Establishment method of phytolacca americana seed EMS mutation library

Abstract

The invention discloses a phytolacca americana seed EMS mutation library establishment method, and belongs to the technical field of mutation library construction.The phytolacca americana seed EMS mutation library establishment method comprises the steps that concentrated sulfuric acid is used for pretreating phytolacca americana seeds, then germination accelerating is conducted on the seeds, and the seeds are treated with an EMS solution when the seeds are white, so that the problems that phytolacca americana seed coats are thick, and the sensitivity to EMS is low are solved; according to the mutagenesis method disclosed by the invention, high-density series allele point mutation can be induced and generated, the mutagenesis frequency is relatively high, and meanwhile, chromosome distortion is less caused. In the mutagenesis process, abundant genetic variation can be more efficiently created, and more possibilities are provided for screening mutants with excellent characters. Besides, the method has a remarkable advantage in the aspect of cost, the required chemical reagent is cheap, the experimental operation process is simple, and complicated equipment and technology are not required like common irradiation. The breeding cost is reduced, the working efficiency is improved, and wide popularization and application are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of mutation library construction, and more particularly to a method for establishing an EMS mutation library of pokeweed seeds. Background Art

[0002] As an invasive plant, pokeweed has attracted much attention in ecological research and environmental remediation. Pokeweed is an efficient cadmium (Cd) hyperaccumulator with significant Cd accumulation characteristics, making it a potential candidate for the remediation of Cd-contaminated soils. In addition, pokeweed has also shown its potential in remediating petroleum-contaminated soils. A patented technical document introduces a method for enhancing the ability of pokeweed to remediate petroleum-contaminated soils. This involves inoculating arbuscular mycorrhizal fungal agents into the pokeweed growth matrix to promote the absorption of mineral nutrients and growth and development of the pokeweed, thereby enhancing its remediation capacity. Therefore, pokeweed plays an important role in ecological research and environmental remediation, providing an effective ecological remediation method for solving soil pollution problems.

[0003] Constructing saturated mutant libraries is a key approach for conducting plant functional genomics and genetic breeding research. Currently, saturated mutant libraries have been constructed and related functional genomics studies have been conducted in model crops such as Arabidopsis thaliana and rice, resulting in the acquisition of a number of valuable innovative germplasm resources. In recent years, ethyl methanesulfonate (EMS) mutagenesis has been successfully applied to soybeans, rice, salvia miltiorrhiza, quinoa, alfalfa, and peas, resulting in the construction of numerous EMS mutant libraries, providing a wealth of material for plant genetic research and variety improvement.

[0004] However, among the known ethyl methanesulfonate (EMS) mutagenesis methods, methods for EMS treatment of pokeweed seeds and construction of an EMS mutant library have not been reported. Furthermore, the thick seed coat of pokeweed, which has strong toughness and protective properties, presents numerous obstacles to EMS mutagenesis. This restricts the full contact between the EMS solution and the cells within the seed, making it difficult for the mutagen to evenly and effectively penetrate key areas within the seed, such as the embryo and endosperm, to exert its effects, making EMS mutagenesis more difficult. Furthermore, pokeweed is less sensitive to EMS than traditional crops such as soybeans, rice, and salvia miltiorrhiza, further hindering the construction of a pokeweed seed mutant library.

[0005] Therefore, how to solve the problem of thick seed coat and low sensitivity to EMS in American pokeweed and provide a method for constructing an EMS mutation library of American pokeweed seeds are technical problems that technicians in this field urgently need to solve. Summary of the Invention

[0006] In view of this, the present invention provides a method for establishing an EMS mutation library of pokeweed seeds, which solves the problems of thick seed coat and low sensitivity to EMS in pokeweed, improves the construction effect, and provides more possibilities for screening mutants with excellent traits.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for establishing an EMS mutation library of pokeweed seeds comprises the following steps:

[0009] (1) Treating seeds with concentrated sulfuric acid: Select pokeweed seeds with high plumpness and soak them in concentrated sulfuric acid for 7-9 minutes;

[0010] (2) Seed germination: The seeds treated in step (1) are subjected to germination treatment until the seeds turn white and are set aside;

[0011] (3) Seed treatment: Place the whitened seeds from step (2) in an ethyl methanesulfonate solution to cover the seeds, shade them, soak and shake for 12-16 hours;

[0012] (4) Termination of reaction: Add sodium thiosulfate solution to terminate the reaction and wash the seeds;

[0013] (5) Sowing: Sow the washed seeds and screen for mutant materials.

[0014] As a preferred technical solution, the concentrated sulfuric acid in step (1) is undiluted concentrated sulfuric acid.

[0015] The seed coat of pokeweed is thick, possessing strong toughness and the ability to protect the seeds. This characteristic limits the EMS solution from fully contacting the cells within the seed, making it difficult for the mutagen to evenly and effectively penetrate key areas within the seed, such as the embryo and endosperm, to exert its effect, resulting in a poor mutagenic effect. By treating pokeweed seeds with concentrated sulfuric acid and precisely controlling the treatment time, a balance between the mutagenic effect and seed damage was achieved. This enhanced seed coat permeability after treatment with concentrated sulfuric acid allowed the EMS solution to penetrate the seed more effectively, while also avoiding excessive damage to the embryonic tissue within the seed and loss of activity, achieving the optimal mutagenic effect.

