Oat EMS mutagenesis method based on PEG synergistic effect and application of oat EMS mutagenesis method

Abstract

The invention discloses an oat EMS mutagenesis method based on a PEG synergistic effect and application of the oat EMS mutagenesis method, and belongs to the technical field of biotechnology breeding. According to the oat EMS mutagenesis method based on the PEG synergistic effect, two oat varieties of Zhongtian 4 and Qinghai 444 are selected, PEG-6000 solutions with different concentrations are adopted for osmotic regulation, and the influence of PEG treatment on seed germination and seedling growth of oat is systematically researched. EMS mutagenic agents with different concentrations are adopted for treatment, a semi-lethal dose is taken as a critical reference, and mutagenesis effects of EMS mutagenesis on the two varieties of avena husks in the germination period and the seedling period are researched. By systematically integrating PEG pretreatment and EMS mutagenesis treatment, a set of EMS mutagenesis technology system capable of remarkably improving the mutagenesis efficiency, the survival rate of mutants and the diversity of variation types is established, and an effective method support is provided for breeding oat germplasm resource creation and genetic improvement.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology breeding, and relates to an oat EMS mutagenesis method based on PEG synergistic effect and application thereof. BACKGROUND

[0002] Oat (Avena sativa L.) is an annual herbaceous plant of the Poaceae family, and the forage oat is generally mainly in the form of husked oat. Variety improvement of the forage oat is of great significance to improving the production efficiency of the animal husbandry. However, the existing mainstream breeding technology has obvious bottlenecks: the traditional hybrid breeding has a long cycle and low efficiency, and the genetic transformation technology based on introduction of exogenous genes has great difficulty in genetic operation and low transformation efficiency due to the complexity of the heterologous hexaploid genome of the oat, and faces the regulatory challenges such as biological safety approval. Therefore, there is an urgent need in the field to develop a breeding technology which does not depend on integration of exogenous genes and can efficiently create new germplasm.

[0003] Chemical mutagenesis breeding can construct a mutant library with a rich spectrum of point mutations in a short period of time, and provide effective support for germplasm innovation and trait mining. In the technology, ethyl methanesulfonate (EMS) is the most widely used and effective chemical mutagen. Compared with other mutagenic methods, EMS can not only induce high-density point mutations, but also cause fewer chromosomal aberrations, and the proportion of dominant mutations induced by EMS is higher, which significantly reduces the difficulty of subsequent mutant screening and identification.

[0004] There are many studies on the use of the chemical mutagen EMS for crop mutagenesis breeding and the construction of a mutant library to create new germplasm resources reported in existing journals and patents, which are as follows:

[0005] The invention patent CN202510641665.9 discloses a method for establishing an EMS mutant library of pokeweed seeds, which is characterized by the following steps: (1) treating the seeds with concentrated sulfuric acid: selecting pokeweed seeds with high fullness, and soaking in concentrated sulfuric acid for 7-9 min; (2) seed germination: treating the seeds treated in step (1) until the seeds are white, and reserving; (3) seed treatment: placing the white seeds in step (2) in an ethyl methanesulfonate solution to immerse the seeds, and performing light-shielding, soaking and shaking treatment for 12-16 h; (4) terminating the reaction: adding sodium thiosulfate solution to terminate the reaction, and washing the seeds; (5) sowing: sowing the washed seeds, and screening mutant materials.

[0006] Invention patent CN202510518049.4 discloses a method for creating new dwarf tomato germplasm by EMS mutagenesis, which is characterized by the following steps: (1) selecting seeds of full-grain, non-injured dwarf tomato, soaking in 1:1 84 disinfectant water, and washing with tap water for 5 times; (2) soaking the seeds of step (1) in EMS-phosphoric acid buffer solution containing 0.4% to 2% concentration, avoiding light and oscillating for 6-36 hours; (3) detoxifying the seeds after mutagenesis in step (2), pouring out the EMS solution and washing with detoxifying agent for 2 times, and washing with tap water for 2 hours; (4) germinating the detoxified seeds in step (3) in a dark incubator, picking out the seeds with germination and white sprouts for planting, and observing and counting the mutagenesis effect; (5) sowing the seeds collected in step (S4), observing the phenotypes of M1 and M2 generations during the whole growth period, counting the proportion of each mutation phenotype in the population, identifying the mutagenesis target traits, and obtaining the EMS mutation population of dwarf tomato.

[0007] Invention patent CN202510299947.5 discloses a breeding method of a new wheat variety with high amylose and high resistant starch, mainly including the following steps: step 1: treating the parent wheat seeds with EMS mutagenesis, and planting the M0 seeds after mutagenesis to obtain M1 seeds harvested according to the strains; step 2: screening the strains to which the M1 seeds with wrinkled and lusterless seed coat belong, and planting to obtain M2 seeds harvested according to the strains; step 3: screening the strains to which at least 90% of the M2 seeds with wrinkled and lusterless seed coat belong, and planting to obtain M3 seeds harvested according to the strains; step 4: I2-KI staining is performed on the M3 seeds, the strains to which the whole grains dyed blue-black are screened, and at least part of the seeds of the strains are detected for amylose and resistant starch, so as to determine the strains with amylose content of at least 45% and / or resistant starch content of at least 6%, so as to obtain a new wheat variety with high amylose and high resistant starch.

[0008] The above prior art does not consider the state of seed germination and its sensitivity, and also does not involve the serious damage and inhibition of chemical mutagen to seed germination and seedling growth. At present, no one has treated the seeds before mutagenesis to adjust the state of the seeds, and no one has treated the material after mutagenesis to improve the seed germination rate and seedling growth, so as to improve the survival rate and mutagenesis efficiency of EMS mutagenesis. In the prior art, the treatment of wheat, quinoa, soybean and other crop seeds with chemical mutagen mainly explores the effects of different mutagenesis time and concentration on seed germination and seedling growth, and selects the semi-lethal condition on this basis. In addition, the research on the treatment of feed oats with EMS mutagenesis does not consider the effect of glume on EMS mutagenesis.

