A breeding method of low concentration EMS mutagenesis and aging window control for germinating seeds
By using germinating seeds as the treatment target, leveraging their physiological sensitivity and time-sensitive window control, and employing low-concentration EMS mutagenesis for timely sowing, the contradiction between mutagenesis efficiency and survival rate, as well as the problem of time-sensitive window control in dry seed EMS mutagenesis, was solved, achieving efficient and controllable breeding results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
- Filing Date
- 2026-04-11
- Publication Date
- 2026-06-05
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Abstract
Description
Technical Field
[0001] This invention relates to a breeding method for low-concentration EMS mutagenesis and timely window control of germinating seeds, belonging to the field of low-concentration EMS mutagenesis technology. Background Technology
[0002] In the field of crop genetic improvement and germplasm innovation, EMS (ethyl methanesulfonate) chemical mutagenesis is an important means of creating point mutant libraries and mining functional genes. Currently, the mainstream EMS mutagenesis breeding techniques both domestically and internationally generally employ direct soaking of dry seeds: fully dried seeds (usually with a moisture content below 12%) are placed in a 0.8%-1.2% EMS solution and soaked at a specific temperature for several to over ten hours, followed by thorough rinsing before sowing. This technique has successfully created a large number of mutant libraries in various crops such as rice, wheat, barley, and rapeseed, and has proven to be an effective method for inducing point mutations. However, existing dry seed EMS mutagenesis techniques have the following significant drawbacks: 1. A prominent contradiction exists between mutagenesis efficiency and seed survival rate; excessively high half-lethal concentrations limit the size of the effective mutant population. Dry seeds have dense seed coats and low cellular metabolic activity, making it difficult for EMS to penetrate quickly and uniformly. To obtain sufficient mutation frequencies, higher concentrations of EMS and extended treatment times are necessary. Studies have shown that the half-lethal concentration for direct mutagenesis of barley dry seeds is as high as 0.8%. When the EMS concentration exceeds 1.0%, the germination rate of many crop seeds often drops below 50%, leading to the death of a large number of treated materials and greatly limiting the size of the effective mutant population that can be screened. 2. Controlling the effective window after mutagenesis is difficult, and the sowing time seriously affects the seedling rate. Current technologies generally neglect the critical impact of the "effective window" from mutagenesis treatment to sowing on seed vigor. Summary of the Invention
[0003] This invention provides a breeding method for low-concentration EMS mutagenesis and time-controlled breeding after mutagenesis in germinating seeds. Using germinating seeds as the treatment object, the method significantly reduces the concentration of EMS by mutagenesis with low concentration EMS and by precisely controlling the time window after mutagenesis. This overcomes the contradiction between mutagenesis efficiency and seed survival rate, and improves seedling rate and vigor.
[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0005] A breeding method for low-concentration EMS mutagenesis and time-controlled breeding of germinating seeds includes the following steps:
[0006] 1) After disinfecting the seeds, place them in 3-5 times their volume of sterile deionized water and soak them at a constant temperature of 25±1℃ until the seeds just begin to show white (the radicle breaks through the seed coat). This will cause the seeds to enter the germination state from dormancy, activate physiological metabolism, soften the seed coat, and obtain germinating seeds.
[0007] 2) The germinating seeds obtained in step 1) were subjected to mutagenesis using a low-concentration EMS working solution, wherein the volume concentration of the EMS working solution was <0.5% (v / v);
[0008] 3) Transfer the seeds obtained in step 2) to 5-10 times their volume of 5% sodium thiosulfate solution, soak for 20-30 minutes, then filter out the seeds and rinse with the cleaning solution until there is no EMS residue. Finally, dry the surface of the seeds.
[0009] 4) Sow the seeds obtained in step 3) within 12 hours after treatment, and then place them in an environment of 25±2℃ and 70%-80% relative humidity to germinate and cultivate seedlings.
