Efficient fraxinus mandshurica mutation breeding method and application
Through efficient ash mutagenesis breeding methods integrating physical, chemical and superimposed mutagenesis technologies, the problems of insufficient genetic variation, long breeding cycle and single germplasm resources in ash breeding are solved, and efficient breeding of ash and rapid screening of new varieties are achieved.
Patent Information
- Application Number
- CN202510237882.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-03
- Publication Date
- 2025-05-09
Smart Images
Figure CN119949243A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a high-efficiency elm induced breeding method and application thereof. Background Art
[0002] Manchurian ash (Fraxinus mandshurica) is a large deciduous tree of the genus Fraxinus in the family Oleaceae. It is often referred to as the "three major hardwood species in Northeast China" together with yellow ash and Juglans mandshurica. It is not only an important and precious timber species in Northeast China, but also a large-diameter timber species in the National Reserve Forest Program. Due to its hard wood and beautiful texture, Manchurian ash is widely used in furniture, construction and other fields. Traditional Manchurian ash breeding is mainly based on natural variation and conventional breeding methods. The breeding cycle is long, and it is difficult to obtain rich genetic variation types in a short period of time. With the rapid development of the forestry industry and ecological construction, new Manchurian ash varieties with the characteristics of "fast growth, high quality and high resistance" are urgently needed.
[0003] Mutation breeding is to use physical, chemical or biological mutagenesis to induce gene mutation or chromosome variation in plant cells, thereby obtaining new germplasm resources. 60 Co-γ-ray radiation is a commonly used method in crop and forest breeding; chemical mutagenesis mainly uses common mutagenic agents such as EMS, colchicine, sodium azide, and 5-azacytidine. The superposition of physical and chemical mutagenesis helps to increase the frequency of mutation or expand the types of mutation.
[0004] In recent years, physical mutagenesis, chemical mutagenesis and superposition mutagenesis techniques have been widely used in forest tree species improvement, and have achieved remarkable results in improving genetic diversity, improving traits and breeding new varieties. However, in the field of elm, the application of related mutagenesis techniques is still in its initial stage, the progress of germplasm improvement is slow, and a complete physical and chemical mutagenesis system has not yet been established, which has affected the breeding of new varieties of "fast-growing, high-quality and highly resistant" elm to a certain extent. Therefore, integrating physical, chemical and superposition mutagenesis techniques to establish a scientific, systematic and efficient elm mutagenesis breeding method will significantly improve the genetic variation efficiency of elm, accelerate the selection and improvement of new varieties, and promote its promotion and application in actual production, which has important scientific value and practical significance. However, the current systematic and multi-mode superposition mutagenesis research on elm is still imperfect, and it is difficult to achieve a high level of phenotypic variation while ensuring the germination rate.
[0005] Therefore, providing an efficient elm breeding method that integrates physical, chemical and superimposed mutagenesis operations is of great significance to accelerate the improvement and promotion of elm variety. Summary of the invention
[0006] The present invention aims to solve the key problems of insufficient genetic variation, long breeding cycle, and single germplasm resources in current ash breeding, and provides an efficient ash mutagenesis breeding method and application. The method significantly improves the genetic variation frequency of ash through multiple technical paths such as single mutagenesis and superposition mutagenesis, combined with optimized mutagenesis dose, concentration, and treatment time, broadens the genetic diversity of germplasm resources, and constructs a scientific, systematic, and repeatable ash mutagenesis breeding technology system.
[0007] The present invention can quickly screen excellent new strains of elm, which have obvious improvements in key traits such as plant height, ground diameter, and compound leaf area. The method significantly improves breeding efficiency, shortens breeding cycles, and reduces economic costs. At the same time, it fills the technical gap in the elm mutagenesis breeding system and solves the technical bottleneck in this field. At the same time, the present invention shows important strategic value in promoting the sustainable development of forestry, maintaining the stability of the ecosystem, and improving the efficient utilization of forest resources. The present invention aims to solve the key problem of "insufficient genetic variation" in existing elm breeding, and proposes an efficient elm mutagenesis breeding method and application. By optimizing the radiation dose, the concentration of chemical reagents, the treatment time and other conditions, efficient mutagenesis of elm seeds is achieved, its genetic diversity is broadened, and excellent new strains are quickly screened.
[0008] To achieve the above object, the present invention provides an efficient ash mutation breeding method and application, which specifically comprises the following steps:
[0009] Step (1): Seed selection and processing
[0010] Preferably, seeds with uniform grain weight, fullness and no diseases are selected from seeds of the ash family, washed with distilled water, and soaked for 24 hours to remove the shriveled seeds.
[0011] Preferably, each rinse lasts for 5 minutes to remove dust and other impurities attached to the surface. During the soaking process, use tweezers to remove bad seeds floating on the water surface to ensure the quality and uniformity of the experimental seeds, and place the soaked seeds in clean gauze to drain the surface water.