[0009] In the prior art, the oat seed is treated by soaking in an EMS solution, which is the most commonly used mutagenesis method. However, the efficient application of the technology to the oat forage mainly with husked oat still faces significant bottlenecks. First, the hard hull wrapped outside the husked oat grain seriously hinders the effective penetration of the EMS solution to the embryo, which is a key limiting factor leading to low mutagenesis efficiency. Secondly, and most importantly, there is a lack of pretreatment technology that can precisely regulate the physiological state of the seed and effectively improve its sensitivity to mutagens. The osmotic pressure of the seed directly affects the cell membrane permeability and physiological activity, thereby determining the penetration efficiency and effect of the mutagen. However, there is still a lack of systematic research on how to make the forage oat seeds enter the ideal water and physiological sensitive state simultaneously before mutagenesis through pretreatment such as osmotic adjustment. As an efficient osmotic regulator, PEG can simulate a mild osmotic adjustment environment, regulate the water potential to control the water absorption process of the seed, and make it enter the "pre-activation" state with active metabolism without germination. This state not only enhances the permeability of the cell membrane, promotes the penetration and absorption of EMS mutagens to the embryo, but also may improve the sensitivity and mutagenesis efficiency of the seed to EMS mutagens by inducing stress response. However, the application of PEG osmotic adjustment as a standardized pretreatment technology to optimize the EMS mutagenesis scheme of forage oat is still a blank at home and abroad.

[0010] Therefore, the present application aims to provide a husked oat EMS mutagenesis technology system with PEG osmotic adjustment as the core pretreatment method. SUMMARY

[0011] In order to overcome the defects in the prior art, the present application provides a PEG synergistic oat EMS mutagenesis method and its application, which is an efficient and stable EMS mutagenesis method based on PEG synergistic regulation. The following technical problems are solved:

[0012] (1) The regulatory effect of PEG pretreatment on the water absorption rule and physiological state of oat seeds is determined, and the mutagenesis sensitive window period is determined;

[0013] (2) The PEG and EMS treatment conditions of suitable concentration and time are screened to realize the synergistic improvement of mutagenesis efficiency and survival rate;

[0014] (3) The penetration of EMS to the embryo is enhanced by PEG pretreatment to improve the mutation frequency.

[0015] The application establishes an EMS mutagenesis technology system that can significantly improve mutagenesis efficiency, mutant survival rate and variation type diversity by system integration of PEG pretreatment and EMS mutagenesis treatment, and provides effective method support for forage oat germplasm resource creation and genetic improvement. PEG solution is introduced for accurate osmotic pretreatment of oat seeds after hulling, so that they can reach the best physiological sensitive state before mutagenesis. This method aims to fundamentally solve the problem of low mutagenesis efficiency caused by inconsistent seed physiological state and uneven penetration of mutagen, and provides key method support for establishing an efficient and stable forage oat EMS mutagenesis breeding technology platform.

[0016] The technical scheme is as follows:

[0017] Firstly, the embodiments of the application provide an oat EMS mutagenesis method based on PEG synergistic effect, comprising the following steps:

[0018] Step 1, PEG treatment of hulled oat seeds: the concentration of PEG treatment is 0-30 %, and the time of PEG treatment is 1.5-24 h;

[0019] The "%" is mass fraction.

[0020] The specific concentration of PEG is 0 %, 10 %, 20 %, 30 %;

[0021] The specific PEG treatment time is 1.5 h, 3 h, 6 h, 12 h, 24 h;

[0022] Step 2, EMS mutagenesis treatment of dehulled oat seeds: the concentration of EMS mutagenesis treatment is 0-1.00 %; the time of EMS mutagenesis treatment is 8-16 h; and the temperature of EMS mutagenesis treatment is 18-22 °C.

[0023] The specific concentration of EMS treatment is 0 %, 0.5 %, 0.75 %, 1 %;

[0024] The specific EMS treatment time is 8 h, 12 h, 16 h.

[0025] Step 3, 4 PEG treatment synergistic EMS mutagenesis treatment germination test, the treatment conditions are 'Zhongtian 4' (10 % PEG 3 h+1 % EMS 16 h, 10 % PEG 6 h+1 % EMS 16 h, 20 % PEG 3 h+1 % EMS 16 h, 30 % PEG 3 h+1 % EMS 16 h), 'Qinghai 444' (10 % PEG 3 h+0.75 % EMS 16 h, 10 % PEG 6 h+0.75 % EMS 16 h, 20 % PEG 3 h+0.75 % EMS 16 h, 30 % PEG 3 h+0.75 % EMS 16 h)

[0026] In the above method, the 4 PEG treatment synergistic EMS mutagenesis treatment further comprises the construction of the M2 generation population, comprising the following steps:

[0027] 1) Select mature and full oat seeds without hulls as mutagenic base materials;

[0028] 2) The seeds are pretreated with 4 different concentrations and times of PEG;

[0029] 3) The PEG pretreated seeds are subjected to optimal EMS mutagenesis treatment, and the mutagenized seeds are M0 generation;

[0030] 4) The M0 generation seeds are planted in a single plant room and the M1 generation is harvested from a single plant;

[0031] 5) The M1 generation seeds are planted according to the row method to form the M2 generation population;

[0032] 6) Agronomic trait determination at the milk ripening stage of the M2 generation;

[0033] 7) Evaluate the agronomic trait performance of each treatment according to the agronomic trait;

[0034] 8) Evaluate the mutagenic effect according to the positive variation rate of the agronomic trait being greater than 130 % of the average value of the control and the negative variation rate being less than 70 % of the average value of the control;

[0035] 9) Evaluate the mutagenic efficiency of each treatment according to the number of M0 generation mutagenized seeds.

[0036] Further, in step 2, the PEG pretreatment conditions are 30 % PEG, and the PEG pretreatment time is 3 h.

[0037] Further, in step 3, the PEG pretreated oat seeds are treated with a 1 % EMS solution by mass percentage concentration for 16 hours to obtain the PEG synergistic EMS mutagenized 'Zhongtian 4' oat seeds.

[0038] Further, in step 3, the PEG pretreated oat seeds are treated with a 0.75% EMS solution for 16 hours to obtain PEG and EMS synergistic mutagenized 'Qinghai 444' oat seeds.

[0039] Further, in step 1, the seeds are treated with shaking in a 150r / min temperature-controlled shaker.

[0040] Further, in steps 2 and 3, the seeds are soaked in distilled water at 4℃ for 3 hours, the distilled water is poured out, and the residual water on the surface of the seeds is cleaned with water-absorbing paper, then EMS-PBS solution is added, and the treated seeds are sealed and wrapped with tin paper and placed in a 150r / min temperature-controlled shaker for light-free shaking mutagenesis.