[0010] After step 4) above is completed, M1 generation plants are obtained, and target mutants are screened through M2 generation families: seeds are harvested from surviving M1 generation plants as individual plants, and each plant is numbered to establish an M2 generation family; field screening and identification of target traits (such as plant height, leaf color, resistance, quality, etc.) are carried out in the M2 generation population; and the genetic stability of the target mutants is verified.
[0011] Step 1 above achieves seed soaking activation and germination state induction, creating conditions for subsequent efficient absorption of low-concentration EMS.
[0012] In step 1) above, 3-5 times the volume of sterile deionized water means that the volume of sterile deionized water is 3-5 times the volume of the seed.
[0013] Step 2 above utilizes the high sensitivity of germinating seeds to EMS, employing low concentrations of EMS for mutagenesis to achieve efficient mutagenesis while ensuring a high survival rate.
[0014] Step 3 above) thoroughly neutralizes and removes residual EMS, terminates the mutagenesis reaction, and eliminates subsequent toxicity. 5-10 times the volume of a 5% sodium thiosulfate solution means the volume of the 5% sodium thiosulfate solution is 5-10 times the volume of the seed. A 5% sodium thiosulfate solution refers to a 5% (w / w) aqueous solution of sodium thiosulfate.
[0015] Step 4 above) involves sowing within the optimal repair window after mutation, using time-sensitive window control to ensure precise sowing and maximize seedling survival rate.
[0016] Unless otherwise specified, all percentages in this application are percentages by mass.
[0017] As one specific implementation scheme, in step 3) above, the cleaning solution is sterile deionized water; the sterile deionized water is used to rinse ≥10 times, each time for 2-3 minutes. After rinsing, the seeds are placed on sterile filter paper and the surface moisture is absorbed. EMS residues can be quickly detected using silver nitrate test paper.
[0018] To mitigate secondary damage to seeds caused by EMS mutagenesis and further improve seedling survival rate, as another specific implementation scheme, in step 3), the cleaning solution is sterile deionized water and a composite cleaning and protective solution. The composite cleaning and protective solution is composed of glutathione, trehalose, polyvinylpyrrolidone, and sterile deionized water, wherein the concentration of glutathione is 0.5-2.0 mmol / L, the concentration of trehalose is 10-50 mmol / L, and the mass concentration of polyvinylpyrrolidone is 0.5%-2.0%. The composite cleaning and protective solution used in this invention contains glutathione, trehalose, and polyvinylpyrrolidone, and is mainly used to reduce the residual toxicity of EMS, alleviate oxidative damage to cells, and improve seed germination and seedling survival rate. Its effect occurs after the mutagenesis reaction has completely terminated, therefore it does not affect the frequency of gene mutations induced by EMS, and can significantly improve the efficiency of obtaining heritable mutants in surviving plants.
[0019] The preparation method of the above-mentioned composite cleaning and protective solution is as follows: glutathione, trehalose and polyvinylpyrrolidone are added sequentially according to their concentrations in sterile deionized water, stirred until completely dissolved, pH adjusted to 6.8-7.0, and sterilized by autoclaving (121℃, 20 minutes) or by filtration through a 0.22 μm filter membrane before use.
[0020] The aforementioned glutathione has the ability to scavenge reactive oxygen species (ROS) and reduce oxidative damage. Trehalose can stabilize cell membranes and protein structures and resist osmotic stress, while polyvinylpyrrolidone can adsorb residual harmful substances and form a protective layer. The combination of these three ingredients can effectively and synergistically promote a significant increase in germination rate, seedling survival rate, and overall health.
[0021] In step 3), the cleaning solution is sterile deionized water and a composite cleaning and protective solution. After soaking in sodium thiosulfate in step 3), the seeds are first cleaned 2-3 times with the composite cleaning and protective solution, each time soaking for 15-20 minutes. Then, the seeds are rinsed 7-8 times with flowing sterile deionized water, each time rinsing for 2-3 minutes.