[0012] Step (2): Treat the seeds of step (1) by single mutagenesis or superposition mutagenesis
[0013] Preferably, the single mutagenesis method is physical mutagenesis or chemical mutagenesis; the physical mutagenesis is 60Co-γ rays, the radiation dose is 25Gy, 50Gy or 75Gy, and the radiation time is 3 hours; the chemical mutagenesis is EMS, colchicine solution, sodium azide and 5-azacytidine, the mutagenesis concentrations are 0.5%, 0.15%, 4mmol / L and 50μmol / L respectively, and the mutagenesis time is 24 hours.
[0014] Preferably, the physical-chemical superposition mutagenesis is to first subject the seeds to physical mutagenesis with 25 Gy or 50 Gy of 60Co-γ rays for 3 hours, and then subject them to chemical mutagenesis for 48 hours; the chemical-chemical superposition mutagenesis is to first soak the seeds in 0.5% EMS solution for 24 hours, rinse them with distilled water, and then transfer them to 4 mmol / L sodium azide solution or 50 μmol / L 5-azacytidine solution and continue soaking for 24 hours.
[0015] Step (3): Field planting and trait investigation
[0016] Preferably, the mutagenized seeds are sown in field test sites, and the germination rate and seedling characteristics are recorded and evaluated.
[0017] Preferably, the field test site is located in a typical elm growing area with suitable temperature, humidity and soil conditions; the seedling stage traits include plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, leaflet area and other trait parameters.
[0018] Step (4): Screening of excellent plants and construction of database
[0019] Preferably, plants with excellent phenotypes are screened and asexually propagated, and a corresponding mutagenic trait database is established.
[0020] Preferably, the excellent phenotypic screening criteria include but are not limited to: higher germination rate, larger plant height, thicker ground diameter, wider compound leaves and other indicators; the screened excellent plants are propagated by asexual propagation, and the propagation methods include but are not limited to cuttings, grafting or tissue culture; the corresponding mutagenic trait database is established, and the key parameters stored in the database include: germination rate, plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, leaflet area, etc., through standardized data management and evaluation system, to provide a scientific basis for the breeding of new elm varieties.
[0021] The present invention also provides an application of efficient elm in polyploid mutation breeding. The present invention also has significant value in creating fast-growing, high-quality, stress-resistant new germplasm of elm and improving the adaptability and stress resistance of elm. The efficient mutation breeding method of the present invention is not only suitable for polyploid mutation breeding, but can also be widely used in creating fast-growing, high-quality, stress-resistant new germplasm, significantly improving the adaptability and stress resistance of elm, thereby providing broader application prospects and value for the genetic improvement and industrial promotion of elm.
[0022] The beneficial effects of the present invention are:
[0023] 1. Systematic system: The present invention systematically studies the growth performance of elm wood under different mutagenesis treatments, and establishes an efficient mutagenesis breeding system suitable for elm wood by optimizing physical and chemical mutagenesis techniques.
[0024] 2. Diversification of mutagenesis strategies: The present invention adopts a combination of multiple mutagenesis methods (single physical mutagenesis, single chemical mutagenesis, physical-chemical superposition mutagenesis and chemical-chemical superposition mutagenesis) to comprehensively evaluate the effects of different mutagenesis methods on the germination rate and phenotypic traits of elm, providing a scientific basis for improving genetic diversity.
[0025] 3. Wide applicability: Through genetic analysis of six phenotypic traits, namely plant height, diameter at ground level, compound leaf length, compound leaf width, compound leaf area and leaflet area at the seedling stage, the present invention screened out ash plants with excellent phenotypic traits, laying a foundation for the selection and breeding of new varieties. At the operational level, the physiological characteristics of ash are closely followed, and the radiation dose and chemical reagent concentration within a certain range are used to take into account a high mutation frequency and an acceptable germination rate and seedling survival rate, so as to give full play to the advantages of mutation breeding in the cultivation of new forest varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a schematic diagram of the leaflet spacing and leaf color variation after elm mutagenesis.
[0027] Figure 2 This is a schematic diagram of the leaf characteristics of elm wood after mutation, including the variation in leaflet number, leaf margin and leaflet morphology.
[0028] Figure 3 This is a schematic diagram of the variation of terminal leaflets after elm wood mutation. DETAILED DESCRIPTION
[0029] The present invention is further explained by means of the accompanying drawings and embodiments.
[0030] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. The test methods in the following examples without specifying specific conditions are usually operated under conventional conditions, and since they do not involve the invention, the steps are not described in detail.
[0031] Example 1 Creation of Manchurian ash mutants using a single mutagenesis method
[0032] (1) Seed selection and processing
[0033] Follow the steps (1) above.
[0034] Seeds of the Manchurian ash family collected in the Dailing area of Heilongjiang Province in September 2021 were selected as the basic materials for this mutagenesis experiment. After vernalization in 2022, these seeds were first screened to ensure that the seeds were full and uniform in size. At the same time, the surface of the seeds was carefully observed through a magnifying glass to remove seeds with diseases, mechanical damage or discoloration.
