Influence factor analysis method for compound mutagenesis treatment of sand-stored germinating seeds of acanthopanax based on orthogonal test and optimized mutagenesis treatment method

Abstract

The invention discloses an influence factor analysis method and an optimized mutagenesis treatment method for compound mutagenesis treatment of sand-stored germinating seeds of acanthopanax based on an orthogonal test. The invention aims to analyze the influence of each factor in compound mutagenesis on the emergence rate of the sand-stored germinating seeds of acanthopanax. The method for analyzing the influence factors of the sand-stored germinating seeds of the acanthopanax comprises the following steps of: 1, determining that gamma-ray and electron beam composite treatment has interaction; 2, taking interaction of 60Co-gamma ray mutagenesis, electron beam mutagenesis and 60Co-gamma ray and electron beam compound mutagenesis as total three factors, setting N levels according to the range of mutagen amount, and constructing an orthogonal table of the two factors having interaction and N levels; 3, carrying out an orthogonal experiment on sand-stored germinating seeds of the acanthopanax root through compound mutagenesis treatment; and 4, analyzing the difference between the F ratio and the F critical value to obtain the significant level of the influence of the two factors and interaction on the relative emergence rate of the sand-stored germinating seeds of the acanthopanax. According to the invention, different mutagenesis factors are found to have a synergistic effect, so that the mutation rate and the mutation spectrum of the acanthopanax senticosus are improved.

Description

Technical Field

[0001] The invention relates to an analysis method of influencing factors of gamma ray and electron beam (e) composite mutagenesis technology for germinating seeds of Acanthopanax senticosus and a composite mutagenesis method. Background Art

[0002] Radiation mutagenesis breeding refers to the use of ionizing radiation such as X-rays, gamma rays, beta particles, neutrons and heavy ions to induce genetic variation in organisms, select excellent mutants from them, and directly or indirectly breed new varieties. The high mutation rate and wide mutation spectrum of radiation mutagenesis technology have unique advantages in germplasm innovation, play an irreplaceable and important role in agricultural production, and have made great contributions to the sustainable development of modern agriculture.

[0003] Different ionizing rays have different physical properties, such as mass, energy, and charge, so the ionization ability, penetration ability, linear energy transfer density (LET), mutagenesis mechanism, and mutagenesis efficiency of different rays vary greatly. However, with the growing demand for improving the efficiency of radiation mutagenesis, the scientific research field continues to explore and discover more diverse physical mutagenic factors and innovative mutagenesis strategies. In this context, the method of composite application of different mutagenic sources has emerged and has become one of the effective ways to significantly increase the mutation frequency. This strategy not only enriches the diversity of mutagenesis methods, but also promotes the discovery of broader and more efficient genetic variation resources.

[0004] 60 Co-γ rays are 60 The photons released by the Co radionuclide during its decay process are massless, chargeless, have an average energy of 1.25 MeV, and have a very short wavelength (10 -8 ~10 -11 cm), has strong penetrating power, and is suitable for various materials such as seeds, branches, tissue culture materials and living plants, as well as the conventional outer packaging of the irradiated materials without affecting its radiation effect. Electron beam is a high-energy electron beam generated by an electron accelerator.

[0005] Acanthopanax senticosus Maxim, also known as "Siberian ginseng", "woody ginseng", "Acanthopanax ginseng", etc., is an edible and medicinal shrub of the Araliaceae family. Due to the after-ripening process of seeds during sexual reproduction of Acanthopanax, it takes 19 months for the seeds to mature and germinate naturally. In order to shorten the germination time, high and low temperature variable temperature stratification treatment is required for 6-7 months after harvest to promote the morphological and physiological after-ripening process of the embryo of Acanthopanax to complete. Therefore, artificially planted Acanthopanax seeds are generally seeds stored in low-temperature sand, and the seeds are generally germinated and white in the spring sowing season. Wet seeds and germinated seeds are more sensitive to radiation than dry seeds due to their different physiological states and water contents (the influence of free radicals produced after water is ionized, etc.), and have lower tolerance doses. When a composite mutagenesis method is used for mutagenesis treatment, due to the interaction between different mutagenic factors, it is necessary to determine the main effect factors and the effects of the interactions. However, the current research on 60 There are few specific studies on the combined use of Co-γ rays and electron beams as mutagenic sources. Therefore, various factors need to be fully considered in practical applications to ensure the effectiveness of the combined mutagenesis technology.