[0041] Further, the PEG and EMS synergistic mutagenesis also includes the step of investigating the agronomic traits of the M2 generation of mutagenized populations at the milk stage to evaluate the field mutagenesis effect and mutagenesis efficiency.

[0042] Further, the method for evaluating the field mutagenesis effect is specifically the difference of any single trait of a single plant relative to the mean value ± 30% of the control group; and the method for evaluating the mutagenesis efficiency is the change range of the number of single plants with any positive or negative variation in any trait of the M2 generation relative to the number of initial mutagenized seeds of the M0 generation.

[0043] Secondly, the application also provides any one of the following a1) to a8):

[0044] a1) the application of the method described in any one of the application to evaluate the field mutagenesis effect and mutagenesis efficiency of the plant height of two oat varieties;

[0045] a2) the application of the method described in any one of the application to evaluate the field mutagenesis effect and mutagenesis efficiency of the total tiller of two oat varieties;

[0046] a3) the application of the method described in any one of the application to evaluate the field mutagenesis effect and mutagenesis efficiency of the effective tiller of two oat varieties;

[0047] a4) the application of the method described in any one of the application to evaluate the field mutagenesis effect and mutagenesis efficiency of the flag leaf area of two oat varieties;

[0048] a5) the application of the method described in any one of the application to evaluate the field mutagenesis effect and mutagenesis efficiency of the main ear length of two oat varieties;

[0049] a6) the application of the method described in any one of the application to evaluate the field mutagenesis effect and mutagenesis efficiency of the stem diameter of two oat varieties;

[0050] a7) the application of the method described in any one of the application to evaluate the field mutagenesis effect and mutagenesis efficiency of the fresh weight of two oat varieties;

[0051] a8) The method of any of the present invention is used to screen PEG synergistic EMS mutagenic treatment for creating high-yield germplasm.

[0052] Further, the two oat varieties are 'Zhongtian No. 4' and 'Qinghai No. 444'.

[0053] Beneficial effects:

[0054] The present invention uses the widely adaptable 'Qinghai No. 444' and 'Zhongtian No. 4' bred by space breeding technology as mutagenic platform material, uses polyethylene glycol (PEG) solution of different concentrations and times to pretreat the unhulled oat seeds, adjusts the water state and osmotic pressure of the seeds to make them enter the key physiological period sensitive to mutagens. On this basis, EMS-PBS solution of appropriate concentration is used for mutagenic treatment. This method effectively regulates the water absorption process and internal physiological state of the seeds by using PEG pretreatment, enhances the penetration and action efficiency of EMS mutagenic agent to the seed embryo, and thus significantly improves the mutation frequency. The present invention constructs a technical system of PEG pretreatment synergistic EMS mutagenesis, which is simple and stable, not only provides an efficient way for the innovation of forage oat germplasm, but also provides a reference for the mutagenic breeding research of other cereal crops. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 Effect of PEG treatment on seed germination of two varieties of oats;

[0056] Figure 2 Effect of PEG treatment on seed germination of two varieties of oats;

[0057] Figure 3 EMS mutagenic effect of two varieties of oats, wherein, Figure 3 A of FIG. 4 is the EMS mutagenic effect of 'Zhongtian No. 4', Figure 3 B of FIG. 4 is the EMS mutagenic effect of 'Qinghai No. 444';

[0058] Figure 4 Effect of PEG pretreatment on EMS mutagenic effect of two varieties of oats, wherein, Figure 4 A of FIG. 4 is the effect of PEG pretreatment on EMS mutagenic effect of 'Zhongtian No. 4', B of FIG. 4 is the effect of PEG pretreatment on EMS mutagenic effect of 'Qinghai No. 444';

[0059] Figure 5 Effect of PEG pretreatment on growth of seedlings after EMS mutagenesis, wherein, Figure 5 A of FIG. 4 is the effect of PEG pretreatment on growth of seedlings after EMS mutagenesis of 'Zhongtian No. 4', Figure 5 B of FIG. 4 is the effect of PEG pretreatment on growth of seedlings after EMS mutagenesis of 'Qinghai No. 444'.

[0060] The small letters in the figure represent the significant difference (P<0.05) of different concentrations at the same processing time. DETAILED DESCRIPTION

[0061] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific embodiments.

[0062] In the following examples, the seeds of the forage oat varieties 'Zhongtian No. 4' and 'Qinghai No. 444' were selected. The seeds of 'Qinghai No. 444' were provided by the College of Animal Husbandry and Veterinary Medicine of Qinghai University, which has the characteristics of wide adaptability. The seeds of 'Zhongtian No. 4' were provided by the Institute of Animal Husbandry and Veterinary Medicine of the Chinese Academy of Agricultural Sciences, which is a new forage oat variety selected by space breeding technology.

[0063] Example 1: Effect of PEG treatment on the germination of seeds of two varieties of hulless oat

[0064] Two hulless oat varieties, 'Zhongtian No. 4' and 'Qinghai No. 444', were selected, and the oat seeds were soaked in different concentrations of PEG-6000 solution. The effects of PEG treatment on the germination and seedling growth of hulless oat seeds were systematically studied. By measuring and statistically analyzing the germination potential, germination rate, seedling length, root length, and other indicators, the specific effects of PEG osmotic regulation on the germination characteristics of seeds of the two varieties of hulless oat were determined. The specific test methods and results are as follows:

[0065] I. Test method

[0066] Uniform and plump hulless oat seeds were selected and soaked in 0 (distilled water as control), 10%, 20%, and 30% PEG (PEG-6000, molecular weight: 6000; purity: >99% molecular biology grade) solutions. The seeds were placed in a 150 r / min temperature-controlled shaker for different time periods (1.5 h, 3 h, 6 h, 12 h, and 24 h). Each group of treatment had 50 seeds, and the germination test was performed in triplicate.

