High purity glyphosate-resistant transgenic corn seed and method for increasing purity thereof

Abstract

The present application relates to the technical field of pesticides (IPC classification A01C1 / 00), and in particular to a high-purity glyphosate-resistant transgenic corn seed and a method for improving purity, the method comprising: seed dressing treatment of corn seeds with a treatment agent, the mass ratio of the treatment agent to the corn seeds being 1:(20-30). The method provided by the present application cannot germinate after seed dressing of seeds without glyphosate-resistant transgenic seeds, thereby removing non-transgenic seeds and improving the purity of glyphosate-resistant transgenic corn seeds.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticides (IPC classification A01C1 / 00), in particular to a high-purity glyphosate-resistant transgenic corn seed and a method for improving the purity thereof. BACKGROUND

[0002] Genetic modification technology is widely used in corn breeding, and glyphosate-resistant transgenic corn varieties have been marketed internationally. China has also approved transgenic corn varieties, including a large proportion of glyphosate-resistant transgenic corn varieties. In the breeding process, the purity of the seed is a key factor for successful breeding. In the prior art, the purity is mainly determined by chemical detection methods, which are based on the differences in the reactions of the shells of different varieties to chemical reagents. However, these methods have the problems of long detection time, complex operation, limited detection types, and susceptibility to environmental factors such as weather. Moreover, they cannot simultaneously detect a large number of samples, and the process of screening high-purity samples after detection is complex.

[0003] Chinese patent CN106576829A discloses a method for identifying glyphosate-resistant corn varieties, which involves soaking corn seeds in a glyphosate aqueous solution with an additive amount of 0.1-2.0% and detecting relevant indicators. However, the solubility of glyphosate in water is limited and unevenly distributed, and the effective additive amount of 0.1-2.0% is too small, resulting in a large error in identification. Chinese patent CN114586496A discloses a method for identifying the glyphosate tolerance of cotton seeds, which involves soaking cotton seeds in a 40-fold, 80-fold, 100-fold, 120-fold, or 140-fold dilution of 33% glyphosate isopropylamine salt. From the perspective of control, seed soaking can kill bacteria carried by the seeds, but the amount of pesticide carried by the seeds is already very small, and the pesticide has little control effect on soil pathogens. Therefore, it is necessary to develop a method for improving purity that is accurate, easy to operate, time-saving, and has a control effect. SUMMARY

[0004] The first aspect of the present application provides a method for improving the purity of glyphosate-resistant transgenic corn seeds, which comprises: seed dressing treatment of corn seeds with a treatment agent, and the mass ratio of the treatment agent to the corn seeds is 1:(20-30).

[0005] In some embodiments, the preparation raw materials of the treatment agent include: glyphosate salt, non-ionic surfactant, and water.

[0006] In some embodiments, the preparation raw materials of the treatment agent include, by mass percentage: 1-60% of glyphosate salt, 0.7-15% of non-ionic surfactant, and the balance of water.

[0007] In some embodiments, the preparation raw material of the treatment agent comprises, by mass percentage, glyphosate salt 5-50%, non-ionic surfactant 0.7-10%, and water in remainder.

[0008] In some embodiments, the non-ionic surfactant comprises at least one of alkyl polyglucoside, fatty alcohol polyoxyethylene ether, and alkyl phenol polyoxyethylene ether.

[0009] In some embodiments, the non-ionic surfactant has a viscosity of 100-200 mPa.s.

[0010] Further, the non-ionic surfactant is alkyl polyglucoside, has a viscosity (20℃) of 200 mPa.s, and is preferably sourced from Nantong Kenhe Chemical Co., Ltd. and has a model number of APG-0810.

[0011] The viscosity of the non-ionic surfactant in the present application is very important for the improvement of purity, and a too high viscosity will affect the dispersibility of glyphosate in water, while a too low viscosity will reduce the adhesion of glyphosate to the surface of the seed and affect the control effect.

[0012] In some embodiments, the glyphosate salt comprises at least one of glyphosate isopropylamine salt, glyphosate amine salt, glyphosate sodium salt, and glyphosate potassium salt.

[0013] Glyphosate is difficult to dissolve in water, and the effective utilization rate of the agent prepared by the water-soluble method in the prior art is very low, and the error in screening the seed is also large. In the present application, the applicant first prepares a glyphosate salt from glyphosate and a specific base, and then further seeds, which on the one hand improves the solubility of glyphosate in water and effectively improves the screening accuracy of the seed purity, and on the other hand, from the control point of view, the seed coating method is more conducive to the control of weeds around the seed than the soaking method in the prior art, and the process of the present application has the effects of improving the purity and controlling the weeds.