[0006] The important reference index of the appropriate dose of radiation mutagenesis is the median lethal dose (LD 50 The median lethal dose is related to the radiation sensitivity of the species, the physiological state of the induced material, and the mode of mutagenesis. The commonly used LD 50 Indicators include germination rate, survival rate, seedling height and fresh weight, etc. Summary of the invention

[0007] The purpose of the present invention is to analyze the influence of various factors in composite mutagenesis on the germination rate of Acanthopanax senticosus germination seeds, determine the half-lethal dose parameters of the composite mutagenesis treatment of Acanthopanax senticosus germination seeds with gamma rays and electron beams, and provide an analysis method of influencing factors of composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal experiments and an optimization mutagenesis treatment method and determine the optimal mutagenic dose (half-lethal dose).

[0008] The method for analyzing the influencing factors of the composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test of the present invention is implemented by the following steps:

[0009] First, the germinating seeds of Acanthopanax senticosus were treated with electron beam mutagenesis at a dose of 10 Gy, and then the germinating seeds of Acanthopanax senticosus were treated with doses of 10 Gy and 20 Gy respectively. 60 Co-γ-ray mutagenesis treatment, taking the relative germination rate as an indicator, the γ-ray dose-relative germination rate curve under the condition of 10Gy electron beam was drawn;

[0010] The germinating seeds of Acanthopanax senticosus were treated with electron beam mutagenesis at a dose of 20 Gy, and then the germinating seeds of Acanthopanax senticosus were treated with 10 Gy and 20 Gy, respectively.60 Co-γ-ray mutagenesis treatment, taking the relative germination rate as an indicator, the γ-ray dose-relative germination rate curve under the condition of 20Gy electron beam was drawn;

[0011] It was found that the γ-ray dose-relative germination rate curve under the condition of electron beam 10Gy and the γ-ray dose-relative germination rate curve under the condition of electron beam 20Gy had an intersection, indicating that there was an interaction (synergistic effect) between the combined treatment of γ-ray and electron beam.

[0012] 2. 60 Co-γ-ray mutagenesis, electron beam (e) mutagenesis, and 60 The interaction of Co-γ-ray and electron beam combined mutagenesis (γ×e) was used as the total three factors, and the 60 The range of Co-γ-ray mutagenic dose and electron beam (e) mutagenic dose, N levels are set according to the range of mutagenic dose, so as to construct an orthogonal table with two factors interacting and N levels;

[0013] Third, based on orthogonal design software, through the orthogonal table with two factors interacting and N level, the relative emergence rate of Acanthopanax senticosus germinating seeds was used as the experimental result, and the orthogonal experiment of compound mutagenesis treatment of Acanthopanax senticosus germinating seeds was carried out to calculate the mean and mean range at each level of the two factors and the interaction, as well as the F ratio (F-ratio) and F critical value of the two factors and the interaction;

[0014] 4. The importance (order) of each factor in affecting the relative germination rate of Acanthopanax senticosus seeds was determined by the size of the mean and range values, and the significance level of the influence of the two factors and their interactions on the relative germination rate of Acanthopanax senticosus seeds was obtained by comparative analysis of the F ratio and the F critical value.

[0015] The optimized composite mutagenesis method for treating germinating seeds of Acanthopanax senticosus is achieved by the following steps:

[0016] 1. Adoption 60 The germination seeds of Acanthopanax senticosus were irradiated with Co-γ rays to induce mutagenesis. 60 The mutagenic dose of Co-γ ray was 20Gy, and the mutagenic Acanthopanax senticosus seeds were obtained;

[0017] 2. The mutagenized Acanthopanax senticosus seeds were subjected to a composite mutagenesis treatment using an electron beam, and the electron beam mutagenesis dose was controlled to be 10 Gy, thereby obtaining the composite mutagenized Acanthopanax senticosus seeds.