[0067] Germination test: The seeds were moistened with distilled water, and the hulless oat seeds soaked in PEG were dispersed in the seed germination bag with the seed furrow facing down. The PEG treatment time and concentration of each portion of oat seeds were numbered and corresponded using a combination of letters and numbers, and were marked on the seed germination bag. The culture dishes were placed in a 2000 lx, 25℃ / 20℃, 16 h / 8 h light cycle incubation condition for germination. The filter paper was checked to ensure it remained moist at all times, and the daily germination of the oat seeds was observed during the incubation process. The initial germination time of the oat seeds was accurately recorded, and the germination potential and germination rate of the oat seeds were calculated. After the test, 10 oat seedlings were randomly selected from each of the three replicates of each treatment and photographed. After photographing, the root length and seedling length of the oat seedlings were measured using Image J. The calculation formulas for the related indicators of oat seed germination are as follows:

[0068] Germination potential (%) = Number of seeds germinated on day 7 / Total number of seeds × 100%;

[0069] Germination rate (%) = Number of seeds germinated on day 10 / Total number of seeds × 100%.

[0070] II. Test Results

[0071] PEG osmotic conditioning treatment allows seeds to absorb water synchronously and slowly, activating metabolic activity in order to create a synergistic effect with subsequent EMS chemical mutagenesis, thereby improving mutagenesis efficiency and breeding results. This study analyzed the effects of different concentrations and durations of PEG treatment on the germination characteristics and seedling growth of two oat varieties, aiming to screen for effective osmotic conditioning conditions. Figure 1 It was found that the germination of 'Zhongtian 4' seeds was more sensitive to PEG treatment than that of 'Qinghai 444', and the germination potential varied significantly under different treatments. After 3 hours of treatment with different concentrations of PEG, the germination potential of 'Zhongtian 4' was significantly higher than that of the control group. Overall, 3 hours was the optimal PEG treatment time, with the best germination potential and germination rate. This result reveals that mild to moderate PEG osmotic regulation for 3 hours has a significant stimulating effect on seed germination, accelerating germination speed and improving uniformity. When the treatment time was extended to 6 hours or more, the germination potential and germination rate of all PEG concentrations decreased significantly, indicating that long-term osmotic regulation had an inhibitory effect on seed vigor. Germination characteristics were relatively stable. Within the 3-hour treatment period, the germination potential and germination rate of all PEG concentrations were not significantly different from the control group, showing strong tolerance. Similar to 'Zhongtian 4', long-term (≥12 hours) PEG treatment also significantly inhibited its germination. However, the germination characteristics of 'Qinghai 444' were relatively stable. Within a 3-hour treatment period, the germination potential and germination rate under all PEG concentrations were not significantly different from the control group, demonstrating strong tolerance. Similar to 'Zhongtian 4', prolonged (≥12h) PEG treatment also significantly inhibited its germination.

[0072] Depend on Figure 2 It was found that when the treatment time was less than 3 h, the inhibition of seedling growth was relatively mild. However, when the treatment time reached 3 h or more, the seedling length and root length of all PEG treatments were significantly lower than those of the control group. This indicates that even mild osmotic regulation (such as 10% PEG for 3 h), which has little effect on germination rate, is sufficient to cause significant inhibition of early seedling growth. Among these, root length was more sensitive to PEG osmotic regulation than seedling length, and the inhibition of root length was more severe at high concentrations (30%) and long treatment times.

[0073] In summary, based on the germination and seedling growth indicators of the two varieties, the results of this test, 3 h was proved to be a key processing time that can effectively trigger the osmotic regulation response and does not cause excessive damage. Based on this, we finally selected four treatment combinations (10% PEG 3 h, 20% PEG 3 h, 30% PEG 3 h and 10% PEG 6 h) for further study. The screening basis is as follows: first, 10% PEG treatment for 3 h and 6 h as a mild osmotic regulation representative, they have a stimulating effect on the germination of 'Zhongtian No. 4' while significantly inhibiting the seedling growth of the two test varieties, indicating that effective osmotic regulation has been produced; among them, 6 h treatment is mainly used to explore the influence of osmotic regulation duration. Second, 20% PEG and 30% PEG treatment for 3 h as a moderate osmotic regulation representative, they not only have a positive effect on the germination of 'Zhongtian No. 4' within the effective window of 3 h, but also have no negative effect on the germination of 'Qinghai 444', and at the same time, they have significantly inhibited the seedling growth of the two varieties, successfully triggering measurable osmotic regulation response. In summary, these four treatments together constitute an osmotic regulation gradient from mild to moderate, and are all within the effective and controllable range. According to the analysis results of the germination test of oat seeds after PEG soaking, four PEG soaking treatment combinations were selected for germination test combined with the best EMS mutagenesis conditions selected.

[0074] Example 2, Mutagenic effect of EMS mutagenesis breeding method on germination and seedling stages of 2 varieties of hulled oat seeds

[0075] With the dehulled 'Zhongtian No. 4' and 'Qinghai 444' hulled oat seeds as the mutagenic platform material, different concentrations of EMS mutagen were used for treatment, with the semi-lethal dose as the critical reference, the mutagenic effect of EMS mutagen concentration and treatment time on the germination and seedling stages of 2 varieties of hulled oat seeds was studied. By comparing and analyzing the tolerance differences of 2 varieties under EMS treatment through statistical germination rate, seedling height, root length and other indicators, reference basis was provided for genetic improvement of hulled oat. The specific test method and results are as follows:

[0076] 1. EMS mutagenesis of 'Zhongtian No. 4' and 'Qinghai 444' oat seeds

[0077] (1) Reagent preparation

[0078] Preparation of phosphate buffer solution (PBS): Dilute 100 mL of 10x PBS buffer solution to 1x PBS, adjust the pH of the buffer to neutral, and add distilled water to 1 L. Among them, 10x PBS buffer solution is provided by Beijing Solaybao Technology Co., Ltd.

[0079] Preparation of EMS-PBS solution: The solution was prepared according to the concentration gradient designed in the experiment, in W / V (EMS mutagen is solute, and the solvent is PBS with pH = 7.0). The EMS mutagen was provided by Beijing Solabio Technology Co., Ltd.

[0080] Preparation of 5% sodium thiosulfate (Na2S2O3) solution: 50 g of anhydrous sodium thiosulfate was added to 1 L of pure water and stirred to dissolve completely, to terminate the mutagenesis reaction and relieve toxicity.