[0022] In step 3) above, the soaking is carried out on a constant temperature shaker at 25±1℃ with a shaker speed of 60-80 rpm.
[0023] In step 4) above, it is preferable to sow the seeds obtained in step 3) within 6 hours after the treatment is completed.
[0024] In step 4) above, sterilized seedling substrate or nutrient soil is used for sowing, and the substrate is kept moist but not waterlogged.
[0025] In step 1) above, the disinfection steps are as follows: Select plump, uniformly sized seeds that are free from pests and diseases, disinfect the surface with 70% ethanol for 60±20 seconds, then disinfect with 2% sodium hypochlorite solution for 15 minutes, and rinse 5 times with sterile deionized water.
[0026] In the above 70% ethanol, % refers to volume percentage. 2% sodium hypochlorite solution refers to an aqueous solution with a sodium hypochlorite content of 2% by mass.
[0027] In step 1) above, the soaking time is determined according to the water absorption characteristics of different crop seeds, with the optimal germination state being when the seeds just begin to sprout (the radicle breaks through the seed coat). For example, barley requires approximately 12-16 hours of soaking; for crops with thicker seed coats (such as rice and rapeseed), the soaking time can be appropriately extended to 24-36 hours.
[0028] In step 1) above, change the water 1-2 times during the soaking period to keep the water clean.
[0029] In step 2) above, the mutagenic buffer used in preparing the EMS working solution is a 0.1 mol / L sodium phosphate buffer (pH 7.0), and the concentration of the EMS working solution is 0.2% - 0.5% (v / v). The 0.1 mol / L sodium phosphate buffer (pH 7.0) is prepared by mixing 0.1 mol / L disodium hydrogen phosphate solution and 0.1 mol / L sodium dihydrogen phosphate solution at a volume ratio of 61:39, and adjusting the pH to 7.0.
[0030] To ensure the treatment effect, as one preferred implementation scheme, step 2) above is as follows: drain the water from the germinating seeds obtained in step 1), transfer them to a mutagenesis container, add EMS working solution for mutagenesis treatment for 8-16 hours, the volume of the working solution should be enough to completely submerge the seeds (usually 3-5 times the seed volume), and the concentration of the EMS working solution is 0.3%-0.4%. At this time, the relative lethality is close to the half-lethal level.
[0031] In step 2) above, the mutagenesis treatment is carried out on a constant temperature shaker at 25±1℃ with a shaker speed of 60-80 rpm to ensure uniform EMS distribution.
[0032] It should be noted that the process in step 2) must be carried out in a fume hood, and the operator should wear protective equipment.
[0033] This invention uses pre-soaked seeds to the germination stage as the treatment target. It utilizes the characteristics of seed coat softening, cell metabolism activation, and significantly increased sensitivity to mutagen to achieve efficient mutagenesis while significantly reducing EMS concentration. Furthermore, it precisely controls the time window from the completion of mutagenesis to sowing to ensure that the seeds are sown within the optimal repair period, thereby maximizing the seedling rate and the efficiency of obtaining mutants.
[0034] The physiological advantages of the germinating seeds in this invention are as follows: Dry seeds have a dense seed coat and low cellular metabolic activity, making it difficult for EMS to penetrate rapidly. After pre-soaking until germination (seeds showing white tips), the seed coat softens, cells absorb water and swell, and DNA replication and repair mechanisms are activated, significantly increasing sensitivity to EMS. The half-lethal concentration (LD50) for direct mutagenesis in dry seeds is approximately 0.8%, while the LDF50 in water-soaked and germinated seeds can be reduced to 0.3%-0.4%, a reduction of over 50%. This provides a physiological basis for achieving efficient mutagenesis using low-concentration EMS.
