Compound mutagenesis method for improving mutagenesis efficiency of feed oat seeds

Abstract

The invention relates to the field of forage grass mutation breeding, and discloses a compound mutagenesis method for improving feed oat seed mutagenesis efficiency, which comprises the following steps: selecting healthy and full feed oat seeds, completing space mutagenesis through spacecraft carrying, and further applying heavy ion, plasma or chemical mutagenesis treatment to obtain the feed oat seed mutagenesis efficiency. The agronomic characters of the milk-ripe stage and the dough stage are observed through single plant field planting, and the mutagenesis efficiency is evaluated according to the difference of the single character index of the mutagenesis single plant relative to + / -30% of the average value of a control group. According to the method, through the synergistic effect of multi-source mutagenesis, the mutagenesis efficiency and the type diversity are remarkably improved, exogenous gene introduction is not involved, and the safety is high. According to the method, a combination mode of space and heavy ion, plasma or chemical mutagenesis can be flexibly selected according to a breeding target, and a technical support is provided for efficient creation of feed oat germplasm resources and new variety breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of forage mutagenesis breeding, and specifically to a compound mutagenesis method for improving the mutagenesis efficiency of forage oat seeds. Background Technique

[0002] As an important means of germplasm innovation, plant mutagenesis breeding induces genetic variation through physical or chemical methods, and more than 3,400 mutant varieties have been cultivated globally. China has achieved remarkable results in this field, with a total of 817 mutant varieties bred, among which major food crops such as rice dominate. However, as a representative of high-quality forage, the research on mutagenesis breeding of forage oats has progressed slowly: the creation of new germplasms still relies on traditional field collection and hybridization breeding, resulting in a lack of breakthrough resources and insufficient genetic diversity; although genomics research has analyzed the genetic basis of some key traits, there are still shortcomings in the large-scale collection of germplasm resources, the precise phenotypic evaluation system, and the efficient genetic transformation technology, which seriously restricts the coordinated improvement of target traits such as high yield and stress resistance.

[0003] When applying existing mutagenesis technologies to forage oats, multiple bottlenecks are faced. Although space mutagenesis has the advantages of high broad-spectrum variation and short breeding cycle, its mutagenesis mechanism and dose effect in oats lack systematic research, and a mature germplasm creation system has not been established; although chemical mutagenesis can break gene linkage, existing research is mostly limited to phenotypic analysis during the germination period, and the tracking of traits throughout the growth period and variety selection are still blank; the dose-phenotype correlation model of physical mutagenesis such as heavy ions and plasma in the field of forage has not been clarified, making it difficult to achieve directional regulation. In addition, traditional single mutagenesis methods generally have problems such as a narrow mutation spectrum and low screening efficiency of beneficial traits, and cannot meet the requirements of coordinated improvement of multiple target traits. Although the above technologies all belong to the category of endogenous gene improvement and have high safety, the existing schemes have not integrated the synergistic effects of multi-source mutagenesis, resulting in a long germplasm creation cycle and difficult enrichment of excellent traits, restricting the industrial application process of forage oat breeding. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the present invention provides a compound mutagenesis method for improving the mutagenesis efficiency of forage oat seeds, solving the problems of low mutagenesis efficiency, narrow mutation spectrum, and difficult screening of excellent traits of forage oats caused by existing single mutagenesis technologies.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A compound mutagenesis method for improving the mutagenesis efficiency of forage oat seeds, comprising the following steps:

[0006] Select mature, healthy, and uniformly plump forage oat seeds as the initial material;

[0007] Perform space mutagenesis treatment on the seeds;

[0008] Subject the seeds treated by space mutagenesis to one or more of plasma mutagenesis, heavy ion mutagenesis or chemical mutagenesis;

[0009] Plant the mutagenized seeds in the field on an individual plant basis and observe the changes in agronomic traits;

[0010] Evaluate the mutagenesis efficiency based on the change range of agronomic trait indicators relative to the control group.

[0011] Preferably, the space mutagenesis treatment is carried out by being carried on a spacecraft.

[0012] Preferably, the step of evaluating the mutagenesis efficiency based on the change range of agronomic trait indicators relative to the control group is to evaluate the mutagenesis efficiency according to the change range that a certain agronomic trait indicator of a mutagenized individual plant ≥ the average value of the control group + 30% and a certain trait indicator of a mutagenized individual plant ≤ the average value of the control group - 30%.

[0013] Preferably, the dose range used for the heavy ion irradiation mutagenesis treatment is 100 Gy to 300 Gy.

[0014] Preferably, the conditions for the plasma treatment are a power of 250 W to 400 W, a gas volume of 10 standard liters per minute to 20 standard liters per minute, and a treatment time of 200 minutes to 400 minutes.

[0015] Preferably, the chemical mutagenesis treatment uses EMS, the treatment concentration is 0.1% to 1%, and the treatment time is 8 hours to 15 hours.