[0081] (2) EMS mutagenesis treatment of hulled oat seeds

[0082] EMS mutagenesis treatment of hulled oat seeds: Hulled oat seeds with full and complete grains were soaked in distilled water at 4 °C for 3 h, the distilled water was poured out, and the seeds were washed with water-absorbing paper to remove residual water on the surface. EMS-PBS solution was added, and four concentrations (0%, 0.50%, 0.75%, and 1.00%) and three time gradients (8 h, 12 h, and 16 h) were set up, forming a total of 12 different treatments. The treatment temperature was 20 °C ± 2 °C, 50 seeds were required for each treatment, and three repeated experiments were conducted. A conical flask was added with 50 mL of EMS-PBS solution with different concentrations, the seeds of each treatment were sealed, wrapped with tin paper (to prevent light decomposition of EMS), and placed in a 150 r / min temperature-controlled shaker for different time (8 h, 12 h, and 16 h) of light-free shaking mutagenesis. All the above operations were carried out in the dark.

[0083] Detoxification process: After mutagenesis, the mutagenized oat seeds needed to be detoxified, and the method was as follows: the mutagenized seeds were drained, 50 mL of prepared Na2S2O3 solution was added, and the seeds were shaken for 30 min to completely terminate the reaction and achieve detoxification. Then the detoxified seeds were poured into a gauze bag and washed with running water for 2 h to obtain the mutagenized seeds.

[0084] Germination test: The seed germination bag was moistened with distilled water, the mutagenized oat seeds were placed in the seed germination bag with the furrow facing down, and each 50 seeds were repeated. The seed germination bag was placed in a 2000 lx, 25 / 20 °C, 16 / 8 h light cycle for germination, and the seed germination bag was checked in time to ensure that it remained moist. The germination standard was that the radicle was equal in length to the seed, and the length of the embryo was about 1 / 2 of the length of the seed. The germination of each treatment was counted.

[0085] (3) Determination of germination indicators

[0086] During the cultivation process, the daily germination of oat seeds was observed, and the initial germination time was accurately recorded. After 7 days of cultivation, the germination potential of the oat seeds was calculated. After 10 days of cultivation, the germination rate of the oat seeds was calculated, and the relative and absolute germination rates were also calculated to determine the median lethal dose. After the experiment, for each of the three replicates of each treatment, 10 oat seedlings were randomly selected from each replicate and photographed. After photographing, the root length and seedling length of the oat seedlings were measured using ImageJ. The calculation formulas for the relevant indicators of oat seed germination are as follows:

[0087] Germination rate (%) = Number of seeds germinated on day 10 / Total number of seeds × 100%;

[0088] Relative germination rate (%) = Germination rate of treated seeds / Germination rate of control seeds × 100%;

[0089] Germination potential (%) = Number of seeds germinated on day 7 / Total number of seeds × 100%;

[0090] Relative germination potential (%) = Germination potential of treated seeds / Germination potential of control seeds × 100%

[0091] II. Test Results

[0092] Germination is an important indicator of mutagenesis efficiency. Figure 3 As shown in section A, under the control condition (0% EMS), the germination rate of oat seeds was 97%. With increasing EMS concentration and treatment time, except for the germination rate of different treatment combinations of 0.5% EMS at which no significant change was observed, the germination potential and germination rate of oat seeds decreased significantly. When the EMS treatment condition was 1% for 16 h, the seed germination rate was 57%, with a relative germination rate of 0.58, approaching the semi-lethal treatment condition. With increasing EMS concentration and treatment time, the root length and seedling length of oat seedlings decreased significantly, and the inhibitory effect was more pronounced with higher concentrations. Under the 1% EMS 16 h treatment condition, the seedling length and root length were half that of the control group, indicating that this EMS treatment condition had a dwarfing effect on oat seedlings. Therefore, the optimal EMS treatment condition for 'Zhongtian No. 4' is 1% EMS treatment for 16 h.

[0093] Depend on Figure 3As known from Table 2, the inhibitory effect of EMS treatment on seed germination and seedling growth of 'Qinghai 444' was similar to that of 'Zhongtian 4'. When treated with low concentration for a short time, the inhibitory effect was not significant, but with the increase of treatment concentration, the seed germination and seedling growth were significantly inhibited. It is worth noting that the root system was more sensitive to EMS, and in all high concentration treatment groups (≥0.75%), the root length was extremely severely inhibited. Under 16 h treatment, the germination rate of seeds treated with 0.75% EMS decreased to about 48%, and the relative germination rate was 0.46, close to the semi-lethal level. Therefore, the optimal EMS treatment condition for 'Qinghai 444' was 0.75% EMS treatment for 16 h.

[0094] Example 3, Effect of PEG treatment on EMS mutagenesis of two oat varieties

[0095] With the dehulled 'Zhongtian 4' and 'Qinghai 444' as the mutagenic platform material, the effect of PEG pretreatment on EMS mutagenesis was explored. Different concentrations and times of PEG pretreatment combined with EMS semi-lethal dose mutagenesis were set up, and the germination characteristics and seedling growth indexes such as germination rate, seedling length, and root length were measured to explore the regulatory effect of PEG pretreatment on EMS mutagenesis of two oat varieties, and to provide a basis for optimizing the mutagenic breeding technology of dehulled oat. The specific test methods and results are as follows:

[0096] I. Test method

[0097] Select uniform and full dehulled oat seeds, according to the PEG treatment germination test, select four PEG treatments combined with the best EMS mutagenesis conditions for germination test, and use the best EMS conditions for mutagenesis treatment. The seeds treated with mutagenesis were placed in seed germination bags and treated with 50 mg / L gibberellin (GA3) for exogenous treatment to promote seed germination. The control (CK) selected seeds without any treatment for germination test, and the treatment condition of seeds soaked in distilled water for 3 h was used as the control of PEG pretreatment. The germination test method and determination index are the same as above.

[0098] II. Test results

[0099] As Figure 4The present study systematically evaluated the effects of different PEG pretreatments on EMS mutagenesis of 'Zhongtian 4' by analyzing four key indicators, including germination energy, germination rate, seedling length, and root length. EMS treatment alone significantly inhibited seed germination and seedling growth. Different PEG treatments alleviated this inhibition to varying degrees when combined with EMS mutagenesis. In terms of the mean, the 20% PEG 3 h combined treatment had the best effect, with the germination energy and germination rate closest to the CK group, and the best effect on promoting root recovery, with the root length significantly higher than the EMS group and closest to the CK level among all treatment groups. The 10% PEG 6 h combined treatment was slightly better than the 10% PEG 3 h treatment group, indicating that extending the treatment time within a certain range may have a positive effect. Therefore, the 10% PEG 6 h and 20% PEG 3 h combined treatments had the most outstanding effects. For example Figure 4 As shown in Figure B, similar to 'Zhongtian 4', EMS treatment alone strongly inhibited the germination activity of 'Qinghai 444' seeds. The germination energy and germination rate of all PEG treatments were significantly higher than those of EMS treatment alone, indicating that PEG pretreatment can effectively alleviate the inhibition of EMS on seed germination. In terms of the mean, the 10% PEG 3 h and 20% PEG 3 h treatments had the best germination effect. The 10% PEG 3 h treatment had the most outstanding effect on seedling length. The 10% PEG 3 h and 20% PEG 3 h treatments had the best effect on root growth.