[0035] Research has revealed that mutagenesis treatment causes DNA damage in seeds, and a crucial "golden window" for physiological damage repair exists within the seed. The inventors believe this is because seeds possess an active DNA repair system during germination. If sowing occurs within this window, the damage can be partially repaired; however, if sowing is delayed, the unrepaired damage accumulates and ultimately leads to seed death. Therefore, precisely controlling this window is crucial for ensuring a high seedling survival rate.
[0036] Existing dry seed mutagenesis techniques for multicellular embryos only induce mutagenesis in a subset of embryonic cells, resulting in an extremely high chimeric proportion (>95%) in the M1 generation. Homozygous mutants can only be obtained through self-pollination in the M2-M3 generations. This study, while still using seeds as the treatment target, leverages the physiological characteristics of cellular metabolic activation and DNA replication initiation during germination, combined with low-concentration EMS treatment, to improve the uniformity of mutagenesis events, thereby reducing the chimeric proportion from the source. Combined with an early screening strategy in the M2 generation, the cycle for obtaining homozygous mutants can be shortened by 1-2 generations, significantly reducing breeding time and screening costs.
[0037] Any techniques not mentioned in this invention are based on existing technologies.
[0038] Compared with existing technologies, the advantages of this invention are as follows:
[0039] 1. This invention is the first to explicitly use "budding seeds" (seeds in the white stage) as the target for mutagenesis treatment. By taking advantage of their physiological sensitivity, the concentration of EMS used can be reduced from the traditional 0.8%-1.2% to below 0.5%, a reduction of more than 50%, thus synergistically solving the contradiction between mutagenesis efficiency and material survival rate.
[0040] 2. This invention proposes and verifies the key parameters of the golden sowing window within 12 hours after mutation, transforming the time window from a vague experience-based operation into a precise and controllable technical indicator, which significantly improves the seedling rate and experimental repeatability.
[0041] 3. By organically integrating the three key steps of "seed soaking and activation, low-concentration EMS treatment, and timely window sowing", a new mutation breeding process that is standardized, replicable, and applicable to a variety of crops has been formed. Detailed Implementation
[0042] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.
[0043] EMS stock solution: Ethyl methanesulfonate (EMS, CAS 62-50-0), purity ≥99%, Sigma-Aldrich. 0.1 mol / L phosphate buffer (pH 7.0) is prepared by mixing solution A and solution B in a volume ratio of 61:39. Solution A: 0.1 mol / L Na₂HPO₄ aqueous solution; Solution B: 0.1 mol / L NaH₂PO₄ aqueous solution.
[0044] Plant materials: The test crops were malting barley variety 'Ganpi 8' (provided by the Institute of Economic Crops and Beer Raw Materials, Gansu Academy of Agricultural Sciences), wheat variety 'Xinnong 979', and rice variety 'Nipponbare'. All seeds were carefully selected, with plump, uniform grains and free from pests and diseases.
[0045] Example 1:
[0046] EMS mutagenesis and breeding of germinating seeds of barley variety 'Ganpi 8':
[0047] 1. Seed pretreatment: Soaking for activation and induction of germination state
[0048] Select 2000 plump, uniformly sized, and disease-free 'Ganpi No. 8' seeds. Disinfect the surface with 70% ethanol for 1 minute, then with 2% sodium hypochlorite solution for 15 minutes, and rinse 5 times with sterile deionized water. Place the disinfected seeds in three times their volume of sterile deionized water and soak in a 25℃ constant temperature incubator. After 14 hours of soaking, approximately 83% of the seeds will have their radicles breaking through the seed coat (showing white), indicating optimal germination. Change the water once during the soaking period to maintain water quality.
[0049] 2. Low-concentration EMS mutagenesis treatment
[0050] EMS working solution preparation: Take the EMS stock solution and dilute it with 0.1 mol / L phosphate buffer (pH 7.0) to 0.4% (v / v) to obtain 0.4% (v / v) EMS working solution. Prepare and use immediately.