[0035] Place the screened seeds in distilled water and rinse them three times, each time for 5 minutes, to remove dust and other impurities attached to the surface. Soak the washed seeds in distilled water for 24 hours to ensure that each seed can fully absorb water and activate the physiological activity of the seeds. During the soaking process, use tweezers to remove bad seeds floating on the water surface to ensure the quality and uniformity of the experimental seeds. Place the soaked seeds in clean gauze to drain the surface water.
[0036] The seeds were divided into 5 groups, groups 1-4 were treated with a single chemical mutagenesis, and group 5 was a control group without any treatment.
[0037] (2) Treating the seeds in step (1) using a single mutagenesis method
[0038] The single mutagenesis method can be either physical mutagenesis or chemical mutagenesis.
[0039] a) Physical mutagenesis
[0040] The seeds treated in step (1) were sent to Harbin Institute of Physics and 60 Co-γ rays were used for physical mutagenesis. The radiation doses were 25Gy, 50Gy and 75Gy, and the radiation time was 3 hours. These doses were verified by previous experiments and can effectively induce gene mutations without affecting the basic activity of seeds.
[0041] b) Chemical mutagenesis
[0042] ①Preparation of chemical mutagen
[0043] In this implementation case, in order to carry out chemical mutagenesis treatment, the following key chemical reagents were prepared, and all operations were carried out under standard laboratory safety conditions to ensure accurate reagent concentrations, uniform solutions, and meet the technical requirements of subsequent mutagenesis experiments.
[0044] 1L phosphate buffer: Prepare by mixing 0.2mol / L sodium dihydrogen phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4) solutions with a pH of 7.4. Weigh 5.93g of sodium dihydrogen phosphate and dissolve it in about 190mL of distilled water, and weigh 38.66g of disodium hydrogen phosphate and dissolve it in about 810mL of distilled water. Mix the two solutions and stir them thoroughly. Use a pH meter to measure the pH value of the solution to ensure that the pH is between 7.2 and 7.4. If the pH deviation is large, use dilute hydrochloric acid (HCl) or sodium hydroxide (NaOH) solution to make slight adjustments until the desired pH range is reached. Transfer the mixed solution to a 1L volumetric flask and dilute to 1L with distilled water. The prepared phosphate buffer is transferred to a clean storage bottle, sealed and stored in a 4°C refrigerator away from light to prevent changes in the buffer system and affect the accuracy of subsequent experiments.
[0045] 0.15% Colchicine Solution (Q): Weigh 1.5g of colchicine powder, dissolve it in 1L of distilled water, and stir thoroughly until it is completely dissolved. Since colchicine has a certain toxicity, the solution must be prepared and used immediately to avoid long-term storage that may reduce the efficacy. During preparation and use, operations should be carried out in a well-ventilated environment, and protective gloves and protective masks should be worn to ensure safe operation.
[0046] 0.5% EMS solution (E): Add 5mL EMS stock solution (about 99% concentration) to 1L phosphate buffer and stir thoroughly. Since EMS (ethyl methanesulfonate) is highly volatile and toxic, the solution should be prepared and used immediately to avoid decomposition and failure. During preparation and use, operations should be carried out in a fume hood, and operators should wear protective gloves and goggles to ensure safety.
[0047] 4mmol / L sodium azide solution (D): Weigh 2.6g of sodium azide crystals and dissolve them in 50mL of phosphate buffer to prepare a mother solution. When using, take 5mL of the mother solution and add it to 1L of phosphate buffer, mix thoroughly, and finally prepare a 4mmol / L working solution. Sodium azide is highly toxic and easily reacts with heavy metal salts to form explosive compounds. Therefore, the preparation and use of the solution must be carried out in a fume hood, away from heavy metal ions. The unused solution must be sealed and stored in a light-proof, low-temperature environment to ensure its safety and stability.
[0048] 50μmol / L 5-azacytidine solution (5): Weigh 0.1221g of 5-azacytidine powder and dissolve it in 50mL of phosphate buffer to prepare a stock solution. When using, take 5mL of the stock solution and add it to 1L of phosphate buffer, mix well, and you can prepare a 50μmol / L working solution. The 5-azacytidine solution must be prepared and used immediately to avoid long-term storage that may reduce the efficacy. During the preparation and use process, it should be carried out under light-proof conditions, and the solution must be stored in a low-temperature environment to ensure its chemical stability.
[0049] ② Grouping and immersion
[0050] The selected seeds are evenly divided into 1-4 groups and placed in 0.15% colchicine solution (bucket No. 1), 0.5% EMS solution (bucket No. 2), 4mmol / L sodium azide solution (bucket No. 3), and 50μmol / L 5-azacytidine solution (bucket No. 4). Each group of seeds is completely immersed in the mutagenic agent to ensure that each seed is in full contact with the agent. Soak for 24 hours at room temperature (25°C) and stir every 3 hours during this period to ensure that the reagent is evenly distributed and the seeds fully absorb the mutagenic agent. After soaking, rinse the seeds thoroughly with running water for 30 minutes to remove the mutagenic agent remaining on the surface to prevent the residual agent from affecting seed germination and subsequent trait observations. Place the cleaned seeds in a cool and ventilated place to drain the surface moisture naturally.
