Astragalus membranaceus breeding method combining space mutation with phenotypic screening

Abstract

The invention relates to the field of astragalus membranaceus breeding, and particularly discloses an astragalus membranaceus breeding method combining space mutagenesis with phenotype screening, which comprises the following steps: S1, seed pretreatment: space mutagenesis: carrying original seeds on a return satellite, exposing the original seeds on an orbit 200-400km away from the ground for 48-72 hours, and receiving a space radiation dose of 2-5Gy; disinfection treatment: soaking the mutagenized seeds in a 3-5% sodium hypochlorite solution for 15-20 minutes, and then washing the seeds with sterile water for 3 times; through the synergistic effect of space mutation and phenotype screening, the limitation that traditional mutation breeding is high in randomness is broken through, the directional screening efficiency of target characters is remarkably improved, and meanwhile stable transmission of excellent hereditary characters is guaranteed; through combination of multi-dimensional phenotypic indexes of root development, biomass accumulation and stress resistance, superior plants with high medicinal component enrichment capacity and wide adaptability are accurately locked, and the problem of comprehensive performance imbalance caused by single character screening is solved.

Description

Technical Field

[0001] The present invention belongs to the field of Astragalus breeding, and specifically relates to a breeding method of Astragalus by space mutation combined with phenotypic screening. Background Art

[0002] As a traditional Chinese medicinal material, the medicinal value of Astragalus membranaceus mainly depends on the active ingredients (such as astragaloside IV, polysaccharides, etc.) accumulated in the roots. At present, the variety selection of Astragalus mostly relies on natural variation or conventional mutagenesis (such as chemical mutagen EMS, γ-ray radiation), and target plants are screened through phenotypic observation.

[0003] Most existing Astragalus breeding studies only focus on a single trait (such as root diameter or biomass), ignoring the coordinated optimization of root system architecture, stress resistance ability and medicinal ingredient synthesis, which is likely to cause the phenomenon of "high yield and low quality". Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a breeding method of Astragalus by space mutation combined with phenotypic screening, so as to solve the problem that most existing Astragalus breeding studies only focus on a single trait (such as root diameter or biomass), ignoring the coordinated optimization of root system architecture, stress resistance ability and medicinal ingredient synthesis, which is likely to cause the phenomenon of "high yield and low quality".

[0005] A breeding method of Astragalus by space mutation combined with phenotypic screening includes the following steps:

[0006] S1. Seed pretreatment:

[0007] Space mutation: The original seeds are carried on a recoverable satellite and exposed in an orbit 200 - 400 km above the ground for 48 - 72 hours, receiving a space radiation dose of 2 - 5 Gy;

[0008] Disinfection treatment: The mutated seeds are soaked in a 3 - 5% sodium hypochlorite solution for 15 - 20 minutes, and then rinsed 3 times with sterile water;

[0009] Germination acceleration treatment: The disinfected seeds are soaked in warm water at 40 ± 2 °C for 6 hours, drained and transferred to a petri dish lined with moist filter paper, and germinated in the dark in a constant temperature incubator at 25 ± 1 °C for 48 hours until the radicle length reaches 1 - 2 mm;

[0010] S2. Standardized planting:

[0011] Sowing conditions: The germinated seeds are sown in the experimental field at a row spacing of 30 cm and a plant spacing of 15 cm, with a soil covering depth of 2 - 3 cm;

[0012] Environmental control: An intelligent greenhouse is used to regulate the day and night temperature to 25 ± 1 °C / 18 ± 1 °C, and the light cycle is 14 hours per day;

[0013] Water and fertilizer management: Irrigate 2 times a week, and keep the soil water content at 70 - 75% of the field capacity;

[0014] S3. Determination of phenotypic indicators:

[0015] Determination of root development: The main root length ≥ 30 cm, the lateral root density ≥ 15 roots / 10 cm, and the root diameter ≥ 0.5 cm;

[0016] Determination of aboveground biomass: The fresh weight per plant ≥ 80 g, and the stem diameter ≥ 4 mm;

[0017] Evaluation of stress resistance: The relative water content of leaves ≥ 70% under drought stress, and the survival rate ≥ 60% under salt stress (treated with 150 mM NaCl);

[0018] S4. Screening of superior plants: Establish a weighted scoring model for root indexes, biomass, and stress resistance, and screen individuals with a total score ≥ 90 points;

[0019] S5. Asexual propagation: Select stem segments of superior plants for in vitro culture;

[0020] S6. Verification of genetic stability: Verify the stability of target traits through continuous cultivation for three generations.