[0016] As a preferred technical solution, the process of the germination treatment in step (2) is as follows: wrap the seeds with moistened gauze and culture them at a constant temperature of 25-30°C until the seeds turn white.

[0017] The present invention uses accelerated germination to address the low sensitivity of pokeweed seeds to EMS. This treatment places the seeds in an active physiological state, increasing cell metabolism and division rates, thereby enhancing their sensitivity to the EMS mutagen and promoting the occurrence of more genetic mutations. However, controlling the degree of germination is crucial. If the germination is over-accelerated, the seed sprouts will have grown longer, and EMS mutagenesis at this stage may cause serious damage to the young shoots, affecting their subsequent growth or even causing their death. On the other hand, if the germination is under-accelerated, the seed cells will be less active, their ability to absorb and respond to EMS will be weak, and the mutagenesis effect will be less than ideal.

[0018] As a preferred technical solution, the ethyl methanesulfonate solution in step (3) is prepared by the following method: using PBS buffer as a dispersant, adding ethyl methanesulfonate to prepare an EMS solution with a concentration of 0.4%, and setting it aside; the rotation speed of the shaking treatment is 200-250rmp.

[0019] The EMS concentration used for seed mutagenesis differs significantly from that used for other plant seed mutagenesis, making it quite challenging. Considering not only the effect of seed coat thickness on mutagenesis, but also the chemical damage to seed cells caused by EMS and the duration of treatment, among other factors, is crucial. Therefore, achieving optimal mutagenesis results through balancing these factors and achieving optimal results is both a key and challenging aspect of pokeweed seed mutagenesis.

[0020] As a preferred technical solution, the mass concentration of sodium thiosulfate in the sodium thiosulfate solution in step (4) is 5%.

[0021] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a method for constructing an EMS mutation library of pokeweed seeds, which solves the problem of thick pokeweed seed coat and low sensitivity to EMS and improves the construction effect. The mutagenesis method of the present invention can induce a high-density series of allele point mutations with a high mutation frequency while causing less chromosomal aberrations. It can more efficiently create rich genetic variations, provide more possibilities for screening mutants with excellent traits, and improve the availability of mutants.

[0023] In addition, it has significant advantages in terms of cost. The chemical reagents required are relatively cheap, and the experimental operation process is relatively simple. It does not require complex equipment and technology like ordinary irradiation. It reduces the cost of breeding, improves work efficiency, and facilitates widespread promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0025] Figure 1 This is a picture of the whitening seeds of American pokeweed.

[0026] Figure 2 Figure 1: Photo of pokeweed seeds treated with EMS solution on a shaker.

[0027] Figure 3 Reference chart for the optimal EMS treatment time for other seeds.

[0028] Figure 4 Figure 2: Various phenotypes of EMS-induced mutants of Pokeweed: (a) newly emerged etiolated individuals; (b) grown-up etiolated individuals; (c) newly emerged albino individuals; (d) albino mosaic individuals; (e) individuals with shrunken cotyledons; (f), (g) individuals with deformed true leaves; (h) individuals with no phenotype difference from the wild type; (i) comparison between etiolated individuals and wild type. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Materials: high-quality pokeweed seeds stored at low temperatures in the laboratory, ethyl methanesulfonate (EMS), concentrated sulfuric acid (analytical grade, Chengdu Kelong Chemical Co., Ltd., grade: HPLC, 95.0%-98.0% CAS7664-93-9), sodium thiosulfate (analytical grade), PBS buffer (phosphate buffered saline), nutrient soil, and flower pots.

[0031] Example 1

[0032] A method for establishing an EMS mutation library of pokeweed seeds comprises the following steps:

[0033] (1) Treatment of seeds with concentrated sulfuric acid: 100 g of plump and undamaged pokeweed seeds were manually selected and placed in concentrated sulfuric acid for 8 min. During the treatment, the container was gently shaken to allow the seeds to fully contact with the concentrated sulfuric acid. After the treatment, the seeds were quickly rinsed repeatedly with clean water until neutral to remove the concentrated sulfuric acid residue.

[0034] (2) Seed germination: Wrap the seeds treated with concentrated sulfuric acid with moist gauze and place them in a constant temperature incubator at 28°C for germination. During the germination process, observe and keep the gauze moist every day until the seeds turn white. Figure 1 As shown;

[0035] (3) Preparation of EMS solution: Take 500 ml of PBS buffer as a dispersant; take 2 ml of ethyl methanesulfonate (EMS), slowly add it to the above PBS buffer, stir thoroughly to mix, and prepare an EMS solution with a concentration of 0.4%; carefully put the white seeds into a centrifuge tube, and completely wrap the centrifuge tube with tin foil to block light; place the centrifuge tube at an angle on a shaking table ( Figure 2 ), set the shaker speed to 200 rpm, pour in the prepared EMS solution to completely immerse the seeds, and shake for 12-16 hours;

[0036] The present invention refers to the treatment concentration and treatment time of other seeds EMS ( Figure 3 ), developed the concentration and time of treatment of American pokeweed seeds with EMS solution;

[0037] (4) Termination of the reaction: After the reaction is completed, 25 ml of 5% sodium thiosulfate solution is added to the centrifuge tube to terminate the reaction. The seeds are then removed from the centrifuge tube and rinsed under running water for 5 min to remove the EMS residue on the seed surface.