[0051] (3) Field planting and trait investigation
[0052] a) Seeding
[0053] At the field test site suitable for the growth of ash, the treated seeds were evenly sown. That is, in April 2022, they were sown in the test field of Maoershan Experimental Forest Farm of Northeast Forestry University in Shangzhi City, Heilongjiang Province. They were sown evenly to ensure that different treatment groups were clearly marked to prevent cross-confusion. And each mutagenesis combination was set up with 3 to 5 repeated controls (including the non-mutated control group). After sowing, keep it moderately moist and check the soil moisture regularly to ensure that the seeds can germinate normally.
[0054] b) Germination rate statistics
[0055] Record seed germination rate, plant height after emergence, ground diameter and other seedling indicators to obtain preliminary data. According to the characteristics of different mutagenesis treatments, the germination rate can be recorded 3 or 4 weeks after sowing, and the germination rate of each group can be counted. The seedlings are continuously observed until they mature, and the differences between each treatment group and the control group are compared and analyzed. (See Table 1 for specific results).
[0056] c) Observation of seedling characteristics
[0057] When the seeds grew into seedlings, the growth conditions and trait variation of each group of seedlings were recorded.
[0058] It is necessary to measure the plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, leaflet area and other traits of each group (see Table 2 for specific results).
[0059] (4) Results
[0060] After statistical analysis, the germination rates of the single chemical mutagenesis groups were as follows: colchicine group (54.21%), EMS group (46.43%), sodium azide group (50.72%), and 5-azacytidine group (54.48%); in the physical mutagenesis group, the germination rate decreased with increasing doses (25Gy→68.55%, 50Gy→47.36%, 75Gy→28.17%).
[0061] Combined with the subsequent experiments of the present invention, single mutagenesis and superposition mutagenesis were counted together to form "Table 1" (sequence numbers 1-7 are single mutagenesis, 8-13 are superposition mutagenesis)
[0062] Table 1 Effects of different mutagenesis methods on germination rate of ash seeds
[0063]
[0064]
[0065] Single chemical mutagenesis (especially 0.15% colchicine and 50μmol / L 5-azacytidine) has a significant effect on the performance of seedling traits such as plant height, ground diameter, and compound leaf area of Fraxinus mandshurica, showing a high mutation rate. The relevant measurement data are presented in the comprehensive statistical table below. The results show that a single chemical mutagenesis method has a significant effect on the trait mutation of ash seeds. Among them, the treatment effects of 0.15% colchicine and 50μmol / L 5-azacytidine are particularly prominent. These treatments not only effectively improved the germination rate of seeds, but also significantly improved the trait performance of ash.
[0066] This discovery provides a clear technical direction and practical basis for the subsequent single chemical mutation breeding of elm.
[0067] Example 2 Creation of Manchurian ash mutants using superposition mutagenesis
[0068] (1) Seed selection and processing
[0069] The same as Example 1. The treated seeds were evenly divided into groups, where groups 1 to 4 were superimposed mutagenesis treatment groups, and group 5 was a control group without any treatment.
[0070] (2) Treating the seeds of step (1) by superposition mutagenesis
[0071] This example uses physical-chemical superposition mutagenesis and chemical-chemical superposition mutagenesis to further enrich the mutation types of ash and increase the mutation rate. To facilitate experimental operations, seeds and mutagenic agents are distinguished and marked in marked barrels. The specific steps are as follows.
[0072] (a) Physical-chemical superposition mutagenesis treatment
[0073] ① Physical mutagenesis treatment
[0074] The full seeds that had been processed (soaked and screened) were divided into two groups, A and B, and sent to the Harbin Institute of Physics for seed 60 Co-γ-ray mutagenesis. 60 Co-γ-ray radiation); Group B seeds: (received 50 Gy 60 Co-γ-ray radiation).
[0075] Each group of seeds was irradiated for 3 hours. This dosage range has been verified by previous experiments and can effectively induce gene mutations while maintaining the basic activity of seeds.
[0076] ②Chemical mutagenesis immersion
[0077] After the physical irradiation, the seeds of group A and group B were washed 3 to 5 times with distilled water to remove the dust or radioactive residues that may be attached to the surface. Then, the two groups of seeds were further divided into four subgroups and placed in different marked buckets corresponding to the chemical mutagenesis solutions (see Example 1 for the specific solution configuration).