[0051] Drain the germinating seeds obtained in step 1 and transfer them to a 500 mL Erlenmeyer flask. Add 0.4% (v / v) EMS working solution, ensuring the solution completely submerges the seeds (approximately 300 mL). Place the Erlenmeyer flask on a 25°C constant-temperature shaker at 80 rpm for 16 hours in the dark.
[0052] 3. Termination of mutagenesis and seed washing
[0053] After completing step 2, immediately transfer the seeds to 5 times their volume of 5% sodium thiosulfate solution and soak them on a shaker at 25°C and 80 rpm for 30 minutes. Then, discard the neutralization solution and rinse the seeds 10 times with running sterile deionized water for 3 minutes each time, until the rinsing solution is odorless and a rapid test with silver nitrate paper shows no EMS residue. Place the washed seeds on sterile filter paper and blot dry the surface moisture.
[0054] 4. Time-sensitive window control for precise sowing.
[0055] After step 3 is completed, sowing should be finished in 6 hours. The sowing substrate is a mixture of sterilized vermiculite and peat moss (1:1 by weight), keeping the substrate moist but not waterlogged. After sowing, place the seeds in an environment of 25±2℃ and 70%-80% relative humidity to promote germination and seedling growth.
[0056] 5. Results Recording and Analysis
[0057] Germination rate statistics: Seven days after sowing, the germination rate of the seeds in the treatment group was 65.7%.
[0058] Seedling survival rate: The seedling survival rate was 61.1% 14 days after sowing.
[0059] M1 generation phenotypic observation: M1 generation plants were observed to exhibit phenotypic mutations such as red stem, short stem, and leaf variation, with the mutation frequency increasing by 3 times compared to control example 1.
[0060] Comparative Example 1
[0061] The difference from Example 1 is that step 1 is omitted. Simultaneously, the concentration of the EMS working solution in step 2 is increased to 0.8% (v / v). That is, this example uses direct soaking of dry seeds. All other procedures are the same as in Example 1. Seven days after sowing, the germination rate was 44.9%.
[0062] Comparative Example 2
[0063] The difference from Example 1 is that the sowing time in step 4) was modified to: sowing completed at 12 hours, 24 hours, and 12 days respectively. All other steps were the same as in Example 1. The results showed that the seedling survival rate was 52.4% after sowing at 12 hours, decreased to 49% after 24 hours, and only 0.8% after 12 days.
[0064] Example 2:
[0065] EMS mutagenesis and breeding of germinating seeds of wheat 'Xinnong 979':
[0066] Three EMS concentration treatment groups (0.2%, 0.4%, and 0.6%) and one water control group (CK) were set up, with 500 seeds treated in each group.
[0067] The processing procedure differs from that in Example 1 in that:
[0068] In step 1, the soaking time is 12 hours;
[0069] In step 2, the concentrations of the EMS working solution were 0.2%, 0.4%, and 0.6%, respectively.
[0070] In step 4, sowing must be completed within 12 hours.
[0071] The rest are the same as in Example 1.
[0072]
[0073] Median lethal concentration (LD50) estimation: According to regression analysis, the LD50 of EMS-induced mutagenesis in germinating wheat 'Xinnong 979' seeds is approximately 0.45%. This is about 40%-50% lower than the existing LD50 of 0.65%-0.90% in dry wheat seeds.
[0074] Example 3:
[0075] EMS mutagenesis and breeding of germinating rice 'Nipponbare' seeds:
[0076] With a fixed EMS concentration of 0.4% and a treatment time of 12 hours, three different sowing time points were set:
[0077] The processing procedure differs from that in Example 1 in that:
[0078] In step 1, the soaking time is 24 hours;
[0079] In step 2, the concentration of the EMS working solution is 0.4%.
[0080] In step 4, the following settings were made: Group T1: sowing within 6 hours after treatment; Group T2: sowing 24 hours after treatment; Group T3: sowing 72 hours after treatment.
[0081] CK group: treated with clean water, sown within 6 hours.
[0082] Each group was treated with 300 seeds.