[0018] The electrons used in the electron beam radiation of the present invention are negative electrons with a charge of 1.6×10 -19 C, has mass, and its rest mass is about 9.1×10 -31kg, the acceleration energy is 1.2MeV, the penetration depth in water is 5mm, and the penetration depth in other objects varies due to their density. Using gamma rays and electron beams to treat crops can make up for the shortcomings of a single radiation source in terms of quantity, quality, penetration ability, and energy distribution in the irradiated material, thereby improving the comprehensiveness and effectiveness of mutagenesis. Due to the synergistic effect between different mutagenic factors, the mutation frequency and mutation spectrum of compound mutagenesis are significantly improved compared with single-factor mutagenesis.

[0019] Traditional single mutagenesis techniques (such as using gamma rays or electron beams alone) often have problems such as low mutagenesis efficiency and narrow mutation spectrum during the mutagenesis process. The present invention achieves complementary advantages and reduces the total mutagenic dose by combining two mutagenic sources with different physical properties, gamma rays and electron beams. The difficulties in the composite mutagenesis technology are to determine the presence or absence of synergistic effects, the contribution of each mutagenic factor in the composite mutagenesis treatment, and the composition of specific mutagenic parameters. The present invention provides a method for determining the composite mutagenesis parameters of Acanthopanax senticosus germinating seeds under a composite irradiation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a relative germination rate test diagram of Acanthopanax senticosus seeds subjected to single factor mutagenesis treatment with gamma rays and electron beams in the embodiment;

[0021] Figure 2 In the embodiment 60 Test diagram of the synergistic (interactive) effect of Co-γ ray and electron beam combined mutagenesis treatment. DETAILED DESCRIPTION

[0022] Specific implementation method 1: The method for analyzing the emergence rate of Acanthopanax senticosus seeds germinating in the composite mutagenesis treatment based on orthogonal test in this implementation method is implemented according to the following steps:

[0023] First, the germinating seeds of Acanthopanax senticosus were treated with electron beam mutagenesis at a dose of 10 Gy, and then the germinating seeds of Acanthopanax senticosus were treated with doses of 10 Gy and 20 Gy respectively. 60 Co-γ-ray mutagenesis treatment, taking the relative germination rate as an indicator, the γ-ray dose-relative germination rate curve under the condition of 10Gy electron beam was drawn;

[0024] The germinating seeds of Acanthopanax senticosus were treated with electron beam mutagenesis at a dose of 20 Gy, and then the germinating seeds of Acanthopanax senticosus were treated with 10 Gy and 20 Gy, respectively. 60 Co-γ-ray mutagenesis treatment, taking the relative germination rate as an indicator, the γ-ray dose-relative germination rate curve under the condition of 20Gy electron beam was drawn;

[0025] It was found that the γ-ray dose-relative germination rate curve under the condition of electron beam 10Gy and the γ-ray dose-relative germination rate curve under the condition of electron beam 20Gy had an intersection, indicating that there was an interaction (synergistic effect) between the combined treatment of γ-ray and electron beam.

[0026] 2. 60 Co-γ-ray mutagenesis, electron beam (e) mutagenesis, and 60 The interaction of Co-γ-ray and electron beam combined mutagenesis (γ×e) was used as the total three factors, and the 60 The range of Co-γ-ray mutagenic dose and electron beam (e) mutagenic dose, N levels are set according to the range of mutagenic dose, so as to construct an orthogonal table with two factors interacting and N levels;

[0027] Third, based on orthogonal design software, through the orthogonal table with two factors interacting and N level, the relative emergence rate of Acanthopanax senticosus germinating seeds was used as the experimental result, and the orthogonal experiment of compound mutagenesis treatment of Acanthopanax senticosus germinating seeds was carried out to calculate the mean and mean range at each level of the two factors and the interaction, as well as the F ratio (F-ratio) and F critical value of the two factors and the interaction;

[0028] 4. The importance (order) of each factor in affecting the relative germination rate of Acanthopanax senticosus seeds was determined by the size of the mean and range values, and the significance level of the influence of the two factors and their interactions on the relative germination rate of Acanthopanax senticosus seeds was obtained by comparative analysis of the F ratio and the F critical value.