[0078] Bucket A1: Group A seeds + 0.15% colchicine solution;
[0079] Bucket A2: Group A seeds + 0.5% EMS solution;
[0080] Bucket A3: Group A seeds + 4mmol / L sodium azide solution;
[0081] Bucket A4: Group A seeds + 50 μmol / L 5-azacytidine solution;
[0082] Bucket B1: Group B seeds + 0.15% colchicine solution;
[0083] Bucket B2: Group B seeds + 0.5% EMS solution;
[0084] Bucket B3: Group B seeds + 4mmol / L sodium azide solution;
[0085] Bucket B4: Group B seeds + 50 μmol / L 5-azacytidine solution;
[0086] The seeds of each subgroup are completely immersed in the corresponding chemical reagent for 48 hours at room temperature. During the soaking process, each bucket is gently stirred for about 5 minutes every 12 hours to ensure that the liquid is in full contact with the seeds. After the soaking, rinse with running water for 30 minutes to completely remove the chemical residue. Finally, spread the seeds in each bucket on a clean gauze, place it in a cool and ventilated place to naturally drain the surface moisture, and mark the number for subsequent statistics and field sowing.
[0087] b) Chemical-chemical superposition mutagenesis
[0088] ①The first chemical mutagenesis
[0089] The pretreated seeds of the same batch were divided into two groups: bucket E1 (prepared for the combination of "0.5% EMS + 4mmol / L sodium azide") and bucket E2 (prepared for the combination of "0.5% EMS + 50μmol / L 5-azacytidine").
[0090] In the first chemical mutagenesis, 0.5% EMS solution was added to buckets E1 and E2 to completely immerse the seeds, and the seeds were soaked at room temperature (about 20°C) for 24 hours. To ensure that the reagent was in full contact with the seeds, they were gently stirred every 6 hours for about 1 minute each time.
[0091] After soaking, rinse the seeds in buckets E1 and E2 with running water for no less than 30 minutes to remove the EMS residue on the surface.
[0092] ② Second chemical mutagenesis
[0093] To achieve the superimposed effect of the two agents, the seeds in bucket E1 were transferred to bucket F1 after rinsing, and the seeds in bucket E2 were transferred to bucket F2, and the corresponding second chemical solutions were added respectively: bucket F1 (4 mmol / L sodium azide solution) and bucket F2 (50 μmol / L 5-azacytidine solution).
[0094] Continue soaking for 24 hours at room temperature, stirring every 6 hours to ensure even distribution of the solution.
[0095] After soaking, rinse the seeds in buckets F1 and F2 with running water for about 30 minutes, spread the rinsed seeds on clean gauze, and drain the surface water naturally. After recording the numbers, you can start field sowing or subsequent experiments.
[0096] (3) Field planting and trait investigation
[0097] The seeds that have completed the superimposed mutagenesis treatment were numbered and brought back to the field test site - Maoershan Experimental Forest Farm of Northeast Forestry University in Shangzhi City, Heilongjiang Province. The seeds in each marked bucket were evenly sown in their corresponding test plots on the same sowing date (such as mid-to-late April), and were divided into zones with the control group (no mutagenesis or only single mutagenesis). A randomized block design was used, with 3 to 5 replicates set for each treatment group.
[0098] (a) Germination rate statistics
[0099] Observe and record the germination rate of each treatment group 3 to 4 weeks after sowing, and make statistics according to the average germination rate of each treatment group, and compare and analyze with the single mutagenesis method and the control group in Example 1. At this stage, the number and robustness of seedlings after emergence are also monitored to prepare for subsequent seedling measurements.
[0100] (b) Observation of seedling characteristics
[0101] When the seedlings grow to maturity, key morphological indicators such as plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, and leaflet area of each group are measured and recorded, and the average value and coefficient of variation are calculated. If conditions permit, the survival of seedlings can also be observed under extreme weather conditions (such as low temperature and drought) to preliminarily evaluate the potential effects of different superimposed mutagenesis treatments on stress resistance.
[0102] (4) Results
[0103] (a) Germination rate and seedling performance
[0104] Compared with a single mutagenesis method, the germination rate of superimposed mutagenesis treatments usually fluctuates to varying degrees. Some combinations (such as the physical-chemical superposition treatment of "50Gy+0.5%EMS") are outstanding in improving the germination rate or accelerating the emergence speed. Other combinations (such as the chemical-chemical superposition treatment of "0.5% EMS+4mmol / L sodium azide") show richer phenotypic variation in the seedling stage, including differences in plant height and compound leaf morphology. However, there are also combinations (such as "25Gy+EMS") that result in a germination rate that is too low or even 0 (see Table 1).
[0105] (b) Trait variation diversity
[0106] In order to more intuitively illustrate the variation range of different superposition combinations in traits such as plant height, ground diameter, and compound leaf area, some key treatment combinations (EMS+D, EMS+5, 25+Q, 50+D, 50+5) were selected in this embodiment to measure the maximum value, minimum value, and coefficient of variation. The results are shown in "Table 2".
[0107] Statistical analysis showed that the superimposed mutagenesis combination was more likely to produce significant variation in plant height, ground diameter and leaf morphology. Some mutants grew faster in the seedling stage, and the leaf width and thickness also changed significantly. This means that superimposed mutagenesis treatment has potential advantages in accelerating ash breeding and enriching trait diversity.