[0083] The rest are the same as in Example 1.
[0084] Sowing and statistics: Sow according to the above time points, and count the germination rate 10 days after sowing and the seedling rate 15 days after sowing.
[0085]
[0086] Time window effect: The seedling survival rate of group T1 (within 6 hours) was 61.5%, that of group T2 (24 hours) dropped to 44.2%, and that of group T3 (72 hours) was only 22.7%.
[0087] Rice suitability: Under the condition of 0.4% EMS treatment for 12 hours, rice germination seeds can achieve a seedling rate of over 60% when sown within 6 hours, which is significantly superior to traditional dry seed mutation (germination rate is often <50%).
[0088] This embodiment fully verifies the universality and crucial role of the "golden planting window" after mutagenesis. Different crops such as rice, wheat, and barley all exhibit the same pattern, indicating that this technology has good cross-species applicability.
[0089] Example 4:
[0090] The difference from Example 1 is that the sterile deionized water used for rinsing in step 3 is replaced with a composite cleaning and protective solution, wherein the composition of the composite cleaning and protective solution is as follows (based on the final concentration of the cleaning solution):
[0091]
[0092] Preparation method of composite cleaning and protective solution: Add glutathione, trehalose and PVP in sterile deionized water according to the concentrations in the table above, stir until completely dissolved, adjust the pH to 7.0, autoclave (121℃, 20 minutes) and set aside.
[0093] Application method of composite cleaning and protective solution: After completing the sodium thiosulfate soaking in step 3, use the composite cleaning and protective solution to perform the subsequent 2-3 cleaning steps, soaking for 15-20 minutes each time. Then rinse the seeds 7 times with running sterile deionized water for 3 minutes each time until the rinsing solution has no odor. Use silver nitrate test paper for rapid detection and there is no EMS residue.
[0094] The remaining steps are the same as in Example 1, and the results are shown in the table below.
[0095]
[0096] As can be seen from the above, the composite cleaning-protecting solution can effectively alleviate the secondary oxidative stress caused by the mutagenesis treatment, and further improve the overall efficiency of the present invention.
[0097] Comparative Example 3
[0098] The difference from Example 4 is that glutathione in the composite cleaning and protective solution was omitted. All other aspects were the same as in Example 4. Germination rate was 66.2%, seedling survival rate was 61.2%, and seedling root length (7 days) was 4.2 cm.
[0099] Comparative Example 4
[0100] The difference from Example 4 is that trehalose was omitted from the composite cleaning and protective solution. All other procedures were the same as in Example 4. Germination rate was 67.1%, seedling survival rate was 61.6%, and seedling root length (7 days) was 4.3 cm.
[0101] Comparative Example 5
[0102] The difference from Example 4 is that polyvinylpyrrolidone was omitted from the composite cleaning and protective solution. All other aspects were the same as in Example 4. Germination rate was 68.3%, seedling survival rate was 62.7%, and seedling root length (7 days) was 4.5 cm.
[0103] The above embodiments illustrate in detail the specific implementation methods, parameter ranges, and application effects of the present invention on different crops. Those skilled in the art can use these teachings to fine-tune the parameters for different crop varieties and breeding objectives; all such adjustments fall within the protection scope of the present invention.
Claims
1. A breeding method for controlling germination by low-concentration EMS mutagenesis and time-dependent window, characterized in that: Includes the following steps: 1) After disinfecting the seeds, place them in 3-5 times their volume of sterile deionized water and soak them at a constant temperature of 25±1℃ until the seeds just begin to sprout. This will cause the seeds to enter the germination state from dormancy, activate physiological metabolism, soften the seed coat, and obtain germinating seeds. 2) The germinating seeds obtained in step 1) were subjected to mutagenesis using a low-concentration EMS working solution, wherein the volume concentration of the EMS working solution was <0.5%; 3) Transfer the seeds obtained in step 2) to 5-10 times their volume of 5% sodium thiosulfate solution, soak for 20-30 minutes, then filter out the seeds and rinse with the cleaning solution until there is no EMS residue. Finally, dry the surface of the seeds. 4) Sow the seeds obtained in step 3) within 12 hours after treatment, and then place them in an environment of 25±2℃ and 70%-80% relative humidity to germinate and cultivate seedlings.