[0029] In the composite mutagenesis process of this embodiment, the γ-ray mutagenesis dose rate is controlled to be 20 Gy / h; the electron beam mutagenesis dose rate is controlled to be 2.5 Gy / s. Based on the actual growth of Acanthopanax senticosus seedlings, the indicator used in this embodiment is the relative emergence rate.

[0030] The relative germination rate in this embodiment is the ratio of the germination rate of the germination seeds of Acanthopanax senticosus treated with the compound mutagenesis to the germination rate of the blank test.

[0031] Specific implementation method 2: This implementation method is different from the specific implementation method 1 in that the Acanthopanax senticosus seeds described in step 1 are pre-treated with stratification and low temperature. The stratification and low temperature treatment process is as follows:

[0032] a. Mixing Acanthopanax senticosus seeds with sand to obtain a seed-sand mixture, placing the seed-sand mixture in a room at 18-20° C., maintaining humidity at more than 60%, and performing stratification treatment on the seed-sand mixture;

[0033] b. After stratification, the temperature of the seed-sand mixture drops to 15-18°C and the treatment is continued for 25-35 days. Then the seed-sand mixture is moved to a 0-5°C environment for low-temperature storage.

[0034] Specific implementation method three: This implementation method is different from specific implementation method two in that the mass ratio of Acanthopanax senticosus seeds to sand in step a is 1:3.

[0035] Specific implementation method 4: This implementation method is different from specific implementation method 2 in that the stacking process in step a is turned over 1-2 times a day.

[0036] Specific implementation method five: This implementation method is different from specific implementation method two in that the lamination treatment time in step a is 40-45 days.

[0037] Specific implementation method 6: This implementation method is different from specific implementation methods 1 or 2 in that step 2 is set 60 The Co-γ-ray mutagenesis dose ranged from 0 to 40 Gy, and the electron beam (e) mutagenesis dose ranged from 0 to 40 Gy.

[0038] Specific implementation method 7: This implementation method is different from specific implementation methods 1 or 2 in that step 2 60 The Co-γ ray mutagenic dose and the electron beam (e) mutagenic dose were both set to 5 levels, namely, when the mutagenic dose was 0 Gy, the factor level was set to 1; when the mutagenic dose was 10 Gy, the factor level was set to 2; when the mutagenic dose was 20 Gy, the factor level was set to 3; when the mutagenic dose was 30 Gy, the factor level was set to 4; when the mutagenic dose was 40 Gy, the factor level was set to 5.

[0039] Specific implementation eight: This implementation is different from specific implementation one or two in that the orthogonal design software described in step three is an orthogonal design assistant.

[0040] Specific implementation method 9: The difference between this implementation method and specific implementation method 1 or 2 is that in step 3, through orthogonal experiment, when the relative emergence rate is 0.49-0.51, it is determined as the half-lethal dose of the compound mutagenesis of Acanthopanax senticosus germination seeds.

[0041] Embodiment 1: The method for analyzing the emergence rate of Acanthopanax senticosus seeds germinating in this embodiment based on the composite mutagenesis treatment of orthogonal test is implemented according to the following steps:

[0042] 1. Mutagenesis process

[0043] The seeds to be mutagenized were the germinated Acanthopanax senticosus seeds with the same genetic background conditions stored in sand, and the γ-ray mutagenization dose rate was 20 Gy / h; the electron beam mutagenization dose rate was 2.5 Gy / s, and during the electron beam mutagenization, the Acanthopanax senticosus seeds were spread in a single layer in an aluminum sample box;

[0044] 2. Detection of radiation sensitivity of single factor mutagenesis of Acanthopanax senticosus seeds

[0045] Using conventional half-lethal dose screening method, gradient design60 In the single-factor mutagenesis experiment with two mutagenic sources, Co-γ ray and electron beam, the relative germination rate was used as an indicator to determine the half-lethal dose; the gradient design doses of the two mutagenic sources were: 0Gy, 10Gy, 20Gy, 30Gy, 40Gy and 50Gy. The relative germination rate curves obtained under different mutagenic doses are shown in Figure 2. Figure 1 As shown;

[0046] Acanthopanax senticosus seed pairs 60 The single-factor mutagenesis responses of Co-γ-ray and electron beam mutagenesis sources are relatively consistent. Figure 1 The median lethal dose of γ-ray mutagenesis calculated from the relative emergence rate was 32.1 Gy, and the median lethal dose of electron beam mutagenesis was 34.7 Gy.