[0021] Preferably, in step S1, the orbital altitude of the space mutagenesis is 300 - 350 km, the exposure time is 60 hours, and the space radiation dose is 3.5 Gy.

[0022] Preferably, in step S2, the water and fertilizer management includes applying 5 g / m of compound fertilizer every two weeks 2 , and the N:P:K ratio of the compound fertilizer is 15:15:15.

[0023] Preferably, in step S3, the time for determination of root development is the 90th day after sowing, and the determination is completed using a root scanner.

[0024] Preferably, the resolution of the root scanner is not less than 4800 dpi.

[0025] Preferably, in step S3, the salt stress treatment lasts for 14 days, and the daily light exposure is 12 hours.

[0026] Preferably, in step S4, the weight distribution of the scoring model is: root index 50%, biomass 30%, and stress resistance 20%.

[0027] Preferably, in step S5, the culture medium for in vitro culture is MS basal medium supplemented with 0.5 mg / L NAA, 1.0 mg / L 6 - BA, 30 g / L sucrose, and 7 g / L agar.

[0028] Preferably, in step S6, the requirements for genetic stability verification are that the coefficient of variation of the target trait among three generations is ≤5%, and the excellent plant rate is ≥95%.

[0029] Preferably, a control group is set during stability verification, and the content of astragaloside IV in the excellent plant group is increased by ≥15% compared with the control group.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Through the synergistic effect of space mutation and phenotypic screening, the limitation of strong randomness in traditional mutation breeding is broken through, the directional screening efficiency of target traits is significantly improved, and the stable transmission of excellent genetic characteristics is ensured at the same time;

[0032] By using multi-dimensional phenotypic indicators of root development, biomass accumulation and stress resistance, excellent plants with strong enrichment ability of medicinal components and wide adaptability are accurately locked, overcoming the problem of imbalance in comprehensive performance caused by single-trait screening;

[0033] Combined with asexual reproduction and multi-generation stability verification, the genetic consistency of mutagenized excellent plants is effectively maintained, and the impact of trait segregation on industrial promotion is avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the breeding method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all 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.

[0036] As Figure 1 shown:

[0037] Example 1

[0038] Step 1: Seed pretreatment

[0039] Space mutation: Carried by a recoverable satellite and exposed at a 200 km orbit for 48 hours, receiving a radiation dose of 2 Gy

[0040] Disinfection treatment: Soak in 3% sodium hypochlorite solution for 15 minutes and rinse 3 times with sterile water

[0041] Germination promotion treatment: Soak the seeds in warm water at 38°C for 6 hours and germinate in the dark at 25°C for 48 hours until the radicle length is 1 mm

[0042] Step 2: Standardized planting

[0043] Row spacing: 30 cm, plant spacing: 15 cm, soil covering depth: 2 cm

[0044] Day and night temperature: 24°C / 17°C, light intensity: 12000 lux, soil water content: 70%

[0045] Compound fertilizer application: 4.8 g / m every two weeks 2 (N:P:K = 15:15:15)

[0046] Step 3: Phenotype determination

[0047] Root development (90th day): Main root length: 30.2 cm, lateral root density: 15 roots / 10 cm, root diameter: 0.51 cm

[0048] Biomass (early flowering stage): Fresh weight per plant: 80.5 g, stem diameter: 4.1 mm

[0049] Stress resistance: Leaf water content after 7 days of drought treatment: 70.3%, survival rate after 150 mM NaCl treatment: 60.2%

[0050] Step 4: Superior plant selection

[0051] Calculate the score: 90.1 points according to the weight (root system 50% / biomass 30% / stress resistance 20%)

[0052] Superior plant rate: 95.0%

[0053] Step 5: Asexual propagation

[0054] Inoculate stem segments on the MS + 0.5 mg / L NAA + 1.0 mg / L 6 - BA medium, cluster bud differentiation rate: 81%

[0055] Step 6: Genetic verification

[0056] Coefficient of variation of main root length after three generations of cultivation: 4.8%, superior plant rate: 95.2%

[0057] Content of astragaloside IV increased by 15.3% compared with the control group

[0058] Example 2

[0059] Step 1: Seed pretreatment

[0060] Space mutation: Exposed at 300 km orbit for 60 hours, received a radiation dose of 3.5 Gy

[0061] Disinfection treatment: Immerse in 4% sodium hypochlorite solution for 18 minutes, rinse with sterile water 3 times

[0062] Germination acceleration treatment: Soak seeds in 40°C warm water for 6 hours, germinate in the dark at 25°C for 48 hours until the radicle length reaches 1.5 mm