2. The breeding method for low-concentration EMS mutagenesis and time-sensitive window control of germinating seeds according to claim 1, characterized in that: In step 3), the cleaning solution is sterile deionized water; the sterile deionized water is used to rinse ≥10 times, each time for 2-3 minutes. After rinsing, the seeds are placed on sterile filter paper and the surface moisture is absorbed.
3. The method for low-concentration EMS mutagenesis and time-controlled breeding of germinating seeds according to claim 1 or 2, characterized in that: In step 3), the cleaning solution is sterile deionized water and a composite cleaning and protective solution. The composite cleaning and protective solution is composed of glutathione, trehalose, polyvinylpyrrolidone and sterile deionized water. The concentration of glutathione is 0.5-2.0 mmol / L, the concentration of trehalose is 10-50 mmol / L, and the mass concentration of polyvinylpyrrolidone is 0.5%-2.0%.
4. The breeding method for low-concentration EMS mutagenesis and time-controlled breeding of germinating seeds according to claim 3, characterized in that: The preparation method of the composite cleaning and protective solution is as follows: glutathione, trehalose and polyvinylpyrrolidone are added sequentially in sterile deionized water according to their concentrations, stirred until completely dissolved, the pH is adjusted to 6.8-7.0, and then autoclaved to obtain the composite cleaning and protective solution.
5. The breeding method for low-concentration EMS mutagenesis and time-controlled breeding of germinating seeds according to claim 3, characterized in that: After soaking in sodium thiosulfate in step 3), use the composite cleaning and protective solution to clean 2-3 times, soaking for 15-20 minutes each time. Then rinse the seeds 7-8 times with running sterile deionized water, rinsing for 2-3 minutes each time.
6. The method for controlling breeding by low-concentration EMS mutagenesis and time-sensitive window in germinating seeds according to claim 1 or 2, characterized in that: In step 3), the soaking is carried out on a constant temperature shaker at 25±1℃ with a shaker speed of 60-80 rpm.
7. The method for controlling breeding by low-concentration EMS mutagenesis and time-sensitive window in germinating seeds according to claim 1 or 2, characterized in that: In step 4), the seeds obtained in step 3) are sown within 6 hours after the treatment is completed.
8. The method for low-concentration EMS mutagenesis and time-controlled breeding of germinating seeds according to claim 1 or 2, characterized in that: In step 1), the disinfection steps are as follows: Select plump, uniformly sized seeds that are free from pests and diseases, disinfect the surface with 70% ethanol for 60±20 seconds, then disinfect with 2% sodium hypochlorite solution for 15 minutes, and rinse 5 times with sterile deionized water.
9. The breeding method for low-concentration EMS mutagenesis and time-controlled breeding of germinating seeds according to claim 1 or 2, characterized in that: In step 2), the solvent used to prepare the EMS working solution is 0.1 mol / L sodium phosphate buffer, and the concentration of the EMS working solution is 0.2% - 0.5%.
10. The breeding method for low-concentration EMS mutagenesis and time-controlled breeding of germinating seeds according to claim 1 or 2, characterized in that: Step 2) involves draining the water from the germinating seeds obtained in step 1), transferring them to a mutagenesis container, and adding EMS working solution for mutagenesis treatment for 8-16 hours. The volume of the working solution should be sufficient to completely submerge the seeds, and the concentration of the EMS working solution should be 0.3% - 0.4%. And / or, in step 2), the mutagenesis treatment is carried out on a constant temperature shaker at 25±1℃ with a shaker speed of 60-80 rpm.