[0047] 3. Experiment on the synergistic (interactive) effect of compound mutagenesis of Acanthopanax senticosus seeds

[0048] Take separately 60 The synergistic effect of the Co-γ-ray dose and the electron beam mutagenesis dose was tested with the relative germination rate as an indicator. The experimental results showed that the synergistic effect of the electron beam was 10Gy and 20Gy. 60 The germination rate curves of Co-γ-ray induced mutagenesis at 10Gy and 20Gy irradiation were compared with those at 20Gy electron beam 60 The germination rate curves of 10Gy and 20Gy irradiation induced by Co-γ-ray cross ( Figure 2 ), indicating the above 60 The combined treatment of Co-γ-ray mutagenesis and electron beam (e) mutagenesis has a synergistic effect;

[0049] 4. Design and analysis of compound mutagenesis half-lethal dose experiment based on orthogonal experimental method

[0050] Based on the radiation sensitivity test results of single-factor mutagenesis of Acanthopanax senticosus seeds and the synergistic effect of compound mutagenesis, an orthogonal table with 2 factors and 5 levels was designed and constructed (factor levels are shown in Table 1). The experimental results obtained by using the orthogonal table for orthogonal experiments are listed in Table 2.

[0051] Table 1 Factor levels of the half-lethal dose test of compound mutagenesis of Acanthopanax senticosus seeds

[0052]

[0053] Table 2 Orthogonal test results

[0054]

[0055]

[0056] Example: Taking Experiment 13 in Table 2 as an example, the experimental process of Experiment 13 is to firstly treat the germinating seeds of Acanthopanax senticosus with a dose of 20 Gy. 60 The germinating seeds of Acanthopanax senticosus were treated with Co-γ rays and then subjected to electron beam mutagenesis at a dose of 20 Gy. The relative germination rate of the germinating seeds of Acanthopanax senticosus was 0.54.

[0057] The research target is the half-lethal dose of combined mutagenesis, that is, the treatment combination with a germination rate of 50%. Since γ-ray and electron beam mutagenesis have an interaction, the experimental results of the orthogonal test are converted into an interaction table (Table 3), and the combination with the relative germination rate closest to the 0.5 level is found, which is the optimal combination. The germination rate of γ3e2 is 0.51, that is, the combined treatment of γ-ray 20Gy + electron beam 10Gy is the optimal combination.

[0058] Table 3 Interaction table

[0059]

[0060]

[0061] Table 4 Orthogonal test variance analysis table

[0062]

[0063] The main influencing factors were determined by the size of the range value, which determined the order of the influence of each factor on the experimental results from large to small; the significance of the influence of each factor on the relative emergence rate was further determined by variance analysis, that is, the F ratio was greater than the F critical value (α=0.01) for a very significant effect, and the F ratio was greater than the F critical value (α=0.05) and less than the F critical value (α=0.01) for a significant effect.

[0064] The analysis of factors γ, e and γ×e in this embodiment is as follows:

[0065] (1) Factor: γ (first data set)

[0066] The sum of squared deviations was 1.791, indicating the contribution of gamma mutagenesis alone to the variability of the response variable.

[0067] F-ratio: 22.202, which is the ratio of the mean square of the γ effect to the mean square of the error, is used to test whether the effect of the γ single factor mutagenesis is significant.

[0068] F critical value: Thresholds of α = 0.05 and α = 0.01 indicate statistical significance of gamma mutagenesis.

[0069] Critical values: 3.260 (α = 0.05 level) and 5.410 (α = 0.01 level), F ratios exceeding these values ​​indicate that the interaction is significant.

[0070] Significance: ** indicates that the interaction is statistically significant.

[0071] (2) Factor: e (second data set)

[0072] The sum of squared deviations was 1.471, indicating the contribution of the e mutagenesis alone to the variability of the response variable.