[0100] Example 4, Field Mutagenesis Effects of PEG Combined with EMS Treatment in M2 Generation

[0101] I. Test Method

[0102] 1. Construction of M1 Population

[0103] Uniform and full hulless oat seeds were selected, and germination test was carried out with different treatment combinations of 'Zhongtian 4' and 'Qinghai 444'. The test design included 2 PEG treatments (10% PEG 6 h, 30% PEG 3 h) and the optimal EMS mutagenesis treatment. 208 seeds of 'Zhongtian 4' and 238 seeds of 'Qinghai 444' were treated in each treatment combination. The hulless oat seeds treated by EMS mutagenesis after 3 h soaking were used as the treatment control. The seeds without any treatment were used as the reference control (CK) and were planted in the culture dish covered with double-layer filter paper under the condition of 2000 lx, 25 / 20 ℃, and 16 / 8 h light cycle. After germination, the seedlings were transplanted into the flowerpot and were grown under the condition of 2000 lx, 25 / 20 ℃, and 16 / 8 h light cycle. Due to the toxic effect of EMS on oat seeds, the whole plant vigor of M1 generation was weak. Watering was carried out in time during the growth period, and Hoggland nutrient solution was used once a week to ensure sufficient water and nutrient supply for the mutagenesis materials. The seeds of M1 generation were collected after selfing and were stored after threshing.

[0104] 2. Construction of M2 population

[0105] The test site was located in Dongcun (101°59′ E, 36°42′ N) of Lushar Town, Huangzhong District, Qinghai Province, with an altitude of 2630 m, an average annual temperature of 4.6 ℃, an annual precipitation of 500-650 mm in previous years, and buckwheat as the previous crop. The total nitrogen content in the plough layer of the test site was 1.56 g·kg -1 , the total phosphorus content was 2.24 g·kg -1 , the ammonium nitrogen content was 0.79 mg·kg -1 , the nitrate nitrogen content was 2.24 mg·kg -1 , and the organic matter content was 17.90 mg·kg -1 . M2 was sown by plant row method, and full and consistent M1 single seed was selected, with 16 seeds planted in each plant row. The row spacing was 40 cm, the plant spacing was 40 cm, the sowing depth was 3-4 cm, and the seeds were sown in holes. Every 10 rows planted 1 row of non-mutagenesis materials as the control. Urea (total nitrogen ≥ 46.0%) 75 kg·hm -2 and diammonium phosphate (total nutrients ≥ 64.0%, in which N ≥ 18.0% and P2O5 ≥ 46.0%) 150 kg·hm -2 were used as base fertilizer before sowing.

[0106] 3. Determination index and method

[0107] In order to compare the EMS mutagenesis effects of different treatment combinations, the variation of M2 generation plants was observed and recorded during the milk ripening period, and the variation type and variation rate were recorded. The observation method of variation plants was as follows:

[0108] Plant height (cm): The vertical distance from the ground to the top of the plant was measured with a tape measure.

[0109] Total tillers (number): The total number of tillers from the plant at 10 cm from the ground.

[0110] Effective tillers (number): The number of tillers that produced a panicle.

[0111] Leaf length (cm): The length from the base of the 2nd leaf to the tip of the leaf.

[0112] Leaf width (cm): The width of the widest part of the 2nd leaf.

[0113] Leaf area (cm 2 ): Leaf length x Leaf width x R, R is the correction factor of oat leaf area 0.8317.

[0114] Panicle length (cm): The length from the panicle neck node to the top of the panicle on the main stem.

[0115] Stem diameter (mm): The diameter of the main stem at the middle of the second internode above the ground was measured with a vernier caliper.

[0116] II. Test Results

[0117] 1. Field appearance evaluation of ‘Zhongtian 4’

[0118] In view of the high randomness of EMS mutagenesis, its phenotype effect spectrum is extremely wide. In order to efficiently screen the extremely variable mutations with breeding value from it, this study adopts the analysis method of setting threshold (positive mutation rate is greater than 130% of the average value of the control, and negative mutation rate is less than 70% of the average value of the control). The purpose is to highlight the significant phenotype variation and avoid the interference of random mutations. In addition, by comparing each group (B-E) treated by PEG combined with EMS mutagenesis with the group (A) treated by simple EMS mutagenesis, the comprehensive effect of PEG combined with EMS treatment on mutagenesis can be accurately evaluated. As shown in Table 1.1, compared with the control group (CK), the average value of each agronomic trait of group A is the lowest, indicating that EMS mutagenesis has an inhibitory effect on plant growth. The coefficient of variation (CV%) of multiple traits in each PEG treatment group is generally higher than that in the CK group, indicating that PEG pretreatment has expanded the range of phenotype variation of the mutagenesis population, creating conditions for screening mutants. Plant height and main ear length are relatively stable traits, with a CV% generally lower than 15%, indicating that they are highly conservative. However, the negative mutation rate of plant height in group D is 5.75%, which can be used to screen short-stalk resources. The number of tillers is the most variable trait, with the highest coefficient of variation (31.88%) in group D, and the highest positive mutation rate (11.66%) in group E, further confirming the advantages of the two groups in inducing tiller-related excellent mutations. The coefficient of variation of effective tiller number in group D is as high as 55.36%, indicating that this treatment has induced extremely rich phenotypic diversity; the positive mutation rate of effective tiller number in group C is 10.98%, which is significantly higher than that in group A (4.35%), indicating that this treatment is more likely to obtain individuals with enhanced effective tiller capacity, thereby creating new germplasm to improve seed yield. The average value of leaf area is inhibited, but groups A and E retain 4.35% and 3.68% of positive mutants respectively, indicating the existence of large-leaf single plants. In the stem diameter trait, the average values of groups C and E are higher than that of CK, and the positive mutation rate of group C is 3.66%, indicating that it may be possible to screen anti-lodging materials with thick and strong stems. The variation range of fresh weight is significantly expanded, and more than 10% of positive mutants are found in groups B, C and E, with the highest positive mutation rate of 11.32% in group C, providing an important basis for screening high-yield single plants.