[0016] Preferably, the mutagenesis treatment is a combined mutagenesis of space mutagenesis and heavy ion irradiation.

[0017] Preferably, the mutagenesis treatment is a combined mutagenesis of space mutagenesis and plasma treatment.

[0018] Preferably, the mutagenesis treatment is a combined mutagenesis of space mutagenesis and chemical mutagenesis.

[0019] Preferably, the mutagenesis treatment is a combined mutagenesis of space mutagenesis, heavy ion irradiation and plasma treatment.

[0020] Preferably, the plant spacing for planting the mutagenized seeds on an individual plant basis is 20 cm to 30 cm, the row spacing is 40 cm to 50 cm, and the plant height, tiller number, stem diameter or biomass are measured at the milk ripening stage and the dough ripening stage respectively to calculate the mutagenesis efficiency.

[0021] The present invention provides a combined mutagenesis method for improving the mutagenesis efficiency of forage oat seeds. It has the following beneficial effects:

[0022] 1. The present invention uses a combined treatment of space mutagenesis and chemical, plasma, or heavy ion mutagenesis, leveraging the synergistic effect of multi-source mutagenesis means to effectively improve the mutagenesis efficiency of forage oat seeds, providing a technical basis for the rapid creation of new germplasms.

[0023] 2. The present invention combines the broad-spectrum mutagenesis effect of the space environment with the specific effects of other mutagenesis methods in the compound mutagenesis, inducing a richer variety of mutants, significantly increasing the mutagenic variation diversity of forage oats, and providing a wider range of germplasm resources for the screening of target traits such as stress resistance, high yield, and high quality.

[0024] 3. The present invention regulates endogenous gene mutations through physical and chemical mutagenesis means without introducing foreign genes, conforming to the technical specifications of non-transgenic breeding, avoiding biosafety disputes, and meeting the requirements of the market and policies for the safety of germplasms. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic flow chart of the method of the present invention;

[0026] Figure 2 is a schematic diagram of the method of an embodiment of the present invention;

[0027] Figure 3 is a schematic diagram of the forward mutagenesis efficiency of the compound mutagenesis of forage oats of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] As Figure 1 shown, the present invention provides a compound mutagenesis method for improving the mutagenesis efficiency of forage oat seeds, belonging to the technical field of forage mutagenesis breeding, involving the combined application of various physical and chemical mutagenesis means to increase the frequency and type diversity of mutations generated in forage oat seeds.

[0030] The method may include the following steps:

[0031] S1. Select seed materials

[0032] Select mature, healthy, plump, and uniformly sized forage oat seeds as the initial materials.

[0033] The seeds should be manually screened to remove damaged, moldy, and immature seeds to ensure the overall physiological activity of the seeds and the complete development of the embryos, providing a stable basis for subsequent mutagenesis treatment. During the seed selection process, visual screening combined with the gravity sedimentation method can be used to further ensure the consistency and uniformity of the seeds.

[0034] S2. Space mutagenesis treatment

[0035] The selected forage oat seeds are subjected to space mutagenesis treatment.

[0036] Space mutagenesis treatment refers to sending the seeds on board a spacecraft (such as a manned spacecraft, space station, or other satellite platform), so that the seeds are subjected to the combined action of complex space environmental factors such as microgravity, high-energy particle radiation, and cosmic rays.

[0037] During the period of being carried, the seeds do not need to be germinated or sprouted, and should be stored in a dry, low-temperature, and sealed state to reduce the interference of external pollution or moisture changes on the mutagenesis effect.

[0038] S3. Composite mutagenesis treatment

[0039] For the seeds after space mutagenesis treatment, one or more composite mutagenesis operations such as plasma treatment, heavy ion irradiation treatment, or chemical mutagenesis treatment are further applied.

[0040] Among them:

[0041] Heavy ion irradiation mutagenesis treatment:

[0042] Using a heavy ion accelerator device, irradiate the seeds after space mutagenesis, and control the dose within the range of 100 Gy to 300 Gy.

[0043] Under preferred conditions, carbon ion beams can be used as the irradiation source, and the irradiation dose can be determined according to the pre-experiment of seed tolerance. During the treatment, the seeds are evenly spread on the irradiation table to ensure uniform irradiation. After irradiation, the seeds should be collected and sealed in time.

[0044] Plasma mutagenesis treatment:

[0045] Using a low-temperature plasma generation device, perform plasma exposure treatment on the seeds, and the treatment conditions are a power of 250 W to 400 W, a gas volume of 10 standard liters per minute to 20 standard liters per minute, and a treatment time of 200 minutes to 400 minutes.

[0046] Helium is used as the carrier gas for plasma treatment to ensure the stability of the plasma environment. During the treatment, the seeds need to be thinly spread on the sample tray and kept rotating or moving to obtain a uniform treatment effect.