[0014] In some embodiments, the pH value of the treatment agent is 6.5-7.5.

[0015] The applicant found in the exploration that when the pH value is lower than 6.5, the treatment agent will precipitate crystals and cannot be further coated on the seed, and when the pH value is too high, the decomposition rate of glyphosate will exceed 5% after one month of storage at room temperature, and the storage stability is poor.

[0016] In some embodiments, the temperature of the seed coating treatment is 25±5℃, and the time is 0.5-5 min.

[0017] The second aspect of the present application provides a high-purity glyphosate-resistant transgenic corn seed, which is obtained by the method and has a purity of 100%.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] 1. The application provides a method for improving the purity of glyphosate-resistant transgenic corn seeds.

[0020] 2. Compared with the prior art, the purity of the screened corn seeds is as high as 100%, and the result accuracy is high.

[0021] 3. The method provided by the application not only improves the purity of the seeds, but also has a preventive effect, and simultaneously inhibits weeds in the surrounding soil.

[0022] 4. The method provided by the application can be used for indoor small-scale testing and outdoor field testing, and the test scale can be flexibly adjusted according to actual needs, and the experimental method is not limited by the environment.

[0023] 5. The application has the advantages of simple operation, easy-to-obtain raw materials, low testing cost, high result accuracy, good stability and suitability for large-scale promotion. DETAILED DESCRIPTION

[0024] Embodiment 1

[0025] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the method comprising: using a treatment agent to treat corn seeds at 25 DEG C for 1 min, and the mass ratio of the treatment agent to the corn seeds is 1:25.

[0026] The preparation raw materials of the treatment agent comprise: glyphosate isopropylamine salt 8.5%, non-ionic surfactant 5%, and water in remainder by mass percentage.

[0027] The non-ionic surfactant is alkyl polyglucoside, the viscosity (20 DEG C) is 200 mPa.s, the source is Nantong Kenhe Chemical Co., Ltd., and the model is APG-0810.

[0028] The preparation method of the treatment agent comprises the following steps: placing water in a stirring kettle, adding isopropylamine while stirring, continuously adding glyphosate raw powder while stirring, adjusting the pH value to 6.5-7.5 by using 5 wt% ice acetic acid aqueous solution after the glyphosate is completely dissolved, and filtering out mechanical impurities by using 200-mesh filter cloth, to obtain the treatment agent, and the stirring speed is 200 r / min.

[0029] Embodiment 2

[0030] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that in embodiment 1, and the difference lies in that

[0031] The raw materials for preparing the treating agent include glyphosate ammonium salt 17%, non-ionic surfactant 10%, and water in balance, in terms of mass percentage. The mass ratio of glyphosate to ammonia in the glyphosate isopropylamine salt is 10:7.

[0032] The preparation method of the treating agent includes placing water in a stirring kettle, adding ammonia water while stirring, continuously stirring to add glyphosate raw powder, adjusting the pH value to 6.5-7.5 with 5wt% ice acetic acid aqueous solution after the glyphosate is completely dissolved, filtering out mechanical impurities with 200-mesh filter cloth, and obtaining the treating agent. The stirring speed is 200r / min.

[0033] Example 3

[0034] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that in Example 1, except that:

[0035] The raw materials for preparing the treating agent include glyphosate sodium salt 40%, non-ionic surfactant 10%, and water in balance, in terms of mass percentage. The mass ratio of glyphosate to sodium hydroxide in the glyphosate isopropylamine salt is 35:5.

[0036] The preparation method of the treating agent includes placing water in a stirring kettle, adding sodium hydroxide while stirring, continuously stirring to add glyphosate raw powder, adjusting the pH value to 6.5-7.5 with 5wt% ice acetic acid aqueous solution after the glyphosate is completely dissolved, filtering out mechanical impurities with 200-mesh filter cloth, and obtaining the treating agent. The stirring speed is 200r / min.

[0037] Example 4

[0038] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that in Example 1, except that:

[0039] The raw materials for preparing the treating agent include glyphosate potassium salt 58%, non-ionic surfactant 3%, and water in balance, in terms of mass percentage. The mass ratio of glyphosate to potassium hydroxide in the glyphosate isopropylamine salt is 50:8.

[0040] The preparation method of the treating agent includes placing water in a stirring kettle, adding potassium hydroxide while stirring, continuously stirring to add glyphosate raw powder, adjusting the pH value to 6.5-7.5 with 5wt% ice acetic acid aqueous solution after the glyphosate is completely dissolved, filtering out mechanical impurities with 200-mesh filter cloth, and obtaining the treating agent. The stirring speed is 200r / min.