[0108] Table 2 Variation range and coefficient of variation of seedling traits in different superimposed mutagenesis combinations
[0109]
[0110]
[0111] The comprehensive germination rate and seedling trait performance showed that under the same experimental conditions, some combinations showed higher coefficients of variation in traits such as plant height and ground diameter. The superimposed mutagenesis method was easier to obtain multiple types of excellent variant plants than the single mutagenesis method, providing more optional mutants for subsequent screening of excellent strains and stress-resistant breeding.
[0112] Example 3 Field planting and trait investigation
[0113] (1) Field trial location and experimental design
[0114] A typical ash growing area was selected, that is, the area met suitable temperature (annual average temperature 68°C), humidity (annual average precipitation 600-800mm) and soil conditions (black soil or brown soil, soil organic matter content>3%).
[0115] The depth of trenching in the field should be kept at 3 to 5 cm so that the seeds can fully contact the soil after sowing.
[0116] Different mutagenesis combinations (including single mutagenesis, physical-chemical superposition mutagenesis, and chemical-chemical superposition mutagenesis) were planted in different areas at the same location for comparison; the control group and the treatment group adopted a randomized block design (RCBD), with 3 to 5 repetitions in each group to ensure the reliability of statistical analysis.
[0117] (2) Investigation of traits at the seedling and maturity stages
[0118] For each treatment, 100 plants were sampled and measured. The germination rate was investigated 3 to 4 weeks after sowing. The length, width, area and leaflet of the compound leaf were measured at maturity (30 to 34 weeks after sowing). The plant height and ground diameter were measured after the plants had completely shed their leaves.
[0119] a) Plant height: the height from the ground to the growing point (cm), measured with a tape measure, with an accuracy of 0.01 cm;
[0120] b) Ground diameter: the diameter of the main stem 1 cm above the ground (mm), measured with a vernier caliper, with an accuracy of 0.01 mm;
[0121] c) Compound leaf length, compound leaf width, compound leaf area, and leaflet area: Use a steel ruler to measure the length and width of the compound leaves with an accuracy of 0.01 cm; use image J software to measure the area of compound leaves and terminal leaflets; use a camera to photograph the leaves of the mutant seedlings, record various leaf traits, and calculate the average value.
[0122] For ease of comparison, the average phenotypic parameters of some major treatments are summarized in Table 3.
[0123] Table 3 Average phenotypic parameters of different induced elm treatments and the control group
[0124]
[0125]
[0126] As shown in Table 3, different mutagenesis treatments produced different degrees of differences in plant height, ground diameter, compound leaf area, etc. Among them, the colchicine group (Q) performed outstandingly in compound leaf area, and the EMS+5 group performed better in plant height and overall growth potential. Although some superposition combinations (such as 25+Q, 50+D, 50+5) may not necessarily have an advantage in germination rate, they can present significant leaf shape variation or special resistance performance, providing more directions for subsequent breeding screening.
[0127] Example 4: Screening of superior plants and construction of database
[0128] (1) Screening for superior phenotypes
[0129] In order to ensure that the new variety selection process has an objective and unified evaluation basis, this embodiment uses local ash families as a control group when screening excellent phenotypes and determining new variety candidates, and refers to the key evaluation indicators and thresholds listed in the "Guidelines for New Varieties of Fraxinus" to construct a scientific screening process. The guidelines set relatively comprehensive standards from the dimensions of morphological characteristics, genetic stability, ecological adaptability, etc., making the new variety selection of the present invention more operational and popularizable.
[0130] (a) Screening indicators and scope
[0131] After completing the field trials and trait measurements in the aforementioned embodiments (Example 1, Example 2, Example 3), all mutated strains were comprehensively evaluated based on key traits such as plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, and terminal leaflet area of the elm seedlings.
[0132] Table 4 Genetic variation parameters of phenotypic traits of ash mutant strains
[0133]
[0134]
[0135] According to the statistics in Table 4, the overall coefficient of variation of the above six phenotypic traits ranged from 8.05% to 32.98%, among which plant height was the highest (32.98%) and compound leaf width was the lowest (8.05%); in other words, plant height was the most likely to differentiate in this mutagenesis population, while compound leaf width was relatively stable. This phenomenon indicates that if breeding focuses on highly variable traits (such as plant height, compound leaf area, and terminal leaflet area), more potential mutant materials can be obtained.
[0136] (b) Screening process and criteria
[0137] Initial screening: Conduct large-scale observations during the seedling stage (usually 6 to 8 weeks or 30 weeks old), combine important growth indicators such as plant height and ground diameter, and eliminate individuals with deformities, serious diseases, or obvious degeneration of traits.
[0138] Re-screening: Six major phenotypic traits (plant height, diameter at ground level, compound leaf length, compound leaf width, compound leaf area, and terminal leaflet area) are accurately measured for the population that passes the initial screening. Combined with the mean, standard deviation, and coefficient of variation of each trait, focus is placed on individuals that perform well and stably in traits with higher variability, such as plant height and compound leaf area.