[0073] F-ratio: 18.236, used to test whether the effect of factor e is significant.

[0074] F critical value: The threshold values ​​of α=0.05 and α=0.01 indicate that the effect of factor e is statistically significant.

[0075] Critical values: Same as above, F ratios exceeding these values ​​indicate that the effect of factor e is significant.

[0076] Significance: Marked as **, indicating that the effect of factor e is statistically significant.

[0077] (3) Factor: γ×e (third data set)

[0078] The sum of squared deviations: 0.338, indicating that the interaction between γ and e contributes little to the variability of the response variable.

[0079] F-ratio: 4.19, indicating that the interaction between γ and e may not be statistically significant.

[0080] F critical value: significant at α=0.05 level, but not significant at α=0.01 level.

[0081] Critical values: Same as above, F ratios exceeding these values ​​indicate significant mutagenicity.

[0082] Significance: * indicates that the interaction is significant at the α=0.05 level.

[0083] Summary: Factor γ and factor e have significant effects on the response variable. The interaction γ×e is significant at the α=0.05 level, but not at the α=0.01 level, indicating that its effect is relatively small.

[0084] Embodiment 2: The method of treating the germination seeds of Acanthopanax senticosus by combined mutagenesis of gamma ray and electron beam in this embodiment is as follows:

[0085] 1. Adoption 60 The germinating seeds of Acanthopanax senticosus were irradiated with Co-γ rays to induce mutation, and the mutated Acanthopanax senticosus seeds were obtained.

[0086] 2. Performing a composite mutagenesis treatment on the mutagenized Acanthopanax senticosus seeds by using an electron beam to obtain composite mutagenized Acanthopanax senticosus sand-stored germination seeds;

[0087] In step one 60 The Co-γ-ray mutagenesis dose is 20 Gy, and the electron beam mutagenesis dose in step 2 is 10 Gy.

[0088] The Acanthopanax senticosus seeds described in Example 1 and Example 2 are all pre-treated with stratification and low temperature treatment, and the stratification and low temperature treatment process is as follows:

[0089] a. Mixing Acanthopanax senticosus seeds with sand to obtain a seed-sand mixture, placing the seed-sand mixture in a room at 18° C., maintaining humidity above 60%, stratifying the seed-sand mixture for 45 days, and turning it twice a day;

[0090] b. The temperature of the seed-sand mixture after stratification treatment drops to 16°C and the treatment is continued for 30 days. The seed-sand mixture is then moved to an environment of 0-5°C for low-temperature storage for 45 days.

[0091] In summary, 60 The combined mutagenesis of Co-γ ray and electron beam on germinating seeds of Acanthopanax senticosus could reduce the total mutagenic dose and increase the mutation rate and mutation spectrum of Acanthopanax senticosus.

Claims

1. A method for analyzing factors affecting the germination of Acanthopanax senticosus seeds by composite mutagenesis based on orthogonal experiments, characterized in that The method for analyzing the influencing factors of the composite mutagenesis treatment of Acanthopanax senticosus germination seeds is implemented by the following steps: First, the germinating seeds of Acanthopanax senticosus were treated with electron beam mutagenesis at a dose of 10 Gy, and then the germinating seeds of Acanthopanax senticosus were treated with doses of 10 Gy and 20 Gy respectively. 60 Co-γ-ray mutagenesis treatment, taking the relative germination rate as an indicator, the γ-ray dose-relative germination rate curve under the condition of 10Gy electron beam was drawn; The germinating seeds of Acanthopanax senticosus were treated with electron beam mutagenesis at a dose of 20 Gy, and then the germinating seeds of Acanthopanax senticosus were treated with 10 Gy and 20 Gy, respectively. 60 Co-γ-ray mutagenesis treatment, taking the relative germination rate as an indicator, the γ-ray dose-relative germination rate curve under the condition of 20Gy electron beam was drawn; It was found that the γ-ray dose-relative germination rate curve under the condition of 10Gy electron beam and the γ-ray dose-relative germination rate curve under the condition of 20Gy electron beam had an intersection, indicating that there was an interaction between the combined treatment of γ-ray and electron beam.