[0038] (5) Sowing: Sow the treated seeds in nutrient pots, identify and screen mutants at various stages of seedling growth and development, and record relevant traits; after the American pokeweed seedlings grow four true leaves, move them into pots filled with nutrient soil. During the growth period, strengthen water and fertilizer management, and regularly weed and control pests and diseases.

[0039] Verification of the mutagenesis effect of the mutagenesis breeding of the present invention

[0040] In order to verify the influence of the mutagenesis treatment of the present invention on the mutagenesis effect, the following groups were set up and the germination rates after treatment were statistically analyzed. The experimental results are shown in Table 1.

[0041] Control group (1): wild-type pokeweed seeds not treated with EMS;

[0042] Control group (2): wild-type pokeweed seeds not treated with EMS;

[0043] Control group (3): wild-type pokeweed seeds not treated with EMS;

[0044] Experimental group: pokeweed seeds treated with EMS in Example 1;

[0045] Table 1 Germination rate of Phytolacca americana seeds after treatment in different groups

[0046]

[0047] Results analysis: As shown in Table 1, the germination rate of the experimental group (EMS treatment) was 52.04%, while the germination rates of the control groups (1), (2), and (3) were 93.75%, 84.38%, and 96.88%, respectively, with an average germination rate of 91.67%. Through such a clear data comparison, it can be intuitively shown that EMS treatment has an inhibitory effect on seed germination, and it can be analyzed that the EMS mutagenesis treatment has achieved the expected semi-lethal effect. When the semi-lethal rate is reached, it means that the other half of the surviving seeds have a higher probability of undergoing gene mutations. It provides rich materials for screening plants with more mutant individual traits.

[0048] At the same time, the mutagenesis rate of the EMS mutagenesis experimental group of this application was statistically analyzed. The total mutagenesis rate was 20-23%. Many mutant traits were involved after mutagenesis, including yellow individuals, albino individuals, albino mosaic individuals, individuals with shrinking cotyledons, individuals with deformed true leaves, and individuals with no phenotype difference from the wild type. Figure 4 In one experiment, there were 178 pots (5.09%) with etiolated individuals, 81 pots (2.31%) with albino individuals, 45 pots (1.29%) with albino chimeras, 39 pots (1.11%) with shrunken cotyledons, and 130 pots (3.71%) with deformed true leaves.

[0049] The mutagenesis method of the present invention can induce a high density of allelic point mutations, with a high mutation frequency and minimal chromosomal aberrations. This means that during the mutagenesis process, a richer genetic variation can be created more efficiently, providing more possibilities for screening mutants with superior traits and improving the availability of mutants. Furthermore, it offers significant cost advantages: the required chemical reagents are relatively inexpensive, and the experimental procedure is relatively simple, unlike conventional irradiation, which requires complex equipment and techniques. This reduces breeding costs, improves work efficiency, and facilitates widespread adoption.

[0050] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0051] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for establishing an EMS mutation library of Phytolacca americana seeds, characterized in that: The steps include: (1) Treating seeds with concentrated sulfuric acid: Select pokeweed seeds with high plumpness and soak them in concentrated sulfuric acid for 7-9 minutes; (2) Seed germination: The seeds treated in step (1) are subjected to germination treatment until the seeds turn white and are set aside; (3) Seed treatment: Place the whitened seeds from step (2) in an ethyl methanesulfonate solution to cover the seeds, shade them, soak and shake for 12-16 hours; (4) Termination of reaction: Add sodium thiosulfate solution to terminate the reaction and wash the seeds; (5) Sowing: Sow the washed seeds and screen for mutant materials.

2. The method for establishing an EMS mutation library of pokeweed seeds according to claim 1, characterized in that: The concentrated sulfuric acid in step (1) is undiluted concentrated sulfuric acid.

3. The method for establishing an EMS mutation library of pokeweed seeds according to claim 1, characterized in that: The process of the germination treatment in step (2) is as follows: wrap the seeds with moistened gauze and culture them at a constant temperature of 25-30° C. until the seeds turn white.

4. The method for establishing an EMS mutation library of pokeweed seeds according to claim 1, characterized in that The ethyl methanesulfonate solution in step (3) is prepared by the following method: using PBS buffer as a dispersant, adding ethyl methanesulfonate to prepare an EMS solution with a concentration of 0.4%, and setting it aside; the rotation speed of the shaking treatment is 200-250 rpm.

5. The method for establishing an EMS mutation library of pokeweed seeds according to claim 1, characterized in that: The mass concentration of sodium thiosulfate in the sodium thiosulfate solution in step (4) is 5%.

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