[0047] Chemical mutagenesis treatment:

[0048] The seeds are chemically treated in an EMS solution at a concentration of 0.1% to 1%, and are shaken in a fume hood for 8 to 15 hours.

[0049] During the treatment process, fresh EMS aqueous solution needs to be prepared and carried out in a well-ventilated environment. After the treatment, the seeds should be soaked in 5% sodium thiosulfate for 0.5 hours to terminate the reaction, and then rinsed with a large amount of flowing water for more than 2 hours to thoroughly remove the residual mutagen and prevent subsequent toxic effects.

[0050] According to the needs, single composite mutagenesis (space + heavy ion, space + plasma, space + chemical) or multiple composite mutagenesis (space + heavy ion + plasma) can be selected for implementation. The application order of each mutagenesis step can be flexibly adjusted according to the experimental design, but it is advisable to give priority to space mutagenesis to ensure the subsequent cumulative mutagenic effect.

[0051] S4. Field planting and trait observation

[0052] The seeds after composite mutagenesis treatment are planted in the field on a single-plant basis.

[0053] The field planting conditions are as follows:

[0054] The plant spacing is controlled between 20 cm and 30 cm;

[0055] The row spacing is controlled between 40 cm and 50 cm;

[0056] Single-seed sowing is adopted to ensure one plant per hole, which is convenient for subsequent single-plant trait observation and data recording;

[0057] During the planting process, normal fertilization, irrigation and pest control are carried out to maintain the consistency of agronomic management and avoid interference of environmental factors on the observation of mutagenic traits.

[0058] Agronomic trait measurements are carried out at the milk-ripe stage and the dough-ripe stage respectively. The main measurement indicators include:

[0059] Plant height (the distance from the ground to the longest spike or the tip of the stem at the top);

[0060] Total tiller number (the total number of all tillers formed by a single plant);

[0061] Effective tiller number (the number of tillers that can produce spikes and set seeds);

[0062] Stem diameter (the average diameter of the basal stem node);

[0063] Biomass (the fresh weight or dry weight of the above-ground part at the time of single-plant harvest).

[0064] Each index needs to be observed and recorded synchronously with the control group that has not undergone mutagenesis treatment.

[0065] S5. Evaluation of mutagenesis efficiency

[0066] The effect of the mutagenesis treatment was evaluated based on the observed agronomic trait data.

[0067] The specific evaluation methods are:

[0068] The individual plant trait index values of the mutagenesis treatment group were compared with the mean values of the same index of the control group;

[0069] If a certain trait index of a single plant in the mutagenesis group is higher or lower than the mean of the control group by more than ±30%, the single plant is deemed to have mutated in the trait;

[0070] The total mutagenesis efficiency was calculated as the ratio of the number of mutant plants to the total number of observed plants;

[0071] Among them, if a certain trait index of a single plant is higher than the mean of the control group + 30% or more, it is counted as a positive mutagenesis efficiency.

[0072] The entire evaluation process can be repeated multiple times to enhance data reliability.

[0073] In the implementation process of the present invention, space environment mutagenesis is carried out by spacecraft, which does not rely on additional energy input, ensuring that the seed morphology and activity are basically maintained. Heavy ion irradiation, plasma treatment and chemical mutagenesis damage or reshape cell DNA from different mechanism levels, thereby increasing the richness of the mutation spectrum. Through the synergistic effect of multi-source mutagenesis, the genetic variation space of forage oats can be effectively expanded, providing a richer germplasm basis for subsequent breeding screening.

[0074] Example 1: Example of implementation of the composite mutagenesis method for fodder oats in the M1 generation

[0075] like Figure 2 As shown, Zhongtian No. 4 forage oats were used as the base material, and mature, healthy seeds with uniform particle size were selected for mutagenesis treatment according to the following steps:

[0076] 1. Mutagenesis combination design:

[0077] Single mutagenesis group:

[0078] Space mutagenesis (onboard Shenzhou 15 spacecraft);

[0079] Heavy ion mutagenesis (carbon ion beam irradiation, dose 200 Gy);

[0080] Plasma induction (power 360 watts, gas volume 15 standard liters per minute, time 300 minutes);

[0081] Chemical mutagenesis (treatment with 0.5% EMS solution for 11 hours).

[0082] Compound mutagenesis group:

[0083] Space + heavy ions;

[0084] Space + plasma;

[0085] Space + chemistry;

[0086] Space + heavy ions + plasma.

[0087] 2. Field planting and observation:

[0088] The seeds of each individual plant after mutagenesis were planted separately (plant spacing 20 cm, row spacing 50 cm), with the non - mutagenized seeds as the control;

[0089] At the milk - ripe stage, plant height, total tillers, effective tillers and stem diameter were measured, and the biomass was measured at the dough - ripe stage;

[0090] Calculation standard for mutagenesis efficiency: If any agronomic trait index of an individual plant differs from the control mean by ±30%, it is determined as a mutation. The total mutagenesis efficiency is the percentage of the number of mutant plants to the total number of observed plants.