[0063] Step 2: Standardized planting

[0064] Row spacing: 30 cm, plant spacing: 15 cm, soil covering depth: 2.5 cm

[0065] Day and night temperature: 25°C / 18°C, light intensity: 13500 lux, soil water content: 73%

[0066] Compound fertilizer application: 5.0 g / m every two weeks 2 (N:P:K = 15:15:15)

[0067] Step 3: Phenotype determination

[0068] Root system development (90th day): Main root length: 34.8 cm, lateral root density: 18 roots / 10 cm, root diameter: 0.63 cm

[0069] Biomass (early flowering stage): Fresh weight per plant: 92.7 g, stem diameter: 4.8 mm

[0070] Stress resistance: Leaf water content after 7 days of drought treatment: 75.6%, survival rate after 150 mM NaCl treatment: 68.4%

[0071] Step 4: Selection of superior plants

[0072] Score calculated by weight: 94.5 points

[0073] Rate of superior plants: 97.8%

[0074] Step 5: Asexual propagation

[0075] Induction rate of stem segments inoculated on the medium: 89%, multiplication coefficient of cluster buds: 3.2

[0076] Step 6: Genetic verification

[0077] Coefficient of variation of the main root length of three generations: 3.1%, rate of superior plants: 98.5%

[0078] Content of astragaloside IV increased by 18.9% compared with the control group

[0079] Example 3

[0080] Step 1: Seed pretreatment

[0081] Spaceflight mutagenesis: Exposed at 400 km orbit for 72 hours, received a radiation dose of 5 Gy

[0082] Disinfection treatment: Soaked in 5% sodium hypochlorite solution for 20 minutes, rinsed 3 times with sterile water

[0083] Germination acceleration treatment: Soaked seeds in 42°C warm water for 6 hours, germinated in the dark at 25°C for 48 hours until the radicle length reached 2 mm

[0084] Step 2: Standardized planting

[0085] Row spacing: 30 cm, plant spacing: 15 cm, soil covering depth: 3 cm

[0086] Day and night temperature: 26 °C / 19 °C, light intensity: 15000 lux, soil water content: 75%

[0087] Compound fertilizer application: 5.2 g / m every two weeks 2 (N:P:K = 15:15:15)

[0088] Step 3: Phenotype determination

[0089] Root system development (on the 90th day): Main root length: 39.1 cm, lateral root density: 22 roots / 10 cm, root diameter: 0.78 cm

[0090] Biomass (at the early flowering stage): Fresh weight per plant: 105.3 g, stem diameter: 5.5 mm

[0091] Stress resistance: Leaf water content: 82.4% after 7 days of drought treatment, survival rate: 73.8% after 150 mM NaCl treatment

[0092] Step 4: Selection of superior plants

[0093] Score calculated according to weight: 98.9 points

[0094] Rate of superior plants: 99.1%

[0095] Step 5: Asexual propagation

[0096] Induction rate of stem segments inoculated on the medium: 93%, multiplication coefficient of cluster buds: 4.5

[0097] Step 6: Genetic verification

[0098] Coefficient of variation of the main root length of three generations: 2.3%, rate of superior plants: 99.6%

[0099] The content of astragaloside IV is increased by 22.7% compared with the control group.

[0100] Experimental example 1:

[0101] I. Experimental scheme

[0102] 1. Experimental design

[0103] Reference group: Example 2 (space mutation + sodium hypochlorite disinfection + comprehensive scoring model)

[0104] Control group 1: Cancel space mutation and use γ-ray radiation (conventional mutation) instead

[0105] Control group 2: Change the concentration and time of the disinfectant (sodium hypochlorite concentration: 2%, soak for 10 minutes)

[0106] Comparative Example 3: Adjust the weight of the scoring model (root system 40%, biomass 40%, stress resistance 20%)

[0107] 2. Experimental Condition Control

[0108] The non-variable parameters such as planting environment, water and fertilizer management, and measurement time are exactly the same as those in Example 2. Each group of experiments was repeated 3 times and the average value was taken.

[0109] 2. Experimental table

[0110]

[0111] 3. Experimental Results Analysis

[0112] 1. Comparative Example 1 (γ-ray mutagenesis)

[0113] Low mutagenesis efficiency: Under γ-ray (150 Gy) treatment, the main root length (28.3 cm) and fresh weight (78.2 g) were significantly lower than those in Example 2 (34.8 cm, 92.7 g), indicating that space mutagenesis has a better effect on promoting the root development of Astragalus.

[0114] Poor genetic stability: the coefficient of variation of three generations is 8.7%, far exceeding the 3.1% of Example 2, indicating that the mutant traits produced by space radiation mutagenesis are more stable.