[0139] Comprehensive evaluation: Consider whether the plants have any abnormalities during their growth (such as chlorosis, extreme leggy growth, etc.), and combine the breeding goals (fast growth, stress resistance or wood quality, etc.) for comprehensive scoring and ranking, and finally determine the superior mutants or superior mutant groups.
[0140] (c) Examples of Typical Excellent Strains
[0141] Certain mutant strains can be selected preferentially if they are significantly higher than the average in plant height (>28 cm) and ground diameter (>3.2 mm), and have larger compound leaf area or terminal leaflet area;
[0142] Although some individual plants are slightly shorter in height, they excel in leaf morphology (wider and larger terminal leaflets) or stress resistance (such as thicker leaves and greater drought resistance), and can also be used as objects for targeted breeding.
[0143] (2) Asexual propagation of excellent plants
[0144] (a) Grafting
[0145] The selected excellent plants or groups are grafted.
[0146] ① Rootstock selection: It is preferred to use species of the same genus with higher affinity with elm (such as local common elm seedlings) as rootstock to improve the grafting survival rate.
[0147] ② Grafting method: Branch grafting or bud grafting (such as bark grafting, tongue grafting, bud grafting, etc.) can be used, and the specific selection depends on the climate, season and labor conditions.
[0148] ③Management points: After grafting, appropriate temperature and humidity conditions must be provided, and the interface must be tightly fitted. It usually takes about 2 to 4 weeks to heal and sprout new shoots.
[0149] (b) Other asexual reproduction methods
[0150] ① Cutting: Cut semi-lignified young branches in the appropriate season, dip them in IBA or NAA, and insert them into a matrix of vermiculite and peat (1:1) to promote rooting in a moisturizing environment.
[0151] ② Tissue culture: obtain explants from the stem tips or petioles of excellent strains, induce callus or clustered buds in improved MS or WPM culture medium, and then differentiate and root to form complete plants.
[0152] All of the above methods can obtain offspring with the same phenotype as the mother in batches within a relatively short period of time, ensuring that excellent traits can be stably transmitted.
[0153] (3) Construction of mutagenic trait database
[0154] (a) Data collection and integration
[0155] ① Individual label: number each excellent plant (such as "25Gy+EMS-A3"), indicating its mutagenesis method, dosage combination, field plot number, etc.
[0156] ② Phenotypic data entry: The actual measured values of the six major traits (plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, terminal leaflet area) and the corresponding statistical parameters (mean, standard deviation, coefficient of variation) are entered into the database.
[0157] ③ Auxiliary information: If drought resistance, cold resistance or disease resistance are found to be outstanding in later tests, they will be additionally noted in the database for subsequent screening and application.
[0158] (b) Database management and application
[0159] ① Classification storage: Create indexes based on mutagenesis type (physical, chemical, superposition), dose level, year, etc. for quick retrieval.
[0160] ②Statistical analysis: Use Excel, Access or special statistical software (such as SPSS, R language, etc.) to conduct variance analysis, cluster analysis or multivariate regression on each trait to explore the correlation between traits or potential excellent genotypes.
[0161] ③Data updating and sharing: Data are continuously updated along the growth cycle (such as annual monitoring and observation after wintering), and visual charts (such as line charts, scatter plots, etc.) are formed to provide a scientific basis for subsequent breeding or production decisions of elm.
[0162] (4) Data analysis and breeding value
[0163] (a) Interpretation of the data in Table 4
[0164] Table 4 shows the genetic variation of 6 main phenotypic traits of ash mutant strains obtained by the present invention under 13 mutagenesis methods, namely, plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, and terminal leaflet area:
[0165] ① The coefficient of variation ranged from 8.05% to 32.98%, with a large range of variation;
[0166] ② The order of coefficient of variation of each trait is: plant height > compound leaf area > terminal leaflet area > ground diameter > compound leaf length > compound leaf width;
[0167] This indicates that plant height, compound leaf area, and terminal leaflet area are more likely to vary and can serve as an important reference for screening excellent mutants.
[0168] (b) Breeding direction
[0169] ① Fast-growing: Pay attention to the plant strains with obvious increases in plant height and ground diameter.
[0170] ② Resistance to adversity: Observe the survival and growth conditions under extreme climatic conditions, and make a comprehensive judgment based on leaf thickness, root system development, etc.
[0171] ③ Polyploidy mutagenesis: If polyploidy is successfully induced using colchicine or a stacking combination, it can be marked in the database and subsequent breeding tests can be performed.
[0172] (5) Implementation Effect
[0173] (a) Rapid propagation and stable inheritance: Through grafting, cuttings or tissue culture, excellent genotypes can be propagated on a large scale in a short period of time while maintaining the original characteristics.