[0119] Table 1 Mutagenic effect of PEG combined with EMS treatment on oat field phenotype at milk ripening stage

[0120]

[0121]

[0122] Note: CK refers to seeds without any treatment; A refers to 'Zhongtian No. 4' seeds soaked in distilled water for 3 h and then treated with EMS mutagenesis; B refers to 'Zhongtian No. 4' seeds treated with 10% PEG for 3 h and then treated with EMS mutagenesis; C refers to 'Zhongtian No. 4' seeds treated with 10% PEG for 6 h and then treated with EMS mutagenesis; D refers to 'Zhongtian No. 4' seeds treated with 20% PEG for 3 h and then treated with EMS mutagenesis; E refers to 'Zhongtian No. 4' seeds treated with 30% PEG for 3 h and then treated with EMS mutagenesis; F refers to 'Qinghai No. 444' seeds soaked in distilled water for 3 h and then treated with EMS mutagenesis; G refers to 'Qinghai No. 444' seeds treated with 10% PEG for 3 h and then treated with EMS mutagenesis; H refers to 'Qinghai No. 444' seeds treated with 10% PEG for 6 h and then treated with EMS mutagenesis; I refers to 'Qinghai No. 444' seeds treated with 20% PEG for 3 h and then treated with EMS mutagenesis; J refers to 'Qinghai No. 444' seeds treated with 30% PEG for 3 h and then treated with EMS mutagenesis.

[0123] In terms of creating high-yield mutants of oats, C treatment and E treatment performed outstandingly. Among them, C treatment had significant advantages in promoting the increase of effective tillers (positive mutation rate 10.98%) and the increase of fresh weight (positive mutation rate 11.32%); while E treatment performed best in inducing the increase of total tillers (positive mutation rate 11.66%). These two treatments provided the most effective breeding materials for breeding new high-yield lines with strong tillering ability and high biomass.

[0124] 2, Field phenotype evaluation of 'Qinghai No. 444'

[0125] This study created rich phenotypic variation in the M2 generation of 'Zhongtian 4' cultivar through combined mutagenesis treatment. The variation characteristics induced by each treatment are analyzed from the perspective of major agronomic traits. Tiller number is a core factor constituting yield, and the mutagenic effects of each treatment on tillering traits differed significantly (Table 1). Treatment F showed the strongest inhibitory effect, significantly reducing the total number of tillers (5.94) and the number of effective tillers (3.93), and exhibiting an extremely high negative mutation rate. Treatments G (10% PEG for 3 h) and H (10% PEG for 6 h) showed intermediate tiller numbers, but in treatment H, due to the extended treatment time, the negative mutation rate of effective tillers increased to 40.88%. Notably, treatment J (30% PEG 3h) exhibited the most pronounced mutagenic effect, with a total number of tillers (10.84) comparable to the control (CK), and the highest positive mutation rate of effective tillers (15.38%) among all treatments. This indicates that this treatment successfully enriched beneficial variations associated with multiple tillers, making it an ideal population for screening high-yielding lines. Fresh weight and flag leaf area are the material basis for yield formation. Treatment I (20% PEG 3h) showed good recovery in fresh weight (282.99 g) and a relatively small coefficient of variation within the population (31.17%), indicating that the variations it created tended towards stable biomass accumulation. Treatment J, on the other hand, demonstrated high mutation potential, with a maximum flag leaf area (103.47 cm²) far exceeding other treatments, and the highest positive mutation rate (15.38%), suggesting the existence of large-leaf mutants within this population. Regarding plant height, treatment F had a mean height of 106.55 cm and a negative variation rate of 7.46%, creating numerous dwarfing variants. Treatment I, on the other hand, had a plant height of 121.72 cm and a CV of 7.57%, showing a smaller variation range. In terms of spike length, treatment J also exhibited a high positive variation capacity, with its maximum value (44.4 cm) significantly higher than other treatments, indicating a greater number of large spike mutants in this treatment. Stem diameter decreased to varying degrees under all treatments, with treatment I showing the best mean stem diameter of 7.60 mm.

[0126] In summary, from the perspective of phenotypic variation, all treatments effectively induced rich phenotypic diversity. Treatment I (20% PEG 3h) showed the best performance in creating variations with excellent overall traits and uniform expression; while treatment J (30% PEG 3h) showed significant advantages in creating high-yield phenotypic variations with breakthrough potential, such as multiple tillers, large ears, and large leaves, and is an important genetic resource for future high-yield breeding.

[0127] 3. Analysis of the mutagenesis efficiency of 'Zhongtian-4'

[0128] In terms of overall mutagenesis efficiency, treatment E significantly outperformed other treatments, reaching 87.98%, indicating that this treatment had the most significant mutagenesis effect on 'Zhongtian 4'. Tiller number and single-plant fresh weight are key factors directly determining oat yield. Table 2 shows that these two traits were most sensitive to the mutagenesis treatments in this experiment, and there were significant differences in mutagenesis efficiency among different treatments. Regarding the induction of tiller number variation, all treatments were more likely to induce mutations of "reduced effective tiller number" (up to 29.81% in treatment E), but treatment E also had the greatest advantage in creating beneficial mutations of "increased" tiller number. The mutagenesis efficiency of treatment E far exceeded that of other treatments, with a mutation rate of "increased effective tiller number" reaching 9.13%, the highest among all treatments, making it the optimal population for screening high-tillering, high-yielding single plants. The mutation rate of increased single-plant fresh weight induced by treatment E was 3.37%. This indicates that its population successfully enriched a large number of mutants with significantly increased biomass, making it a key material for high-yield breeding.