[0091] 3. Experimental results of M1 generation:

[0092] The experimental results are shown in Table 1 and Table 2.

[0093] Table 1 Mutagenesis efficiency of different mutagenic treatments of forage oats in M1 generation

[0094]

[0095] Table 2 Positive mutagenesis efficiency of different mutagenic treatments of forage oats in M1 generation

[0096]

[0097]

[0098] Figure 3 Shows the schematic diagram of positive mutagenesis efficiency of different mutagenic treatments of forage oats in M1 generation. The experimental results show that:

[0099] 1. Comparison of total mutagenesis efficiency

[0100] The total mutagenesis efficiency of the space + chemistry combined mutagenesis in M1 generation is 100%, higher than that of other treatment groups;

[0101] Among the single - mutagenesis groups, the total mutagenesis efficiency of chemical mutagenesis (91.49%) and heavy - ion mutagenesis (88.46%) in M1 generation is relatively high, while the efficiency of pure space mutagenesis (54.14%) is relatively low.

[0102] 2. Characteristics of positive mutagenesis efficiency

[0103] The positive mutagenesis efficiency of the M1 generation by space + plasma composite mutagenesis was 21.74%, 22.98% and 22.36% respectively in the total tiller number, effective tiller number and biomass indexes;

[0104] The positive efficiency of single plasma mutagenesis (total tiller number 18.94%, effective tiller number 22.73%) was the second, and the positive efficiency of heavy ion or chemical mutagenesis was lower.

[0105] 3. Effect differences of composite mutagenesis combinations

[0106] The total mutagenesis efficiency of the M1 generation by space + heavy ion + plasma composite treatment was 70.83%, lower than that of the single heavy ion or chemical mutagenesis group;

[0107] The phenotypic differences of the M1 generation of different mutagenesis combinations indicate that the selection of mutagenesis methods has a significant impact on the mutation types and efficiencies.

[0108] The data of this embodiment show that the combined application of space mutagenesis and chemical mutagenesis is outstanding in the mutagenesis efficiency of the M1 generation, while the combined treatment of space and plasma has more potential in the positive mutations of agronomic traits such as tiller number. The effect differences of different mutagenesis combinations provide an experimental basis for the selection of composite schemes guided by breeding goals.

[0109] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds, characterized in that, It includes the following steps: Select mature, healthy, and uniformly plump forage oat seeds as the initial materials; Conduct space mutagenesis treatment on the seeds; Conduct one or more of plasma mutagenesis, heavy ion mutagenesis, or chemical mutagenesis on the seeds that have undergone space mutagenesis treatment; Plant the mutagenized seeds in the field on a single-plant basis and observe the changes in agronomic traits; Evaluate the mutagenesis efficiency based on the change range of agronomic trait indicators relative to the control group.

2. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, characterized in that, The step of evaluating the mutagenesis efficiency based on the change range of agronomic trait indicators relative to the control group is to evaluate the mutagenesis efficiency according to the change range where a certain agronomic trait indicator of a mutagenized single plant ≥ the average value of the control group + 30% and a certain trait indicator of a mutagenized single plant ≤ the average value of the control group - 30%.

3. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, wherein The dose range used for the heavy ion irradiation mutagenesis treatment is 100 Gy to 300 Gy.

4. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, characterized in that, The conditions for the plasma treatment are a power of 250 watts to 400 watts, a gas flow rate of 10 standard liters per minute to 20 standard liters per minute, and a treatment time of 200 minutes to 400 minutes.

5. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, characterized in that, The chemical mutagenesis treatment uses ethyl methanesulfonate (EMS), with a treatment concentration of 0.1% to 1% and a treatment time of 8 hours to 15 hours.

6. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, wherein The mutagenesis treatment is a combined mutagenesis of space mutagenesis and heavy ion irradiation.

7. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, characterized in that, The mutagenesis treatment is a combined mutagenesis of space mutagenesis and plasma treatment.

8. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, characterized in that, The mutagenesis treatment is a combined mutagenesis of space mutagenesis and chemical mutagenesis.

9. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, characterized in that, The mutagenesis treatment is a combined mutagenesis of space mutagenesis, heavy ion irradiation, and plasma treatment.

10. The composite mutagenesis method for improving the mutagenesis efficiency of forage oat seeds according to claim 1, characterized in that, The plant spacing for single-plant planting of the mutagenized seeds is 20 cm to 30 cm, and the row spacing is 40 cm to 50 cm. The plant height, tiller number, stem diameter, and biomass are measured at the milk ripening stage and the dough ripening stage respectively to calculate the mutagenesis efficiency.

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