[0174] (b) Refined management: key trait data (including coefficient of variation, mean value, standard deviation, etc.) are entered into a unified database to facilitate tracking and comparison during the breeding process of each generation.
[0175] (c) Significant breeding application prospects: Highly variable plant height, compound leaf area and other indicators indicate great breeding potential. If further testing and screening are carried out for rapid growth and stress resistance, it is expected that high-quality new germplasm will be created to better meet the needs of forest breeding and industrialization.
[0176] This implementation case fully combines phenotypic data with asexual propagation and database management, so that polyploid mutation breeding of ash and the creation of fast-growing, high-quality, and stress-resistant new germplasm are both provided with operational technical support. At the application level, the excellent strains screened from the database can be promoted on a large scale or further breeding experiments can be carried out.
[0177] In summary, the present invention provides an efficient elm induced breeding method and its application, which overcomes the technical difficulties in existing elm breeding, such as insufficient genetic variation, long breeding cycle, and single germplasm resources. By integrating physical, chemical and superposition mutagenesis techniques, the present invention significantly improves the genetic variation efficiency of elm, broadens its genetic diversity, and provides a scientific, systematic and repeatable technical system for the rapid screening of excellent new elm strains. This method not only has significant economic benefits, but also has broad application prospects. It can promote technological progress in the field of elm breeding and meet the needs of the forestry industry and ecological construction for new elm varieties. The technical solution of the present invention has important scientific value and practical significance.
[0178] The specific embodiments of the present invention are only for illustrating the technical solutions of the present invention. Any modification, replacement and improvement made within the concept and technical scope of the present invention shall fall within the protection scope of the present invention.
Claims
1. An efficient mutation breeding method for ash, characterized in that: The method comprises the following steps: (1) Seed selection and treatment: Select seeds with uniform weight, fullness and no diseases from the ash family, rinse with distilled water, soak for 24 hours, and remove the empty seeds; (2) treating the seeds of step (1) by a single mutagenesis method or a superposition mutagenesis method: the single mutagenesis method is physical mutagenesis or chemical mutagenesis, and the superposition mutagenesis method is physical-chemical superposition mutagenesis or chemical-chemical superposition mutagenesis; (3) Field planting and trait investigation: Sow the mutagenized seeds at the field test site, and record and evaluate the germination rate and seedling traits; (4) Screening of superior plants and construction of database: Screening and asexual propagation of plants with superior phenotypes, and establishing a corresponding database of mutagenic traits.
2. The high-efficiency mutation breeding method of ash according to claim 1, characterized in that: The physical mutagenesis in step (2) is carried out by 60Co-γ ray, the radiation dose is 25Gy, 50Gy or 75Gy, and the radiation time is 3 hours.
3. The high-efficiency mutation breeding method of ash according to claim 1, characterized in that: The chemical mutagenesis in step (2) is carried out by using EMS, colchicine solution, sodium azide and 5-azacytidine, with the mutagenesis concentrations being 0.5%, 0.15%, 4mmol / L and 50μmol / L respectively, and the mutagenesis time being 24 hours.
4. The high-efficiency mutation breeding method of Manchurian ash according to claim 1, characterized in that: The physical-chemical superposition mutagenesis in step (2) is as follows: firstly, the seeds are subjected to 25Gy or 50Gy 60Co-γ ray physical mutagenesis for 3 hours, and then chemical mutagenesis for 48 hours.
5. The high-efficiency mutation breeding method of Manchurian ash according to claim 1, characterized in that: The chemical-chemical superposition mutagenesis in step (2) is as follows: first, the seeds are soaked in 0.5% EMS solution for 24 hours, then rinsed with distilled water, and then transferred to 4 mmol / L sodium azide solution or 50 μmol / L 5-azacytidine solution for further soaking for 24 hours.
6. The high-efficiency mutation breeding method of Manchurian ash according to claim 1, characterized in that: The field test site in step (3) is located in a typical elm growing area and has suitable temperature, humidity and soil conditions; the seedling stage traits include plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, leaflet area and other trait parameters.
7. The high-efficiency mutation breeding method of Manchurian ash according to claim 1, characterized in that: The excellent phenotype screening criteria in step (4) include, but are not limited to, higher germination rate, larger plant height, thicker ground diameter, wider compound leaves, and the like; the selected excellent plants are propagated by asexual propagation, and the propagation methods include, but are not limited to, cuttings, grafting, or tissue culture; The corresponding mutagenic trait database is established, and the key parameters stored in the database include: germination rate, plant height, ground diameter, compound leaf length, compound leaf width, compound leaf area, leaflet area, etc. Through standardized data management and evaluation system, a scientific basis is provided for the breeding of new elm varieties.
8. Application of the high-efficiency ash mutation breeding method according to any one of claims 1 to 7 in ash polyploid mutation breeding.
9. Use of the high-efficiency elm mutation breeding method according to any one of claims 1 to 7 in creating fast-growing, high-quality, stress-resistant new elm germplasm and improving the adaptability and stress resistance of elm.