[0129] In conclusion, treatment E is the optimal approach for creating high-yielding mutants of 'Zhongtian 4'. Its M2 generation population simultaneously achieved high mutagenesis efficiency and a high rate of beneficial mutations in both tiller number and individual plant biomass, representing valuable genetic resources for a significant leap in yield potential. Subsequent breeding efforts should focus on targeted screening of beneficial mutant plants within this population. Therefore, based on field phenotypic analysis of the M2 generation, the optimal mutagenesis conditions for 'Zhongtian 4' are 30% PEG treatment for 3 h followed by 1.00% EMS treatment for 16 h.

[0130] 4. Analysis of the mutagenesis efficiency of 'Qinghai 444'

[0131] Table 3 shows the phenotypic mutations induced by different PEG and EMS treatments in the M2 population of Qinghai 444. The data show that each treatment induced extensive phenotypic variation, especially in yield traits such as tillering and biomass, with a very high mutation efficiency, and significant differences between different treatments. Treatment J had an absolute advantage in inducing "total tiller increase", with a mutation efficiency of 9.24%, the highest among all treatments. It provided the richest resources for screening "multiple tiller" high-yield germplasm. In addition to tiller number, treatment J also showed strong positive mutagenic ability in other yield-related traits. The "fresh weight increase" mutation efficiency induced by treatment J was 1.68%, indicating that the population contained a large number of individuals with significantly increased biomass, providing an adequate material basis for high yield. The "flag leaf area increase" and "main ear length increase" mutation efficiencies were 6.72% and 2.10%, respectively, which helped to screen for strains with higher photosynthetic efficiency, more adequate grain filling, and higher seed yield. Treatment I had an "effective tiller increase" mutation efficiency of 8.82%. This indicates that although its overall mutation efficiency was lower than that of treatment J, it was still an important source for screening high-yield mutants, especially as a complementary population to increase genetic diversity.

[0132]

[0133] From the breeding goal of screening high-yield germplasm, treatment J is the best choice, with the highest mutation efficiency of "fresh weight increase" per plant, reaching 1.68%. It successfully directs the mutation direction to key traits required for high yield, such as "tiller increase" and "fresh weight increase", creating the highest frequency and most diverse library of high-yield potential mutants. It is recommended that future breeding work focus on the M2 population of treatment J, concentrating on screening excellent individuals with significantly increased tiller number and biomass, which is expected to quickly obtain new lines with outstanding yield traits. Therefore, based on the field phenotype analysis of the M2 generation, the best mutation conditions for Qinghai 444 are 30% PEG treatment for 3 h, followed by 0.75% EMS treatment for 16 h.

[0134] In summary, the best PEG pretreatment conditions for the two oat varieties are 30% for 3 h, and the best EMS treatment conditions are 0.75%-1% EMS treatment for 16 h. Under these conditions, the positive mutation efficiency of yield-related traits in the M2 generation is the highest.

Claims

1. A PEG synergy based oat EMS mutagenesis method, characterized in that, It comprises the following steps: Step 1, select mature, full 'Zhongtian 4', 'Qinghai 444' hulless oat seeds as mutagenic platform material; Step 2, PEG pretreatment of oat seeds, PEG pretreatment concentration is 0-30 %, PEG pretreatment time is 1.5-24 h; Step 3, EMS mutagenesis treatment of two varieties of hulless oat seeds; the EMS mutagenesis treatment concentration is 0-1.00 %; the EMS mutagenesis treatment time is 8-16 h; the EMS mutagenesis treatment temperature is 18-22 °C.

2. The method of claim 1, wherein: In step 2, the PEG pretreatment condition is 30 % PEG, and the PEG pretreatment time is 3 h.

3. The method of claim 1, wherein: In step 3, the PEG pretreated oat seeds are treated with 1 % EMS solution by mass percentage concentration for 16 hours to obtain PEG and EMS mutagenized 'Zhongtian 4' oat seeds.

4. The method of claim 1, wherein: In step 3, the PEG pretreated oat seeds are treated with 0.75 % EMS solution by mass percentage concentration for 16 hours to obtain PEG and EMS mutagenized 'Qinghai 444' oat seeds.

5. The method of claim 1, wherein: In step 1, the shaking treatment is carried out in a 150 r / min temperature-controlled shaker.

6. The method of claim 1, wherein: In steps 2 and 3, the seeds are soaked in distilled water at 4 °C for 3 h, the distilled water is poured out, the residual water on the surface of the seeds is washed with water-absorbing paper, the EMS-PBS solution is added, the seeds of each group are sealed, wrapped with tin paper, and placed in a 150 r / min temperature-controlled shaker for light-free shaking mutagenesis.

7. The method of claim 1, wherein: The PEG and EMS mutagenesis further comprises the step of investigating the agronomic traits of the M2 generation mutagenized population at the milk stage, evaluating the field mutagenesis effect and mutagenesis efficiency.

8. The method of claim 7, wherein: The method for evaluating the field mutagenesis effect is that the difference of any single trait of a single plant relative to the mean value ± 30 % of the control group; the method for evaluating the mutagenesis efficiency is that the change range of the number of single plants with positive and negative variation of any single trait of the M2 generation relative to the number of initial mutagenized seeds of the M0 generation.

9. Any one of the following a1) to a8) applications: a1) Application of the method of any one of claims 1-7 to evaluate the field mutagenesis effect and mutagenesis efficiency of plant height of two oat varieties; a2) Application of the method of any one of claims 1-7 to evaluate the field mutagenesis effect and mutagenesis efficiency of total tiller of two oat varieties; a3) Application of the method of any one of claims 1-7 to evaluate the field mutagenesis effect and mutagenesis efficiency of effective tiller of two oat varieties; a4) Application of the method of any one of claims 1-7 to evaluate the field mutagenesis effect and mutagenesis efficiency of flag leaf area of two oat varieties; a5) Application of the method of any one of claims 1-7 to evaluate the field mutagenesis effect and mutagenesis efficiency of main ear length of two oat varieties; a6) Application of the method of any one of claims 1-7 to evaluate the field mutagenesis effect and mutagenesis efficiency of stem diameter of two oat varieties; a7) Application of the method of any one of claims 1-7 to evaluate the field mutagenesis effect and mutagenesis efficiency of fresh weight of two oat varieties; a8) Application of the method of any one of claims 1-7 to screen PEG and EMS mutagenesis treatment for creating high-yield germplasm.

10. Use according to claim 9, characterized in that, The two oat varieties are 'Zhongtian 4' and 'Qinghai 444'. The two oat varieties are 'Zhongtian 4' and 'Qinghai 444'.

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