[0115] Conclusion: The genetic variation induced by space mutagenesis through the composite space environment (microgravity + radiation) is more suitable for the morphological construction of Astragalus.

[0116] 2. Comparative Example 2 (low concentration disinfection)

[0117] Incomplete disinfection: 2% sodium hypochlorite treatment resulted in an increase in the seed infection rate, and the salt stress survival rate (61.3%) was lower than that of Example 2 (68.4%), indicating that insufficient disinfection intensity affected the plant's stress resistance.

[0118] The superior strain rate decreased: the superior strain rate of 89.6% was 8.2% lower than that of Example 2 (97.8%), which proved that immersion in 4% sodium hypochlorite for 18 minutes was the balance point between disinfection effect and safety.

[0119] 3. Comparative Example 3 (Weight Adjustment)

[0120] Screening deviation: After increasing the biomass weight to 40%, the increase in astragaloside IV of the superior strain (16.8%) was lower than that of Example 2 (18.9%), indicating that the original weight model (root system 50%) was more conducive to the accumulation of medicinal ingredients.

[0121] Stability impact: The coefficient of variation of 4.3% still meets the requirements, but is better than comparison example 1-2, indicating that the weight adjustment did not destroy the genetic stability, but the comprehensive screening effect decreased.

[0122] IV. Core Conclusions

[0123] Irreplaceability of Space Mutation: Compared with γ-rays, space mutation shows significant advantages in promoting root development (+22.9%) and stability (coefficient of variation -64.4%).

[0124] Strictness of Disinfection Parameters: Deviations in sodium hypochlorite concentration and time lead to a decrease in stress resistance and excellent strain rate, and parameters need to be strictly followed.

[0125] Scientific Nature of Scoring Model: The original weight distribution (root system 50%) is more in line with the core indicators of the medicinal value of Astragalus membranaceus (root development and secondary metabolite accumulation).

[0126] Experimental Example 2

[0127] I. Experimental Design

[0128] 1. Experimental Group and Control Group

[0129] Experimental Group: Example 2 (space mutation + multi-dimensional phenotype screening + stability verification)

[0130] Control Group A: Traditional γ-ray mutation (150 Gy) + single trait screening (only root length ≥ 30 cm)

[0131] Control Group B: Space mutation (same as the experimental group) but without systematic screening (only randomly selected plants for reproduction)

[0132] 2. Control of Experimental Variables

[0133] Seed Source: Seeds of the same batch of the original species of Astragalus membranaceus. Cultivation Environment: An intelligent greenhouse is uniformly used (temperature, light, and humidity parameters are the same as those in Example 2).

[0134] Detection Indicators: Main root length, fresh weight per plant, salt stress survival rate, excellent strain rate, and content of astragaloside IV

[0135] II. Experimental Results

[0136]

[0137] III. Creativity Analysis

[0138] 1. Verification of Technical Synergy Effect

[0139] Control Group B (only space mutation): Although the main root length (33.7 cm) is close to that of the experimental group, the fresh weight per plant (85.2 g) and salt stress survival rate (53.6%) are significantly lower, indicating that beneficial mutations generated by mutation cannot be effectively locked without systematic screening.

[0140] Advantages of the experimental group: Through the synergistic effect of multi-dimensional phenotypic screening (root system + biomass + stress resistance), the superior plant rate (97.8%) was increased by 10% compared with the control group B, and the astragaloside content increased by 52.4% (18.9% vs 12.4%), proving that the screening model of the present invention can directionally enrich composite excellent traits.

[0141] 2. Comparison with traditional technology (control group A)

[0142] Limitations of single trait screening: Although the control group A met the root length requirement (31.5 cm), other indicators deteriorated across the board (fresh weight -26.2%, salt stress survival rate -38.5%), resulting in insufficient increase in the final medicinal ingredients (9.7% vs 18.9%), revealing that traditional methods cannot balance the contradiction between yield and quality.

[0143] Stability breakthrough: The coefficient of variation of the three generations of the experimental group (3.1%) was reduced by 75% compared with the control group A (12.5%), indicating that the "mutation-screening-verification" technology chain of the present invention significantly improves genetic stability and solves the problem of easy degeneration of conventional mutagenesis breeding traits.

[0144] IV. Conclusion

[0145] The present invention achieves the following creative breakthroughs through the technical synergy of expanding genetic diversity through space mutagenesis and directional enrichment of superior strains through multi-dimensional phenotypic screening:

[0146] Balance of traits: Synchronously optimize root development, biomass accumulation and stress resistance, overcoming the defects of traditional methods that "give and take care of one thing while neglecting another";

[0147] Screening accuracy: The weight distribution of the scoring model (50% for the root system) is highly consistent with the synthesis requirements of medicinal ingredients, avoiding the waste of resources in random screening;

[0148] Industrial feasibility: Standardized environmental control and genetic stability verification to ensure the large-scale replication capability of the technical solution.