2. 60 Co-γ-ray mutagenesis, electron beam mutagenesis, and 60 The interaction of Co-γ-ray and electron beam combined mutagenesis was taken as the total three factors, and the 60 The range of Co-γ-ray mutagenic dose and electron beam mutagenic dose is set according to the range of mutagenic dose, so as to construct an orthogonal table with two factors interacting and N levels; Third, based on orthogonal design software, through the orthogonal table with two factors interacting and N level, the relative emergence rate of Acanthopanax senticosus germinating seeds was used as the experimental result, and the orthogonal experiment of compound mutagenesis treatment of Acanthopanax senticosus germinating seeds was carried out to calculate the mean and mean range at each level of the two factors and the interaction, as well as the F ratio and F critical value of the two factors and the interaction; 4. The importance and priority of each factor in affecting the relative germination rate of Acanthopanax senticosus seeds were determined by the size of the mean and range values. The significance level of the influence of the two factors and their interactions on the relative germination rate of Acanthopanax senticosus seeds was obtained by comparative analysis of the F ratio and the F critical value.

2. The method for analyzing influencing factors of composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 1, characterized in that The Acanthopanax senticosus seeds described in step 1 are pre-treated with stratification and low temperature treatment, and the stratification and low temperature treatment process is as follows: a. Mixing Acanthopanax senticosus seeds with sand to obtain a seed-sand mixture, placing the seed-sand mixture in a room at 18-20° C., maintaining humidity at more than 60%, and performing stratification treatment on the seed-sand mixture; b. After stratification, the temperature of the seed-sand mixture drops to 15-18°C and the treatment is continued for 25-35 days. Then the seed-sand mixture is moved to a 0-5°C environment for low-temperature storage.

3. The method for analyzing the influencing factors of the composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 2, characterized in that The mass ratio of Acanthopanax senticosus seeds to sand in step a is 1:

3.

4. The method for analyzing the influencing factors of the composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 2, characterized in that During the stratification process in step a, the soil is turned over 1-2 times a day.

5. The method for analyzing the influencing factors of the composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 2 is characterized in that The stratification treatment time in step a is 40-45 days.

6. The method for analyzing influencing factors of composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 1, characterized in that In step 2, set 60 The Co-γ-ray mutagenesis dose ranged from 0 to 40 Gy, and the electron beam mutagenesis dose ranged from 0 to 40 Gy.

7. The method for analyzing influencing factors of composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 1, characterized in that Step 2 60 The Co-γ-ray mutagenic dose and the electron beam mutagenic dose were both set to 5 levels, namely, when the mutagenic dose was 0 Gy, the factor level was set to 1; when the mutagenic dose was 10 Gy, the factor level was set to 2; when the mutagenic dose was 20 Gy, the factor level was set to 3; when the mutagenic dose was 30 Gy, the factor level was set to 4; when the mutagenic dose was 40 Gy, the factor level was set to 5.

8. The method for analyzing influencing factors of composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 1, characterized in that The orthogonal design software described in step three is the orthogonal design assistant.

9. The method for analyzing influencing factors of composite mutagenesis treatment of Acanthopanax senticosus germination seeds based on orthogonal test according to claim 1, characterized in that In step three, through orthogonal experiments, when the relative germination rate is 0.49-0.51, it is determined to be the half-lethal dose of the compound mutagenesis of Acanthopanax senticosus germination seeds.

10. The optimized method for treating germination seeds of Acanthopanax senticosus by compound mutagenesis is characterized in that The method for treating the germinating seeds of Acanthopanax senticosus by compound mutagenesis is achieved by the following steps:

1. Adoption 60 The germination seeds of Acanthopanax senticosus were irradiated with Co-γ rays to induce mutagenesis. 60 The mutagenic dose of Co-γ ray was 20Gy, and the mutagenic Acanthopanax senticosus seeds were obtained; 2. The mutagenized Acanthopanax senticosus seeds were subjected to a composite mutagenesis treatment using an electron beam, and the electron beam mutagenesis dose was controlled to be 10 Gy, thereby obtaining the composite mutagenized Acanthopanax senticosus seeds.

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