to claim 1, characterized in that: The control seed in step (1) is N131.

7. The method for rapidly identifying the purity of rapeseed seed resistance to ALS herbicides according to claim 1, characterized in that: The rapeseed seeds to be tested in step (1) are ALS-resistant rapeseed seeds.

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