[0041] Comparative Example 1

[0042] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that of example 1, except that the preparation raw materials of the treatment agent include: glyphosate isopropylamine salt 1.5%, non-ionic surfactant 5%, and water in remainder. The mass ratio of glyphosate and isopropylamine in the glyphosate isopropylamine salt is 1:0.5.

[0043] Comparative example 2

[0044] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that of example 1, except that the preparation raw materials of the treatment agent include: glyphosate isopropylamine salt 2%, non-ionic surfactant 5%, and water in remainder. The mass ratio of glyphosate and isopropylamine in the glyphosate isopropylamine salt is 1:1.

[0045] Comparative example 3

[0046] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that of example 1, except that the non-ionic surfactant is replaced by an anionic surfactant (sodium dodecyl benzene sulfonate).

[0047] Comparative example 4

[0048] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that of example 1, except that the viscosity of the non-ionic surfactant is 1000 mPa.s, and the model is APG-0814 from Nantong Xianhe Chemical Co., Ltd.

[0049] Comparative example 5

[0050] A method for improving the purity of glyphosate-resistant transgenic corn seeds, the specific implementation manner is the same as that of example 1, except that the pH value is adjusted to 5.5-6.0 by using 5 wt% ice acetic acid aqueous solution.

[0051] Performance test

[0052] In comparative example 1, the content of isopropylamine is too low, which leads to that glyphosate cannot be completely dissolved, and the treatment agent is unqualified, so it is not involved in the performance test.

[0053] In comparative example 2, the content of isopropylamine is too high, and the amount of ice acetic acid aqueous solution used is excessive, which leads to that the content of glyphosate is lower than expected, and the treatment agent is unqualified, so it is not involved in the performance test.

[0054] The corn seeds used in the present application are from Jilin Hengda Seed Co., Ltd., and specifically are glyphosate-resistant transgenic corn seeds.

[0055] (1) The corn seeds obtained in the examples and comparative examples were planted in soil as male parent and female parent, and the seedling rate was calculated 14 days later. The seeds were detected after harvesting to determine the purity of the hybrid seeds, and the results are shown in Table 1. The male control and female control were seeds without any treatment.

[0056] Seedling rate = number of seedlings / total number of seeds * 100%

[0057] Purity of hybrid seeds = number of glyphosate-resistant seeds / total number of seeds * 100%

[0058] Detection method of seed purity: rapid test paper detection method was used.

[0059] The test paper used for detection was produced by Atron Company, and the model was GENE TRUETM, anti-herbicide transgenic protein BAR detection reagent strip.

[0060] Specific operation procedure:

[0061] 1. Seed sample treatment: The seed sample was crushed into powder with a mortar or other means, and was ready for use.

[0062] 2. Add the powder to be detected into the reaction tube, and add pure water at a solid-liquid ratio of 1:5, and shake well.

[0063] 3. After the solid precipitates, hold the handle end of the test paper vertically downward, and insert the sample end into the supernatant in the reaction tube. The liquid level should not exceed the bottom color line. After the liquid automatically wets the upper end of the reaction window, the test paper can be taken out and placed on a clean plane. After 10 minutes, the reaction result is read.

[0064] 4. Result determination: only one red band appears in the quality control line, and no red band appears in the detection line, which is determined as negative, and the seed is non-glyphosate-resistant transgenic seed; one red band appears in the quality control line, and one red band appears in the detection line, which is determined as positive, and the seed is glyphosate-resistant transgenic seed; other abnormal results are invalid results.

[0065] Table 1

[0066]

[0067]

Claims

1. A method of improving the purity of a glyphosate-resistant transgenic corn seed, comprising, The method comprises: seed dressing treatment of corn seeds by using a treatment agent, and the mass ratio of the treatment agent to the corn seeds is 1:(20-30); The preparation raw material of the treatment agent comprises, in percentage by mass: glyphosate salt 5-50%, non-ionic surfactant 0.7-10%, and water in remainder; The viscosity of the non-ionic surfactant is 100-200 mPa.s; The glyphosate salt comprises at least one of glyphosate isopropylamine salt, glyphosate amine salt, glyphosate sodium salt and glyphosate potassium salt; The pH value of the treatment agent is 6.5-7.

5.

2. The method of increasing the purity of a seed of a glyphosate-resistant transgenic corn plant according to claim 1, wherein, The non-ionic surfactant comprises at least one of alkyl polyglucoside, fatty alcohol polyoxyethylene ether and alkyl phenol polyoxyethylene ether.

3. The method of increasing the purity of seeds of a glyphosate-resistant transgenic corn according to claim 1, wherein, The seed dressing treatment is performed at a temperature of 25±5 DEG C for 0.5-5 min.

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