[0149] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0150] In the accompanying drawings of the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments are involved. For other structures, reference may be made to the general design. Without conflict, the same embodiment and different embodiments of the present invention may be combined with each other.

[0151] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A breeding method for Astragalus membranaceus by space mutation combined with phenotypic screening, characterized in that, It includes the following steps: S1. Seed pretreatment: Space mutation: The original seeds are carried on a recoverable satellite and exposed in an orbit 200 - 400 km above the ground for 48 - 72 hours, receiving a space radiation dose of 2 - 5 Gy; Disinfection treatment: The mutated seeds are soaked in a 3 - 5% sodium hypochlorite solution for 15 - 20 minutes, and then rinsed 3 times with sterile water; Germination promotion treatment: The disinfected seeds are soaked in warm water at 40 ± 2°C for 6 hours, drained and then transferred to a petri dish lined with moist filter paper, and germinated in the dark in a constant temperature incubator at 25 ± 1°C for 48 hours until the radicle length reaches 1 - 2 mm; S2. Standardized planting: Sowing conditions: The germinated seeds are sown in the experimental field with a row spacing of 30 cm and a plant spacing of 15 cm, and the soil covering depth is 2 - 3 cm; Environmental control: An intelligent greenhouse is used to regulate the day and night temperature to 25 ± 1°C / 18 ± 1°C, and the light cycle is 14 hours per day; Water and fertilizer management: Irrigate 2 times a week to keep the soil water content at 70 - 75% of the field water holding capacity; S3. Determination of phenotypic indicators: Root development determination: The main root length ≥ 30 cm, the lateral root density ≥ 15 roots / 10 cm, and the root diameter ≥ 0.5 cm; Determination of aboveground biomass: The fresh weight per plant ≥ 80 g, and the stem diameter ≥ 4 mm; Stress resistance evaluation: The relative water content of leaves ≥ 70% under drought stress, and the survival rate ≥ 60% under salt stress (treated with 150 mM NaCl); S4. Selection of superior plants: Establish a weighted scoring model for root index, biomass, and stress resistance, and select individuals with a total score ≥ 90 points; S5. Asexual propagation: Select the stem segments of superior plants for in vitro culture; S6. Verification of genetic stability: Verify the stability of the target traits through continuous cultivation for three generations.

2. The Astragalus membranaceus breeding method by space mutation combined with phenotypic screening according to claim 1, wherein In step S1, the orbital height of the space mutation is 300 - 350 km, the exposure time is 60 hours, and the space radiation dose is 3.5 Gy.

3. The Astragalus membranaceus breeding method by space mutation combined with phenotypic screening according to claim 1, characterized in that, In step S2, the water and fertilizer management includes applying 5 g / m of compound fertilizer every two weeks 2 , and the N:P:K ratio of the compound fertilizer is 15:15:

15.

4. The astragalus breeding method by space mutation combined with phenotypic screening according to claim 1, characterized in that, In step S3, the time for root development determination is the 90th day after sowing, and the determination is completed using a root scanner.

5. The astragalus breeding method combining space mutation and phenotypic screening according to claim 4, characterized in that The resolution of the root scanner is not less than 4800 dpi.

6. The method for breeding Astragalus membranaceus by space mutation combined with phenotypic screening according to claim 1, wherein In step S3, the salt stress treatment lasts for 14 days, with 12 hours of light per day.

7. The Astragalus membranaceus breeding method by space mutation combined with phenotypic screening according to claim 1, characterized in that, In step S4, the weight distribution of the scoring model is: root index 50%, biomass 30%, and stress resistance 20%.

8. The Astragalus membranaceus breeding method by space mutation combined with phenotypic screening according to claim 1, characterized in that, In step S5, the culture medium for in vitro culture is MS basal medium supplemented with 0.5 mg / L NAA, 1.0 mg / L 6 - BA, 30 g / L sucrose, and 7 g / L agar.

9. The method for breeding Astragalus membranaceus by space mutation combined with phenotypic screening according to claim 1, characterized in that, In step S6, the genetic stability verification requires that the coefficient of variation of the target traits among three generations ≤ 5%, and the superior plant rate ≥ 95%.

10. The astragalus breeding method by space mutation combined with phenotypic screening according to claim 9, characterized in that, When verifying stability, a control group is set, and the content of astragaloside IV in the superior plant group is increased by ≥ 15% compared with the control group.

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